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Role of NMDA receptor signaling in the regulation of inflammatory gene expression after focal brain ischemia
Abstract
Inflammatory mediators are involved in the pathogenesis of focal ischemic brain damage. In this study we used quantitative reverse transcriptase-polymerase chain reaction to analyze the spatiotemporal pattern of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and inducible nitric oxide synthase (iNOS) expression in focal ischemia of the rat brain. Focal ischemia of the rat parietal cortex was induced noninvasively by photothrombosis of cortical microvessels. In a proportion of the animals NMDA receptor signaling was blocked by the noncompetitive receptor antagonist MK-801. Within 4 h after ischemia we found induction of TNF-alpha and IL-1beta mRNA not only in the infarcts but also in all representative tissue samples removed from noninfarcted frontal, lateral, and occipital cortex of the ipsilateral, but not contralateral hemisphere. Contrastingly, the expression of iNOS mRNA remained restricted to the evolving infarcts. Pretreatment with MK-801 strongly inhibited remote cytokine expression (mean reduction by 80% relative to vehicle treated animals at 4 h; P<0.001) whereas in the lesions only partial reductions in the expression of IL-1beta and iNOS mRNA were found. Our data for the first time demonstrate remote cytokine induction following focal brain ischemia and suggest that NMDA receptor-mediated signaling can activate inflammatory gene expression independently from the occurrence of neuronal cell death.
... 18 A relationship between cytokines and glutamate toxicity has been observed in excitotoxicity models; brain damage induced by local application of NMDA increased the expression of TNF-α and IL-1β ( Figure 2). 121,122 NMDARs are widely expressed on microglia cells, and their activation may lead to neuronal death, following an extensive release of pro-and antiinflammatory factors like IL-1β and IL-4. [123][124][125] Also, the observation that concentrations of both IL-1b and TNF-α are reduced under the influence of NMDA receptor antagonists independently of neuronal death, 122 and excitotoxicity is reduced by administration of IL-1 receptor antagonists 126 further reinforces the concept that excitotoxicity and proinflammatory cytokines are not mutually exclusive. ...
... 121,122 NMDARs are widely expressed on microglia cells, and their activation may lead to neuronal death, following an extensive release of pro-and antiinflammatory factors like IL-1β and IL-4. [123][124][125] Also, the observation that concentrations of both IL-1b and TNF-α are reduced under the influence of NMDA receptor antagonists independently of neuronal death, 122 and excitotoxicity is reduced by administration of IL-1 receptor antagonists 126 further reinforces the concept that excitotoxicity and proinflammatory cytokines are not mutually exclusive. ...
Acute organophosphorus compound (OP) poisoning induces symptoms of the cholinergic crises with the occurrence of severe epileptic seizures. Seizures are induced by hyperstimulation of the cholinergic system, but are enhanced by hyperactivation of the glutamatergic system. Overstimulation of muscarinic cholinergic receptors by the elevated acetylcholine causes glutamatergic hyperexcitation and an increased influx of Ca ²⁺ into neurons through a type of ionotropic glutamate receptors, N ‐methyl‐ d ‐aspartate (NMDA) receptors (NMDAR). These excitotoxic signaling processes generate reactive oxygen species, oxidative stress, and activation of the neuroinflammatory response, which can lead to recurrent epileptic seizures, neuronal cell death, and long‐term neurological damage. In this review, we illustrate the NMDAR structure, complexity of subunit composition, and the various receptor properties that change accordingly. Although NMDARs are in normal physiological conditions important for controlling synaptic plasticity and mediating learning and memory functions, we elaborate the detrimental role NMDARs play in neurotoxicity of OPs and focus on the central role NMDAR inhibition plays in suppressing neurotoxicity and modulating the inflammatory response. The limited efficacy of current medical therapies for OP poisoning concerning the development of pharmacoresistance and mitigating proinflammatory response highlights the importance of NMDAR inhibitors in preventing neurotoxic processes and points to new avenues for exploring therapeutics for OP poisoning.
... Preclinical data suggest that a significant association exists between NMDA receptor blockade and decreased inflammation. For instance, it was reported that MK-801 administration decreases neuroinflammation in the brain by inhibiting microglial activation and reducing the production of pro-inflammatory cytokines [66][67][68]. However, there is no evidence whether NMDA receptor blockade in the hippocampus can attenuate memory impairment, anxiety-and depression-like symptoms via decreased inflammation in the hippocampus of rats with sporadic AD. ...
... Consistent with this notion, a study conducted by Liu et al. showed that treatment with NMDA receptor antagonist MK-801 altered the morphology of activated astrocytes and microglia and subsequently suppressed the expression of IL-6, TNF-α and IL-1β at both the mRNA and protein levels in the spinal cord of morphine-tolerant rats [67]. Pretreatment with MK-801 was also found to inhibit the expression of TNF-α and IL-1β in rats with focal brain ischemia [68]. Moreover, stimulation of cultured microglia with NMDA was shown to significantly increase the release of TNF-α [107]. ...
Many patients with sporadic Alzheimer's disease (AD) suffer from memory impairment, anxiety- and depression. The systemic utility of N-Methyl-d-Aspartate (NMDA) receptor antagonists has been shown to be potential therapeutic target for memory loss in AD. However, there is no evidence that shows whether NMDA receptor antagonists have the same effects when these blockers are directly used within the brain regions including hippocampus. It might be an urgent to further explore the therapeutic role of NMDA receptor antagonists in behavioral abnormalities such as anxiety and depression in AD. The aim of this study was to determine whether blockade of the hippocampal NMDA receptors could attenuate neurobehavioral abnormalities in rats with sporadic AD. Twelve days after AD induction by streptozotocin (STZ), animals received either vehicle or MK-801 (NMDA receptor antagonist) in the hippocampus for 10 days. Two or five days after the last MK-801 treatment, spatial memory, anxiety- and depression-related behaviors, and inflammatory cytokines (interleukin-(IL)-6, IL-1β and tumor necrosis factor (TNF)-α) were evaluated. Our findings indicated that STZ treatment significantly elevated hippocampal inflammation, impaired spatial memory, and increased anxiety- and depression-related symptoms in rats. Interestingly, the hippocampal NMDA receptor blockade improved these neurobehavioral phenotypes and decreased inflammatory cytokines in the hippocampus of STZ-treated rats. Hippocampal NMDA receptors might be involved in neurobehavioral abnormalities via inflammation in sporadic AD.
... The spinal cord recruits more neutrophils into a mechanically injured area than an equivalent area of similarly injured brain tissue (Schnell et al. 1999a), suggesting that there may be some differences between areas of the CNS in their responses to injury. Following a spinal cord contusion injury (Streit et al. 1998) or focal ischaemia in the parietal cortex (Jander et al. 2000), TNFa and IL-ip rtiRNAs are increased transiently, returning to baseline values within 24 hours. The direct microinjection of these proinflammatory cytokines (TNFa or IL-ip) into the brain and spinal cord also results in a differential inflammatory response, with a more pronounced neutrophil recruitment in the spinal cord than the brain (Schnell et al. 1999b). ...
p>A novel model of SCI has been generated in which microinjection of the vasoconstricting peptide endothelin-1 (ET-1) into the ventral grey matter of the rat spinal cord is used to generate an atraumatic focal ischaemic lesion. Within 15 minutes of microinjection of 15pmol ET-1, blood flow in the spinal cord is reduced by 90%, the remains below baseline values for at least one hour. Neurons and astrocytes are destroyed by the ischaemia within 6 hours, and there is widespread astrocyte activation in adjacent areas. There is a profound acute inflammatory response characterised by peak recruitment of neutrophils at 24 hours, and macrophages are present in the grey and white matter from 3 days to at least 21 days after microinjection. Macrophage numbers are maintained in the white matter from 7 to 21 days, whereas in the grey matter the number of macrophages decreases at this time.
Histological evidence of axonal injury, in the form of amyloid precursor protein (APP) positive axon profiles and ‘end-bulb’-like structures, is present 24 hours and 3 days after microinjection of ET-1 but not vehicle. Examination of the ventral white matter axons 3 days after microinjection of ET-1 using electron microscopy revealed substantial injury to the axoplasm but less injury to myelin sheaths. Microinjection of the excitotoxin N -methyl-D-aspartate into the ventral grey matter of the spinal cord resulted in more extensive myelin injury and sparing of axoplasm, which suggests that the axonal injury seen after ET-1 is not solely due to release of glutamate from injured neurons and is likely to be the result of ischaemia.
This model presents a novel opportunity to study the mechanisms of secondary injury, particularly with respect to axonal injury, in an atraumatic setting. All the characteristics of conventional SCI models are reproduced but without paralysis of the animals or the involvement of direct mechanical injury.</p
... Indeed, cortical infusion of NMDA increased the expression of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and Interleukin-1beta (IL-1b) [105]. Accordingly, administration of an NMDA antagonist reduced both IL-1b and TNF-α levels [106]; on the other hand, an interleukin-1 receptor antagonist reduced excitotoxicity [107]. Glia and immune cells control pro-inflammatory activity driven by "on" and "off" signals originating from neurons. ...
Disturbances in the glutamatergic system have been increasingly documented in several neuropsychiatric disorders, including autism spectrum disorder (ASD). Glutamate-centered theories of ASD are based on evidence from patient samples and postmortem studies, as well as from studies documenting abnormalities in glutamatergic gene expression and metabolic pathways, including changes in the gut microbiota glutamate metabolism in patients with ASD. In addition, preclinical studies on animal models have demonstrated glutamatergic neurotransmission deficits and altered expression of glutamate synaptic proteins. At present, there are no approved glutamatergic drugs for ASD, but several ongoing clinical trials are currently focusing on evaluating in autistic patients glutamatergic pharmaceuticals already approved for other conditions. In this review, we provide an overview of the literature concerning the role of glutamatergic neurotransmission in the pathophysiology of ASD and as a potential target for novel treatments.
... 10 Under physiological conditions, the substance of IL-1β was in a low level. 11 The expression of IL-1β messenger RNA (mRNA) increases rapidly following brain injury, which has a regulatory effect on inflammation and immune response. 12 Tumor necrosis factor-α (TNF-α), a multifunctional pro-inflammatory cytokine, was released by microglia and astrocytes. ...
Abstract Currently, there is no effective therapy for traumatic brain injury (TBI). Therefore, this study was conducted to determine the protective effect of Lu Tong Ke Li (LTKL), a Chinese medicine, for TBI in experimental animals. The TBI rat model was induced using the modified Feeney's protocol. The rats were divided into four groups: Sham group, Control group, LTKL lower‐dose group (LTL, 2 g/kg/day, p.o.), and LTKL higher‐dose group (LTH, 4 g/kg/day, p.o.). The Neurological Severity Score (NSS) was used to examine neurological function. Magnetic resonance imaging was performed to check the brain tissue lesions in rats. Cell apoptosis in the damaged area was evaluated using the Terminal deoxynucleotidyl transferase deoxy‐UTP‐nick end labeling assay. Reverse‐transcription polymerase chain reaction was used to investigate the expression of inflammatory cytokines, including tumor necrosis factor‐α (TNF‐α), interleukin 1β (IL‐1β), and interleukin 10 (IL‐10). The TBI rat model was successfully constructed. Neurological function was enhanced at 14, 21, and 28 days post TBI in the LTH groups, indicated by gradually decreased NSS scores. Administration of LTH led to fewer brain defects in the damaged area, and the number of apoptosis cells in the brain injury area markedly decreased. LTKL treatment led to upregulation of IL‐10 expression and downregulation of TNF‐α and IL‐1β expressions at the molecular level. LTKL can improve the neurobehavior of TBI. The neuroprotective effect was probably related to regulation of inflammation cytokines. Our results provide crucial evidence of the potentially useful application of LTKL in the therapy of TBI in clinic practice in the future.
... 46 In brain ischemia, iNOS is expressed by activated microglia and astrocytes. 47 Melatonin's protective effect on the iNOS levels that is increased in brain ischemia has been previously demonstrated by us and others. 8,48,49 However, in those studies, the prophylactic or early effect of melatonin was investigated in contrast to the present study which the melatonin's long-term therapeutic and restorative effect was investigated. ...
Objectives:
Post-ischemic inflammation leads to apoptosis as an indirect cause of functional disabilities after the stroke. Melatonin may be a good candidate for the stroke recovery because of its anti-inflammatory effects. Therefore, we investigated the effect of melatonin on inflammation in the functional recovery of brain by evaluating ipsilesional and contralesional alterations.
Materials and methods:
Melatonin (4 mg/kg/day) was intraperitoneally administered into the mice from the 3rd to the 55th day of the post-ischemia after 30 min of middle cerebral artery occlusion.
Results:
Melatonin produced a functional recovery by reducing the emigration of the circulatory leukocytes and the local microglial activation within the ischemic brain. Overall, the expression of the inflammation-related genes reduced upon melatonin treatment in the ischemic hemisphere. On the other hand, the expression level of the inflammatory cytokine genes raised in the contralateral hemisphere at the 55th day of the post-ischemia. Furthermore, melatonin triggers an increase in the iNOS expression and a decrease in the nNOS expression in the ipsilateral hemisphere at the earlier times in the post-ischemic recovery. At the 55th day of the post-ischemic recovery, melatonin administration enhanced the eNOS and nNOS protein expressions.
Conclusions:
The present molecular, biological, and histological data have revealed broad anti-inflammatory effects of melatonin in both hemispheres with distinct temporal and spatial patterns at different phases of post-stroke recovery. These outcomes also established that melatonin act recruitment of contralesional rather than of ipsilesional.
... [2,7,8] MK-801 (dizocilpine), an uncompetitive NMDAR antagonist, downregulates expression of inflammatory genes. [9] Moreover, administration of dizocilpine decreased cardiovascular inflammation following Mg 2+ deficiency in rats. [6] Another NMDAR antagonist, dextromethorphan, attenuated lung inflammation induced by heat in rats. ...
... Situés entre les cellules endothéliales et les neurones, les astrocytes contribuent au maintien de l'intégrité de la BHE grâce à leurs pieds astrocytaires qui entourent les cellules endothéliales (Risau & Wolburg, 1990 ;Abbott et al., 2006) Par ailleurs, pendant la NI, la capacité de recapture du glutamate par les astrocytes est fortement altérée, amplifiant l'excitotoxicité et les dommages neuronaux (Viviani et al., 2014). Expérimentalement, l'excitotoxicité provoquée par un excès de glutamate stimule la production de médiateurs pro-inflammatoires tels que l'IL-1β et le TNF-α (Hagan et al., 1996 ;Pearson et al., 1999 ;Jander et al., 2000 ;Viviani et al., 2014 ;Kochanek et al., 2015). ...
Le traumatisme crânien (TC) est un problème majeur de santé publique pour lequel il n'y a encore aucun traitement à l'heure actuelle. La neuro-inflammation (NI) post-traumatique et la microglie, principale actrice du système immunitaire du système nerveux central (SNC), sont de plus en plus incriminées dans la survenue des lésions de la substance blanche (LSB) à l'origine des troubles neuropsychocomportementaux post-traumatiques. Le suivi et la modulation de l'activation microgliale se révèlent donc être des enjeux scientifiques majeurs. Le but de notre travail a été d'étudier l'utilisation de la translocator protein (TSPO) comme marqueur, car elle est décrite comme spécifique de la microglie activée, à l'aide d'un modèle murin de TC développé au laboratoire. Pour cela, une technique d'imagerie en tomographie par émission de positons (TEP) a été utilisée à l'aide d'un radiotraceur ciblant TSPO, ainsi que différentes techniques d'analyses cellulaires (immunohisto- et cytochimie ; cytométrie en flux). Nos études ont révélé que l'utilisation d'un radiotraceur spécifique de TSPO en TEP ne permet pas de détecter l'activation microgliale que ce soit à 1, 3, 7 ou 14 jours après le TC dans notre modèle expérimental, temps auxquels les précédentes études du laboratoire ont déjà mis en évidence une activation microgliale. Les méthodes immunohistochimiques n'ont pas révélé de colocalisation de TSPO avec les microglies mais ont mis en évidence une importante expression endothéliale de TSPO, que les souris aient subi, ou non, un TC et ceci quel que soit le temps après le TC. Les études immunocytochimiques ont confirmé l'expression de TSPO par les cellules endothéliales d'une part, et par les microglies d'autre part. Enfin, les études de cytométrie en flux ont confirmé l'augmentation de l'expression de TSPO dans les microglies suite à un TC. Cependant, elles ont également révélé que les microglies représentent seulement 44 à 58% des cellules exprimant TSPO selon l'état de la souris et le temps après le TC (entre 1 et 3 jours post-TC). La protéine TSPO est notamment exprimée dans toutes les cellules immunitaires infiltrantes, et de façon plus importante que dans la microglie. En conclusion, le manque de spécificité de TSPO, ainsi que son polymorphisme décrit chez l'Homme, remettent en question son utilisation comme marqueur de suivi de la NI d'origine microgliale. De manière plus générale, la faible intensité de la NI induite par notre modèle de TC et la faible résolution de la TEP remettent en question l'utilisation globale de la TEP lors d'une NI microgliale de faible intensité.
... The authors suggested potential mechanisms, such as blood-brain barrier disruption-mediated parenchymal edema causing secondary vascular compression as well as inflammatory brain damage involving cytotoxic radicals, microglial activation, and cytokine release. [259][260][261] The major strengths of the photothrombosis model include reproducible infarct size and location as well as a relatively simple surgical procedure and low operative mortality. The infarct size can be controlled by changing the concentration of Rose Bengal and the duration of light illumination. ...
Despite recent advances in recanalization therapy, mechanical thrombectomy will never be a treatment for every ischemic stroke because access to mechanical thrombectomy is still limited in many countries. Moreover, many ischemic strokes are caused by occlusion of cerebral arteries that cannot be reached by intra-arterial catheters. Reperfusion using thrombolytic agents will therefore remain an important therapy for hyperacute ischemic stroke. However, thrombolytic drugs have shown limited efficacy and notable hemorrhagic complication rates, leaving room for improvement. A comprehensive understanding of basic and clinical research pipelines as well as the current status of thrombolytic therapy will help facilitate the development of new thrombolytics. Compared with alteplase, an ideal thrombolytic agent is expected to provide faster reperfusion in more patients; prevent re-occlusions; have higher fibrin specificity for selective activation of clot-bound plasminogen to decrease bleeding complications; be retained in the blood for a longer time to minimize dosage and allow administration as a single bolus; be more resistant to inhibitors; and be less antigenic for repetitive usage. Here, we review the currently available thrombolytics, strategies for the development of new clot-dissolving substances, and the assessment of thrombolytic efficacies in vitro and in vivo .
