No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Inducible Nitric Oxide Synthase Gene Expression in Vascular Cells After Transient Focal Cerebral Ischemia
Abstract
We investigated whether inducible nitric oxide synthase (iNOS) is expressed after transient cerebral ischemia and, if so, we sought to define the temporal profile and cellular localization of the expression and the role of iNOS in the mechanism of ischemic brain injury.
The middle cerebral artery in rats was occluded for 2 hours by an intraluminal filament. The occurrence of transient ischemia and reperfusion was confirmed by laser-Doppler flowmetry (n = 5). iNOS message in the ischemic neocortex was determined by reverse-transcription polymerase chain reaction. iNOS enzymatic activity was assessed by citrulline assay. The cellular localization of iNOS expression was determined by immunohistochemistry.
iNOS mRNA was maximally expressed in postischemic brain at 12 hours and was not present at 4 days (n = 3 per time point). iNOS mRNA was not observed in the contralateral cerebral cortex. iNOS enzymatic activity developed in the postischemic brain between 12 and 24 hours (P < .05) and subsided at 4 days (n = 4 to 8 per time point). iNOS immunoreactivity in the ischemic region was restricted to the wall of capillaries and of larger blood vessels at 12 to 24 hours. In regions of early necrosis, inflammatory cells were iNOS positive. Treatment with the iNOS inhibitor aminoguanidine (n = 5; 100 mg/kg IP, BID for 4 days), starting 6 hours after ischemia, reduced infarct size in neocortex by 36 +/- 7% in comparison with vehicle-treated controls (n = 5) (P < .05).
Transient focal ischemia leads to iNOS expression in postischemic brain. However, the spatial and temporal patterns of expression differ from those occurring in permanent ischemia: iNOS is induced earlier and predominantly in vascular cells rather than in neutrophils. Thus, the temporal profile and localization of postischemic iNOS expression depend on the nature of the ischemic insult. The finding that aminoguanidine reduces infarct size adds further support to the hypothesis that postischemic iNOS expression contributes to ischemic brain damage.
... Vasorelaxation of brain microvessels is a feature of some diseases including chronic hypertension, diabetes, hypercholesterolemia, subarachnoid hemorrhage (SAH), and ischemia [41,117,125]. NO is also involved in regulating the cerebral circulation during hypercapnia [126,127] and focal [115,[128][129][130] or global brain ischemia [127,[131][132][133][134][135]. Furthermore, arginine-derived NO mediates the powerful effects of CO 2 on cerebral circulation. ...
... Due to its vascular effect, NO might improve tissue perfusion and exert a protective action. Overproduction, either by activation of nNOS by excitatory amino acids [137], or by induction of iNOS in glial, vascular, or blood cells [132][133][134] during the ischemic episodes, might be deleterious. Mice with eNOS gene knockout exhibit a decrease in vascular relaxation. ...
... Thorns and collaborators suggest that iNOS plays a role in the formation of NFT [141]. Iadecola and coworkers propose that iNOS contributes to ischemic brain damage [133]. The catalytic activities of iNOS enzymes or mRNA expression are evident in brain tissue after 2 hours of transient focal ischemia or 1-2 days after permanent focal ischemia [132,134]. ...
Mitochondrial dysfunction may be a principal underlying event in aging, including age-associated brain degeneration. Mitochondria provide energy for basic metabolic processes. Their decay with age impairs cellular metabolism and leads to a OPEN ACCESS Pharmaceuticals 2010, 3 159 decline of cellular function. Alzheimer disease (AD) and cerebrovascular accidents (CVAs) are two leading causes of age-related dementia. Increasing evidence strongly supports the theory that oxidative stress, largely due to reactive oxygen species (ROS), induces mitochondrial damage, which arises from chronic hypoperfusion and is primarily responsible for the pathogenesis that underlies both disease processes. Mitochondrial membrane potential, respiratory control ratios and cellular oxygen consumption decline with age and correlate with increased oxidant production. The sustained hypoperfusion and oxidative stress in brain tissues can stimulate the expression of nitric oxide synthases (NOSs) and brain endothelium probably increase the accumulation of oxidative stress products, which therefore contributes to blood brain barrier (BBB) breakdown and brain parenchymal cell damage. Determining the mechanisms behind these imbalances may provide crucial information in the development of new, more effective therapies for stroke and AD patients in the near future.
... NOS2, a key player in the post-ischemic inflammatory cascade (Dirnagl et al., 1999;Garcia-Bonilla et al., 2014), is expressed from hours to several days after MCAO in rodents (Iadecola et al., 1995;Grandati et al., 1997). Inhibiting NOS2 has an extended therapeutic window and induces long-lasting protection (Iadecola et al., 1995(Iadecola et al., , 1996Garcia-Bonilla et al., 2014). Post-ischemic NOS2 is expressed in leukocytes and brain endothelial cells of rodents and humans (Iadecola et al., 1996;Nathan, 1997;Forster et al., 1999;Niwa et al., 2001;Garcia-Bonilla et al., 2014), though decreasing NOS2 expression in leucocytes alone is insufficient to improve stroke outcomes (Garcia-Bonilla et al., 2014). ...
... Inhibiting NOS2 has an extended therapeutic window and induces long-lasting protection (Iadecola et al., 1995(Iadecola et al., , 1996Garcia-Bonilla et al., 2014). Post-ischemic NOS2 is expressed in leukocytes and brain endothelial cells of rodents and humans (Iadecola et al., 1996;Nathan, 1997;Forster et al., 1999;Niwa et al., 2001;Garcia-Bonilla et al., 2014), though decreasing NOS2 expression in leucocytes alone is insufficient to improve stroke outcomes (Garcia-Bonilla et al., 2014). ...
... These combined results suggest that intravenous miR-122 mimic acted on both blood leucocytes and BMVECs from the luminal sides of vessels, and are consistent with previous reports that only combined deletion of NOS2 in blood cells and BMVECs prevents brain injury after ischemic stroke in rats (Garcia-Bonilla et al., 2014). Since NOS2 is a key player in the post-ischemic inflammatory cascade, and inhibiting NOS2 has an extended therapeutic window out to at least 6 h (Iadecola et al., 1995(Iadecola et al., , 1996Garcia-Bonilla et al., 2014), it suggests that miR-122 mimic could have a broad therapeutic window to treat stroke. ...
Aim: Our previous study demonstrated miR-122 mimic decreased NOS2 expression in blood leucocytes and improved stroke outcomes when given immediately after middle cerebral artery occlusion (MCAO) in rats. Since NOS2 is associated with neuro-inflammation in stroke and decreasing NOS2 expression alone in leucocytes is insufficient to improve stroke outcomes, we hypothesized that miR-122 mimic may also decrease NOS2 expression in brain microvascular endothelial cells (BMVECs) even at extended time windows.
Methods: We administered PEG-liposome wrapped miR-122 mimic (2.4 mg/kg, i.v.) 0 or 6 h after MCAO, and assessed stroke volume and NOS2 expression in BMVECs 24 h following MCAO in rats. Luciferase reporter assays were used to determine if miR-122 binds to 3′ untranslated regions (3′UTR) of NOS2.
Results: The data showed that miR-122 mimic decreased infarct volumes and decreased MCAO-induced NOS2 over-expression in BMVECs. However, miR-122 did not bind to 3′UTR of NOS2 in the luciferase assays.
Conclusion: The data show the 6-h period of therapeutic efficacy of miR-122 mimic which could relate to indirect knockdown of NOS2 in both BMVECs and leucocytes.
... A number of investigators have studied NOS expression in other models of cerebral ischemia (14,18,32). Accumulating evidence indicates early increases in NOS-1 (neuronal) and NOS-3 (endothelial) (49) and a delayed increase in NOS-2 (inducible) expression (18,19), accompanied by increased NO production (21). ...
... A number of investigators have studied NOS expression in other models of cerebral ischemia (14,18,32). Accumulating evidence indicates early increases in NOS-1 (neuronal) and NOS-3 (endothelial) (49) and a delayed increase in NOS-2 (inducible) expression (18,19), accompanied by increased NO production (21). To determine how NOS is altered after SAH, we studied changes in NOS protein lev-els after SAH by examining the amount of active NOS, its kinetic parameters, and the number of NOS-1-and NOS-2positive cells at different time intervals after experimental SAH. ...
... They found newly expressed NOS-2 to be enzymatically active, and because its inhibition ameliorated SAH-induced vasoconstriction, they suggested that NOS-2 contributes directly to delayed vasoconstriction after SAH (34). In other ischemic injury models, increased expression of NOS-2 (12 h to 2 or 4 d) is accompanied by large increases in NO production and is deleterious (18,19). In the present study, NOS enzymatic data indicated that the increase in total NOS-2-positive cells 1 hour after SAH was not accompanied by an increase in total tissue content of active NOS. ...
... Povećan nivo NO dovodi do inhibicije enzima uključenih u regulaciju metabolizma i DNK sinteze [12,[34][35][36]. Pokazano je da tokom ABI dolazi do povećane produkcije NO [37][38][39]. ...
According to the World Health Organization, 15 million people per year are affected by stroke. The most common cause of stroke is brain ischemia, which occurs in almost 85% of cases. Ischemia caused by thromboembolism is defined as permanently or temporarily decreased blood flow which prevents an adequate delivery of oxygen, glucose and other important nutrients, leading progressively to metabolic changes and cell apoptosis. Carotid endarterectomy (CEA) can cause hypoxic - ischemic states of the brain or acute brain ischemia (ABI) leading eventually to stroke. The main cause of ABI as a result of CEA is cerebral hypoperfusion caused by clamping of carotid arteries, when hypoxia occurs.. Hypoxia per se is one of the triggers of complex physiological responses in the body, including the release of various mediators of inflammation. One of these inflammatory mediators is nitric oxide (NO), a free radical which has numerous physiological effects and also plays an important role in the immune response of the organism. However, NO may be very harmful and cause cell and tissue damage. The lack of literature data on the role of endothelial NOS (eNOS) and inducible NOS (iNOS) during CEA, as well as the mechanisms of their regulation in ischemic conditions, suggest that intensifying future research in this field is very important. An insight into molecular mechanisms of iNOS activity and expression regulation will certainly help to develop new therapeutic strategies for treating harmful effects of free radicals, especially uncontrolled production of NO.