... Since it has been recently reported that a-syn impairs visuospatial learning, striatal LTP and NMDAmediating transmission [53], we assessed glial activation in the striatum of 'double-hit' Tg mice. There is a close link between neuroinflammation and NMDA receptors [54][55][56][57] and A53T + LPS-treated mice had visuospatial learning impairment in the MWM. We found opposite responses by microglial and astroglial cells in the striatum. ...
Aims
Parkinson’s disease and related disorders are devastating neurodegenerative pathologies. Since α‐synuclein was identified as a main component of Lewy bodies and neurites, efforts have been made to clarify the pathogenic mechanisms of α‐synuclein's detrimental effects. α‐synuclein oligomers are the most harmful species and may recruit and activate glial cells. Inflammation is emerging as a bridge between genetic susceptibility and environmental factors co‐fostering Parkinson’s disease. However, direct evidence linking inflammation to the harmful activities of α‐synuclein oligomers or to the Parkinson’s disease behavioural phenotype is lacking.
Methods
To clarify whether neuroinflammation influences Parkinson’s disease pathogenesis, we developed: (i) a “double‐hit” approach in C57BL/6 naïve mice where peripherally administered lipopolysaccharides were followed by intracerebroventricular injection of an inactive oligomer dose; (ii) a transgenic “double‐hit” model where lipopolysaccharides were given to A53T α‐synuclein transgenic Parkinson’s disease mice.
Results
Lipopolysaccharides induced a long‐lasting neuroinflammatory response which facilitated the detrimental cognitive activities of oligomers. LPS‐activated microglia and astrocytes responded differently to the oligomers. with microglia activating further and acquiring a proinflammatory M1 phenotype, while astrocytes atrophied. In the transgenic “double‐hit” A53T mouse model, lipopolysaccharides aggravated cognitive deficits and increased microgliosis. Again, astrocytes responded differently to the double challenge. These findings indicate that peripherally‐induced neuroinflammation potentiates the α‐synuclein oligomer’s actions and aggravates cognitive deficits in A53T mice.
Conclusions
The fine management of both peripheral and central inflammation may offer a promising therapeutic approach to prevent or slow down some behavioural aspects in α‐synucleinopathies.
... The obtained results indicate that PHHC causes excessive activation of maternal immune system, resulting in the increased blood levels of proin flammatory cytokines, such as IL 1β, that can cross the fetoplacental barrier and cause neurodegeneration and other long term disorders in the brain of the offspring [31 33]. An increased content of IL 1β could be due to the induction of its expression facilitated by the glutamate NMDA receptors, as it was demonstrated in a number of studies on focal brain ischemia [34,35]. Based on the sig nificance of NMDA receptor signaling in the HC neuro toxicity, its involvement in the development of PHHC associated conditions cannot be excluded. ...
Prenatal hyperhomocysteinemia (PHHC) in pregnant rats was induced by chronic L-methionine loading, resulting in a significant increase in the L-homocysteine content both in the mothers’ blood and blood and brain of fetuses. Significant decrease in the weight of the placenta, fetus, and fetal brain was detected by the morphometric studies on day 20 of pregnancy. PHHC also activated maternal immune system due to the increase in the content of proinflammatory inter-leukin-1β in the rat blood and fetal part of the placenta. PHHC elevated the levels of the brain-derived neurotrophic factor (BDNF, 29 kDa) and nerve growth factor (NGF, 31 kDa) precursors in the placenta and the content of the BDNF isoform (29 kDa) in the fetal brain. The content of neuregulin 1 (NRG1) decreased in the placenta and increased in the fetal brain on day 20 of embryonic development. An increase in the caspase-3 activity was detected in the brains of fetuses subjected to PHHC. It was suggested that changes in the processing of neurotrophins induced by PPHC, oxidative stress, and inflammatory processes initiated by it, as well as apoptosis, play an important role in the development of brain disorders in the offspring.
... First, histopathological characterization of brains from rats that survived acute OP-induced SE have shown that neuroinflammatory responses typically coincide spatially and temporally with neurodegeneration (Ferchmin et al., 2014;Kim et al., 1999 Fogal and Hewett, 2008). Specifically, IL-1β and TNF-α can hinder astrocytic uptake of synaptic glutamate and increase NMDA receptor activity, actions that promote excitotoxic neuronal cell death ( Jander et al., 2000;Lawrence et al., 1998;Vezzani et al., 2013). These interactions are particularly important in the context of acute OP intoxication in which excessive cholinergic stimulation due to AChE inhibition increases glutamatergic signaling (Mcdonough and Shih, 1997). ...
... Authors explained that this gradual loss of function of NMDA receptor within the temporal lobe region may contribute to some of the cognitive deficits observed in patients with AD [349]. Instead, Jander et al. suggested that NMDA receptormediated signaling can activate inflammatory gene expression independently from the occurrence of neuronal cell death [350]. ...
Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease and prion disease are not timely and effectively treated using conventional therapies. This emphasizes the need for alternative therapeutic approaches. In this respect, gene-based therapies have been adopted as potentially feasible alternative therapies, where the microRNA (miRNA) approach has experienced a great explosion in recent years. Because miRNAs have been shown to be implicated in the pathogenesis of several diseases including neurodegenerative diseases, they are intensely studied as candidates for diagnostic and prognostic biomarkers, as predictors of drug response and as therapeutic agents. In this review, we evaluate the feasibility of both direct and indirect miRNA mimics and inhibitors toward the regulation of neurodegenerative-related genes both in vivo and in vitro models, highlight the advantages and drawbacks associated with miRNA-based therapy, and summarize the relevant techniques and approaches attempted to deliver miRNAs to the central nervous system for therapeutic purposes, with particular regard to the exosomes. Additionally, we describe a new approach that holds great promise for the treatment of a wide range of diseases including neurodegenerative disorders. This approach is based on addressing the incorporation of miRNAs into exosomes to increase the quantity and quality of miRNA packed and delivered to the central nervous system and other sites of action.
... The mechanisms that are responsible for the elevated content of IL-1β in both the ipsi-and contralateral hippocampi are probably different. In the VH of the ischemic hemisphere, they may be related to glutamate excitotoxicity, because it induces IL-1β expression [30], whereas in the contralateral hemisphere it is related to corticosteroid signaling. On the other hand, the enhanced levels of IL-1β and corticosterone in the VH of the ischemic hemisphere suggest that both neuroinflammation and corticosteroid signaling are involved in the mechanisms of cellular damage and death in this region of the hippocampus. ...
Most ischemic strokes are caused by the occlusion of the middle cerebral artery (MCAO), which results in focal brain lesions in different areas of the neocortex. Secondary damage develops in brain regions located out of the infarct area, including the hippocampus. Hippocampal lesion may lead to cognitive impairments and post-stroke depression. Here, we studied the time course of changes in the levels of corticosterone and proinflammatory cytokine interleukine-1β (IL-1β) in the blood and hippocampus of rats after transient focal brain ischemia. Activation of the hypothalamo–pituitary–adrenal axis, which causes a release of corticosterone into blood, was observed at the early stage after MCAO and was accompanied by the presence of the stress hormone in the hippocampi of both the ischemic and contralateral hemispheres. We show for the first time that this effect was observed only in the ventral hippocampus (VH) but not in the dorsal hippocampus (DH). MCAO induced accumulation of the proinflammatory cytokine IL-1β, which coexisted with the elevated level of corticosterone at the early and delayed stages after reperfusion and was also observed in the VH of both hemispheres. Our data show that the VH is more vulnerable to remote damage induced by MCAO compared to the DH and corticosteroid response and neuroinflammation may be detected in the VH of both ischemic and contralateral hemispheres.
... The neuronal protective potential of theaflavin was dose dependently and the effect of 20 mg/kg theaflavin was similar to that of nimodipine. Rats subjected to cerebral ischemia-reperfusion showed typical markers of cerebral inflammation and oxidative/ni-trosative injury including leukocyte infiltration into the infarct area (enhanced MPO activity), upregulation of adhesion molecules (ICAM-1), and induction of prooxidative enzymes (COX-2 and iNOS) [36,37]. Ischemia activates a cascade that leads to the induction and expression of genes in a variety of cell types throughout the central nervous system (CNS). ...
Theaflavin, a major constituent of black tea, possesses biological functions such as the antioxidative, antiviral, and anti-inflammatory ones. The purpose of this study was to verify whether theaflavin reduces focal cerebral ischemia injury in a rat model of middle cerebral artery occlusion (MCAO). Male Sprague-Dawley rats were anesthetized and subjected to 2 hours of MCAO followed 24 hours reperfusion. Theaflavin administration (5, 10, and 20 mg/kg, IV) ameliorated infarct and edema volume. Theaflavin inhibited leukocyte infiltration and expression of ICAM-1, COX-2, and iNOS in injured brain. Phosphorylation of STAT-1, a protein which mediates intracellular signaling to the nucleus, was enhanced 2-fold over that of sham group and was inhibited by theaflavin. Our study demonstrated that theaflavin significantly protected neurons from cerebral ischemia-reperfusion injury by limiting leukocyte infiltration and expression of ICAM-1, and suppressing upregulation of inflammatory-related prooxidative enzymes (iNOS and COX-2) in ischemic brain via, at least in part, reducing the phosphorylation of STAT-1.
... Inflammatory mediators, especially IL-1β and TNF-α could induce glutamate release by neurons and glial cells, contributing to neurotoxicity mediated mainly through NMDA receptors over-activation [70][71][72]. Besides preventing neuronal damage by direct blockage of NMDA receptors activity, growing evidence supports an immunomodulatory effect of MK801 treatment during CNS insults including focal brain ischemia and spinal cord injury [27,[73][74][75]. In this scenario, we found a balance between TH1/TH2 response in frontal cortex and hippocampus of MK801 treated infected mice 10 days following cessation of CQ therapy. ...
Cerebral malaria (CM) is a life-threatening complication of Plasmodium falciparum infection, which can result in long-term cognitive and behavioral deficits despite successful anti-malarial therapy. Due to the substantial social and economic burden of CM, the development of adjuvant therapies is a scientific goal of highest priority. Apart from vascular and immune responses, changes in glutamate system have been reported in CM pathogenesis suggesting a potential therapeutic target. Based on that, we hypothesized that interventions in the glutamatergic system induced by blockage of N-methyl-D-aspartate (NMDA) receptors could attenuate experimental CM long-term cognitive and behavioral outcomes. Before the development of evident CM signs, susceptible mice infected with Plasmodium berghei ANKA (PbA) strain were initiated on treatment with dizocilpine maleate (MK801, 0.5 mg/kg), a noncompetitive NMDA receptor antagonist. On day 5 post-infection, mice were treated orally with a 10-day course chloroquine (CQ, 30 mg/kg). Control mice also received saline, CQ or MK801 + CQ therapy. After 10 days of cessation of CQ treatment, magnetic resonance images (MRI), behavioral and immunological assays were performed. Indeed, MK801 combined with CQ prevented long-term memory impairment and depressive-like behavior following successful PbA infection resolution. In addition, MK801 also modulated the immune system by promoting a balance of TH1/TH2 response and upregulating neurotrophic factors levels in the frontal cortex and hippocampus. Moreover, hippocampus abnormalities observed by MRI were partially prevented by MK801 treatment. Our results indicate that NMDA receptor antagonists can be neuroprotective in CM and could be a valuable adjuvant strategy for the management of the long-term impairment observed in CM.
... We propose that at least part of the protecting effect of NMDA-R blockers may be attributable to their therapeutic effect to reduce BBB breakdown within the periischemic/peri-injured brain. However, other non-neuronal mechanisms, including downregulation of cytokines and reduced injury-induced inflammation ( Jander et al., 2000), may contribute to the observed effect. The failure of NMDA-R antagonists as neuroprotectants in clinical trials ( Morris et al., 1999;Ikonomidou and Turski, 2002) might be attributable to variability between patients in the extent of BBB damage ( ). ...
Unlabelled:
The blood-brain barrier is a highly selective anatomical and functional interface allowing a unique environment for neuro-glia networks. Blood-brain barrier dysfunction is common in most brain disorders and is associated with disease course and delayed complications. However, the mechanisms underlying blood-brain barrier opening are poorly understood. Here we demonstrate the role of the neurotransmitter glutamate in modulating early barrier permeability in vivo Using intravital microscopy, we show that recurrent seizures and the associated excessive glutamate release lead to increased vascular permeability in the rat cerebral cortex, through activation of NMDA receptors. NMDA receptor antagonists reduce barrier permeability in the peri-ischemic brain, whereas neuronal activation using high-intensity magnetic stimulation increases barrier permeability and facilitates drug delivery. Finally, we conducted a double-blind clinical trial in patients with malignant glial tumors, using contrast-enhanced magnetic resonance imaging to quantitatively assess blood-brain barrier permeability. We demonstrate the safety of stimulation that efficiently increased blood-brain barrier permeability in 10 of 15 patients with malignant glial tumors. We suggest a novel mechanism for the bidirectional modulation of brain vascular permeability toward increased drug delivery and prevention of delayed complications in brain disorders.
Significance statement:
In this study, we reveal a new mechanism that governs blood-brain barrier (BBB) function in the rat cerebral cortex, and, by using the discovered mechanism, we demonstrate bidirectional control over brain endothelial permeability. Obviously, the clinical potential of manipulating BBB permeability for neuroprotection and drug delivery is immense, as we show in preclinical and proof-of-concept clinical studies. This study addresses an unmet need to induce transient BBB opening for drug delivery in patients with malignant brain tumors and effectively facilitate BBB closure in neurological disorders.
... Others [53] observed that KET can suppress the induction of NF-kappaB and TNF-a in endotoxin-treated rats and attenuated LPS-induced up-regulation of iNOS in rat tissues [54,55]. Evidence [56] indicates that KET has hepatoprotective effects, mediated at least in part through a reduction in COX-2 and iNOS. All these data confirm the anti-inflammatory effect of KET at subanaesthetic doses, as demonstrated in the present work that, at least partly, is due to the NMDA receptor antagonism [57]. ...
Ketamine (KET), a NMDA antagonist, exerts an antidepressant effect at subanesthetic doses and possesses analgesic and anti-inflammatory activities. We evaluated the involvement of KET antinociceptive and anti-inflammatory effects with its antidepressant action. Male Swiss mice were subjected to formalin, carrageenan-induced paw oedema and forced swimming tests, for assessing antinociceptive, anti-inflammatory and antidepressant effects. The treatment groups were: control, KET (2, 5 and 10 mg/kg), lithium (LI: 5 mg/kg) and KET2+LI5 combination. Immunohistochemistry analyses (TNF-alpha, iNOS, COX-2 and GSK3) in oedematous paws were performed. KET5 and KET10 reduced licking times in neurogenic (22 and 38%) and inflammatory (67 and 78%) phases of the formalin test, respectively, as related to controls. While LI5 inhibited the 2(nd) phase by 24%, the licking time was inhibited by 26 and 59% in the KET2+LI5 group (1(st) and 2(nd) phases). Furthermore, oedema volumes were reduced by 37 and 45% in the KET5 and KET10 groups, respectively. Oedema reductions were 29% in the LI5 group and 48% in the KET2+LI5 group. In the forced swimming test, there were 23, 38 and 53% decreases of the immobility time in KET2, KET5 and KET10 groups, respectively. While LI5 caused no significant effect, decreases of 52% were observed with KET2+LI5. KET also decreased TNF-alpha, iNOS, COX-2 and GSK3 immunostainings in oedematous paws, effects intensified with KET2+LI5. We showed that KET presents antinociceptive and anti-inflammatory effects associated to its antidepressant response. Furthermore, our results indicate the close involvement of GSK3 inhibition and blockade of inflammatory responses, in the antidepressant drug effect. This article is protected by copyright. All rights reserved.
... LDH ¼ lactate dehydrogenase; NMDA ¼ N-methyl D-aspartate. toxicity (Jander et al., 2000;Kawasaki et al., 2008;Gao et al., 2015). Others studies have demonstrated that only Ca 2þ entering the neuron through NMDARs can cause the calcium-mediated neurotoxicity (Sattler et al., 1998;Lau and Tymianski, 2010). ...
Excessive glutamate release causes overactivation of N-methyl d-aspartate receptors (NMDARs), leading to excitatory neuronal damage in cerebral ischemia. Hydroxysafflor yellow A (HSYA), a compound extracted from Carthamus tinctorius L., has been reported to exert a neuroprotective effect in many pathological conditions, including brain ischemia. However, the underlying mechanism of HSYA's effect on neurons remains elusive. In the present study, we conducted experiments using patch-clamp recording of mouse hippocampal slices. In addition, we performed Ca²⁺ imaging, Western blots, as well as mitochondrial-targeted circularly permuted yellow fluorescent protein transfection into cultured hippocampal neurons in order to decipher the physiological mechanism underlying HSYA's neuroprotective effect.
Through the electrophysiology experiments, we found that HSYA inhibited NMDAR-mediated excitatory postsynaptic currents without affecting α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and γ-aminobutyric acid A-type receptor-mediated currents. This inhibitory effect of HSYA on NMDARs was concentration dependent. HSYA did not show any preferential inhibition of either N-methyl d-aspartate receptor subtype 2A- or N-methyl d-aspartate receptor subtype 2B- subunit-containing NMDARs. Additionally, HSYA exhibits a facilitatory effect on paired NMDAR-mediated excitatory postsynaptic currents. Furthermore, HSYA reduced the magnitude of NMDAR-mediated membrane depolarization currents evoked by oxygen-glucose deprivation, and suppressed oxygen-glucose deprivation–induced and NMDAR-dependent ischemic long-term potentiation, which is believed to cause severe reperfusion damage after ischemia. Through the molecular biology experiments, we found that HSYA inhibited the NMDA-induced and NMDAR-mediated intracellular Ca²⁺ concentration increase in hippocampal cultures, reduced apoptotic and necrotic cell deaths, and prevented mitochondrial damage. Together, our data demonstrate for the first time that HSYA protects hippocampal neurons from excitotoxic damage through the inhibition of NMDARs. This novel finding indicates that HSYA may be a promising pharmacological candidate for the treatment of brain ischemia.