... Nevertheless, damaged brain cells release factors that promote macrophage polarization towards a pro-inflammatory M1 phenotype, remaining in this state and releasing proinflammatory factors for days to weeks later [44]. Furthermore, these cells may produce toxic levels of NO mostly via the iNOS isoform, which is markedly induced after ischemic injury both in animal models [45,46] and in patients suffering from stroke [47]. Thus, it could be stated that ROS-induced inflammatory response enhances ROS production, in a pathophysiological vicious cycle. ...
The underlying mechanism of cerebral injury occurring in patients with acute ischemic stroke involves a key pathophysiological role of oxidative stress. Thus, reactive oxygen species are related to the development of brain edema, calcium overload, mitochondrial dysfunction, excitotoxicity, iron release and inflammation. Nevertheless, although experimental studies have tested the use of antioxidants as an adjuvant therapy in this setting, clinical data and randomized trials are still lacking. Current approved pharmacological therapy is aimed at reperfusion strategies; however, the therapeutic window is limited and also challenged by the injury known to result from the reperfusion. We have recently defined a timecourse occurrence of pathological events triggered by reperfusion-dependent increased reactive oxygen species, thus suggesting the beneficial role of the pertinent use of antioxidants. The present study was aimed to support the hypothesis that an enhanced antioxidant neuroprotection could be achieved by the use of two or more antioxidants opportunely provided to ischemic stroke patients focused against the specific mechanism occurring throughout the pathophysiological process. From this paradigm,using an underexplored therapeutic principle, it could be suggested that antioxidant-based therapy is a novel, promising, safe, available and cost-effective strategy against the deleterious effects of ischemic stroke that needs to be further studied in clinical protocols.
... Neutrophil intracellular metabolism is important for stroke pathogenesis, with neutrophil-mediated oxidative stress being implicated in detrimental effects (Bonaventura et al., 2019;Jickling et al., 2015). Production of NO by activated infiltrating neutrophils, mediated by iNOS, induces brain damage in the middle cerebral artery occlusion (MCAO) model of ischemic stroke (IS) (Garcia-Bonilla et al., 2014;Iadecola, Zhang, Casey, Clark, & Ross, 1996). In accordance, post-mortem analysis of human brains from patients who died of IS identified extensive iNOS expression in neutrophils located in the infarcted area, which correlated with protein nitration, suggesting the presence of nitrosative stress mediated by iNOS-expressing neutrophils (Forster, Clark, Ross, & Iadecola, 1999). ...
Neuroinflammatory and neurodegenerative diseases are characterized by the recruitment of circulating blood‐borne innate and adaptive immune cells into the central nervous system (CNS). These leukocytes sustain the detrimental response in the CNS by releasing pro‐inflammatory mediators that induce activation of local glial cells, blood–brain barrier (BBB) dysfunction, and neural cell death. However, infiltrating peripheral immune cells could also dampen CNS inflammation and support tissue repair. Recent advances in the field of immunometabolism demonstrate the importance of metabolic reprogramming for the activation and functionality of such innate and adaptive immune cell populations. In particular, an increasing body of evidence suggests that the activity of metabolites and metabolic enzymes could influence the pathogenic potential of immune cells during neuroinflammatory and neurodegenerative disorders. In this review, we discuss the role of intracellular metabolic cues in regulating leukocyte‐mediated CNS damage in Alzheimer's and Parkinson's disease, multiple sclerosis and stroke, highlighting the therapeutic potential of drugs targeting metabolic pathways for the treatment of neurological diseases.
image
... www.nature.com/scientificreports/ expression of COX-2, iNOS, and TNF-α after 12-24 h in rats [56][57][58][59] . Prior research has shown that COX-2 contributes to brain damage by producing toxic prostanoids and reactive oxygen species, while iNOS generates nitric oxide in large amounts resulting in DNA damage and oxidative stress 60 . ...
Oleic acid (OA) is released from brain phospholipids after cerebral ischaemia; however, its role in ischaemic injury remains unknown. We hypothesised that OA has neuroprotective effects after cerebral ischaemia, which may be exerted through peroxisome proliferator-activated receptor gamma (PPAR-γ) activation, since OA is an endogenous ligand of PPAR-γ. The effects of OA administration were evaluated in rodent models of middle cerebral artery occlusion (MCAO), photothrombosis, and four-vessel occlusion (4-VO). We determined the time window of therapeutic opportunity and examined the ability of the PPAR-γ antagonist GW9662 to reverse OA’s protective effects after MCAO. We found that OA administration decreased the MCAO-induced infarct volume and functional deficits, photothrombosis-induced infarct volume, and 4-VO-induced hippocampal neuronal death. Additionally, OA was highly efficacious when administered up to 3 h after MCAO. Pre-treatment with GW9662 abolished the inhibitory effects of OA on the infarct volume and immunoreactivity of key inflammatory mediators in the ischaemic cortex. Our results indicate that OA has neuroprotective effects against transient and permanent focal cerebral ischaemia, as well as global cerebral ischaemia. It may have therapeutic value for the ischaemic stroke treatment with a clinically feasible therapeutic window. The OA-mediated neuroprotection might be attributable to its anti-inflammatory actions through PPAR-γ activation.
... FN-evoked neuroprotection also seems to suppress the inflammatory response, which plays an important role in the protective effects. Expression of inducible nitric oxide synthase (iNOS) by cerebral microvessels and leukocytes is one of the components of the inflammatory reaction to ischemia (Iadecola et al., 1995a;Iadecola et al., 1995b;Nagafuji et al., 1995;Iadecola et al., 1996;Cobbs et al., 1997;Galea et al., 1998c;Cernak et al., 2001), which while important for reparative processes after brain damage, can also exacerbate it (see (Barone and Parsons, 2000;Iadecola and Alexander, 2001;Morganti-Kossmann et al., 2002)). FN stimulation 48 h prior to MCAO reduces induction of iNOS mRNA and expression of active iNOS in brain microvessels, and the infiltration of macrophages into the territory which is salvaged (Galea et al., 1998a). ...
Massive research efforts to develop effective neuroprotective therapy against stroke until now produced unsatisfactory results. It has been suggested that monotherapeutic approaches may not be sufficient. Investigations over the last three decades convincingly demonstrated the existence of powerful endogenous protective mechanisms. One of the innate protective mechanisms includes several brain structures, which when activated render the brain tolerant to various damaging stimuli. The best studied today is the cerebellar fastigial nucleus, neurons of which when activated, initiate coordinated multifactorial response providing long lasting neuroprotection. Numerous protective mechanisms induced by fastigial nucleus stimulation and other conditioning maneuvers are shared. In this review we summarize current knowledge of the neurogenic neuroprotection system related to the cerebellar fastigial nucleus and its commonalities with other forms of conditioning. Unveiling the systemic neuroprotective mechanisms will allow development of therapeutic approaches targeted toward activation/amplification of innate protective multifactorial mechanisms.
... iNOS participates in the immune response mainly contributing to induce apoptosis in transformed cells and pathogens. In brain iNOS is expressed by astrocytes, microglia and vascular cells releasing NO during hours or days [7,48,49]. ...
Nitric oxide (NO) works as a retrograde neurotransmitter in synapses, allows the brain blood flow and also has important roles in intracellular signaling in neurons from the regulation of the neuronal metabolic status to the dendritic spine growth. Moreover NO is able to perform post-translational modifications in proteins by the S-nitrosylation of the thiol amino acids, which is a physiological mechanism to regulate protein function. On the other hand, during aging and pathological processes the behavior of NO can turn harmful when reacts with superoxide anion to form peroxynitrite. This gaseous compound can diffuse easily throughout the neuronal membranes damaging lipid, proteins and nucleic acids. In the case of proteins, peroxynitrite reacts mostly with the phenolic ring of the tyrosines forming nitro-tyrosines that affects dramatically to the physiological functions of the proteins. Protein nitrotyrosination is an irreversible process that also yields to the accumulation of the modified proteins contributing to the onset and progression of neurodegenerative processes such as Alzheimer's disease or Parkinson's disease.
... Furthermore, iNOS expression in the hippocampal CA1 subfield at 7 days time point was significantly increased compared to 2 days time point and injured neurons were observed in a larger area in the hippocampal CA1 subfield. It was reported that iNOS, expressed at late stages after ischemia [46], participated in 1 3 the later processes of neurotoxicity. Some previous studies indicated that IH preconditioning induced brain ischemia tolerance [19,47]. ...
Previous studies have shown that intermittent hypobaric hypoxia (IH) preconditioning protected neurons survival from brain ischemia. However, the mechanism remains to be elucidated. The present study explored the role of nitric oxide (NO) in the process by measuring the expression of NO synthase (NOS) and NO levels. Male Wistar rats (100) were randomly assigned into four groups: sham group, IH + sham group, ischemia group and IH + ischemia group. Rats for IH preconditioning were exposed to hypobaric hypoxia mimicking 5000 m high-altitude (PB = 404 mmHg, PO2 = 84 mmHg) 6 h/day, once daily for 28 days. Global brain ischemia was established by four-vessel occlusion that has been created by Pulsinelli. Rats were sacrificed at 7th day after the ischemia for neuropathological evaluation by thionin stain. In addition, the expression of neuronal NOS (nNOS), inducible NOS (iNOS), and NO content in the hippocampal CA1 subfield were measured at 2nd day and 7th day after the ischemia. Results revealed that global brain ischemia engendered delayed neuronal death (DND), both nNOS and iNOS expression up-regulated, and NO content increased in the hippocampal CA1 subfield. IH preconditioning reduced neuronal injury induced by the ischemia, and prevented the up-regulation of NOS expression and NO production. In addition, L-NAME + ischemia group was designed to detect whether depressing NO production could alleviate the DND. Pre-administration of L-NAME alleviated DND induced by the ischemia. These results suggest that IH preconditioning plays a protective role by inhibiting the over expression of NOS and NO content after brain ischemia.
... MB could attenuate the negative effect of the hyperoxia by acting as an antioxidant to reduce free radical injury (Bruchey and Gonzalez-Lima 2008), and by enhancing energy production at a time when there is a significant percentage of occluded capillaries (Clifton and Leikin 2003). Low-dose MB suppresses inducible nitric oxide synthase, which plays a role in inflammatory pathways and is overexpressed in transient brain ischemia and contributes to early necrosis (Iadecola et al. 1996). ...
Introduction
Ischemic stroke is a global burden that contributes to the disability and mortality of millions of patients. This study aimed to evaluate the efficacy of combined MB (methylene blue) and NBO (normobaric hyperoxia) therapy in experimental ischemic stroke.
Methods
Rats with transient (60 min) MCAO (middle cerebral artery occlusion) were treated with: (1) air + vehicle (N = 8), (2) air + MB (N = 8), (3) NBO + vehicle (N = 7), and (4) NBO + MB (N = 9). MB (1 mg/kg) was administered at 30 min, again on days 2, 7, and 14 after stroke. NBO was given during MRI (30–150 min) on day 0, and again 1 h each during MRI on subsequent days. Serial diffusion, perfusion and T2 MRI were performed to evaluate lesion volumes. Foot‐fault and cylinder tests were performed to evaluate sensorimotor function.
Results
The major findings were: (1) NBO + MB therapy showed a greater decrease in infarct volume compared to NBO alone, but similar infarct volume compared to MB alone, (2) NBO + MB therapy accelerated sensorimotor functional recovery compared to NBO or MB alone, (3) Infarct volumes on day 2 did not change significantly from those on day 28 for all four groups, but behavioral function continued to show improved recovery in the NBO + MB group.
Conclusions
These findings support the hypothesis that combined NBO + MB further improves functional outcome and reduces infarct volume compared to either treatment alone and these improvements extended up to 28 days.
... 126,127 FN-evoked neuroprotection also seems to suppress the body's inflammatory response, a response that plays an important role in posthypoxic neuronal damage. The expression of inducible nitric oxide synthase (iNOS) by cerebral microvessels and leukocytes is one of the components of the inflammatory reaction, [128][129][130][131][132][133] which, while important for reparative processes after brain damage, can also exacerbate it. [134][135][136] FN stimulation 48 h prior to MCA occlusion reduces the induction of iNOS messenger ribonucleic acid (mRNA) and the expression of active iNOS in brain microvessels and, consequently, the infiltration of macrophages into the territory salvaged. ...
Stimulation of the ophthalmic branch of the trigeminal nerve can trigger a complex response known as the diving response (DR). Universally expressed in all animals, the DR is targeted to enable them to survive anoxic conditions. In the response, severe hypoxia or anoxia, complex multisystem and multilevel changes diminish oxygen consumption and activate endogenous protective mechanisms. The salient features of the DR are apnea, bradycardia, and peripheral vasoconstriction. Bradycardia decreases myocardial oxygen consumption, peripheral vasoconstriction limits both the blood supply and oxygen use by "nonvital" organs. Although the brain's blood supply remains stable, neurogenic mechanisms diminish brain activity in order to decrease brain metabolism. Along with the slowing of oxygen consumption, neurogenic neuroprotective mechanisms are activated to increase the brain's tolerance of hypoxia at system and cellular levels.
... However, endothelial nitric oxide synthase is localized primarily in endothelium and endothelial nitric oxide synthase -derived NO, via peroxynitrite, has been associated with blood-brain barrier leakage, cerebral hemorrhage and edema [11][12][13]. In rodent, inducible nitric oxide synthase is expressed from ~12 h to several days after IR and its immunoreactivity is present mainly in inflammatory cells and endothelium [14][15][16]. Therefore, the focus of this acute IR study is to investigate the mechanisms of nNOS regulation for developing stroke therapy. ...
Stroke is associated with significant morbidity and mortality due to the limited neuroregeneration capacity of the injured brain. Other than thrombolysis in the acute phase of the disease by tissue plasminogen activator (tPA), which offers only a short window of treatment (~3 hours), an ideal stroke therapy is not available mainly because of limited understanding of the mechanisms of neuroregeneration and functional recovery in the chronic phase. Yet many drug therapies, including S-nitrosoglutathione (GSNO), have been shown to provide neuroprotection against acute disease in animal models of transient cerebral ischemia reperfusion (IR) and permanent ischemia. GSNO was also effective in stimulating neuroregeneration-related factors in the chronic phase of the disease. In this short review, we assess the evidence supporting exogenous administration of GSNO after experimental stroke as a means to stimulate neuroregeneration and aid in functional recovery via stabilization of the HIF-1α/VEGF pathway.
... Within minutes of the vascular occlusion, multiple cascades of inflammatory events are initiated. These cascades include infiltration and accumulation of neutrophils and macrophages (10)(11)(12)(13); platelet accumulation (1,(13)(14)(15); adhesion molecule up-regulation, such as ICAM-1, P-selectin, and E-selectin (14,(16)(17)(18)(19)(20)(21); blood-brain barrier destruction (1,22,23); cytokine production including IL-1, IL-8, tumor necrosis factoralpha (TNF-␣), and platelet activating factor (PAF) (14,(24)(25)(26)(27)(28); increased nitric oxide levels (1,21,(29)(30)(31)(32)(33)(34); and cyclooxygenase up-regulation (1,(35)(36)(37)(38)(39)(40). ...
Background
The complement cascade plays a deleterious role in multiple models of ischemia/reperfusion (I/R) injury, including stroke. Investigation of the complement cascade may provide a critical approach to identifying neuroprotective strategies that can be effective at clinically relevant time points in cerebral ischemia. This review of the literature describes the deleterious effects of complement activation in systemic I/R models and previous attempts at therapeutic complement inhibition, with a focus on the potential role of complement inhibition in ischemic neuroprotection. Translation of these concepts into ischemic stroke models and exploration of related neuroprotective strategies are also reviewed. Summary of ReviewWe performed a MEDLINE search to identify any studies published between 1966 and 2001 dealing with complement activation in the setting of I/R injury. We also searched for studies demonstrating up-regulation of any complement components within the central nervous system during inflammation and/or ischemia. Conclusions
The temporal and mechanistic overlap of the complement cascade with other biochemical events occurring in cerebral I/R injury is quite complex and is only beginning to be understood. However, there is compelling evidence that complement is quite active in the setting of acute stroke, suggesting that anticomplement strategies should be further investigated through genetic analysis, nonhuman primate models, and clinical investigations.
... [101] Transient global cerebral ischemia upregulates NOS-2 in rodents. [102][103][104] Although direct examination of the effects of ischemia on NOS-2 expression in the context of SAH does not exist, ischemic injury presents a plausible source for NOS-2 elevation following SAH. ...
Aneurysmal subarachnoid hemorrhage (SAH) typically carries a poor prognosis. Growing evidence indicates that overabundant production of nitric oxide (NO) may be responsible for a large part of the secondary injury that follows SAH. Although SAH modulates the activity of all three isoforms of nitric oxide synthase (NOS), the inducible isoform, NOS-2, accounts for a majority of NO-mediated secondary injuries after SAH. Here, we review the indispensable physiological roles of NO that must be preserved, even while attempting to downmodulate the pathophysiologic effects of NO that are induced by SAH. We examine the effects of SAH on the function of the various NOS isoforms, with a particular focus on the pathological effects of NOS-2 and on the mechanisms responsible for its transcriptional upregulation. Finally, we review interventions to block NOS-2 upregulation or to counteract its effects, with an emphasis on the potential therapeutic strategies to improve outcomes in patients afflicted with SAH. There is still much to be learned regarding the apparently maladaptive response of NOS-2 and its harmful product NO in SAH. However, the available evidence points to crucial effects that, on balance, are adverse, making the NOS-2/NO/peroxynitrite axis an attractive therapeutic target in SAH.
... The brain is particularly vulnerable to oxidative stress on account of its high reactive oxygen levels, the presence of polyunsaturated fatty acids, transition metals in ionic form and low amounts of antioxidants 37 . Earlier it was reported that aberrant production of NO leads to peroxy-nitrite production can irreversibly damage lipid and protein synthesis causing cell death 38 . NO enhances glutamate excito-toxicity. ...
Neurotropic viruses induce neurodegeneration either directly by activating host death domains or indirectly through host immune response pathways. Chandipura Virus (CHPV) belonging to family Rhabdoviridae is ranked among the emerging pathogens of the Indian subcontinent. Previously we have reported that CHPV induces neurodegeneration albeit the root cause of this degeneration is still an open question. In this study we explored the role of microglia following CHPV infection. Phenotypic analysis of microglia through lectin and Iba-1 staining indicated cells were in an activated state post CHPV infection in cortical region of the infected mouse brain. Cytokine Bead Array (CBA) analysis revealed comparatively higher cytokine and chemokine levels in the same region. Increased level of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), Nitric Oxide (NO) and Reactive Oxygen species (ROS) in CHPV infected mouse brain indicated a strong inflammatory response to CHPV infection. Hence it was hypothesized through our analyses that this inflammatory response may stimulate the neuronal death following CHPV infection. In order to validate our hypothesis supernatant from CHPV infected microglial culture was used to infect neuronal cell line and primary neurons. This study confirmed the bystander killing of neurons due to activation of microglia post CHPV infection.
... Upon stroke onset, NO level is remarkably increased within half hour from less than 10 nM to more than 1 µM [109], which is mainly derived from nNOS and eNOS [110][111][112]. In brain microvessels, iNOS activity was induced, and a large amount of NO was produced within 4 hours after cerebral ischemia [113]. Increased iNOS activity lasted for at least 24 hours, continuously over-producing NO [114,115]. ...
Tissue plasminogen activator (t-PA) is the only FDA-approved drug for acute ischemic stroke treatment, but its clinical use is limited due to the narrow therapeutic time window and severe adverse effects, including hemorrhagic transformation (HT) and neurotoxicity. One of the potential resolutions is to use adjunct therapies to reduce the side effects and extend t-PA's therapeutic time window. However, therapies modulating single target seem not to be satisfied, and a multitarget strategy is warranted to resolve such complex disease. Recently, large amount of efforts have been made to explore the active compounds from herbal supplements to treat ischemic stroke. Some natural compounds revealed both neuro- and bloodbrain- barrier (BBB)-protective effects by concurrently targeting multiple cellular signaling pathways in cerebral ischemia-reperfusion injury. Thus, those compounds are potential to be one-drug-multi-target agents as combined therapy with t-PA for ischemic stroke.
In this review article, we summarize current progress about molecular targets involving in t-PA-mediated HT and neurotoxicity in ischemic brain injury. Based on these targets, we select 23 promising compounds from currently available literature with the bioactivities simultaneously targeting several important molecular targets. We propose that those compounds merit further investigation as combined therapy with t-PA. Finally, we discuss the potential drawbacks of the natural compounds' studies and raise several important issues to be addressed in the future for the development of natural compound as an adjunct therapy.