... 16,18 Focal ischemia in rat cortex induced the expression of IL-1ß mRNA in the infarct zone and remote areas of the ipsilateral hemisphere. 20 Early application of the antagonist, IL-1 receptor 1 antagonist (IL-1RA) reduces the infarct volume and is, therefore, neuroprotective. 18 Interestingly, activation of the inflammasome and the release of IL-1ß is stimulated by high extracellular potassium concentration, 19 a stimulus which can elicit CSD. ...
During brain damage and ischemia, the cytokine interleukin-1ß is rapidly upregulated due to activation of inflammasomes. We studied whether interleukin-1ß influences cortical spreading depolarization, and whether lipopolysaccharide, often used for microglial stimulation, influences cortical spreading depolarizations. In anaesthetized rats, cortical spreading depolarizations were elicited by microinjection of KCl. Interleukin-1ß, the IL-1 receptor 1 antagonist, the GABAA receptor blocker bicuculline, and lipopolysaccharide were administered either alone or combined (interleukin-1ß + IL-1 receptor 1 antagonist; interleukin-1ß + bicuculline; lipopolysaccharide + IL-1 receptor 1 antagonist) into a local cortical treatment area. Using microelectrodes, cortical spreading depolarizations were recorded in a non-treatment and in the treatment area. Plasma extravasation in cortical grey matter was assessed with Evans blue. Local application of interleukin-1ß reduced cortical spreading depolarization amplitudes in the treatment area, but not at a high dose. This reduction was prevented by IL-1 receptor 1 antagonist and by bicuculline. However, interleukin-1ß induced pronounced plasma extravasation independently on cortical spreading depolarizations. Application of lipopolysaccharide reduced cortical spreading depolarization amplitudes but prolonged their duration; EEG activity was still present. These effects were also blocked by IL-1 receptor 1 antagonist. Interleukin-1ß evokes changes of neuronal activity and of vascular functions. Thus, although the reduction of cortical spreading depolarization amplitudes at lower doses of interleukin-1ß may reduce deleterious effects of cortical spreading depolarizations, the sum of interleukin-1ß effects on excitability and on the vasculature rather promote brain damaging mechanisms.
... Tissue regions that surround the ischemic infarct, which are at risk of ongoing tissue damage are often termed the peri-infarct zone (PIZ) (Carmichael, 2005;Katsman et al., 2003). In addition to the spread of toxic products from the ischemic infarct into the PIZ, glial and macrophage activation from the ensuing inflammatory response often occur in this region and may also contribute to ongoing cell death Katsman et al., 2003;Schroeter et al., 1997;Jander et al., 2000;Stoll et al., 1998;Nowicka et al., 2008;Braun et al., 1996). The PIZ is also a major site of brain plasticity associated with spontaneous recovery and compensation after stroke (Carmichael, 2006;Murphy and Corbett, 2009). ...
... There are still questions to be answered in this pathway. Jander et al. (2000) identified a role for glutamate receptors in the induction of Figure 25. Schematic diagram illustrating the effects of HI-induced IL-1 increases on NF-κB activation. ...
... An accumulation of experimental data suggests that NMDARs antagonists prevent ischemic neuronal injury following transient global ischemia and reduce infarct volumes following focal ischemic insults. The excitotoxic hypothesis states that the excitatory amino acid neurotransmitter L-glutamate has neurotoxic properties that can be attenuated by antagonism of the NMDARs [88,89]. ...
Abstract N-methyl-D-aspartate ionotropic glutamate receptor (NMDARs) is a ligand-gated ion channel that plays a critical role in excitatory neurotransmission, brain development, synaptic plasticity associated with memory formation, central sensitization during persistent pain, excitotoxicity and neurodegenerative diseases in the central nervous system (CNS). Within iGluRs, NMDA receptors have been the most actively investigated for their role in neurological diseases, especially neurodegenerative pathologies such as Alzheimer's and Parkinson's disease. It has been demonstrated that excessive activation of NMDA receptors (NMDARs) plays a key role in mediating some aspects of synaptic dysfunction in several CNS disorders, so extensive research has been directed on the discovery of compounds that are able to reduce NMDARs activity. This review discusses the role of NMDARs on neurological pathologies and the possible therapeutic use of agents that target this receptor. Additionally, we delve into the role of NMDARs in Alzheimer's and Parkinson's diseases and the receptor antagonists that have been tested on in vivo models of these pathologies. Finally we put into consideration the importance of antioxidants to counteract oxidative capacity of the signaling cascade in which NMDARs are involved.
... Numerous studies have implicated NMDA-type glutamate receptors in various CNS pathologies. Inhibition of NMDA receptor stimulation was suggested as a treatment for a wide variety of CNS pathologies ranging from neurodegenerative diseases such as Huntington's, Parkinson's (Steece-Collier et al., 2000) and HIV dementia (Epstein, 1998) to epilepsy (Sagratella, 1995), brain ischemia (Williams et al., 2000;Jander et al., 2000), chronic pain and drug and alcohol addiction (Bisaga and Popik, 2000). ...
This chapter explores that the exact mechanisms that lead to demyelination, axonal damage, and death of oligodendrocytes in multiple sclerosis (MS) are still unknown. Among the mechanisms implicated are contact with cytotoxic immune cells, antibodies, and soluble mediators especially proinflammatory cytokines. It discusses that in glutamate excitotoxicity, agonist binding to ionotropic glutamate receptors leads to influx of sodium and calcium ions; a cell membrane depolarizing mechanism. Overstimulation, ion influxes, and membrane depolarization is then conducive to the activation of destructive processes, such as interruption of electrolyte and fluid balance, phospholipase and protease activation, and formation of free radicals and activation of cell death pathways. The chapter also reviews the recent findings that expand the mechanisms of glutamate excitotoxicity from gray matter diseases to MS, a white matter disease. For glutamate excitotoxicity in white matter to be a valid mechanism of damage, the presence of glutamate receptors and one or more of these elements are required.
... Ischemic condition accelerates the release of excitatory amino acids, nitric oxide, and free radicals, and induces microglial activation and programmed cell death [23][24][25]. iNOS is mainly expressed in astrocytes and activated microglia during focal brain ischemia [26]. iNOS knockout mice reduce brain damage after ischemia and the increased expression of iNOS contributes to neuronal injury [25]. ...
Nitric oxide (NO) is generated by three different NO synthase (NOS) isoforms, endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS). It is known that eNOS produces NO, which exerts a protective effect, while iNOS produces NO with neurotoxic effects. Ferulic acid preserves neuronal cells against from cerebral ischemia and glutamate-induced excitotoxicity. This study confirmed the neuroprotective effect of ferulic acid and investigated the levels of three NOS isoforms in focal cerebral ischemia with or without ferulic acid. Rats were immediately treated with ferulic acid (100 mg/kg, i.v.) after middle cerebral artery occlusion (MCAO). Brains tissues were collected at 24 h after the onset of occlusion. The expressions of these three isoforms in cerebral ischemia with ferulic acid were analyzed using Western blot technique. Ferulic acid treatment significantly decreases the number of TUNEL-positive cells in the cerebral cortex against MCAO injury. The levels of eNOS decreased in MCAO-operated animals, while ferulic acid treatment attenuated the MCAO-induced decrease of eNOS. However, iNOS and nNOS expression levels increased during MCAO, and ferulic acid prevented injury-induced increase of these isoforms. Thus, these findings suggest that the up- and down modulation of three isoforms by ferulic acid is associated with a neuroprotective mechanism.
... Moreover, increased immunoreactivity against iNOS following transient ischemia was shown to correlate with a decrease of nNOS in the hippocampus, which is concomitant with an increased neurogenesis [116, 129]. Numerous works showed that ischemia-induced neurogenesis in DG involves the activation of NMDA receptors [130], which is simultaneous to increased iNOS expression [131, 132] (Table 3). However, in a study regarding the effects of NO in cell proliferation, both nNOS- and iNOS-derived NO increases neurogenesis following seizures in the DG of adult rats [133]. ...
The finding that neural stem cells (NSCs) are able to divide, migrate, and differentiate into several cellular types in the adult brain raised a new hope for restorative neurology. Nitric oxide (NO), a pleiotropic signaling molecule in the central nervous system (CNS), has been described to be able to modulate neurogenesis, acting as a pro- or antineurogenic agent. Some authors suggest that NO is a physiological inhibitor of neurogenesis, while others described NO to favor neurogenesis, particularly under inflammatory conditions. Thus, targeting the NO system may be a powerful strategy to control the formation of new neurons. However, the exact mechanisms by which NO regulates neural proliferation and differentiation are not yet completely clarified. In this paper we will discuss the potential interest of the modulation of the NO system for the treatment of neurodegenerative diseases or other pathological conditions that may affect the CNS.
... Ketamine interacts with many other receptors, such as dopaminergic, serotonergic, and other receptors (44). Neuronal proliferation in the hippocampus may also be affected by NMDA blockadeinduced potential changes in the local microenvironments, including cytokine secretion, activation or deactivation of neurons and astrocytes (45). ...
High doses or prolonged exposure to ketamine increase neuronal apoptosis in the developing brain, although effects on neural stem progenitor cells remain unexplored. This study investigated dose- and time-dependent responses to ketamine on cell death and neurogenesis in cultured rat fetal cortical neural stem progenitor cells.
Laboratory-based study.
University research laboratory.
Sprague-Dawley rats.
Neural stem progenitor cells were isolated from the cortex of Sprague-Dawley rat fetuses on embryonic day 17. In dose-response experiments, cultured neural stem progenitor cells were exposed to different concentrations of ketamine (0-100 µM) for 24 hrs. In time-course experiments, neural stem progenitor cells cultures were exposed to 10 µM ketamine for different durations (0-48 hrs).
Apoptosis and necrosis in neural stem progenitor cells were assessed using activated caspase-3 immunostaining and lactate dehydrogenase assays, respectively. Proliferative changes in neural stem progenitor cells were detected using bromo-deoxyuridine incorporation and Ki67 immunostaining. Neuronal differentiation was assessed using Tuj-1 immunostaining. Cultured neural stem progenitor cells were resistant to apoptosis and necrosis following all concentrations and durations of ketamine exposure tested. Ketamine inhibited proliferation with decreased numbers of bromo-deoxyuridine-positive cells following ketamine exposure to 100 µM for 24 hrs (p<.005) or 10 µM for 48 hrs (p< .01), and reduced numbers of Ki67-positive cells following exposure to ketamine concentration>10 µM for 24 hrs (p<.001) or at 10 µM for 48 hrs (p<.01). Ketamine enhanced neuronal differentiation, with all ketamine concentrations increasing Tuj-1-positive neurons (p<.001) after 24-hrs of exposure. This also occurred with all exposures to 10 µM ketamine for >8 hrs (p<.001).
Clinically relevant concentrations of ketamine do not induce cell death in neural stem progenitor cells via apoptosis or necrosis. Ketamine alters the proliferation and increases the neuronal differentiation of neural stem progenitor cells isolated from the rat neocortex. These studies imply that ketamine exposure during fetal or neonatal life may alter neurogenesis and subsequent brain development.
... After traumatic SC injury, MK801 treatment reduced inflammatory reactions including enhanced iNOS expression and improved function recovery (Esposito et al. 2011). A similar phenomenon was also observed after hypoxic brain injury (Jander et al. 2000). Our results suggest similar NMDAR-mediated NOS expression is involved in interneurons for the mediation of PDN of type 2 diabetes. ...
Activation of the neuronal-glial network in the spinal cord dorsal horn (SCDH) mediates various chronic painful conditions. We studied spinal neuronal-astrocyte signaling interactions involved in the maintenance of painful diabetic neuropathy (PDN) in type 2 diabetes. We used the db/db mouse, an animal model for PDN of type 2 diabetes, which develops mechanical allodynia from 6 to 12 wk of age. In this study, enhanced substance P expression was detected in the presynaptic sensory fibers innervating lamina I-III in the lumbar SCDH (LSCDH) of the db/db mouse at 10 wk of age. This phenomenon is associated with enhanced spinal ERK1/2 phosphorylation in projection sensory neurons and regional astrocyte activation. In addition, peak phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptor (NMDAR), along with upregulation of neuronal and inducible nitric oxide synthase (nNOS and iNOS) expression were detected in diabetic mice. Expression of nNOS and iNOS was detected in both interneurons and astrocytes in lamina I-III of the LSCDH. Treatment with MK801, an NMDAR inhibitor, inhibited mechanical allodynia, ERK1/2 phosphorylation, and nNOS and iNOS upregulation in diabetic mice. MK801 also reduced astrocytosis and glial acidic fibrillary protein upregulation in db/db mice. In addition, N(G)-nitro-L-arginine methyl ester (L-NAME), a nonspecific NOS inhibitor, had similar effects on NMDAR signaling and NOS expression. These results suggest that nitric oxide from surrounding interneurons and astrocytes interacts with NMDAR-dependent signaling in the projection neurons of the SCDH during the maintenance of PDN.
... 108,109 Rapidly evolving ischemic cell death occurs in the irradiated cortical bed, as measured by nonselective indicators of cell death, such as terminal deoxynucleoti-dyl transferase-mediated biotinylated uridine triphosphate nick end labeling (TUNEL) staining, or indicators of apoptotic progression, such as cytoplasmic cytochrome c. 110,111 T cells infiltrate the edge of the lesion, followed by microglial/macrophage activation and both local and distant cytokine production in cortex. 112,113 The advantages of this model are the small size of the infarcts, the ability to place the infarct within distinct functional subdivisions of cortex, and the minimal surgical manipulation of the animal. This model has recently been modified in the mouse so that rose-bengal is administered intraperitoneally, further streamlining the technique. ...
Rodent stroke models provide the experimental backbone for the in vivo determination of the mechanisms of cell death and neural repair, and for the initial testing of neu-roprotective compounds. Less than 10 rodent models of focal stroke are routinely used in experimental study. These vary widely in their ability to model the human disease, and in their application to the study of cell death or neural repair. Many rodent focal stroke models produce large infarcts that more closely resemble malignant and fatal human infarction than the average sized human stroke. This review focuses on the mechanisms of ischemic damage in rat and mouse stroke models, the relative size of stroke generated in each model, and the purpose with which focal stroke models are applied to the study of ischemic cell death and to neural repair after stroke.
Fluorosis is a widespread endemic disease. Reports have shown that high fluoride causes the dysfunction of central nervous system (CNS) in animals. The neurotoxicity of fluoride may be related to the activation of microglia. Moreover, numerous studies have found that exercise facilitates the plasticity of structure and function in CNS, partly owing to the regulation of microglia activation. The present study was conducted to explore the effect of exercise on the microglial activation of hippocampus in fluorosis mice. One hundred adult female Institute of Cancer Research (ICR) mice were randomly divided into 4 groups: control group (group C, distilled water by gavage); exercise group (group E, distilled water by gavage and treadmill exercise); fluoride group [group F, 24 mg/kg sodium fluoride (NaF) by gavage]; fluoride plus exercise group (group F + E, 24 mg/kg NaF by gavage and treadmill exercise). After 8 weeks, hippocampal morphological structure, microglial activation and RNA transcriptome of mice in each group were evaluated by hematoxylin and eosin (HE) staining, Nissl staining, immunohistochemistry (IHC), quantitative real time PCR (QRT-PCR) and transcriptome sequencing. We discovered that the number of M1-type microglia in fluorosis-mice hippocampus was significantly increased when compared to group C; group F + E showed a decrease in the number of M1-type microglia with the comparison to group F. In addition, the hippocampal transcriptome analysis showed that 576 differential expression genes (DEG) were confirmed in group F, compared to group C, and 670 DEG were differently expressed in group F + E when compared to group F. Gene Ontology (GO) analysis showed that changed genes were implicated in regulation of transcription, DNA-templated, integral component of membrane and adenosine triphosphate (ATP) binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of 670 DEG was helpful to find neuroactive ligand-receptor interaction pathway. In conclusion, these results indicate that treadmill running inhibits the excessive activation of microglia in hippocampus of the fluoride-toxic mice, accompanied with the alteration of neuroactive ligand-receptor interaction pathway.
Cerebral endothelial cells accomplish numerous tasks connected to the maintenance of homeostasis of the central nervous system. They create a barrier between the central nervous system and peripheral blood and regulate mechanotransduction, vascular permeability, rheology, thrombogenesis, and leukocyte adhesion. In pathophysiological conditions (e.g., stroke or ischemia-reperfusion injury) the endothelial functions are impaired, leading to increased vascular permeability, vascular inflammation, leukocyte-endothelium interactions, and transendothelial migration, driving CNS inflammation and neuronal destruction. This review describes the current knowledge on the regulatory roles of endothelial cells in neuroinflammatory processes.
Objective: This study was focused on screening leech extracts to identify those with little or no anti-coagulation effect or with significant anti-endothelial dysfunction activity.
Methods: Different leech extracts were prepared by enzymolysis and microbial transformation and their cytotoxicity were measured by MTT assay. The effect of different leech extracts on mRNA expression of coagulation-related factors (PAI, vWF, tPA, PS, TFPI, TM) was quantified by RT-PCR. After identifying a leech extract with little anti-coagulatory effect, RT-PCR was then used to assess the effect of this extract on the mRNA expression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, IL-6).
Results: 8 leech extracts were obtained, including 4 enzymatic extracts (LP, PHL, PTHL, CEHL) and 4 Lactobacillus metabolites (MRS, MRS-1, MRS-2, and MRS-3). Following optimization of conditions using MTT assays, we treated EA.hy926 cells with 0, 12.5, 25, 50 μg/mL of LP, PTHL, CEHL, MRS, MRS-1 or MRS-3 extract for 24 h. We found that PHL and MRS-1 had no significant effect on coagulation-related factors. Furthermore, treatment with 50 μg/mL PHL resulted in significant decreases in ET-1, iNOS, MCP-1, and IL-6 mRNA expression by 28.06%, 33.30%, 19.80%, and 52.34%, respectively.