... 216 In addition to VEGF, a host of signaling molecules including CCL2, angiopoietin 2 (Ang2), and nitric oxide are released following injury, and can inhibit expression of tight junction proteins and thereby exacerbate vasogenic edema formation. [217][218][219][220][221] Endothelial basement membrane proteins and tight junction proteins also can be lost following CNS injury through protease degradation. Following injury, matrix metalloproteinase (MMP) activity increases through de novo expression and activation of latent MMPs, resulting in degradation of basement membrane and tight junction proteins. ...
Advancements in molecular biology have led to a greater understanding of the individual proteins responsible for generating cerebral edema. In large part, the study of cerebral edema is the study of maladaptive ion transport. Following acute CNS injury, cells of the neurovascular unit, particularly brain endothelial cells and astrocytes, undergo a program of pre- and post-transcriptional changes in the activity of ion channels and transporters. These changes can result in maladaptive ion transport and the generation of abnormal osmotic forces that, ultimately, manifest as cerebral edema. This review discusses past models and current knowledge regarding the molecular and cellular pathophysiology of cerebral edema.
... A151 reduced mature caspase-1 ( Fig 2B) and NLRP3 (Fig 2D), but did not affect ASC, AIM2, NLRP1 or NLRC4 (data not shown). Further it is known that IL-1β can induce the expression of iNOS [48] and that iNOS can influence stroke-induced cellular damage [49]. We therefore analyzed iNOS levels and found that A151 reduced the levels of iNOS expression (Fig 2E). ...
The immune system plays a fundamental role in both the development and pathobiology of stroke. Inflammasomes are multiprotein complexes that have come to be recognized as critical players in the inflammation that ultimately contributes to stroke severity. Inflammasomes recognize microbial and host-derived danger signals and activate caspase-1, which in turn controls the production of the pro-inflammatory cytokine IL-1β. We have shown that A151, a synthetic oligodeoxynucleotide containing multiple telemeric TTAGGG motifs, reduces IL-1β production by activated bone marrow derived macrophages that have been subjected to oxygen-glucose deprivation and LPS stimulation. Further, we demonstrate that A151 reduces the maturation of caspase-1 and IL-1β, the levels of both the iNOS and NLRP3 proteins, and the depolarization of mitochondrial membrane potential within such cells. In addition, we have demonstrated that A151 reduces ischemic brain damage and NLRP3 mRNA levels in SHR-SP rats that have undergone permanent middle cerebral artery occlusion. These findings clearly suggest that the modulation of inflammasome activity via A151 may contribute to a reduction in pro-inflammatory cytokine production by macrophages subjected to conditions that model brain ischemia and modulate ischemic brain damage in an animal model of stroke. Therefore, modulation of ischemic pathobiology by A151 may have a role in the development of novel stroke prevention and therapeutic strategies.
... However, endothelial nitric oxide synthase is localized primarily in endothelium and endothelial nitric oxide synthase -derived NO, via peroxynitrite, has been associated with blood-brain barrier leakage, cerebral hemorrhage and edema [11][12][13]. In rodent, inducible nitric oxide synthase is expressed from ~12 h to several days after IR and its immunoreactivity is present mainly in inflammatory cells and endothelium [14][15][16]. Therefore, the focus of this acute IR study is to investigate the mechanisms of nNOS regulation for developing stroke therapy. ...
Stroke immediately sets into motion sustained excitotoxicity and calcium dysregulation, causing aberrant activity in neuronal nitric oxide synthase (nNOS) and an imbalance in the levels of nitric oxide (NO). Drugs targeting nNOS-originated toxicity may therefore reduce stroke-induced damage. Recently, we observed that a redox-modulating agent of the NO metabolome, S-nitrosoglutathione (GSNO), confers neurovascular protection by reducing the levels of peroxynitrite, a product of aberrant NOS activity. We therefore investigated whether GSNO-mediated neuroprotection and improved neurological functions depend on blocking nNOS/peroxynitrite-associated injurious mechanisms using a rat model of cerebral ischemia reperfusion (IR).
IR increased the activity of nNOS, the levels of neuronal peroxynitrite and phosphorylation at Ser(1412) of nNOS. GSNO treatment of IR animals decreased IR-activated nNOS activity and neuronal peroxynitrite levels by reducing nNOS phosphorylation at Ser(1412). The Ser(1412) phosphorylation is associated with increased nNOS activity. Supporting the notion that nNOS activity and peroxynitrite are deleterious following IR, inhibition of nNOS by its inhibitor 7-nitroindazole or reducing peroxynitrite by its scavenger FeTPPS decreased IR injury. GSNO also decreased the activation of AMP Kinase (AMPK) and its upstream kinase LKB1, both of which were activated in IR brain. AMPK has been implicated in nNOS activation via Ser(1412) phosphorylation. To determine whether AMPK activation is deleterious in the acute phase of IR, we treated animals after IR with AICAR (an AMPK activator) and compound c (an AMPK inhibitor). While AICAR potentiated, compound c reduced the IR injury.
Taken together, these results indicate an injurious nNOS/peroxynitrite/AMPK cycle following stroke, and GSNO treatment of IR inhibits this vicious cycle, resulting in neuroprotection and improved neurological function. GSNO is a natural component of the human body, and its exogenous administration to humans is not associated with any known side effects. Currently, the FDA-approved thrombolytic therapy suffers from a lack of neuronal protective activity. Because GSNO provides neuroprotection by ameliorating stroke's initial and causative injuries, it is a candidate of translational value for stroke therapy.
Nitric oxide (NO·) is produced by many different cell types in the brain, including neurons, vascular endothelial cells, glial cells, and astrocytes. Although the neuronal nitric oxide synthase (nNOS) isoform accounts for the majority of NOS in the brain, all three isoforms are present and serve as potential sources of NO· production under physiological and pathological conditions (1,2). Therefore, precise tools and carefully designed experiments are necessary to elucidate the functions and effects of this ubiquitous molecule. In this chapter, we summarize the evidence to date that NO· and NOS isoforms play important roles in the pathophysiology of cerebral ischemia.
The susceptibility of the brain to ischaemic injury dramatically limits its viability following interruptions in blood flow. However, data from studies of dissociated cells, tissue specimens, isolated organs and whole bodies have brought into question the temporal limits within which the brain is capable of tolerating prolonged circulatory arrest. This Review assesses cell type-specific mechanisms of global cerebral ischaemia, and examines the circumstances in which the brain exhibits heightened resilience to injury. We suggest strategies for expanding such discoveries to fuel translational research into novel cytoprotective therapies, and describe emerging technologies and experimental concepts. By doing so, we propose a new multimodal framework to investigate brain resuscitation following extended periods of circulatory arrest.
Quantitative human mRNA data are derived from post-mortem or biopsied tissue. Confounding factors, RNA degradation, poor replication and a large variance are often cited, however, as objections to the data's reliability. At issue is whether post-mortem mRNA represents an ordered system and to what degree non-specific factors contribute to the measurements. I developed statistical methods and validated them by measuring 25 mRNA transcripts in an animal model of ischaemia. In the process I discovered novel increases for 3 genes in rats with ischaemic damage: leukaemia inhibitory factor, nestin and galanin mRNA. Additionally, I discovered that reference genes known as "housekeepers"' do not always act as steady-state controls and that the precise value of a test gene response varies according to the baseline choice of reference gene. Once optimised, I applied the analytical methods to human post-mortem brains. I used TaqMan™ real-time RT-PCR to measure 13 mRNAs in 513 cortical samples taken from 90 Alzheimer's disease and 81 control brains. Despite a high variance and confounding factors such as brain pH, I found strong geometric relations between the mRNA transcripts up to and beyond 100 hours autopsy delay. Where a postmortem brain had a high/low level of one mRNA, the same brain invariably had a high/low level of other mRNAs; correlated order is present and provides a means of isolating any mRNA change due specifically to disease. I measured mRNA levels of β-Secretase (BACE), GSK 3 and the isoforms of APP/APRP in the AD and control brains. After adjustment for age of death, brain pH, and gender, there was no change in the mRNA levels for either BACE or GSK 3α mRNA (p = 0.354 and p = 0.054 respectively). There was a change, however, in the ratio of KPI+ to KPI- mRNA isoforms of APP/APRP. Three separate probes, designed only to recognise KPI+ mRNA, each gave increases of between 28 and 50% in AD brains relative to controls (p = 0.002). There was no change in the mRNA levels of KPI-(APP 695) (p = 0.898). Therefore, whilst I KPI- mRNA levels remained level between AD and control brains, the KPI+ species were seen to increase specifically in the AD brains.
We reported previously that Stachybotrys microspora triprenyl phenol-7 (SMTP-7) showed potential thrombolytic, anti-inflammatory and anti-oxidant effects that account for its excellent pharmacological activity such as having a wider therapeutic time window than tissue plasminogen activator (t-PA) and a significant protection against hemorrhage. The aim of the present study was to evaluate and compare the effect of a new series of SMTPs in the acetic acid-induced embolic cerebral infarct mouse model. Thrombotic occlusion was produced in mice by inducing the transfer of acetic acid-induced thrombi from the right common carotid artery into the brain. SMTPs were evaluated by their effect on reducing infarct area, neurological score and edema. Furthermore, plasmin formation, anti-inflammatory and anti-oxidant activities were assessed by fibrin zymography, measuring pro-inflammatory gene expression, and thiobarbituric acid reactive substances (TBARS) assay, respectively. Treatment with either SMTP-22 or SMTP-43 (10mg/kg), which have similar plasmin formation, anti-inflammatory and anti-oxidant activities to SMTP-7, resulted in reduced infarct area, neurological score and edema. Coexistence of all these three activities appears to be important for the treatment of embolic infarction because SMTP-6, SMTP-25, and SMTP-44D (10mg/kg), which are each missing at least one of the three functions, were not as effective. Therefore, these results indicate that SMTP-22 and SMTP-43 have potential as medicinal compounds for the treatment of embolic cerebral infarction.
OBJECTIVE
Nitric oxide (NO) is a universal mediator of biological effects in the brain. It has been implicated in the pathophysiological processes of traumatic brain injury. Understanding its pathophysiological role in vivo requires an understanding of the cellular sources and tissue compartments of the differentially regulated NO synthase (NOS) isoforms. This study was undertaken to investigate the cellular sources and tissue compartments of NO produced after experimental brain contusions in rats, by analysis of the early expression of the three isoforms of NOS, i.e., the inducible, endothelial, and neuronal isoforms.
METHODS
Focal brain contusions were produced in 24 rats using a weight-drop model. The animals were killed 6, 12, 24, 36, or 48 hours after trauma. Sections were analyzed by immunohistochemical and immunofluorescence analyses. Double staining assays were used to define which cells produced the different NOS isoforms.
RESULTS
Increases in endothelial NOS-, inducible NOS (iNOS)-, and neuronal NOS-positive cells were detectable by 6 hours after trauma. Endothelial NOS and iNOS levels peaked at 6 and 12 hours, respectively. Expression of neuronal NOS initially increased to a peak at 12 hours but then decreased to a level lower than that in control samples at 36 hours. Endothelial NOS was expressed exclusively in endothelial cells, whereas iNOS was expressed in neutrophils and macrophages. Neuronal NOS was predominantly detected in neurons but was also unexpectedly detected in polymorphonuclear cells.
CONCLUSION
In this model, the most striking finding regarding NO-producing enzymes was the expression of iNOS in polymorphonuclear cells and macrophages, cells that invade injured brain tissue. iNOS is thus implicated as a therapeutic target in contusional injuries. This pattern of NOS expression cannot be generalized to all types of brain injuries. The different compartments and cells that can produce NO are differentially regulated; therefore, compartmentalization can explain why NO is beneficial or detrimental, depending on the circumstances. A knowledge of different potential sites and sources of NO is required for any attempts to interfere with the pathophysiological properties of NO. (46;177;2000)
Subarachnoid hemorrhage (SAH) results from the rupture of an intracranial aneurysm, and the first consequent events are increased intracranial pressure (ICP), reduced cerebral perfusion pressure (CPP), and decreased cerebral blood flow (CBF). The resultant hypoxic state alters autoregulation, ionic homeostasis, and excitotoxicity as well as initiates secondary injuries such as cytotoxic edema, blood-brain barrier (BBB) disruption, inflammation, and apoptotic cell death. Inflammation persists through hemorrhage degradation in the subarachnoid space. Several different aspects of the inflammatory response have been demonstrated in stroke pathogenesis, including cellular response (e.g., leukocyte adherence and microglia activation), expression of adhesion molecules (e.g., selectins, integrins, and immunoglobulin superfamily), production of inflammatory mediators (e.g., cytokines, nitric oxide/nitric oxide synthase (NO/NOS), and free radicals), and accumulation of platelet aggregates. Since all of these inflammatory aspects lead to brain edema and cell death, inflammation could be a particularly important target for designing therapeutic strategies against secondary injuries after SAH. Given these inflammatory contributions could be seen in large vessels, a plethora of research has been intended to reduce cerebral vasospasm (CVS) after SAH. The main research field, however, is moving toward studying early brain injury (EBI) because some human research demonstrated the morphological alleviation of CVS alone might not improve the functional recovery in patients after SAH. This chapter provides the current knowledge of the inflammatory response, translational research, and human clinical trials in SAH as well as discusses emerging opportunities for novel therapeutic strategies for clinical management of SAH.
The cerebral dysfunction such as deterioration of memory remains serious complications of ischemic reperfusion injury. Long-term potentiation (LTP) has been widely studied as a form of synaptic plasticity that represents a cellular mechanism of learning and memory. Numerous processes and molecules are reported to be involved in LTP mechanisms, and some elements including neurotrophic and transcription factors are likely to be common with those involved in neural death after cerebral ischemia. Nitric oxide (NO) is a molecule which has crucial roles in neuronal injury. In our study, we focused on LTP formation as a functional response to cerebral ischemia and elucidated the implication of NO production in LTP formation in the rat hippocampus following transient cerebral ischemia. NO production was evaluated by oxidative NO metabolite levels determined using in vivo brain microdialysis. Transient cerebral ischemia produced a marked inhibition of LTP in both Schaffer-CAl synapses and perforant path-dentategyrus synapses. The increase in hippocampal NO production was observed to precede LTP inhibition. Direct or indirect inhibition of an inducible NO synthase (iNOS) rescued ischemia-induced LTP inhibition. Centrally administered bacterial endotoxin, lipopolysaccharide, which is known to induce iNOS expression, could mimic the time-course changes in hippocampal NO production observed after ischemic insult. These findings suggest that iNOS-derived NO is partly responsible for the ischemia-induced impairment of LTP in the rat hippocampus.
Acute ischaemic stroke is a major cause of morbidity and mortality. The most effective treatment is early reperfusion, although less than half of patients who are finally treated obtain permanent benefits. The combination of rapid reperfusion and neuroprotective therapies would be of added value for maximizing the beneficial effects of rapid reperfusion and to reduce the harmful consequences of reperfusion injury, including the no-reflow phenomenon, after recanalization. Given the relevance of oxidative stress in the physiopathology of brain ischaemia, the use of antioxidant molecules, such as uric acid, could translate into effective neuroprotective effects. Uric acid is the most potent natural antioxidant, and its exogenous administration is neuroprotective and has synergistic effects when administered alongside alteplase, a thrombolytic drug. In humans, uric acid therapy is safe and has measurable neuroprotective effects, especially in patients with pre-treatment hyperglycaemia or in women. Given the encouraging preclinical and clinical data regarding the potential neuroprotective effects of uric acid administration in combination with reperfusion in acute brain ischaemia, the development of adequately powered pivotal confirmatory clinical trials is warranted.
OBJECTIVE: Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury after brain ischemia, and decreased levels of NO have been implicated in the pathogenesis of vasospasm after subarachnoid hemorrhage (SAH). In this study, we measured the ventricular cerebrospinal fluid (CSF) NO levels in patients with SAH and correlated the levels with clinical grade and middle cerebral artery velocities measured with transcranial Doppler ultrasound.
METHODS: All patients with spontaneous SAH documented on computed tomography and with an external ventricular drain inserted within 24 hours of hemorrhage were included in the study. A total of 16 patients were studied between August 1999 and August 2000. CSF was collected serially at the time of surgery and subsequently at daily intervals. It was collected during the time that the external ventricular drain remained patent and in situ. NO levels were measured by photometric analysis by using a nitrite/nitrate assay kit (Cayman Chemical, Ann Arbor, MI).
RESULTS: The peak NO level in patients with SAH ranged from 9.96 to 168.16 μmol, with a median of 36.93 μmol. The levels were significantly elevated as compared with the control group (5.16 μmol, P < 0.05). The median NO level in patients with poor-grade SAH was 67.14 μmol as compared with 27.42 μmol in patients with good-grade hemorrhage (P < 0.05). No correlation was seen between CSF NO levels and middle cerebral artery velocities. The median NO level was 33.2 μmol in patients with a poor outcome as compared with 30.25 μmol in patients with a good outcome (P > 0.05).
CONCLUSION: This study showed that NO levels are elevated after spontaneous SAH, and the degree of elevation is higher in patients with poor-grade SAH.
OBJECTIVE : Nitric oxide is a universal mediator of biological effects in the brain, and it has been implicated in the pathophysiological processes of traumatic brain injury. Experimental studies have indicated posttraumatic up-regulation of the three different isoforms of nitric oxide synthase (NOS) (i.e., inducible NOS [iNOS], endothelial NOS, and neuronal NOS) after brain trauma. This study was undertaken to investigate the cellular sources and tissue compartments of nitric oxide produced by human patients undergoing surgical treatment for contusional brain injuries.
METHODS : Contused brain tissue specimens from eight consecutive patients who underwent surgical treatment for brain contusions 3 hours to 5 days after trauma were evaluated in immunohistochemical analyses. Double-staining assays were used to define which cells produced the different isoforms.
RESULTS : Increases in iNOS-positive cells were detectable within 6 hours after trauma, with a peak at 8 to 23 hours. Expression of iNOS after trauma was detected in neurons, macrophages, neutrophils, astrocytes, and oligodendrocytes. The cellular sources of iNOS differed at different times after trauma. No detectable difference in the expression of the neuronal or endothelial isoforms was observed for trauma patients, compared with control subjects.
CONCLUSION : iNOS expression was up-regulated in a time-dependent manner in human brain tissue obtained from patients undergoing surgical treatment for contusional trauma. Our human data largely parallel experimental findings in rats, indicating that such trauma models are relevant for experimental studies and treatment trials.
Neurological disorders are diseases of the brain, the spine, and the nerves that connect them. There are more than 600 diseases of the nervous system, such as brain tumors, epilepsy, Parkinson’s disease, and stroke as well as less familiar ones such as frontotemporal dementia, Alzheimer’s disease, and other dementias; cerebrovascular diseases including stroke, migraine, and other headache disorders; multiple sclerosis; neuroinfections; brain tumors; traumatic disorders of the nervous system such as brain trauma; and neurological disorders as a result of malnutrition. More than a decade of research worldwide has shown that berries support cognitive health by protecting nerves and help brain cells communicate with each other and improve the flexibility of nerve structures. Berries help nerves tolerate stress, including the stress of toxic exposure. They also support the healthy function of glial cells in the brain, essential for optimum brain function. Polyphenols, namely, anthocyanins, found in berries may slow cognitive decline through antioxidant and anti-inflammatory properties in experimental animals. Based on the previous reports, this review explains the beneficial effects of the phytochemicals present in nine varieties of berries on neurodegenerative diseases.
Tissue plasminogen activator (t-PA) is the only FDA approved drug for acute ischemic stroke treatment, but its clinical use is limited due to the narrow therapeutic time window and severe adverse effects, including hemorrhagic transformation (HT) and neurotoxicity. One of the potential resolutions is to use adjunct therapies to reduce the side effects and extend t-PA's therapeutic time window. However, therapies modulating single target seems not satisfying, and a multi-target strategy is warranted to resolve such complex disease. Recent years, large amounts of efforts have been made to explore the active compounds from herbal supplements to treat ischemic stroke. Some of the natural compounds revealed both neuro- and blood-brain-barrier (BBB)-protective effects by concurrently targeting on multiple cellular signaling pathways in cerebral ischemia-reperfusion injury. Thus, those compounds are potential one-drug-multi-target agents to be combined with t-PA in treatment of ischemic stroke. In this review article, we summarize current progress in understanding the molecular targets involving in t-PA-mediated HT and neurotoxicity in ischemic brain injury. Based on those targets, we select 23 promising compounds from currently available literature, which revealed their bioactivities simultaneously targeting several important molecular targets. We propose that those compounds merit further investigation as combined therapy with t-PA. Finally, we discussed the potential drawbacks of the natural compounds' studies, and raised several important issues to be addressed in future development of natural compounds as adjunct therapy.