Conclusions: In the present study, we found that PHL, a pepsin hydrolysate of leech with little anti-coagulatory effect, could significantly suppress TNF-α induced mRNA overexpression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, and IL-6). These results provide a reliable experimental basis for identifying new anti-atherosclerosis therapeutics for long term use and with minimal bleeding side effects.
The relationship between stress challenges and adverse health outcomes, particularly for the development of affective disorders, is now well established. The highly conserved neuroimmune mechanisms through which responses to stressors are transcribed into effects on males and females have recently garnered much attention from researchers and clinicians alike. The use of animal models, from mice to guinea pigs to primates, has greatly increased our understanding of these mechanisms on the molecular, cellular, and behavioral levels, and research in humans has identified particular brain regions and connections of interest, as well as associations between stress-induced inflammation and psychiatric disorders. This review brings together findings from multiple species in order to better understand how the mechanisms of the neuroimmune response to stress contribute to stress-related psychopathologies, such as major depressive disorder, schizophrenia, and bipolar disorder.
Except in response to localized conditions of infection or inflammation, polymorphonuclear (PMN) leukocytes and cells of the monocyte/macrophages lineage do not invade the brain parenchyma. Rather, the brain is a transit organ for these leukocyte subclasses. Importantly, the microvasculature provides the scaffolding for the cellular inflammatory response to ischemia. Both the humoral (i.e., cytokine, chemokine) and cellular components of the inflammatory response interact with stimulated microvessels to initiate post-ischemic vascular and tissue injury. In the ischemic basal ganglia, very early changes in microvessel integrin-extracellular matrix (ECM) interactions, PA and MMP release, and vascular cell activation occur in temporal and spatial association with the development of severe neuron injury. Specific changes in microvessel integrity and permeability (50, 94), loss of basal lamina integrity (92, 93), simultaneous decreases in specific endothelial cell and astrocyte integrins (207, 223), and changes in astrocyte ultrastructure (54, 78) occur during this early period. Selected events (e.g., VEGF upregulation) occur along with early evidence of inflammatory cell activation. Moreover, activation of cerebral microvessels within the ischemic core regions is evident within 1–2 hours following middle cerebral artery occlusion (MCA:O) in primate species. These events suggest relationships at multiple levels involving microvessel integrin-matrix responses, inflammatory cell invasion, and cellular activation to effect vascular, glial, and neuron injury following focal cerebral ischemia.
Microglia, the resident immune cells of the brain, perform elaborate surveillance in which they physically interact with neuronal elements. A novel form of microglia–neuron interaction named microglial process convergence (MPC) toward neuronal axons and dendrites has recently been described. However, the molecular regulators and pathological relevance of MPC have not been explored. Here, using high-resolution two-photon imaging in vivo and ex vivo, we observed a dramatic increase in MPCs after kainic acid– or pilocarpine-induced experimental seizures that was reconstituted after glutamate treatment in slices from mice. Interestingly, a deficiency of the fractalkine receptor (CX3CR1) decreased MPCs, whereas fractalkine (CX3CL1) treatment increased MPCs, suggesting that fractalkine signaling is a critical regulator of these microglia–neuron interactions. Furthermore, we found that interleukin-1β was necessary and sufficient to trigger CX3CR1-dependent MPCs. Finally, we show that a deficiency in fractalkine signaling corresponds with increased seizure phenotypes. Together, our results identify the neuroglial CX3CL1–CX3CR1 communication axis as a modulator of potentially neuroprotective microglia–neuron physical interactions during conditions of neuronal hyperactivity.
Prolonged abuse of methylphenidate (MPH) often causes neuronal damage. Topiramate (TPM) has neuroprotective properties, but its mechanism of action remains unclear. The current study evaluates in vivo role of various doses of TPM (10, 30, 50, 70 and 100 mg/kg) and its possible mechanisms against MPH-induced hippocampal oxidative stress, inflammation and apoptosis, in absence and presence of different receptor agonists and antagonists. Domoic acid (DOM) as AMPA/kainate receptor agonist, bicuculline (BIC) as GABAA receptor antagonist, ketamine (KET) as NMDA receptor antagonist, yohimbine (YOH) as ɑ2 adrenergic receptor antagonist and haloperidole (HAL) as D2 dopamine receptor antagonist was used. Open Field Test (OFT) was used to investigate the disturbances in motor activity. Hippocampal oxidative, anti-oxidant and inflammatory parameters and apoptotic factors were studied. Expressions of BDNF at gene and protein levels were also evaluated. Crystal violet staining was performed to determine neuronal cell density. TPM (70 and 100 mg/kg) reduced MPH-induced rise in lipid peroxidation, oxidized form of glutathione (GSSG), IL-1β and TNF-α levels, Bax expression and motor activity disturbances. In addition, TPM treatment increased BDNF gene and protein expressions, Bcl-2 expression, the level of reduced form of glutathione (GSH) and activities of enzymes superoxide dismutase, glutathione peroxidase and glutathione reductase.TPM also inhibited MPH-induced hippocampal degeneration. Pretreatment of animals with DOM, BIC, KET and YOH inhibited TPM-induced neuroprotection and increased oxidative stress, neuroinflammation, neuroapoptosis and neurodegeneration while reducing BDNF expressions. Thus, TPM by interacting with AMPA/kainate, GABAA, NMDA and ɑ2-adrenergic receptors improves BDNF expression and acts as a neuroprotective agent against MPH-induced neurodegeneration.
Studies of inflammatory mediators have established the tumor micro-environment as a driver of oncogenesis. This inflammatory milieu often precedes cancer, however recent data also point to the ability of oncogenic changes to induce inflammatory responses that are later harnessed by the tumor to survive and proliferate. In this review, we propose that the IDH1 mutation, present in the majority of low-grade gliomas (LGGs), initiates an inflammatory cascade that is ultimately hijacked by the tumor. Glioma infiltrating macrophages and microglia (GIMs) are polarized to the M2 phenotype, subverting the host's adaptive immune response, and fostering a tumor milieu ripe for angiogenesis, migration, and metastasis. As data continue to expand the role of inflammation in low-grade gliomas, new molecular pathways may emerge as therapeutic targets that offer a window of opportunity to intervene before the malignant transformation (MT) of LGGs occurs.
Apoptosis, or programmed cell death, is a complex and strictly regulated cellular event. A prerequisite for apoptosis is the existence of highly conserved death programs within the cell genome. The induction of apoptosis is triggered by various intrinsic or extrinsic death signals. In brain these include, excitatory amino acids (EAA), calcium fluxes, free radicals, release of apoptogenic factors from damaged mitochondrion, binding of ligands to corresponding cell surface death receptors, lipid signaling molecules, and other toxins. Moreover, apoptosis can also be triggered by the loss of survival signals. Once initiated, organized cascades consisting of intracellular proenzymes, coenzymes, scaffold and chaperon proteins, and related molecules are set into motion. These pathways converge to a committed point, whereupon effector enzymes are activated that simultaneously lead to cytoskeletal disintegration, nuclear condensation, and digestion of DNA. Finally, the engulfment of the dead cell or cell fragments by phagocytosis peacefully terminates the death program. Incomplete execution of programmed cell death may redirect the cell to necrosis, which is an unfavorable event for the organism as a whole. Integrated cell death programs have been detected in all multi-cellular and unicellular organisms, including bacteria, and have been characterized in cell culture and in vivo models.
Focal impairment, or cessation of blood flow to the brain, restricts the delivery of substrates, most importantly oxygen and glucose, and thereby impairs maintenance of ionic gradients. This is followed by depolarization of neurons and glia that release excitatory amino acids (glutamate) into the extracellular space and accumulate Ca2+ (reviewed in Dirnagl et al. (1999)). Ca2+ is a universal second messenger leading to production of proteolytic enzymes and free-radical species, and activation of glutamate receptors. In the center of the ischemic territory, where the flow reduction is most severe, these processes induce rapid cell death. A significant proportion of neurons, however, dies by an internal program of self-destruction, designated apoptosis or programmed cell death (Bredesen (1995)). Apoptotic neurons are intermingled with necrotic neurons in the core of infarctions. In the boundary zone, apoptotic cell death is ongoing during the first week after focal ischemia (Li et al. (1995); Braun et al. (1996); Isenmann et al. (1998)). Accordingly, several studies using modem imaging techniques provided evidence for infarct growth during the first few days after cerebral ischemia (Marchal et al. (1996); Beaulieu et al. (1999)). In experimental animals, mediators of the immune system appear to play an essential role in this secondary infarct growth. Mice lacking interferon regulatory factor (IRF), a nuclear transcription factor, developed similar infarct volumes at 24 hours, but significant differences in favor of the knock-out animals became evident at day 3 (Iadecola et al. (1999)).
Stroke due to focal brain ischemia is the leading cause of persistent neurological disability in modern western societies. Current thrombolysis therapy is only effective within a narrow time window of 3 to 6 hours. Substantial evidence however indicates that the evolution of ischemic brain damage is a dynamic process extending well into subacute stages of days to weeks after the insult. Lesion-associated inflammation is an integral part of the cellular pathobiology of brain infarction. Within hours after ischemia resident brain macrophages/microglia are activated and produce a wide range of potentially harmful mediators. During later stages, activated monocytes and macrophages migrate from the blood stream into infarcted brain tissue. Brain inflammation may cause secondary infarct growth and inhibit the regenerative capacity of injured brain tissue, but can also mediate neuroprotection and lesion repair. Inflammatory cell recruitment can now be monitored using iron oxide nanoparticles as cell-specific contrast agents for magnetic resonance imaging. These new imaging techniques will contribute to the development of therapies targeting neuroinflammation in ischemic stroke.
Objective:
To investigate the effects of preconditioning and postconditioning with isoflurane on pro-inflammatory cytokines and lipid peroxidation in focal cerebral ischemic/reperfusion (I/R) injury in rats.
Methods:
Thirty-two Sprague-Dawley (SD) rats were randomly divided into four groups: control group, model group, isoflurane preconditioning group and isoflurane postconditioning group, with 8 rats in each group. Rats in control group did not receive any challenge. In rats of model group right middle cerebral artery occlusion (MCAO) was conducted for 90 minutes. Rats in isoflurane preconditioning group received 2% isoflurane exposure for 30 minutes 24 hours before MCAO for 90 minutes. Rats in isoflurane postconditioning group were given 60-minute 2% isoflurane exposure after reperfusion of right MCAO. Twenty-four hours after the procedure, all rats were anesthetized with isoflurane, and blood sample taken from the heart was centrifuged, and the pro-inflammatory cytokines, including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and lipid peroxidation products such as malonaldehyde (MDA) and superoxide dismutase (SOD) were determined. The mRNA and protein expression levels of matrix metalloproteinase (MMP-2, MMP-9), tight junction protein Calaudin-5 and Occludin were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot.
Results:
Compared with control group, serum levels of IL-1β, TNF-α and MDA were elevated and activity of SOD decreased in rats of model group (IL-1β: 76.81±11.14 ng/L vs. 52.43 ± 8.86 ng/L, TNF-α: 64.93 ± 10.81 ng/L vs. 33.64 ± 7.94 ng/L, MDA: 8.63 ± 1.42 μmol/L vs. 4.14 ± 0.98 μmol/L, SOD: 0.95 ± 0.21 U/L vs. 2.36 ± 0.80 U/L, all P<0.05). After isoflurane preconditioning and postconditioning, compared with model group, the levels of IL-1β, TNF-α and MDA were lowered, while activity of SOD was increased (IL-1β: 54.37 ± 9.06 ng/L, 56.82 ± 8.67 ng/L vs. 76.81 ± 11.14 ng/L, TNF-α: 43.72 ± 6.16 ng/L, 39.49 ± 9.34 ng/L vs. 64.93 ± 10.81 ng/L, MDA: 5.65 ± 0.83 μmol/L, 5.82 ± 0.78 μmol/L vs. 8.63 ± 1.42 μmol/L, SOD: 1.64 ± 0.47 U/L, 1.71 ± 0.52 U/L vs. 0.95 ± 0.21 U/L, all P<0.05). Focal cerebral I/R injury could lead to an increased expression of MMP accompanied with a decreased expression of tight junction protein. Compared with model group, after isoflurane preconditioning and postconditioning, it was found that there were decreased mRNA and protein expression of MMP-2 and MMP-9 (MMP-2 mRNA: 1.25 ± 0.08, 1.32 ± 0.12 vs. 2.48 ± 0.26, MMP-2 protein: 1.56 ± 0.09, 1.50 ± 0.08 vs. 2.12 ± 0.11; MMP-9 mRNA: 1.26 ± 0.13, 1.20 ± 0.12 vs. 2.74 ± 0.28, MMP-9 protein: 1.53 ± 0.04, 1.51 ± 0.05 vs. 2.23 ± 0.09, all P<0.05) and increased levels of Calaudin-5 and Occludin (Claudin-5 mRNA: 0.40 ± 0.08, 0.38 ± 0.06 vs. 0.28 ± 0.03, Claudin-5 protein: 0.80 ± 0.06, 0.81 ± 0.07 vs. 0.39 ± 0.02; Occludin mRNA: 0.54 ± 0.07, 0.50 ± 0.08 vs. 0.26 ± 0.06, Occludin protein: 0.64 ± 0.06, 0.69 ± 0.05 vs. 0.49 ± 0.02, all P<0.05).
Conclusions:
Preconditioning and postconditioning with isoflurane can lower the levels of pro-inflammatory cytokines and the degree of lipid peroxidation, and lower the hydrolytic activity of MMP to the tight junction protein in cerebral tissue, thereby decrease the loss of tight junction protein and alleviate I/R injury.
Emerging evidences underline the ability of several environmental contaminants to induce an inflammatory response within the central nervous system, named neuroinflammation. This can occur as a consequence of a direct action of the neurotoxicant to the CNS and/or as a response secondary to the activation of the peripheral inflammatory response. In both cases, neuroinflammation is driven by the release of several soluble factors among which pro-inflammatory cytokines. Il-1β and TNF-α have been extensively studied for their effects within the CNS and emerged for their role in the modulation of the neuronal response, which allow the immune response to integrate with specific neuronal functions, as neurotransmission and synaptic plasticity. In particular, it has been evidenced a potential detrimental link between these cytokines and the glutamatergic system that seems to be part of increased brain excitability and excitotoxicity occurring in different pathological conditions. Aim of this mini-review will be to present experimental evidence on the way IL-1β and TNF-α impact neurons, focusing on the glutamatergic signalling, to provide a perspective on novel pathways possibly involved in environmental contaminants neurotoxicity.
There is increasing evidence that inflammatory processes play a central role in atherosclerosis and in secondary infarct growth after focal cerebral ischaemia. Focal cerebral ischaemia is often the result of arterio-arterial thromboembolism arising from plaques in the internal carotid artery (ICA). In the ICA, the extent of inflammatory infiltration by T cells and macrophages, and the expression of matrix metalloproteinase-9 in high grade stenoses, correlate with clinical features of plaque destabilisation.
Within the CNS, focal ischaemia induces a strong inflammatory response, with recruitment of granulocytes, T cells and macrophages which is facilitated by early upregulation of cell adhesion molecules. In experimental animals, anti-adhesion strategies have led to a dramatic reduction of stroke volumes; however, these strategies have failed to be effective in humans.
‘Immunological’ transcription factors and inducible nitric oxide synthase are upregulated in focal ischaemia and contribute to secondary infarct growth between 24 and 72 hours after the initial insult. The cytokines interleukin-1β and tumour necrosis factor-α are induced prior to inflammation. Functionally, these cytokines exert both neurotoxic and neuroprotective effects after cerebral ischaemia.
At present, immunological strategies targeted at a single immunomodulator for the treatment of stroke are hampered by an incomplete understanding of the complex cellular and molecular interactions that lead to divergent functional effects of inflammatory cells and immunological mediators after focal ischaemia.
Inflammation is an innate immune response to infection or tissue damage that is designed to limit harm to the host, but contributes significantly to ischemic brain injury following stroke. The inflammatory response is initiated by the detection of acute damage via extracellular and intracellular pattern recognition receptors, which respond to conserved microbial structures, termed pathogen-associated molecular patterns or host-derived danger signals termed damage-associated molecular patterns. Multi-protein complexes known as inflammasomes (e.g. containing NLRP1, NLRP2, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4, AIM2 and/or Pyrin), then process these signals to trigger an effector response. Briefly, signaling through NLRP1 and NLRP3 inflammasomes produces cleaved caspase-1, which cleaves both pro-IL-1β and pro- IL-18 into their biologically active mature pro-inflammatory cytokines that are released into the extracellular environment. This review will describe the molecular structure, cellular signaling pathways and current evidence for inflammasome activation following cerebral ischemia, and the potential for future treatments for stroke that may involve targeting inflammasome formation or its products in the ischemic brain.
Currently there are few effective therapeutic interventions for the acute treatment of ischaemic stroke. The few that do exist are primarily thrombolytics such as recombinant tissue plasminogen activator, which reperfuse the ischaemic brain region but do not otherwise aid in neuroprotection. One of the principal explanations as to why the vast majority of neuroprotective drug trials have failed is that the drugs’ therapeutic windows were too narrow to be clinically effective. The inflammatory reaction post-stroke makes a significant contribution to the neurological deterioration witnessed in patients. Since inflammatory mediators have been shown to be upregulated from 1 h to 3 months post-stroke, they represent an ideal target to exploit for therapeutic interventions as their ‘actions’ can potentially be halted within a clinically relevant therapeutic window. There is also mounting evidence that ischaemic stroke can be exacerbated by a predisposition to chronic inflammation and autoimmunity. This review details the role of inflammation and autoimmunity in stroke and how mediators of these pathways can be developed for therapeutic interventions. Recommendations are also made for an ideal therapy regime for acute ischaemic stroke, utilizing currently available, clinically proven treatments. Lastly, novel strategies for future experimental and clinical stroke research are discussed.