The vast majority of studies on stroke therapy have focused on protecting the neuron from hypoxic/ischemic injury. While undoubtedly important, stroke is a vascular disorder affecting not only neurons, but numerous other cell types in the brain including astrocytes, microglia, and vascular cells (endothelium and smooth muscle). In fact, the only effective treatment for ischemic stroke is a vascular one—dissolution of the clot with tissue plasminogen activator and rapid recanalization of an occluded vessel. The failure of every neuroprotective agent to make it into clinical trials highlights the complexity of ischemic stroke pathophysiology that involves inflammation, oxidative stress, and both macro- and micro-vascular dysregulation that causes brain injury itself and exacerbates the primary insult (i.e., secondary brain injury). The vasculature in the brain has a central role in defining stroke injury since the core infarction is dependent on the depth and duration of ischemia. In addition, any neuroprotective therapy for stroke depends on a patent and functional vasculature, further highlighting the importance of vascular protection as an important therapeutic approach to limiting stroke damage.
Analysis of pathological biochemical reactions involved in the mechanisms of acute focal brain ischemia suggests that there are tight correlations between all metabolic cascades and that one can observe their time correspondence and mutual regulation via a unified signal system including common systems of cellular messengers.
The rigorous investigation of cerebral ischemia requires the use of physiologically controlled, clinically relevant animal models. Rodent models of both global and focal cerebral ischemia have been widely applied to study stroke mechanisms and are advantageous from the standpoints of within-strain homogeneity, low animal costs, suitability for extensive physiological monitoring, brain size large enough to permit morphological and biochemical / molecular studies, and (in the case of transgenic or knockout mice) the ability to study specific gene functions (44). Large-animal ischemia models have also been developed and are useful in corroborating insights gained from rodent studies (66).
Many previous studies have shown that reactive oxygen radicals play important roles in the pathophysiology of various neurological disorders (8, 56, 81). Experimental ischemia and reperfusion models, such as transient focal / global ischemia model in rodents, have been well studied and the accumulated evidence suggest the involvement of oxygen radicals in the pathogenesis of their ischemic lesion. In these models, cerebral blood flow (CBF) is reduced in brain regions that are supplied with oxygen by the occluded vessels. Reoxygenation during reperfusion provides oxygen as a substrate for numerous enzymatic oxidation reactions. In this review, the mechanisms of formation / clearance and intracellular signalling pathways of oxygen radicals after cerebral ischemia / reperfusion will be discussed.
There is a growing body of evidence indicating that cerebral ischemic damage is associated with a local inflammatory reaction, which contributes to the development of ischemic brain injury. We review evidence that inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are involved in the mechanisms by which ischemia-induced inflammation contributes to tissue damage. In rodents, as in humans, iNOS is expressed in inflammatory cells infiltrating the ischemic brain and in cerebral blood vessels. Administration of iNOS inhibitors 24h after ischemia reduces the size of the infarct produced by occlusion of the rat middle cerebral artery (MCA). Furthermore, “knockout” mice lacking the iNOS gene have smaller infarcts than wild-type mice. COX-2 is expressed in the postischemic brain with a time course similar to that of iNOS; and it is present in ischemic neurons, inflammatory cells, and blood vessels. Administration of the selective COX-2 inhibitor NS-398 reduces infarct size. Furthermore, NO produced by iNOS activates COX-2, thereby increasing the production of toxic prostanoids and free radicals. The evidence suggests that NO synthesized by iNOS, in part through reaction products of COX-2, contributes to the expansion of the infarct that occurs during the late postischemic period. Thus, the interaction between iNOS and COX-2 plays an important role in the late stages of cerebral ischemic damage. Delayed administration of iNOS and COX-2 inhibitors may be a useful therapeutic strategy to target selectively the progression of ischemic brain injury.
There is increasing evidence that cerebral ischemic injury occurs at a slower pace than previously believed. Although in areas of severe ischemia tissue damage occurs relatively rapidly, in regions of less-severe ischemia, damage develops over the course of many hours or even days. In this chapter, we review data indicating that inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are upregulated following focal cerebral ischemia and that the products of their reaction contribute to the delayed progression of ischemic brain damage. Administration of iNOS and COX-2 inhibitors may be a useful therapeutic strategy to selectively target the progression of the brain damage that takes place during the post-ischemic period.
This chapter discusses the roles of excitotoxins and free radicals in the mechanisms of acute brain damage, mainly ischemic injury, trauma, and neurodegenerative disorders including Huntington's, Parkinson's, and Alzheimer's disease. Excitotoxin-induced neuronal damage is mediated by several Ca²⁺ mediated pathways. The spectrin and microtubule associated protein-2 (MAP-2) are degraded long before delayed neuronal death in the postischemic brain. Xanthine oxidase and monoamine oxidase are two important enzymes potentially causing free radical-induced brain pathology. Xanthine oxidase is synthesized as xanthine dehydrogenase, which transfers electrons to the electron carrier NAD⁺. In ischemic conditions, a calcium-dependent protease converts this dehydrogenase to its oxidase form, which transfers electrons to oxygen instead of to NAD⁺ and produces superoxide. Dopamine turnover in dopamine nerve terminals may generate excessive amounts of H2O2 by monoamine oxidase, which has been hypothesized as a possible cause of neurodegeneration in Parkinson's disease. Although free radicals are damaging chemical substances, biological systems have the machinery to defend against this type of injury. Defenses include iron-binding proteins, the metabolic enzymes such as superoxide dismutases (SOD), catalases, or glutathione peroxidases.
Cells in brain and other tissues respond to ischemic insults with diverse changes in gene expression. The distribution and cellular specificity of such responses in brain differ considerably after global and focal ischemia and are strongly dependent on the duration and severity of blood flow deficits. This review initially focuses on inducible transcription factors encoded by cellular immediate-early genes (cIEGs) of the fos/jun/krox multigene families, and the stress protein, hsp72, which is the most thoroughly studied member of the 70-kDa heat shock protein family in the central nervous system (CNS). It will be demonstrated that induction of cIEGs constitutes a lowthreshold response to brief depolarization, whereas hsp72 induction is more closely associated with potentially injurious stimuli in both global and focal ischemia models. Discrepancies between patterns of mRNA and protein expression will demonstrate the influence of postischemic protein synthesis deficits on mRNA translation. Within this framework, consideration will then be given to other ischemia-induced gene products, including other transcription factors and stress proteins, as well as neurotrophic factors, components of oxidative defense mechanisms, and gene products thought to be involved in regulating cell death. Emphasis is placed on neuronal gene expression, as changes in other cell types may be viewed as components of reactive responses considered in Chapters 9 and 10. A recent review provides a compact summary of postischemic changes in gene expression that may complement this presentation (Koistinaho and Hökfelt, 1997).
Stroke remains one of the major causes of death and disability throughout the world (American Heart Association, 1991). More than 80% of all strokes are a result of cerebral ischemia (Mohr et al., 1978). Global cerebral ischemia involves the entire brain and occurs during cardiac arrest or severe systemic hypotension. Focal cerebral ischemia affects restricted brain regions and occurs in a wide variety of clinical settings but is most commonly a result of cerebral vascular atherosclerosis. Focal ischemia is more frequent than global ischemia.
The major excitatory transmitter in the mammalian central nervous system is glutamate, which exerts its signaling actions through the stimulation of ionotropic and metabotropic receptors (Watkins et al. 1981; Mayer and Westbrook 1987; Nakanishi and Masu 1994). Under pathological conditions, glutamate receptor overactivation can trigger neuronal death, a phenomenon known as excitotoxicity (Lucas and Newhouse 1957; Olney 1969). Incentive for developing practical methods for blocking excitotoxicity arises from its implication in several acute and chronic neurological disease states. While recent clinical trials aimed at blocking excitotoxicity in stroke patients have been disappointing, there are several plausible reasons for these trial failures, including specific study design issues, treatment side effects, and a need to achieve concurrent block of parallel injury pathways. In our view, the case for antiexcitotoxic approaches in stroke remains open, and there are other possible disease targets yet to be explored. Ongoing delineation of the cellular and molecular underpinnings of excitotoxicity has led to the progressive unveiling of countermeasures, aimed at attenuating presynaptic glutamate release, postsynaptic receptor activation, the movement or action of cation second messengers, or downstream intracellular injury cascades. The excitotoxicity concept itself may need to be expanded, to encompass the death of oligodendrocytes as well as neurons, and ionic derangements besides Ca2+ overload.
Recent evidence indicates that thrombolysis may be an effective therapy for the treatment of acute ischemic stroke. However, the reperfusion of ischemic brain comes with a price. In clinical trials, patients treated with thrombolytic therapy have shown a 6% rate of intracerebral hemorrhage, which was balanced against a 30% improvement in functional outcome over controls. Destruction of the microvasculature and extension of the infarct area occur after cerebral reperfusion. We have reviewed the existing data indicating that an inflammatory response occurring after the reestablishment of circulation has a causative role in this reperfusion injury. The recruitment of neutrophils to the area of ischemia, the first step to inflammation, involves the coordinated appearance of multiple proteins. Intercellular adhesion molecule-1 and integrins are adhesion molecules that are up-regulated in endothelial cells and leukocytes. Tumor necrosis factor-alpha, interleukin-1, and platelet-activating factor also participate in leukocyte accumulation and subsequent activation. Therapies that interfere with the functions of these factors have shown promise in reducing reperfusion injury and infarct extension in the experimental setting. They may prove to be useful adjuncts to thrombolytic therapy in the treatment of acute ischemic stroke.
Nitric oxide (NO) overproduction has been demonstrated from different NO-synthase overexpression or hyperactivity after brain ischemia. Here, we examined the effects of inhibition of NO overproduction on brain infarction, cerebrovascular damage and expression of claudin-5 and zonula occludens-1 (ZO-1) in striatum of ischemic brain. The experiment was performed in three groups of rats; sham, control ischemia and ischemic treatment. Brain ischemia was induced by 60 min of middle cerebral artery occlusion (MCAO) followed by 24 hours of reperfusion. Treated rats received L-NAME 30 min before induction of ischemia (1 mg/kg, i.p.). Infarct volume and histopathological changes of ischemic striatum were assessed by TTC and LFB staining methods, respectively. Ultimately, quantitative RT-PCR was used for assessment of claudins-5 and ZO-1 expression. MCAO in the control group induced infarction (135 ± 25 mm3) at large areas of striatum in accompany with neuronal damages, whereas L-NAME significantly reduced infarction (87 ± 16 mm3) and neuronal injuries. The mRNA of ZO-1 and claudin-5 decreased in ischemic striatum, whereas inhibition of NO overproduction by L-NAME attenuated this reduction for these genes. Our findings indicated that NO overproduction after brain ischemia plays a crucial role in neuronal damage especially at striatal regions. Hence, inhibition of excessive NO production may save striatal cerebrovascular integrity of ischemic brain.
An increasing body of evidence suggests that cerebrovascular dysfunction and microvessel disease precede the evolution of hallmark pathological features that characterise Alzheimer's disease (AD), consistent with a causal association for onset or progression. Recent studies, principally in genetically unmanipulated animal models, suggest that chronic ingestion of diets enriched in saturated fats and cholesterol may compromise blood-brain barrier (BBB) integrity resulting in inappropriate blood-to-brain extravasation of plasma proteins, including lipid macromolecules that may be enriched in amyloid-β (Aβ). Brain parenchymal retention of blood proteins and lipoprotein bound Aβ is associated with heightened neurovascular inflammation, altered redox homeostasis and nitric oxide (NO) metabolism. Therefore, it is a reasonable proposition that lipid-lowering agents may positively modulate BBB integrity and by extension attenuate risk or progression of AD. In addition to their robust lipid lowering properties, reported beneficial effects of lipid-lowering agents were attributed to their pleiotropic properties via modulation of inflammation, oxidative stress, NO and Aβ metabolism. The review is a contemporary consideration of a complex body of literature intended to synthesise focussed consideration of mechanisms central to regulation of BBB function and integrity. Emphasis is given to dietary fat driven significant epidemiological evidence consistent with heightened risk amongst populations consuming greater amounts of saturated fats and cholesterol. In addition, potential neurovascular benefits associated with the use of hypolipidemic statins, probucol and fenofibrate are also presented in the context of lipid-lowering and pleiotropic properties.
Ischemic stroke triggers an inflammatory reaction in the affected area, which progresses for days to weeks after the onset of symptoms. There is evidence that selected aspects of such inflammatory processes contribute to the progression of ischemic brain injury, leading to worsening of the tissue damage and exacerbation of neurologic deficits. Therefore, interventions aimed at suppressing postischemic inflammation offer attractive therapeutic strategies for human stroke, with a potentially wide therapeutic window. A large body of work has addressed the inflammatory process in the postischemic brain. 1 2 3 4 5 In this chapter, we review the basic cellular and molecular features of postischemic inflammation, focusing on recent advances and insights on the potential mechanisms by which such inflammation influences stroke outcome. We then analyze the potential therapeutic implications of modulators of specific inflammatory targets from the perspective of near-future translational approaches.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
The promoter of the murine gene encoding inducible nitric oxide synthase (iNOS) contains an NF-kappa B site beginning 55 base pairs upstream of the TATA box, designated NF-kappa Bd. Reporter constructs containing truncated promoter regions, when transfected into macrophages, revealed that NF-kappa Bd is necessary to confer inducibility by bacterial lipopolysaccharide (LPS). Oligonucleotide probes containing NF-kappa Bd plus the downstream 9 or 47 base pairs bound proteins that rapidly appeared in the nuclei of LPS-treated macrophages. The nuclear proteins bound to both probes in an NF-kappa Bd-dependent manner, but binding was resistant to cycloheximide only for the shorter probe. The proteins binding both probes reacted with antibodies against p50 and c-rel but not RelB; those binding the shorter probe also reacted with anti-RelA (p65). Pyrrolidine dithiocarbamate, which acts as a specific inhibitor of NF-kappa B, blocked both the activation of the NF-kappa Bd-binding proteins and the production of NO in LPS-treated macrophages. Thus, activation of NF-kappa Bd/Rel is critical in the induction of iNOS by LPS. However, additional, newly synthesized proteins contribute to the NF-kappa Bd-dependent transcription factor complex on the iNOS promoter in LPS treated mouse macrophages.
The expression of interleukin-6 (IL-6) mRNA in the focal ischemic rat cortex was studied by means of Northern hybridization. IL-6 mRNA was induced after permanent occlusion of the middle cerebral artery, reached a significant level at 3 h, and peaked at 12 h, i.e., ~ 10-fold increase in the ischemic zone compared with the nonischemic cortex or sham-operated controls. The increased IL-6 mRNA was elevated for at least 24 h. Low levels of IL-6 mRNA were detected in sham-operated rats or in the contralateral nonischemic cortex. The expression of c-fos and zif268 mRNAs, two early response genes, was rapid (increased by 1 h postischemia) and transient (returned to basal levels by 24 and 12 h, respectively), clearly having different kinetic patterns from that of IL-6 mRNA. The early response kinetic pattern of c-fos and zif268 mRNAs in focal ischemia suggests their transcriptional regulatory roles in response to ischemic insult, while the delayed induction pattern of IL-6 mRNA suggests a role for this pleiotropic cytokine in the inflammatory response to the focal ischemic damage of the brain.Keywords: c-fos; Focal brain ischemia; Inflammation; Interleukin-6; Stroke; zif268
Nitric oxide (NO) is synthesized by a number of cells from a guanidino nitrogen atom of L-arginine by the action of either constitutive or inducible NO synthases, both of which form citrulline as a co-product. We have determined the source of the oxygen in both NO and in citrulline formed by the constitutive NO synthase from the vascular endothelium and brain and by the inducible NO synthase from the murine macrophage cell line J774. All these enzymes incorporate molecular oxygen both into NO and into citrulline. Furthermore, activated J774 cells form NO from omega-hydroxyl-L-arginine, confirming the proposal that this compound is an intermediate in the biosynthesis of NO.
Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.
Production of nitric oxide (NO) by macrophages is important for the killing of intracellular infectious agents. Interferon
(IFN)-gamma and lipopolysaccharide stimulate NO production by transcriptionally up-regulating the inducible NO synthase (iNOS).
Macrophages from mice with a targeted disruption of the IFN regulatory factor-1 (IRF-1) gene (IRF-1-/- mice) produced little
or no NO and synthesized barely detectable iNOS messenger RNA in response to stimulation. Two adjacent IRF-1 response elements
were identified in the iNOS promoter. Infection with Mycobacterium bovis (BCG) was more severe in IRF-1-/- mice than in wild-type
mice. Thus, IRF-1 is essential for iNOS activation in murine macrophages.
Interferon gamma (IFN-gamma) interacts synergistically with bacterial lipopolysaccharide (LPS) to induce transcription of iNOS, the isoform of nitric oxide synthase whose activity is independent of elevated Ca2+ and exogenous calmodulin. To define a cis-acting element mediating IFN-gamma-dependent synergy, we made deletions in iNOS promoter constructs fused to reporter genes, transfected RAW 264.7 macrophages, and treated the cells with IFN-gamma and/or LPS. This analysis implicated the region from positions -951 to -911, a cluster of four enhancer elements known to bind IFN-gamma-responsive transcription factors, including an interferon regulatory factor binding site (IRF-E) at nucleotides -913 to -923. Site-specific substitution of two conserved nucleotides within IRF-E in the context of the full-length iNOS promoter ablated IFN-gamma's contribution to synergistic enhancement of transcription. Electromobility shift assays performed with a probe containing IRF-E revealed the existence of a complex in nuclei of RAW 264.7 macrophages that was present only after treatment with IFN-gamma, which reacted specifically with anti-IRF-1 immunoglobulin G and which included a species migrating at 40-45 kD, consistent with the apparent molecular weight of murine IRF-1. Thus, the synergistic contribution of IFN-gamma to transcription of iNOS in RAW 264.7 macrophages requires that IRF-1 bind to IRF-E in the iNOS promoter. In conjunction with the work of Kamijo et al. (Kamijo, R., H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, K. S. Im, T. Kimura, S. Green et al. 1994. Science [Wash. DC]. 263:1612), these findings identify iNOS as the first gene that requires IRF-1 for IFN-gamma-dependent transcriptional regulation.
We studied the effect of focal cerebral ischemia on inducible (iNOS) and constitutive (cNOS) nitric oxide synthase enzymatic activities in the affected brain. The middle cerebral artery (MCA) was occluded in spontaneously hypertensive rats. Animals were killed 1, 2, 4, and 7 days later. cNOS and iNOS enzymatic activities were determined in the infarcted cortex using the assay of Bredt and Snyder. cNOS was assayed in the presence of calcium, whereas iNOS was assayed in the absence of calcium and in the presence of tetrahydrobiopterin. The validity of the iNOS assay was verified in rats treated with bacterial lipopolysaccharide. In these animals, the magnitude of the induction of iNOS enzymatic activity in lung, spleen, and brain paralleled the expression of iNOS mRNA, assessed by reverse-transcription polymerase chain reaction. After MCA occlusion, calcium-dependent (cNOS) activity was markedly reduced only in lesioned cerebral cortex at days 1-7 (p < 0.001; analysis of variance and Tukey's test). In contrast to cNOS, calcium-independent (iNOS) activity was induced substantially in the infarct (p < 0.005) but not in the contralateral intact cortex (p > 0.05). iNOS activity peaked at day 2 and was not different from baseline at day 7 (p > 0.05). No NADPH diaphorase-positive neurons were observed in the area of the lesion at days 1-7. Macrophages appeared at day 2 and invaded the infarcted tissue by day 7. At this time, numerous glial fibrillary acidic protein-positive astrocytes were observed within the lesion. The results suggest that the decline in calcium-dependent (cNOS) activity reflects loss of NOS neurons within the lesion.(ABSTRACT TRUNCATED AT 250 WORDS)
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.