Recent studies have indicated that minocycline, a microglia inhibitor, could potentially be used as an antinociceptive agent in pain management, although the underlying mechanisms remain elusive. In this study, we investigated the extent to which minocycline could influence pain behavior in association with the expression of the N-methyl-D-aspartic acid receptor 1 (NMDAR1) in a rat L5 spinal nerve ligation (SNL) model. We observed that the intrathecal injection of minocycline significantly attenuated mechanical allodynia in a rat SNL model from day 1 postinjection and persisted for at least 18 days. We also observed that the expression of NMDAR1 was increased in the spinal dorsal horn at 8 days after SNL, which could be partly inhibited through the intrathecal injection of minocycline. These findings suggest that the attenuation of allodynia in the SNL model following minocycline administration might be associated with the inhibited expression of NMDAR1 and, therefore, might play an important role in the minocycline-mediated antinociception.
Tanshinone IIA is a good candidate for treating cerebral ischemia, but its short half-life and poor permeability across the blood-brain-barrier (BBB) limit its curative efficacy. In this study, we successfully developed cationic bovine serum albumin-conjugated tanshinone IIA PEGylated nanoparticles (CBSA-PEG-TIIA-NPs). A cerebral ischemia rat model was established to evaluate the treatment efficacy and protective mechanism of CBSA-PEG-TIIA-NPs. CBSA-PEG-TIIA-NPs showed the mean particle size 118 ± 14 nm with drug loaded ratio and encapsulation efficiency 5.69 ± 0.6% and 83.2 ± 2.6%, respectively. The pharmacokinetics demonstrated that CBSA-PEG-TIIA-NPs could significantly prolong circulation time and increase plasma concentration compared with intravenously administrated TIIA solution. The biodistribution and brain uptake study confirmed that CBSA-PEG-TIIA-NPs possessed better brain delivery efficacy with a high accumulation in brain. CBSA-PEG-TIIA-NPs obviously ameliorated infarct volume, neurological deficit and histopathological severity. Treatment with CBSA-PEG-TIIA-NPs markedly inhibited the levels of the MPO, TNF-α, IL-1β and IL-6. Furthermore, CBSA-PEG-TIIA-NPs significantly decreased the mRNA expressions of iNOS and p38MAPK, upregulated PPARγ expression, and inhibited the protein levels of iNOS, GFAP and p38MAPK phosphorylation. These results demonstrated that CBSA-PEG-TIIA-NPs possessed remarkable neuroprotective effects on ischemic stroke through modulation of inflammatory cascades and neuronal signal pathways involved in cerebral ischemia.
Pyrroloquinoline quinone PQQ is a naturally occurring redox cofactor that acts as an essential nutrient, antioxidant, and redox modulator. PQQ has been demonstrated to oxidize the redox modulatory site of N-methyl-d-aspartic acid (NMDA) receptors. Such agents are known to be neuroprotective in experimental stroke models. However, there is not report about the therapeutic effect of PQQ on neuropathic pain. We tested the effects of oral administration of PQQ on neuropathic pain of rats with chronic constriction injury (CCI) of the sciatic nerve. The repeated oral administration of PQQ (20 and 40mg/kg, once a day for 4 weeks, from day 1 after the injury) attenuated both thermal and mechanical hyperalgesia, and also attenuated the muscle atrophy. The anti-hyperalgesic activity of PQQ was associated with a significant reduction of pro-inflammatory mediators such as tumor necrosis factor alpha (TNF-α) and lipid peroxide malondialdehyde (MDA) levels. In the present investigation, PQQ is shown to have analgesic effect which was found in the first time, probably through reducing the release of pro-inflammatory mediator and inhibiting oxidative stress.
Headache caused by overuse of symptomatic medications to treat headache represents a challenging clinical problem. Recent studies exploring both the mechanisms of migraine headache and neuroplastic changes following sustained exposure to analgesic drugs suggest insights regarding the pathophysiology of medication overuse headache. In this review, changes that occur following chronic morphine that may be particularly relevant to the induction of medication overuse headache will be discussed. Peripherally, these changes include increased expression of calcitonin gene-related peptide (CGRP) in primary afferent neurons. Centrally, they include increased descending facilitation from the rostral ventromedial medulla and increased excitatory neurotransmission at the level of the dorsal horn. Interestingly, many of these same changes, including increased CGRP levels and peripheral and central sensitization, are apparent in inflammatory pain states. Recent revelations into the mechanisms of migraine headache suggest that neurogenic inflammation leads to both peripheral and central sensitization in the migraine sufferer. Furthermore, CGRP plays a prominent role in initiating vasodilation of the intracranial blood vessels and subsequent headache. Given the many parallels between the effects of chronic morphine exposure and processes that occur during migraine, it is evident that overuse of symptomatic medications to treat headache could lead to worsening of symptoms. In animals, the neural adaptations that occur after sustained morphine exposure are manifested behaviorally as an increased sensitivity to both tactile and thermal stimulation. In the headache sufferer, these adaptations could lead to medication overuse headache.
The aim of this article was to explore the mechanism of injury in rat retina after constant low-level helium-neon (He-Ne)
laser exposure and therapeutic effects of MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, on laser-induced retinal
injury. He-Ne laser lesions were created in the central retina of adult Wistar Kyoto rats and were followed immediately by
intraperitoneal injection of MK-801 (2 mg/kg) or saline, macroscopical and microscopical lesion were observed by funduscope
and light microscope. Ultrastructural changes of the degenerating cells were examined by electron microscopy. Photoreceptor
apoptosis was evaluated by TdT-mediated dUTP nick end-labeling (TUNEL). mRNA levels were measured by in situ hybridization
and NMDA receptor expression was determined by immunohistochemistry. Laser induced damage was histologically quantified by
image-analysis morphometry. Electroretinograms (ERGs) were recorded at different time point after the cessation of exposure
to constant irradiation. There was no visible bleeding, exudation or necrosis under funduscope. TUNEL and electron microscopy
showed photoreceptor apoptosis after irradiation. MK-801-treated animals had significantly fewer TUNEL-positive cells in the
photoreceptors than saline-treated animals after exposure to laser. In situ hybridization (ISH) showed that the NMDAR mRNA
level of MK-801-treated rats decreased in the inner plexiform layer 6 h after the cessation of exposure to constant irradiation
when compared with that of saline-treated rats. So did Immunohistochemistry (IHC). Electroretinogram showed that b-wave amplitudes of MK-801-treated group were higher than that of saline-treated group after laser exposure. These findings
suggest that Low level laser may cause the retinal pathological changes under given conditions. High expression of NMDAR is
one of the possible mechanisms causing experimental retinal laser injury of rats. MK-801 exhibits the therapeutic effect due
to promote the recovery of structure and function of injured retina.
It is widely accepted that interleukin-1beta (IL-1beta), a cytokine produced not only by immune cells but also by glial cells and certain neurons influences brain functions during infectious and inflammatory processes. It is still unclear, however, whether IL-1 production is triggered under nonpathological conditions during activation of a discrete neuronal population and whether this production has functional implications. Here, we show in vivo and in vitro that IL-1beta gene expression is substantially increased during long-term potentiation of synaptic transmission, a process considered to underlie certain forms of learning and memory. The increase in gene expression was long lasting, specific to potentiation, and could be prevented by blockade of potentiation with the N-methyl-D-aspartate (NMDA) receptor antagonist, (+/-)-2-amino-5-phosphonopentanoic acid (AP-5). Furthermore, blockade of IL-1 receptors by the specific interleukin-1 receptor antagonist (IL-1ra) resulted in a reversible impairment of long-term potentiation maintenance without affecting its induction. These results show for the first time that the production of biologically significant amounts of IL-1beta in the brain can be induced by a sustained increase in the activity of a discrete population of neurons and suggest a physiological involvement of this cytokine in synaptic plasticity.
Repetitive spreading depression (SD) waves, involving depolarization of neurons and astrocytes and up-regulation of glucose
consumption, is thought to lower the threshold of neuronal death during and immediately after ischemia. Using rat models for
SD and focal ischemia we investigated the expression of cyclooxygenase-1 (COX-1), the constitutive form, and cyclooxygenase-2
(COX-2), the inducible form of a key enzyme in prostaglandin biosynthesis and the target enzymes for nonsteroidal anti-inflammatory
drugs. Whereas COX-1 mRNA levels were undetectable and uninducible, COX-2 mRNA and protein levels were rapidly increased in
the cortex, especially in layers 2 and 3 after SD and transient focal ischemia. The cortical induction was reduced by MK-801,
an N-methyl-d-aspartic acid-receptor antagonist, and by dexamethasone and quinacrine, phospholipase A2 (PLA2) inhibiting compounds. MK-801 acted by blocking SD whereas treatment with PLA2 inhibitors preserved the wave propagation. NBQX, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-receptor
antagonist, did not affect the SD-induced COX-2 expression, whereas COX-inhibitors indomethacin and diclofenac, as well as
a NO synthase-inhibitor, NG-nitro-l-arginine methyl ester, tended to enhance the COX-2 mRNA expression. In addition, ischemia induced COX-2 expression in the
hippocampal and perifocal striatal neurons and in endothelial cells. Thus, COX-2 is transiently induced after SD and focal
ischemia by activation of N-methyl-d-aspartic acid-receptors and PLA2, most prominently in cortical neurons that are at a high risk to die after focal brain ischemia.
Glutamate excitotoxicity mediated by the AMPA/kainate type of glutamate receptors damages not only neurons but also the myelin-producing cell of the central nervous system, the oligodendrocyte. In multiple sclerosis, myelin, oligodendrocytes and some axons are lost as a result of an inflammatory attack on the central nervous system. Because glutamate is released in large quantities by activated immune cells, we expected that during inflammation in MS, glutamate excitotoxicity might contribute to the lesion. We addressed this by using the AMPA/kainate antagonist NBQX to treat mice sensitized for experimental autoimmune encephalomyelitis, a demyelinating model that mimics many of the clinical and pathologic features of multiple sclerosis. Treatment resulted in substantial amelioration of disease, increased oligodendrocyte survival and reduced dephosphorylation of neurofilament H, an indicator of axonal damage. Despite the clinical differences, treatment with NBQX had no effect on lesion size and did not reduce the degree of central nervous system inflammation. In addition, NBQX did not alter the proliferative activity of antigen-primed T cells in vitro, further indicating a lack of effect on the immune system. Thus, glutamate excitotoxicity seems to be an important mechanism in autoimmune demyelination, and its prevention with AMPA/kainate antagonists may prove to be an effective therapy for multiple sclerosis.
Multiple sclerosis is an immune-mediated disorder of the central nervous system leading to progressive decline of motor and sensory functions and permanent disability. The therapy of multiple sclerosis is only partially effective, despite anti-inflammatory, immunosuppresive and immunomodulatory measures. White matter inflammation and loss of myelin, the pathological hallmarks of multiple sclerosis, are thought to determine disease severity. Experimental autoimmune encephalomyelitis reproduces the features of multiple sclerosis in rodents and in nonhuman primates. The dominant early clinical symptom of acute autoimmune encephalomyelitis is progressive ascending muscle weakness. However, demyelination may not be profound and its extent may not correlate with severity of neurological decline, indicating that targets unrelated to myelin or oligodendrocytes may contribute to the pathogenesis of acute autoimmune encephalomyelitis. Here we report that within the spinal cord in the course of autoimmune encephalomyelitis not only myelin but also neurons are subject to lymphocyte attack and may degenerate. Blockade of glutamate AMPA receptors ameliorated the neurological sequelae of autoimmune encephalomyelitis, indicating the potential for AMPA antagonists in the therapy of multiple sclerosis.
The contribution of the immune system to the pathogenesis of ischemic lesions is still uncertain. We have analyzed leukocyte infiltration in photochemically induced focal ischemia of the rat parietal cortex by immunocytochemistry. Between 1 and 2 days after photothrombosis, CD5+ T cells adhered to subpial and cortical vessels and infiltrated the ischemic lesion prior to macrophages. By day 3 numerous T cells and some macrophages, whose number increased further between day 3 and day 7, had infiltrated the border zone around the lesion sparing the center. In addition, CD5-/CD8+ lymphocytes that probably represent natural killer cells were found. Intercellular adhesion molecule-1 (ICAM-1) was expressed on endothelial cells on days 1 and 2 and in the border zone on infiltrating leukocytes from day 3 to day 7. Starting on day 7, macrophages infiltrated the core of the lesion to remove debris. When the entire lesion was covered by macrophages at day 14, the number of T cells had decreased and ICAM-1 immunoreactivity was no longer found in or around the infarct. In conclusion, our study shows that ischemic lesions can lead to a local immune reaction in the CNS. Thus, blocking of lymphocyte-derived cytokines or cell adhesion molecules may provide a new approach to confining the sequelae of stroke.
Cerebral ischemia is followed by a local inflammatory response that is thought to participate in the extension of the tissue damage occurring in the postischemic period. However, the mechanisms whereby the inflammation contributes to the progression of the damage have not been fully elucidated. In models of inflammation, expression of the inducible isoform of nitric oxide synthase (iNOS) is responsible for cytotoxicity through the production of large amounts of nitric oxide (NO). In this study, therefore, we sought to establish whether iNOS is expressed in the ischemic brain. Rats were killed 6 h to 7 days after occlusion of the middle cerebral artery. iNOS expression in the ischemic area was determined by reverse-transcription polymerase chain reaction. Porphobilinogen deaminase mRNA was detected in the same sample and used for normalization. In the ischemic brain, there was expression of iNOS mRNA that began at 12 h, peaked at 48 h, and returned to baseline at 7 days (n = 3/time point). iNOS mRNA expression paralleled the time course of induction of iNOS catalytic activity, determined by the citrulline assay (17.4 +/- 4.4 pmol citrulline/micrograms protein/min at 48 h; mean +/- SD; n = 5 per time point). iNOS immunoreactivity was seen in neutrophils at 48-96 h after ischemia. The data provide molecular, biochemical, and immunocytochemical evidence of iNOS induction following focal cerebral ischemia. These findings, in concert with our recent demonstration that inhibition of iNOS reduces infarct volume in the same stroke model, indicate that NO production may play an important pathogenic role in the progression of the tissue damage that follows cerebral ischemia.
Certain cytokines have been reported to exert neurotrophic actions in vivo and in vitro. In the present study, we investigated the possible neuroprotective actions of the cytokine human recombinant interleukin-1 beta (hrIL-1 beta) against excitatory amino acid (EAA)-induced neurodegeneration in cultured primary cortical neurons. Brief (15 min) exposure of cultures to submaximal concentrations of glutamate, NMDA, AMPA, or kainate caused extensive neuronal death (approximately 70% of all neurons). Neuronal damage induced by the EAAs was significantly reduced (up to 70%) by pretreatment with 500 ng/ml (6.5 x 10(3) U/ml) hrIL-1 beta for 24 hr. The neuroprotective effect of hrIL-1 beta was reversed by coapplication of an IL-1 receptor antagonist (IL-1ra, 50 micrograms/ml). Neuroprotective actions of hrIL-1 beta were also reduced by administration of a neutralizing monoclonal antibody to NGF (65% inhibition). In concordance, the neurotoxic actions of EAAs were significantly reduced (by 40%) after pretreatment with NGF (100 ng/ml for 48 hr). Furthermore, an additive neuroprotective effect of approximately 75% was observed when cultures were exposed to a combination of hrIL-1 beta and NGF. In contrast, exposure of cultures to high concentrations hrIL-1 beta alone (100 micrograms/ml, 1.3 x 10(6) U/ml) for periods up to 72 hr resulted in neurotoxicity, which was reversed by IL-1ra (1 mg/ml). These findings suggest that hrIL-1 beta can limit EAA-induced neuronal damage. These effects appear to be may be mediated, at least in part, via NGF. These findings may be relevant to the understanding of neurodegenerative diseases.
Tumor necrosis factor-alpha (TNF) markedly stimulates the synthesis and secretion of immunoreactive nerve growth factor (NGF) in quiescent mouse fibroblasts, which is a result of increase in the NGF mRNA level. NGF produced by TNF-treated fibroblasts has a molecular mass of 13 kDa on SDS-polyacrylamide gel electrophoresis, which is consistent in size with the subunit of mouse beta-NGF, and induces neurite outgrowth in paravertebral sympathetic neurons. Several peptide growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor also stimulate NGF production in the cells, but not platelet-derived growth factor. The dose responses of TNF and bFGF to stimulate NGF production in the cells are, respectively, similar to those to induce cell proliferation. However, no correlation is observed between the ability of these growth factors to stimulate NGF production and that to induce cell proliferation. Thus, the stimulation of NGF production in the cells seems to be a specific activity of TNF and some other growth factors. TNF stimulates the synthesis and secretion of NGF also in other cells such as human glioblastoma cells. These findings suggest that TNF plays a role in regulating neuronal cell function through an indirect mechanism by which it stimulates NGF production in glial cells and fibroblasts.
Inducible nitric oxide synthase (iNOS), an enzyme that produces toxic amounts of nitric oxide, is expressed in a number of brain pathologies, including cerebral ischemia. We used mice with a null mutation of the iNOS gene to study the role of iNOS in ischemic brain damage. Focal cerebral ischemia was produced by occlusion of the middle cerebral artery (MCA). In wild-type mice, iNOS mRNA expression in the post-ischemic brain begun between 24 and 48 hr peaked at 96 hr and subsided 7 d after MCA occlusion. iNOS mRNA induction was associated with expression of iNOS protein and enzymatic activity. In contrast, mice lacking the iNOS gene did not express iNOS message or protein after MCA occlusion. The infarct and the motor deficits produced by MCA occlusion were smaller in iNOS knockouts than in wild-type mice (p < 0.05). Such reduction in ischemic damage and neurological deficits was observed 96 hr after ischemia but not at 24 hr, when iNOS is not yet expressed in wild-type mice. The decreased susceptibility to cerebral ischemia in iNOS knockouts could not be attributed to differences in the degree of ischemia or vascular reactivity between wild-type and knockout mice. These findings indicate that iNOS expression is one of the factors contributing to the expansion of the brain damage that occurs in the post-ischemic period. iNOS inhibition may provide a novel therapeutic strategy targeted specifically at the secondary progression of ischemic brain injury.
Cerebral ischemia induces a rapid and dramatic up-regulation of tumor necrosis factor (TNF) protein and mRNA, but the cellular sources of TNF in the ischemic brain have not been defined. The diverse activities of TNF are mediated via ligand interaction with two distinct receptors, p55 and p75, which activate separate intracellular signal transduction pathways, leading to distinct biological effects. Since the effects of cerebral ischemia on TNF receptor (TNFR) expression are unknown, we examined the cellular localization and protein expression of TNF and its two receptors in the rat cerebral cortex in response to permanent middle cerebral artery (MCA) occlusion. The results indicate that focal. cerebral ischemia up-regulates expression of TNF and both TNFRs within the ischemic cortex. The most abundant type of TNF immunoreactivity (IR) was a punctate and filamentous pattern of transected cellular processes; however, cell bodies of neurons, astrocytes, and microglia, as well as infiltrating polymorphonuclear (PMN) leukocytes also showed TNF IR. Brain vasculature displayed TNF IR not only within endothelial cells but also in the perivascular space. MCA occlusion induced significant up-regulation of TNF receptors, with p55 IR appearing within 6 hr, significantly before the appearance of p75 IR at 24 hr after the onset of ischemia. Since p55 has been implicated in transducing cytotoxic signalling of TNF, these results support the proposed injurious role of excessive TNF produced during the acute response to cerebral ischemia.
The molecular mechanism(s) of N-methyl-D-aspartate (NMDA) neuroprotective properties were investigated in primary cultures of cerebellar granule cell neurons. Granule cells express the neurotrophin receptor TrkB but not TrkA or TrkC. In these cells, the TrkB ligand brain-derived neurotrophic factor (BDNF) prevents glutamate toxicity. Therefore, we have tested the hypothesis that NMDA activates synthesis and release of BDNF, which may prevent glutamate toxicity by an autocrine loop. Exposure of granule cells for 2 and 5 min to a subtoxic concentration of NMDA (100 microM) evoked an accumulation of BDNF in the medium without concomitant changes in the intracellular levels of BDNF protein or mRNA. The increase in BDNF in the medium is followed by enhanced TrkB tyrosine phosphorylation, suggesting that NMDA increases the release of BDNF and therefore the activity of TrkB receptors. To examine whether BDNF and TrkB signaling play a role in the NMDA-mediated neuroprotective properties, neurons were exposed to soluble trkB receptor-IgG fusion protein, which is known to inhibit the activity of extracellular BDNF, and to K252a, a tyrosine kinase inhibitor. Both compounds blocked the NMDA-mediated TrkB tyrosine phosphorylation and subsequently its neuroprotective properties. We suggest that NMDA activates the TrkB receptor via a BDNF autocrine loop, resulting in neuronal survival.
A focal infarction produced by occlusion of the middle cerebral artery (MCAO) in spontaneously hypertensive rats induced expression of inducible nitric oxide synthase (iNOS) mRNA, measured by competitive reverse transcription-polymerase chain reaction. The mRNA appeared simultaneously in the ischemic core and penumbra at 8 h, peaked between 14 and 24 h, and disappeared by 48 h. At 24 h, inducible nitric oxide synthase (iNOS)-like immunoreactivity was present in the endothelium of cerebral microvessels and in scattered cells, probably representing leukocytes or activated microglia. Electrical stimulation of the cerebellar fastigial nucleus (FN) for 1 h, 48 h before MCAO, reduced infarct volumes by 45% by decreasing cellular death in the ischemic penumbra. It also reduced by >90% the expression of iNOS mRNA and protein in the penumbra, but not core, and decreased by 44% the iNOS enzyme activity. We conclude that excitation of neuronal networks represented in the cerebellum elicits a conditioned central neurogenic neuroprotection associated with the downregulation of iNOS mRNA and protein. This neuroimmune interaction may, by blocking the expression of iNOS, contribute to neuroprotection.
Murine cortical cultures containing both neurons and glia (days in vitro 13-15) were exposed to periods of oxygen-glucose deprivation (5-30 min) too brief to induce neuronal death. Cultures "preconditioned" by sublethal oxygen-glucose deprivation exhibited 30-50% less neuronal death than controls when exposed to a 45-55 min period of oxygen-glucose deprivation 24 hr later. This preconditioning-induced neuroprotection was specific in that neuronal death induced by exposure to excitotoxins or to staurosporine was not attenuated. Neuroprotection was lost if the time between the preconditioning and severe insult were decreased to 7 hr or increased to 72 hr and was blocked if the NMDA antagonist 100 microM 3-((D)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid was applied during the preconditioning insult. This was true even if the duration of preconditioning was increased as far as possible (while still remaining sublethal). A similar preconditioning effect was also produced by sublethal exposure to high K+, glutamate, or NMDA but not to kainate or trans-1-aminocyclopentane-1, 3-dicarboxylic acid.
Cerebral ischemia induces a rapid and dramatic up-regulation of tumor necrosis factor (TNF) protein and mRNA, but the cellular sources of TNF in the ischemic brain have not been defined. The diverse activities of TNF are mediated via ligand interaction with two distinct receptors, p55 and p75, which activate separate intracellular signal transduction pathways, leading to distinct biological effects. Since the effects of cerebral ischemia on TNF receptor (TNFR) expression are unknown, we examined the cellular localization and protein expression of TNF and its two receptors in the rat cerebral cortex in response to permanent middle cerebral artery (MCA) occlusion. The results indicate that focal. cerebral ischemia up-regulates expression of TNF and both TNFRs within the ischemic cortex. The most abundant type of TNF immunoreactivity (IR) was a punctate and filamentous pattern of transected cellular processes; however, cell bodies of neurons, astrocytes, and microglia, as well as infiltrating polymorphonuclear (PMN) leukocytes also showed TNF IR. Brain vasculature displayed TNF IR not only within endothelial cells but also in the perivascular space. MCA occlusion induced significant up-regulation of TNF receptors, with p55 IR appearing within 6 hr, significantly before the appearance of p75 IR at 24 hr after the onset of ischemia. Since p55 has been implicated in transducing cytotoxic signalling of TNF, these results support the proposed injurious role of excessive TNF produced during the acute response to cerebral ischemia.
In this study we examined the time course of apoptotic cell death after photochemically induced focal ischemia of the rat
cerebral cortex. For unequivocal differentiation between apoptosis and necrosis two criteria of programmed cell death were
used: terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and morphological evidence
of fragmentation and marginalization of nuclei. After photothrombosis, many TUNEL-positive cells were found within the infarct
region from 12 h to 3 days. By day 6 they were preferentially located in the boundary zone of the infarct, and by day 14 they
had disappeared. A high proportion of TUNEL-positive cells displayed fragmentation or marginalization of their nuclei, indicating
apoptosis. Neurons, but not T cells and macrophages, were apoptotic. Inflammatory infiltrates were in close contact to apoptotic
neurons throughout the infarct areas at day 1 and in the boundary zone between days 2 and 6 after photothrombosis. In summary,
our study shows that neuronal apoptosis after cerebral ischemia is a prolonged process to which leukocyte-derived cytokines
may contribute. In contrast to autoimmune diseases of the nervous system, termination of the local inflammatory response after
cerebral ischemia does not involve apoptosis.
The inducible or “immunological” isoform of nitric oxide synthase (iNOS) is induced in many cell types by inflammatory stimuli
and synthesizes toxic amounts of NO. In rodent models of focal cerebral ischemia, iNOS is expressed in neutrophils invading
the injured brain and in local blood vessels. Studies with iNOS inhibitors and iNOS null mice indicate that NO produced by
iNOS contributes to ischemic brain injury. In the present study, we sought to determine whether iNOS is also expressed in
the human brain after ischemic stroke. Studies were conducted using immunohistochemistry on autopsy brains with neuropathological
evidence of acute cerebral infarction. iNOS immunoreactivity was observed in neutrophils infiltrating the ischemic brain and
in blood vessels within the ischemic territory. iNOS-positive cells also were immunoreactive for nitrotyrosine, reflecting
protein nitration by NO-derived peroxynitrite and nitrites. iNOS or nitrotyrosine immunoreactivity was not detected outside
the region of the infarct. These observations provide evidence that iNOS is expressed in the human brain after ischemic infarction
and support the hypothesis that iNOS inhibitors may be useful in the treatment of ischemic stroke in humans.
Considerable evidence implicates nitric oxide (NO) in the pathological events following cerebral ischemia and, depending on the enzyme/cell source, NO is considered to be either damaging or protective. As a role for the enzyme nitric oxide synthase (NOS)-2 in permanent focal ischemia is not clear, we examined its expression following permanent middle cerebral artery occlusion in mice. At 24 h after occlusion, NOS-2 was expressed in cells infiltrating the infarct, while at later times, there was also expression in astrocytes around the infarct. To reveal a role for NO derived from this source, we compared infarct size in male and female mice with littermates in which the NOS-2 gene was disrupted. No differences were found between gender and genotype at 24 h. At 72 h, the infarct was increased in male mice, but not in females or in either gender with the gene disruption. These results suggest that NOS-2 plays a role in the later development of the infarct in male mice. Female mice are protected either against the damaging effects of NO, or because NOS-2 expression/activity is modulated by steroids.
Focal cerebral ischemia elicits a strong inflammatory response involving early recruitment of granulocytes and delayed infiltration of ischemic areas and the boundary zones by T cells and macrophages. Infiltration of hematogenous leukocytes is facilitated by an upregulation of the cellular adhesion molecules P-selectin, intercellular adhesion molecule-1 and vascular adhesion molecule-1 on endothelial cells. Blocking of the leukocyte/endothelial cell adhesion process significantly reduces stroke volume after transient, but not permanent middle cerebral artery occlusion. In the infarct region microglia are activated within hours and within days transform into phagocytes. Astrocytes upregulate intermediate filaments, synthesize neurotrophins and form glial scars. Local microglia and infiltrating macrophages demarcate infarcts and rapidly remove debris. Remote from the lesion no cellular infiltration occurs, but astroglia and microglia are transiently activated. Astrocytic activation is induced by spreading depression. In focal ischemia neurons die acutely by necrosis and in a delayed fashion by programmed cell death, apoptosis. Proinflammatory cytokines such as tumor necrosis factor-α and interleukin-1β are upregulated within hours in ischemic brain lesions. Either directly or via induction of neurotoxic mediators such as nitric oxide, cytokines may contribute to infarct progression in the post-ischemic period. On the other hand, inflammation is tightly linked with rapid removal of debris and repair processes. At present it is unclear whether detrimental effects of inflammation outweigh neuroprotective mechanisms or vice versa. In global ischemia inflammatory responses are limited, but micro- and astroglia are also strongly activated. Glial responses significantly differ between brain regions with selective neuronal death and neighbouring areas that are more resistent to ischemic damage.
This study examined the time course of mRNA levels of the proinflammatory cytokines interferon-gamma (IFNgamma), interleukin-1beta (IL1beta), interleukin-12 (IL12; p40 subunit), and the immunosuppressant interleukin-10 (IL10) by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) in rats with actively induced experimental autoimmune neuritis (EAN) and in distal stumps of crushed sciatic nerves undergoing Wallerian degeneration. In EAN IFNgamma- and IL1beta-mRNA peaked at the onset and acute phase of clinical disease. IL12p40-mRNA was upregulated later than IFNgamma-mRNA in the late acute phase from days 15 to 21. IL10-mRNA appeared concomitantly with the proinflammatory cytokines at day 11, but persisted at high levels into the clinical recovery phase. After nerve crush both IL1beta- and IL10-mRNA were rapidly upregulated in the distal stump at day 1 and slowly declined over the next 2 weeks. Significant levels of mRNA for IFNgamma could be found at days 4 and 7, whereas IL12p40-mRNA showed a biphasic induction. We provide evidence for a concomitant induction of pro- and anti-inflammatory cytokines in EAN. Moreover, the rapid upregulation in Wallerian degeneration suggests a more general role of cytokines in the biology of the peripheral nerve.
Interleukin-1 (IL-1) synthesis in the brain is stimulated by mechanical injury and IL-1 mimics some effects of injury, such as gliosis and neovascularization. We report that neuronal death resulting from focal cerebral ischaemia (middle cerebral artery occlusion, 24 h) is significantly inhibited (by 50%) in rats injected with a recombinant IL-1 receptor antagonist (IL-1ra, 10 micrograms, icv 30 min before and 10 min after ischaemia). Excitotoxic damage due to striatal infusion of an NMDA-receptor agonist (cis-2,4-methanoglutamate) was also markedly inhibited (71%) by injection of the IL-1ra. These data indicate that endogenous IL-1 is a mediator of ischaemic and excitotoxic brain damage, and that inhibitors of IL-1 action may be of therapeutic value in the treatment of acute or chronic neuronal death.
It has become clear that the neurotransmitter glutamate does not confine its excitatory effects to central nervous system neurons but interacts also with glial cells. Neurons and glia share the same types of ionotropic and metabotropic glutamate receptors except for the N-methyl-D-aspartate receptor, which is not found on glia. Applied on cultured glial cells, glutamate regulates the opening of receptor channels, activates second messengers, and causes the release of neuroactive compounds. Although glutamate and glutamate receptors confer on cultured glia the ability to receive and emit signals, it remains to be established whether glial signaling takes place in vivo. The chick Bergmann glial cells provide a unique experimental system with which to test the contribution of glial glutamate receptors to neuronal electrical activity. These cells are the exclusive carriers in the cerebellum of functional kainate receptors. The synaptic location of these receptors, their ion channel properties, and their regulation by phosphorylation reactions suggest that glial kainate receptors play a role in regulating synaptic efficacy and plasticity. If proved, this concept may require a modification of the anatomical and functional definition of a synapse to include a glial component as well.
We have used a photochemical reaction in vivo to induce reproducible thrombosis leading to cerebral infarction in rats. After the intravenous injection of rose bengal, a potent photosensitizing dye, an ischemic lesion was formed by irradiating the left parietal convexity of the exposed skull for 20 minutes with green light (560 nm) from a filtered xenon arc lamp. Animals were allowed to survive from 30 minutes to 15 days after irradiation. Early microscopic alterations within the irradiated zone included the formation of thrombotic plugs and adjacent red blood cell stasis within pial and parenchymal vessels. Scanning electron microscopy revealed frequent platelet aggregates adhering to the vascular endothelium, often resulting in vascular occlusion. Carbon-black brain perfusion demonstrated that occlusion of vascular channels progressed after irradiation and was complete within 4 hours. Histopathological examination at 1, 5, and 15 days revealed that the associated infarct evolved reproducibly through several characteristic stages, including a phase of massive macrophage infiltration. Although cerebral infarction in this model is initiated by thrombosis of small blood vessels, the fact that the main pathological features of stroke are consistently reproduced should permit its use in assessing treatment regimens. Further, the capability of producing infarction in preselected cortical regions may facilitate the study of behavioral, functional, and structural consequences of acute and chronic stroke.
Emerging data indicate that neurotrophic factors and cytokines utilize similar signal transduction mechanisms. Although neurotrophic factors can protect CNS neurons against a variety of insults, the role of cytokines in the injury response is unclear. We now report that TNF beta and TNF alpha (1-100 ng/ml) can protect cultured embryonic rat hippocampal, septal, and cortical neurons against glucose deprivation-induced injury and excitatory amino acid toxicity. The elevation of intracellular calcium concentration ([Ca2+]i) induced by glucose deprivation, glutamate, NMDA, or AMPA was attenuated in neurons pretreated with TNF beta. The mechanism whereby TNFs stabilize [Ca2+]i may involve regulation of the expression of proteins involved in maintaining [Ca2+]i homeostasis, since both TNF beta and TNF alpha caused a 4- to 8-fold increase in the number of neurons expressing the calcium-binding protein calbindin-D28k. These data suggest a neuroprotective role for TNFs in the brain's response to injury.
Nitric oxide (NO) produced by the constitutive NO synthase (cNOS) in neurons has been implicated in mediating excitotoxic neuronal death. In our murine cortical cell culture system, NMDA neurotoxicity was not blocked by addition of the NOS inhibitors, NG-nitro-L-arginine or aminoguanidine. However, following activation of inducible NOS in astrocytes by interleukin-1 beta plus interferon-gamma, NMDA but not kainate neurotoxicity was markedly potentiated. This selective potentiation of NMDA neurotoxicity was blocked by NOS inhibition or antioxidants (superoxide dismutase/catalase or Tempol) and could be mimicked by NO generators (SIN-1 or SNAP) or the oxygen radical generator, pyragallol. These results raise the possibility that NO production by astrocytes may contribute to NMDA receptor-mediated neuronal death, perhaps through interaction with oxygen radicals.
This study describes local immune responses in cerebral ischemia induced by permanent occlusion of the middle cerebral artery (MCAO) in the rat. The temporal and spatial pattern of leukocyte infiltration was characterized immunocytochemically using monoclonal antibodies against CD5, a pan T cell marker, against CD4 and CD8 for subtyping of T lymphocytes, and ED1, a marker for macrophages. CD5+ T cells were present in some animals on the pial surface at day 1 and with increasing numbers mainly at the edges of the infarcts at days 3 and 7. By day 14 their number had significantly decreased. Subtyping of T lymphocytes revealed that CD4+ helper/inducer T cells were rare, while CD8+ lymphocytes were abundant. Moreover, CD8+ lymphocytes outnumbered CD5+ T cells indicating the presence of CD5-/CD8+ natural killer (NK) cells. ED1+ macrophages primarily infiltrated the core of the infarct starting on day 1. Infiltrating leukocytes expressed leukocyte function associated antigen-1 and MHC class I and II antigens. Early after infarction, increased expression of the intercellular adhesion molecule-1 was found on vessels and leukocytes. In conclusion, this study shows that lymphocytes enter the nervous system not only in autoimmune diseases, but also in response to primarily 'non-immune' neuronal damage such as stroke.
Postischemic cerebral inflammation may contribute to ischemic cell damage. Intercellular adhesion molecule-1 (ICAM-1) is a glycoprotein expressed on endothelial cells that facilitates leukocyte adhesion. We investigated the effect of administration of an anti-ICAM-1 antibody (1A29) on ischemic cell damage after transient (2-hour) or permanent middle cerebral artery (MCA) occlusion in the Wistar rat.
Groups studied were as follows: (1) transient MCA occlusion: rats were subjected to 2 hours of MCA occlusion, and after 1 hour of reperfusion they were treated with 1A29 (n = 11) or an isotype control antibody (n = 9); and (2) permanent MCA occlusion: rats were treated with 1A29 (n = 9) or an isotype control antibody (n = 7) 2 hours after onset of MCA occlusion. All animals were killed 1 week after onset of ischemia. Brain sections were stained with hematoxylin and eosin for histological evaluation.
Significant reductions (P < .05) in both volume (44%) of the ischemic lesion and weight loss were found in animals subjected to transient MCA occlusion and treated with 1A29 compared with vehicle-treated animals. In contrast, in animals subjected to permanent MCA occlusion the lesion and the temporal profile of body weight were not altered by 1A29 administration.
Ischemic cell damage is promoted by postischemic inflammatory response after 2 hours of transient MCA occlusion, and ischemic cell damage is reduced by administration of an anti-ICAM-1 antibody during reperfusion.
Tumor necrosis factor-alpha (TNF-alpha) is a cytokine with diverse proinflammatory actions, including endothelial leukocyte adhesion molecule expression. Since leukocytes infiltrate into ischemic brain lesions, the present study was conducted to examine whether TNF-alpha messenger RNA (mRNA) and peptide are expressed in the brain after experimental focal stroke and before leukocyte accumulation.
TNF-alpha mRNA and protein expression were monitored in the ischemic and nonischemic cerebral cortex of rats after focal ischemia produced by permanent middle cerebral artery occlusion. The effect of TNF-alpha administered by microinjection into the brain cortex on leukocyte adherence to brain capillaries was also studied.
Induction of TNF-alpha mRNA, normalized to a standard reference rat macrophage TNF-alpha mRNA, was detected as early as 1 hour after middle cerebral artery occlusion. TNF-alpha mRNA was elevated by 3 hours (29 +/- 6% versus 2 +/- 1% in sham-operated rats) only in the ischemic cortex, with peak expression at 12 hours (104 +/- 8%; P < .01). Five days after middle cerebral artery occlusion, TNF-alpha mRNA levels in ischemic cortex were still significantly elevated (38 +/- 5%; P < .05). Also, TNF-alpha mRNA expression was greater in the ischemic cortex of spontaneously hypertensive rats than in normotensive rats (P < .05). Double-labeling, immunohistochemical studies revealed the presence of TNF-alpha protein localized within nerve fibers in the evolving infarct at 6 and 12 hours after ischemia and further expression in the tissues immediately adjacent to the infarct 24 hours after ischemia. After 5 days, the neuronally localized peptide had diminished greatly, but macrophages located within the infarcted tissues were immunoreactive. Cortical microinjections of TNF-alpha (10 ng in 1 microL) produced a significant neutrophil adherence/accumulation in capillaries and small blood vessels 24 hours later.
These results represent the first demonstration that focal cerebral ischemia in rats results in elevated TNF-alpha mRNA and protein in ischemic neurons. The neuronal expression of peptide appears to facilitate the infiltration of inflammatory cells that can further exacerbate tissue damage in cerebral ischemia and might contribute to increased sensitivity and risk in focal stroke.
Interleukin-1 beta is a proinflammatory cytokine produced by blood-borne and resident brain inflammatory cells. The present study was conducted to determine if interleukin-1 beta mRNA was produced in the brain of rats subjected to permanent focal ischemia.
Rat interleukin-1 beta cDNA, synthesized from stimulated rat peritoneal macrophage RNA by reverse transcription and polymerase chain reaction and cloned in plasmid Bluescript KS+, was used to evaluate the expression of interleukin-1 beta mRNA in cerebral cortex from spontaneously hypertensive rats and normotensive rats subjected to permanent middle cerebral artery occlusion. Interleukin-1 beta mRNA was quantified by Northern blot analysis and compared with rat macrophage RNA standard. To correct for gel loading, blots were also analyzed with cyclophilin cDNA, which encodes an abundant, conserved protein that was unchanged by the experimental conditions.
Interleukin-1 beta mRNA produced in the ischemic zone was significantly increased from 6 hours to 120 hours, with a maximum of 211 +/- 24% of interleukin-1 beta reference standard, ie, 0.2 ng stimulated rat macrophage RNA, mRNA compared with the level in nonischemic cortices (4 +/- 2%) at 12 hours after ischemia (P < .01; n = 6). Interleukin-1 beta mRNA at 12 hours after ischemia was markedly elevated in hypertensive rats over levels found in two normotensive rat strains. Neurological deficits were also apparent only in the hypertensive rats.
Brain interleukin-1 beta mRNA is elevated acutely after permanent focal ischemia and especially in hypertensive rats. These data suggest that this potent proinflammatory and procoagulant cytokine might have a role in brain damage following ischemia.
The purposes of this study were to determine whether cortical spreading depression occurs outside of the infarct produced by photothrombotic vascular occlusion, and also the direction of spreading. Focal cerebral thrombotic infarction was produced by irradiating the exposed skull of anesthetized rats with green light (560 nm) following systemic injection of rose bengal dye. At proximal sites (approximately 2 mm anterior to the infarct border), transient, severe hyperemic episodes (THEs) lasting 1-2 min were intermittently recorded. THE frequency was greatest in the first hour and declined over a 3-h period. THEs were accompanied (and usually preceded) by a precipitous rise in [K+]0 (from approximately 3 to > 40 mM) and were associated with increases in local tissue oxygen tension (tPO2). Following the rise in [K+]0, clearance of [K+]0 to its pre-THE baseline preceded baseline recovery of CBF. These data indicate that THEs were reactive to physiologic events resembling cortical spreading depression (CSD), which provoked increased demand for oxygen and blood flow, and which spread from proximal sites to areas more distal (approximately 4 mm) from the rim of the evolving infarct. MK-801 (1 mg/kg, i.v.) inhibited subsequent CSD-like episodes. We conclude that photothrombosis-induced ischemia provoked CSD which was triggered either within the infarct core or in the infarct rim and spread to more distal sites. Whether multiple episodes of CSD during infarct generation are responsible for the remote consequences of focal brain injury remains to be determined.
This study investigated astroglial responses after focal cerebral ischemia in the rat cortex induced by photothrombosis. Astrocyte activation was studied at various time points by immunocytochemistry for glial fibrillary acidic protein (GFAP) and vimentin (VIM). We found a dual astrocytic response to focal ischemia: In the border zone of the infarct, GFAP-positive astrocytes were present within 2 days and persisted for 10 weeks. These astrocytes additionally expressed VIM. Remote from the ischemic lesion, cortical astrocytes of the entire ipsilateral hemisphere transiently expressed GFAP, but not VIM, beginning on day 3 after photothrombosis. This response had disappeared on day 14. By recording DC potentials, five to seven spreading depressions (SD) could be detected on the cortical surface during the first 2 h after photothrombosis. Treatment with MK801, a non-competitive NMDA-receptor antagonist, completely abolished SD and remote ipsilateral astrocytic activation, while the reaction in the border zone of the infarct remained unchanged. Functionally, persistent astrocytosis around the infarct might be induced by leukocyte-derived cytokines, while NMDA-receptor-mediated SD might cause remote responses.
Acute neutrophil (PMN) recruitment to postischemic cardiac or pulmonary tissue has deleterious effects in the early reperfusion period, but the mechanisms and effects of neutrophil influx in the pathogenesis of evolving stroke remain controversial. To investigate whether PMNs contribute to adverse neurologic sequelae and mortality after stroke, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of stroke, we used a murine model of transient focal cerebral ischemia consisting of intraluminal middle cerebral artery occlusion for 45 min followed by 22 h of reperfusion. PMN accumulation, monitored by deposition of 111In-labeled PMNs in postischemic cerebral tissue, was increased 2.5-fold in the ipsilateral (infarcted) hemisphere compared with the contralateral (noninfarcted) hemisphere (P < 0.01). Mice immunodepleted of neutrophils before surgery demonstrated a 3.0-fold reduction in infarct volumes (P < 0.001), based on triphenyltetrazolium chloride staining of serial cerebral sections, improved ipsilateral cortical cerebral blood flow (measured by laser Doppler), and reduced neurological deficit compared with controls. In wild-type mice subjected to 45 min of ischemia followed by 22 h of reperfusion, ICAM-1 mRNA was increased in the ipsilateral hemisphere, with immunohistochemistry localizing increased ICAM-1 expression on cerebral microvascular endothelium. The role of ICAM-1 expression in stroke was investigated in homozygous null ICAM-1 mice (ICAM-1 -/-) in comparison with wild-type controls (ICAM-1 +/+). ICAM-1 -/- mice demonstrated a 3.7-fold reduction in infarct volume (P < 0.005), a 35% increase in survival (P < 0.05), and reduced neurologic deficit compared with ICAM-1 +/+ controls. Cerebral blood flow to the infarcted hemisphere was 3.1-fold greater in ICAM-1 -/- mice compared with ICAM-1 +/+ controls (P < 0.01), suggesting an important role for ICAM-1 in the genesis of postischemic cerebral no-reflow. Because PMN-depleted and ICAM-1-deficient mice are relatively resistant to cerebral ischemia-reperfusion injury, these studies suggest an important role for ICAM-1-mediated PMN adhesion in the pathophysiology of evolving stroke.
Brain injury, as occurs in stroke or head trauma, induces a dramatic increase in levels of tumor necrosis factor-alpha (TNF), but its role in brain injury response is unknown. We generated mice genetically deficient in TNF receptors (TNFR-KO) to determine the role of TNF in brain cell injury responses. Damage to neurons caused by focal cerebral ischemia and epileptic seizures was exacerbated in TNFR-KO mice, indicating that TNF serves a neuroprotective function. Oxidative stress was increased and levels of an antioxidant enzyme reduced in brain cells of TNFR-KO mice, indicating that TNF protects neurons by stimulating antioxidant pathways. Injury-induced microglial activation was suppressed in TNFR-KO mice, demonstrating a key role for TNF in injury-induced immune response. Drugs that target TNF signaling pathways may prove beneficial in treating stroke and traumatic brain injury.
Induction of tumor necrosis factor α was studied in the brain of rats after focal cerebral ischaemia by occlusion of the left middle cerebral artery. Using a specific antisense riboprobe for situ hybridization histochemistry, cells positive for tumor necrosis factor α messenger RNA were detected within 30 min in the brain regions known to be necrotic within one to two days after onset of ischaemia. Their number increased over a time period of 1–8 h and then declined. Only a few tumor necrosis factor α messenger RNA positive cells could be detected four days after the onset of ischaemia. Reverse-transcription polymerase chain reaction experiments showed that maximal increase of tumor necrosis factor α messenger RNA level in the ischaemic brain hemisphere occurred 3 h after occlusion of the middle cerebral artery. Immunocytochemical experiments using an anti-tumor necrosis factor α antibody showed the presence of tumor necrosis factor α immunopositive cells as early as 30 min after occlusion of the middle cerebral artery in the same brain regions where tumor necrosis factor α messenger RNA positive cells were detected. Tumor necrosis factor α positive cells were highly abundant in the infarcted brain 8–24 h, but only few of them were detectable four days after the onset of ischaemia. Specificity of the anti-tumor necrosis factor α antibody and of the induction of tumor necrosis factor α protein was confirmed by western blot analysis. Tumor necrosis factor α messenger RNA- and protein-positive cells were also detected in the watershed zone and in some structures of the contralateral brain hemisphere. According to their morphology, tumor necrosis factor α-positive cells could be identified as microglial cells and macrophages at different states of activation. This assumption was further confirmed by double-labeling studies using the isolectin B4 from Griffonia simplicifolia, a specific microglial/macrophage cell marker.
In the surroundings of focal ischemic lesions, repetitive spreading depression (SD)-like depolarizations occur. These depolarizations are triggered by the anoxic release of potassium and excitatory amino acids from the infarct core, and they are propagated over the whole hemisphere at a speed of approximately 3 mm/min. The associated fluid shifts can be detected by diffusion-weighted magnetic resonance imaging (MRI) and correlate with an aggravation of the metabolic disturbance. In the peripheral, normally perfused brain regions of the infarcted hemisphere, the metabolic workload of SD is coupled to a parallel increase of blood flow, ensuring undisturbed oxygen supply. In the periinfarct penumbra, in contrast, the reduced hemodynamic capacity of the collateral system prevents adequate oxygenation and results in episodes of tissue hypoxia. Periinfarct SDs induce expression of immediate early genes in all brain regions except the ischemic core, i.e, in the penumbra and the surrounding normal brain tissue. In the penumbra, the hypoxic episodes evoked by SDs produce an additional stress response that is reflected by the expression of stress proteins and the suppression of global protein synthesis. In the most severely ischemic parts of the penumbra, periinfarct depolarizations may turn into terminal depolarization, resulting in a stepwise expansion of the infarct core. Postischemic application of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor antagonists suppresses periinfarct depolarizations, reverses the penumbral suppression of protein synthesis, and reduces infarct size. These observations demonstrate that periinfarct depolarizations aggravate focal ischemic injury and suggest that therapeutic suppression of these depolarizations minimizes infarct size.
The original notion that the brain represented an "immune-privileged" organ lacking the capability to produce an inflammatory response to an injury, would appear no longer tenable. Indeed, accumulating evidence during the last decade has shown that the CNS can mount a well-defined inflammatory response to a variety of insults including trauma, ischemia, transplantation, viral infections, toxins as well as neurodegenerative processes. Many aspects of this centrally-derived inflammatory response parallel, to some extent, the nature of such a reaction in the periphery. Through the recent application of molecular biological techniques, new concepts are rapidly emerging as to the molecular mechanisms associated with the development of brain injury. In particular, the importance of cytokines, especially TNF alpha and IL-1 beta, as well as adhesion molecules, has been emphasized in the propagation and maintenance of a CNS inflammatory response. This review will summarize recent observations as to the involvement of these inflammatory mediators in CNS injury and lay claim to the possibility that inhibitors of peripheral inflammation may also be of benefit in treating CNS injuries such as stroke, head trauma, Alzheimer's disease and multiple sclerosis.
A series of experiments was performed to determine the role of interleukin (IL)-1 in the induction of tolerance to global ischemia in Mongolian gerbils. In Group I, a 2-min "preconditioning" ischemia protected CA1 hippocampal neurons in gerbils subjected to 3.5 min ischemia 3 days later. CA1 neuronal density was: sham, 171 +/- 3/mm; 3.5 min ischemia, 30 +/- 30/mm; 2 and 3.5 min ischemia 162 +/- 6/mm. Experiments in Group II addressed the role of IL-1 in the induction of tolerance by sublethal ischemia. Arterial IL-1 alpha and IL-1 beta became elevated between 1 and 3 days after a 2-min ischemic exposure. IL-1 alpha was: sham, 6.4 +/- 0.6 ng/ml; and 2-day, 10.2 +/- 1.2 ng/ml. IL-1 beta was: sham, 6.4 +/- 0.5 ng/ml; and 2-day, 17.3 +/- 2 ng/ml. Recombinant human IL-1 receptor antagonist (IL-1ra) i.p. blocked ischemic tolerance induction by 2-min preconditioning ischemia: 2-min ischemia + vehicle, 162 +/- 6/mm; and 2-min ischemia + IL-1ra, 67 +/- 17/mm. Experiments in Group III assessed the capacity of IL-1 to induce tolerance to brain ischemia. IL-1 alpha i.p. (0, 10, 20 micrograms/kg) for 3 days prior to 3.5-min forebrain ischemia provided significant CA1 neuroprotection in a dose-dependent manner: 2 +/- 2, 68 +/- 83, and 129 +/- 42/mm, respectively. IL-1 beta (15 micrograms/kg) in combination with either IL-1ra (100 mg/kg) or IL-1ra vehicle i.p. on the same schedule demonstrated a significant CA1 neuroprotection that could be nullified by IL-1ra: IL-1 beta + IL-1ra vehicle, 153 +/- 16/mm; and IL-1 beta + IL-1ra, 67 +/- 36/mm. Recognition that tolerance arises from stimulation of a known receptor (IL-1RI) permits molecular analysis of the intracellular signaling that is critical for production of that state.
The cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha, produced by glial cells within the brain, appear to contribute to the neuropathogenesis of several inflammatory neurodegenerative diseases; however, little is known about the mechanism underlying cytokine-induced neurotoxicity. Using human fetal brain cell cultures composed of neurons and glial cells, we investigated the injurious effects of IL-1beta and TNF-alpha, cytokines which are known to induce nitric oxide (NO) production by astrocytes. Although neither cytokine alone was toxic, IL-1beta and TNF-alpha in combination caused marked neuronal injury. Brain cell cultures treated with IL-1beta plus TNF-alpha generated substantial amounts of NO. Blockade of NO production with a NO synthase inhibitor was accompanied by a marked reduction (about 45%) of neuronal injury, suggesting that NO production by astrocytes plays a role in cytokine-induced neurotoxicity. Addition of N-methly-D-aspartate (NMDA) receptor antagonists to brain cell cultures also blocked IL-1beta plus TNF-alpha-induced neurotoxicity (by 55%), implicating the involvement of NMDA receptors in cytokine-induced neurotoxicity. Treatment of brain cell cultures with IL-1beta plus TNF-alpha was found to inhibit [3H]-glutamate uptake and astrocyte glutamine synthetase activity, two major pathways involved in NMDA receptor-related neurotoxicity. These in vitro findings suggest that agents which suppress NO production or inhibit NMDA receptors may protect against neuronal damage in cytokine-induced neurodegenerative diseases.
We examined by immunohistochemistry the expression of ionotropic glutamate receptor subunits (GluRs) in glial cells of the rat dorsal hippocampus 3 to 28 days after transient forebrain ischemia. In general, the expression of GluRs at all time points studied underwent a drastic reduction that was primarily restricted to the CA1 region. In addition to the disappearance of GluRs as a result of neuronal cell death, we observed their expression in reactive glial cells. The time course of expression and the subunits involved were different for astrocytes and microglia. Reactive astrocytes exhibited kainate, GluR5-7, and N-methyl-D-aspartate (NMDA), NR2A/B, receptor subunits, both of which were maximally expressed approximately 4 weeks after ischemia. In contrast, reactive microglia expressed GluR4 and NR1 subunits, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and NMDA receptor subtypes, respectively, with maximal expression observed between 3 and 7 days after ischemia. These results demonstrate that specific types of GluRs are expressed in reactive glial cells after ischemia and that, overall, their expression levels peak around or after the periods of maximal astrogliosis and microgliosis. Thus, modulation of GluR expression may be one of the molecular components accompanying the gliotic process.
Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine that rapidly upregulates in the brain after injury. The present study was designed to explore the pathophysiological significance of brain TNF-alpha in the ischemic brain by systematically evaluating the effects of lateral cerebroventricular administration of exogenous TNF-alpha and agents that block the effects of TNF-alpha on focal stroke and by examining the potential direct toxic effects of TNF-alpha on cultured neurons to better understand how TNF-alpha might mediate stroke injury.
TNF-alpha (2.5 or 25 pmol) was administered intracerebroventricularly to spontaneously hypertensive rats 24 hours before permanent or transient (80 minutes and 160 minutes) middle cerebral artery occlusion (MCAO). Animals were examined 24 hours later for neurological deficits and ischemic hemisphere necrosis and swelling. In some of these studies, neutralizing anti-TNF-alpha monoclonal antibody (mAb) (60 pmol) was injected intracerebroventricularly 30 minutes before exogenous TNF-alpha (25 pmol). In addition, the effects of blocking endogenous TNF-alpha on permanent focal ischemic injury were determined with the use of either mAb (60 pmol) or soluble TNF receptor I (sTNF-RI) (0.3 or 0.7 nmol) administered intracerebroventricularly 30 minutes before and 3 and 6 hours after MCAO. Finally, the direct neurotoxic effects of TNF-alpha were studied in cultured rat cerebellar granule cells exposed to TNF-alpha (10 to 2000 U/mL for 6 to 24 hours), and neurotransmitter release, glutamate toxicity, and oxygen radical toxicity were studied.
TNF-alpha increased the percent hemispheric infarct induced by permanent MCAO in a dose-related manner from 13.1 +/- 1.3% (vehicle) to 18.9 +/- 1.7% at 2.5 pmol (P < .05) and 27.1 +/- 1.3% at 25 pmol (P < .0001). The high dose of TNF-alpha increased ischemia-induced forelimb deficits from 1.6 +/- 0.2 to 2.3 +/- 0.2 (P < 0.1). TNF-alpha (2.5 pmol) also increased the infarction induced by 80 or 160 minutes of transient MCAO from 1.9 +/- 0.9% to 4.3 +/- 0.4% (P < .01) and from 14.2 +/- 1.3% to 21.6 +/- 2.2% (P < .05), respectively. The exacerbation of infarct size, swelling, and neurological deficit after exogenous TNF-alpha was reversed by preinjection of 60 pmol mAb. Blocking endogenous TNF-alpha also significantly reduced focal ischemic brain injury. Treatment with 60 pmol mAb before and after permanent MCAO significantly reduced infarct size compared with control (nonimmune) antibody treatment by 20.2% (P < .05). Reduced brain infarction also was produced by brain administration of 0.3 nmol (decreased 18.2%) or 0.7 nmol (decreased 26.1%, P < .05) sTNF-RI before and after focal stroke. The intracerebroventricular administration of TNF-alpha or sTNF-RI did not alter brain or body temperature, blood gases or pH, blood pressure, blood glucose, or general blood chemistry. In cultured cerebellar granule cells, the application of TNF-alpha did not directly affect neurotransmitter release or glutamate or oxygen free radical toxicity.
These studies demonstrate that exogenous TNF-alpha exacerbates focal ischemic injury and that blocking endogenous TNF-alpha is neuroprotective. The specificity of the action(s) of TNF-alpha was demonstrated by antagonism of its effects with specific anti-TNF-alpha tools (ie, mAb and sTNF-RI). TNF-alpha toxicity does not appear to be due to a direct effect on neurons or modulation of neuronal sensitivity to glutamate or oxygen radicals and apparently is mediated through nonneuronal cells. These data suggest that inhibiting TNF-alpha may represent a novel pharmacological strategy to treat ischemic stroke.
Cytokines are recognized to play an important role in acute stroke. Tumor necrosis factor-alpha (TNF) is one of the pro-inflammatory cytokines and is expressed in ischemic brain. We hypothesized that TNF might play a role in the regulation of tolerance to ischemia when administered prior to the ischemic episode. We studied the effects of pretreatment of TNF administered intravenously, intraperitoneally, or intracisternally in mice that were subjected to middle cerebral artery occlusion (MCAO) 48 h later. MCAO was performed in BALB/C mice by direct cauterization of distal MCA, which resulted in pure cortical infarction. A significant reduction in infarct size was noted in mice pretreated by TNF at the dose of 0.5 microgram/mouse (p < 0.01) intracisternally. At the doses used in this study, administration of TNF by intravenous or intraperitoneal routes was not effective. Immunohistochemical analysis of brains subjected to 24 h of MCAO revealed a significant decrease in CD11b immunoreactivity after TNF pretreatment compared with control MCAO. Preconditioning with TNF affects infarct size in a time- and dose-dependent manner. TNF induces significant protection against ischemic brain injury and is likely to be involved in the signaling pathways that regulate ischemic tolerance.
In the brain large amounts of nitric oxide are produced in response to various pathological stimuli such as infectious agents, ischemia and trauma. Although it is known that endothelial cells can express the inducible isoform of nitric oxide synthase (iNOS) upon activation, the impact of different cytokines on iNOS expression in rat microvascular endothelial cells remains unclear. We now investigated iNOS mRNA expression and enzyme activity in primary cell cultures of rat microvascular brain endothelial cells after treatment with the proinflammatory cytokines interleukin-1β (IL-1β), Tumor necrosis factor-α (TNF-α), and interferon-gamma (IFN-γ) alone or in combination. Cells were characterized by immunocytochemistry staining for von-Willebrand-factor and the rat brain endothelial antigen recognized by monoclonal antibody Ox2. iNOS-enzyme activity was determined by measurement of nitrite in the supernatants of cell culture using the Griess-reaction. In addition mRNA expression was analysed by RT-PCR with iNOS and IL-1β specific primers. All cells in the endothelial cell culture were found to express the antigenic phenotype vWF+Ox2+/Ox43-, thus identifying the cells as rat brain endothelial cells of microvascular origin. IL-1β was the only cytokine that as a single stimulus induced iNOS mRNA expression and iNOS-enzyme activity in these endothelial cells. All combinations of two cytokines, including that of TNF-α and IFN-γ - or the triple combination led to expression of iNOS-mRNA and active protein. Cell activation by the combination of TNF-α + IFN-γ led to an early expression of IL-1β by the endothelial cells suggesting iNOS induction as a consequence of endogenous IL-1β production under this challenge. The experiments prove that rat brain microvascular endothelial cells express iNOS and produce large amounts of NO under inflammatory conditions. Furthermore, our results indicate a decisive role of IL-1β in iNOS expression and NO generation.
Cortical spreading depression (CSD) protects hippocampal and cortical neurons from an otherwise lethal ischemic insult delivered days later. The present study was undertaken to evaluate changes in the expression of BDNF following CSD, distinct from lesion effects and its possible involvement in delayed ischemic tolerance. CSD was elicited by KCl application and a cortical lesion was made by hyperosmolar NaCl application. BDNF mRNA was examined by in situ hybridization and Northern blot up to 7 days post-CSD. BDNF protein content was measured by ELISA. In the cortex, BDNF protein was mildly elevated despite minimal increases of mRNA in the NaCl lesion group. CSD specifically up-regulated BDNF mRNA at 4 h, followed by a delayed secondary increase at 2-3 days. BDNF protein exhibited smaller biphasic increases at 24 h and 3-7 days post-CSD which were significantly higher than the NaCl lesion group. In the hippocampus, BDNF protein levels showed a delayed decrease in both groups independent of mRNA changes, but CSD specifically delayed this decrease. Thus, CSD can alter BDNF levels independent of lesion effects. The increased BDNF following CSD in the cortex is consistent with the involvement of BDNF in cortical ischemic tolerance. BDNF could not, however, be directly related to ischemic tolerance in the hippocampus.
Inflammatory processes involving reactive microglia, e.g., those associated with beta-amyloid containing neuritic and core plaques in Alzheimer's disease, appear to contribute to neuronal degeneration in the CNS. The fact that increased nerve growth factor (NGF) protein levels were found throughout brains of Alzheimer's disease patients led us to investigate neurotrophin synthesis in a human microglial cell line showing typical properties of human microglial cells, including expression of neurotrophins such as NGF, as well as the NGF receptor trkA and the low-affinity neurotrophin receptor p75. We found that the cytokines interleukin-1beta and tumor necrosis factor-alpha synergistically stimulate microglial NGF transcription and protein release. Moreover, exposure of microglial cells to complement factor C3a induces NGF expression. To assess the role of the transcription factor nuclear factor-kappaB (NF-kappaB) in inflammatory mediator-induced microglial NGF expression, the effect of the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) was analyzed. In the presence of PDTC, a dose-dependent inhibition of cytokine-activated NGF expression occurred. In contrast, the C3a-dependent stimulation of NGF synthesis was not influenced by PDTC. In addition, microglial neurotoxicity-mediating beta-amyloid peptides A beta(1-40) and A beta(1-42) failed to alter NGF synthesis, whereas A beta(25-35) specifically induced NF-kappaB-dependent microglial NGF expression. In conclusion, inflammatory signals (cytokines and complement factors), as well as A beta(25-35), are potent stimulators of human microglial NGF synthesis involving NF-kappaB-dependent and -independent mechanisms. Microglial secretion of neurotrophins appears to be involved in early processes of neuronal regeneration.
In order to study structural alterations which occur after a defined unilateral cortical infarct, the hindlimb region of the rat cortex was photochemically lesioned. The infarcts caused edema restricted to the perilesional cortex which affected allocortical and isocortical areas differently. Postlesional changes in cytoskeletal marker proteins such as microtubule-associated protein 2, non-phosphorylated (SMI32) and phosphorylated (SMI35, SMI31 and 200,000 mol. wt) neurofilaments and 146,000 mol. wt glycoprotein Py as well as changes in proteoglycans visualized with Wisteria floribunda lectin binding (WFA) were studied at various time points and related to glial scar formation. The results obtained by the combination of these markers revealed six distinct regions in which transient, epitope-specific changes occurred: the core, demarcation zone, rim, perilesional cortex, ipsilateral thalamus and contralateral homotopic cortical area. Within the core immunoreactivity for microtubule-associated protein 2 and SMI32 decreased and the cellular components showed structural disintegration 4 h post lesion, but partial recovery of somatodendritic staining was seen after 24 h. Microtubule-associated protein 2 and SMI32 persisted up to days 7 and 5 respectively in the core, whereas the number of glial fibrillary acidic protein- and WFA-positive cells decreased between days 7 and 14. The demarcation zone showed a dramatic loss of immunoreactivity for all epitopes 4 h post lesion which was not followed by a phase of recovery. In the inner region of the demarcation zone there was an invasion and accumulation of non-neuronal WFA-positive cells which formed a tight capsule around the core. Neuronal immunoreactivities for microtubule-associated protein 2, SMI31 and Py as well as astrocytic glial fibrillary acidic protein increased strongly within an approximately 0.4-1.0 mm-wide rim region directly bordering the demarcation zone. Py immunoreactivity increased significantly in the perilesional cortex, whereas glial fibrillary acidic protein-positive astrocytes became transiently more numerous in the entire lesioned hemisphere including strongly enhanced immunoreactivity in the thalamus by days 5-7 post lesion. Glial fibrillary acidic protein immunoreactivity increased in the corpus callosum and the homotopic cortical area of the unlesioned hemisphere by days 5-7. In this homotopic area additional changes in SMI31 immunoreactivity occurred. Our results showed that a cortical infarct is not only a locally restricted lesion, but leads to a variety of cytoskeletal and other structural changes in widely-distributed functionally-related areas of the brain.
Since ischemic insults lead to a deregulation of nitric oxide production which contributes to delayed neuronal death, we investigated changes in the distribution and amount of nitric oxide synthases I and II and in the appearance of nitrotyrosine caused by small, well-defined photothrombic lesions (2 mm in diameter) in the somatosensory cortex of rats. Four hours after lesioning, cell loss was evident in the core of the lesion and no nitric oxide synthase was present within this area, indicating that neurons expressing nitric oxide synthase I were lost or that nitric oxide synthase I was degraded. No increase in the number of neurons expressing nitric oxide synthase I was visible in the area surrounding the lesion, nor in other parts of the brain. One day after lesioning, NADPH-diaphorase- and nitric oxide synthase II-positive leucocytes had invaded the perilesional cortex and were accumulated in injured blood vessels. By two to three days post-lesion, layer V and VI pyramidal neurons, microglia, astrocytes and invading leucocytes had become strongly immunoreactive for nitric oxide synthase II within a perilesional rim. The number of cells expressing nitric oxide synthase I remained stable. Nitric oxide synthase II immunoreactivity and related NADPH-diaphorase had decreased by seven days post-lesion in most animals. However, the number of activated microglia or macrophages and astrocytes, as revealed by other markers, remained elevated. In addition, nitrotyrosine immunoreactivity was evident in the blood vessels close to the lesion, as well as in the ipsilateral hippocampus and thalamus.
Functional recovery after stroke is partly due to cortical reorganization on a structural as well as a functional level. Recent investigations have shown that the excitability of brain areas surrounding cortical ischemic lesions is increased, probably due to a down-regulation of gamma-aminobutyric acid-receptor activity. There is some evidence that these changes might increase the susceptibility of the lesioned brain for adaptive changes and recovery. Here, we investigated the propensity for the induction of long-term potentiation (LTP) in the surround of experimentally induced focal cortical infarcts in rat somatosensory cortex in vitro. By using standard paradigms, LTP induction was found to be facilitated ipsilaterally in slices of lesioned animals 1 week after lesion induction. In homotopic contralateral areas, LTP was not different from control values. As LTP is commonly associated with plasticity and learning, the results provide further evidence for the lesion-induced amplification of network plasticity, as it is required for the reshaping of cortical circuits by timely training procedures.
Tumor necrosis factor alpha (TNFalpha) is a pleiotrophic cytokine with diverse proinflammatory actions. Focal cerebral ischemia induces rapid and dramatic increases in TNFalpha levels within and surrounding the focus of damaged brain both in striatum and cortex. The actions of TNFalpha during cerebral ischemia may be related to the cell types which deliver and/or accept TNFalpha signals. However, the cellular sources of TNFalpha following cerebral ischemia have not been fully elucidated. The present study was designed to determine the cellular localization of TNFalpha following permanent middle cerebral artery occlusion (MCAO) in mice. As judged by immunohistochemistry, TNFalpha expression in the ischemic hemisphere was increased at 3 h following MCAO, peaked at 6 to 12 h, and decreased at 24 h. Double immunostaining for TNFalpha and neuron specific enolase (NSE) or glial fibrillary acidic protein (GFAP) showed that TNFalpha positive neurons were observed in both the ischemic core and perifocal region, while TNFalpha positive astrocytes were observed in the outer cortical layer, the corpus callosum, the molecular layer of the hippocampus, and periventricular areas. The presence of TNFalpha immunoreactivity in neurons and nerve fibers following MCAO suggests that TNFalpha expressed in ischemic neurons might be delivered via axonal transport, while TNFalpha immunoreactivity in astrocyte end-feet and ependymal cells following MCAO suggests that TNFalpha may be involved in blood-brain barrier disruption and the initiation of inflammation in the brain.
Cerebral ischaemia leads to profound glial activation and leukocyte infiltration into the infarct area. In this study, we provide evidence for a dual macrophage response in focal ischaemic lesions of the rat brain. We show that a considerable proportion of macrophages in the ischaemic lesions express the CD8alphabeta heterodimer to date only described on CD8+ T cells. As known from other lesion paradigms, CD4+ macrophages were also present. Interestingly, CD8- and CD4-expressing macrophages formed two non-overlapping subpopulations. CD8+ macrophages reached their maximum during the first week with pronounced downregulation thereafter whereas CD4+ cells persisted at high levels into the second week. In contrast to cerebral ischaemia, macrophages in the spleen and in Wallerian degeneration after optic nerve axotomy expressed CD4, but not CD8. In experimental autoimmune encephalomyelitis, CD8 was mainly associated with T cells and very weakly detectable on some ramified cells resembling activated microglia. In conclusion, we show that cerebral ischaemia triggers an unusual inflammatory response characterized by the appearance of CD8+/CD4- macrophages that might exert specific functions in the pathogenesis of ischaemic brain damage.
Experimental autoimmune encephalomyelitis (EAE) is a model of autoimmune central nervous system (CNS) disease that is mediated by autoreactive Th1 cells secreting the proinflammatory cytokine interferon (IFN)-gamma. Interleukin (IL)-12 in its heterodimeric p35/p40 isoform and the recently described cytokine IL-18 potently induce T cell production of IFN-gamma. Interleukin-1beta converting enzyme (ICE) is required to convert IL-18 precursor protein into its biologically active mature form. In this study, we used semiquantitative reverse transciptase-polymerase chain reaction to determine steady state levels of IL-12, IL-18, and ICE mRNA in the spinal cord of Lewis rats at different stages of EAE. In control rats, we found significant IL-18, ICE, and IL-12p35, but not IL-12p40 mRNA expression. IL-18 mRNA increased during the acute stage of EAE together with a marked induction of ICE mRNA. IL-12p35 mRNA levels did not change significantly throughout the course of EAE. Surprisingly, the peak expression of IL-12p40 mRNA was delayed by several days relative to the peak of T cell infiltration and IFN-gamma mRNA synthesis. Our data implicate the IL-18/ICE pathway in the amplification of Th1-mediated immune responses in the CNS but suggest a different, so far undefined role of endogenous IL-12 in the late effector phase of EAE.