Nitric oxide, a potent vasodilator and an inhibitor of platelet aggregation, may be beneficial in the early stages of focal cerebral ischemia as it may facilitate collateral blood flow to the ischemic territory. Accordingly, the effect of inhibition of nitric oxide synthesis on cerebral ischemic damage may vary depending on the timing of the inhibition relative to the induction of ischemia. We therefore studied the time course of the effect of nitric oxide synthesis inhibition on focal cerebral ischemic damage. The middle cerebral artery was permanently occluded in spontaneously hypertensive rats and the nitric oxide synthase (NOS) inhibitor nitro-L-arginine methyl ester (L-NAME) was administered systemically (3 mg/kg) < 5 min or 2, 3, or 6 h later. Arterial pressure, rectal temperature, plasma glucose, and hematocrit were monitored. Infarct volume was determined on thionin-stained sections 24 h after induction of ischemia. NOS activity was determined in cerebellum from the conversion of L-[3H]arginine to L-[3H]citrulline. Administration of L-NAME < 5 min after arterial occlusion increased the infarct volume by 23 +/- 14% (mean +/- SD; p < 0.05, analysis of variance), while administration of L-NAME at 2 or 6 h did not affect the size of the infarct (p > 0.05). L-NAME administration 3 h after induction of ischemia reduced neocortical infarct size by 14 +/- 11% (p < 0.05). L-NAME decreased cerebellar NOS activity comparably in all groups (range 16-25%). We conclude that the effects of inhibition of nitric oxide synthesis on focal cerebral ischemic damage are time dependent.(ABSTRACT TRUNCATED AT 250 WORDS)
Macrophages can become activated to kill both tumor cells and a variety of microbes. Results here show that synthesis of nitric oxide (NO), a mediator of many macrophage cytotoxic functions, was greatly increased when cells of the mouse macrophage cell line RAW 264.7 were costimulated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), compared to LPS alone. This increase paralleled increases in cytotoxicity. Northern analysis showed that increased production of NO was preceded by markedly enhanced expression of mRNA for the inducible form of macrophage NO synthase (mac-NOS), which catalyzes the synthesis of NO. Cycloheximide inhibited the induction of mac-NOS mRNA by IFN-gamma and LPS, indicating that expression of this mRNA required de novo protein synthesis. Elevated expression of mac-NOS mRNA was not due to an increase in its stability. Rather, the combination of IFN-gamma and LPS induced a much higher rate of transcription of the mac-NOS gene than did stimulation with LPS alone. These results provide one explanation of why priming and triggering stimuli, such as IFN-gamma and LPS, respectively, synergistically activate macrophages and may be applicable to explaining how IFN-gamma augments NO-dependent microbicidal activity in macrophages as well.
The aim of this study was to characterize the microglial and astroglial reactions to degeneration of (a) hippocampal CA1 pyramidal cells and dentate hilar neurons induced by cerebral ischemia and (b) CA3 pyramidal cells and dentate hilar neurons induced by intraventricular injections of kainic acid (KA). The microglial reactions to ischemia, as monitored by histochemical staining for the enzyme nucleoside diphosphatase (NDPase) and immunohistochemical staining for the complement type 3 receptor (CR3), could be divided into (1) initial and generalized, but transient, reactions which also included areas devoid of subsequent neural degeneration and (2) protracted, degeneration-specific reactions in the areas with neural degeneration. Due to more widespread hippocampal involvement a similar distinction was not possible after KA lesions. After both ischemia and KA application the protracted degeneration-specific reactions were characterized by increased NDPase/CR3 reactivity and prominent morphological changes. In the dentate hilus, reactive microglial cells clustered around the degenerating hilar neurons. In stratum radiatum of CA1, reactive microglial cells transformed into either (1) "rod cells," aligned along the postischemic, degenerating pyramidal cell dendrites, followed by subsequent transformation into ameboid-like cells, or (2) "bushy" cells, in response to degeneration of Schaffer collaterals induced by KA lesioning of CA3 pyramidal cells. Within stratum radiatum of the KA-lesioned CA3, where both dendrites and axons were degenerating, the microglial cells developed into stellate cells with thickened, retracted processes and plump cell bodies. These cells were supplemented by rounded macrophage-like cells. Astroglial reactions, monitored by immunohistochemical staining for the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin (VIM), and the normal plasma constituent immunoglobulin G (IgG), showed an initial and generalized astroglial immunoreactivity for IgG, which paralleled the initial and transient microglial reactions, while the reactive changes in GFAP and VIM immunohistochemistry paralleled the protracted, degeneration-specific reactions with regard to timing, strength, and distribution. In the KA-lesioned CA3, the most prominent finding was a prompt loss of astroglial GFAP immunoreactivity corresponding to the degenerating pyramidal cell layer and the adjacent mossy fiber layer. The results strongly indicate that stimuli other than neural degeneration initiated the activation of both microglial and astroglial cells, which then upon further activation by actual neuronal damage and degeneration adjust according to which neuronal structures were undergoing degeneration.
The cytokine tumor necrosis factor (TNF-alpha) is a pleotrophic polypeptide that plays a significant role in brain immune and inflammatory activities. TNF-alpha is produced in the brain in response to various pathological processes such as infectious agents [e.g., human immunodeficiency virus (HIV) and malaria], ischemia, and trauma. TNF-alpha mRNA is rapidly produced in response to brain ischemia within 1 h, reaches a peak at 6-12 h post ischemia, and subsides 1-2 days later. TNF-alpha mRNA expression corresponds in a temporal fashion to other cytokines such as interleukin (IL)-6, cytokine-induced neutrophil chemoattractant (KC), and IL-1 and precedes the infiltration of inflammatory cells into the injured zone. TNF-alpha is present early in neuronal cells in and around the ischemic tissue (penumbra), yet at later time points, the peptide is found in macrophages in the infarcted tissue. TNF-alpha has been demonstrated to cause expression of proadhesive molecules on the endothelium, which results in leukocyte accumulation, adherence, and migration from capillaries into the brain. Furthermore, TNF-alpha activates glial cells, thereby regulating tissue remodeling, gliosis, and scar formation. Thus, evidence is emerging in support of a role for TNF-alpha in injury induced by infectious, immune, toxic, traumatic, and ischemic stimuli. TNF-alpha promotes inflammation by stimulation of capillary endothelial cell proinflammatory responses and thereby provides leukocyte adhesion and infiltration into the ischemic brain. The evidence generated so far suggests that agents that suppress TNF-alpha's production or actions will reduce leukocyte infiltration into ischemic brain regions and thereby diminish the extent of tissue loss.
We studied whether delayed posttreatment with the nitric oxide donor 3-morpholinosydnonimine (SIN-1) is effective in reducing the size of the infarct produced by occlusion of the middle cerebral artery (MCA) in spontaneously hypertensive rats (SHRs). SHRs were anesthetized with halothane and intubated transorally. The left MCA was occluded at the level of the inferior cerebral vein. Cerebral blood flow (CBF) was monitored in the ischemic hemisphere by a laser-Doppler flowmeter, and an electroencephalogram (EEG) was recorded. SIN-1 was infused into the left internal carotid artery for 60 min starting 3, 15, 30, 60 or 120 min after MCA occlusion. The hypotension associated with SIN-1 administration was controlled by i.v. administration of phenylephrine. At the end of the infusion, rats were extubated and allowed to recover. Infarct size was measured 24 h later on thionein-stained coronal brain sections by computer-assisted planimetry. SIN-1 infusion 3 min after MCA occlusion enhanced the recovery of CBF and EEG amplitude and reduced the size of the infarct by 30 +/- 11% (p < 0.05, analysis of variance). The reduction in infarct size by SIN-1 was still seen when SIN-1 was administered 15, 30, and 60 min after MCA occlusion (p < 0.05). However, administration of SIN-1 2 h after MCA occlusion did not affect the size of the infarct (p > 0.05). We conclude that posttreatment with SIN-1 is effective in reducing focal ischemic damage if this agent is administered up to 60 min after MCA occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)
We studied whether administration of nitric oxide (NO) donors reduces the ischemic damage resulting from middle cerebral artery (MCA) occlusion in spontaneously hypertensive rats (SHRs). In halothane-anesthetized and ventilated SHRs, the MCA was occluded. CBF was monitored using a laser-Doppler flowmeter. Three to five minutes after MCA occlusion, the NO donors sodium nitroprusside (SNP; 3 mg/kg/h) or 3-morpholino-sydnonimine (SIN 1; 1.5-6 mg/kg/h) were administered into the carotid artery for 60 min. As a control, the effect of papaverine (3.6 mg/kg/h), a vasodilator that acts independently of NO, was also studied. The hypotension evoked by these agents was counteracted by intravenous infusion of phenylephrine. At the end of the infusion, rats were allowed to recover. Stroke size was determined 24 h later in thionin-stained sections. In sham occluded rats, SNP (n = 5), SIN 1 (n = 5), and papaverine (n = 5) produced comparable increases in CBF (p > 0.05 from vehicle). After MCA occlusion, SNP (n = 5) and SIN 1 (n = 5), but not papaverine (n = 5), enhanced the recovery of CBF (p < 0.05 from vehicle) and reduced the size of the infarct by 28 +/- 12 and 32 +/- 7%, respectively (mean +/- SD; p < 0.05 from vehicle). To determine whether NO donors could act by inhibiting platelet aggregation, we studied the effect of SNP on collagen-induced platelet aggregation. Intracarotid administration of SNP (3 mg/kg/h for 60 min) did not affect platelet aggregation to collagen, suggesting that the protective effect of NO donors was not due to inhibition of platelet function. We conclude that NO donors increase CBF to the ischemic territory and reduce the tissue damage resulting from focal ischemia. The protective effect may result from an increase in CBF to the ischemic territory, probably the ischemic penumbra. These findings suggest that NO donors may represent a new therapeutic strategy for the management of acute stroke.
Nitric oxide is a messenger molecule, mediating the effect of endothelium-derived relaxing factor in blood vessels and the cytotoxic actions of macrophages, and playing a part in neuronal communication in the brain. Cloning of a complementary DNA for brain nitric oxide synthase reveals recognition sites for NADPH, FAD, flavin mononucleotide and calmodulin as well as phosphorylation sites, indicating that the synthase is regulated by many different factors. The only known mammalian enzyme with close homology is cytochrome P-450 reductase.
The development of fibrinolytic agents such as streptokinase and recombinant tissue type plasminogen activator (r-TPA) and other modalities of treatment in acute ischemic stroke, has raised the need for a more precise knowledge of the pathophysiology of the acute phases of ischemic stroke as it pertains to prediction of clinical outcome. In a prospective analysis, 80 patients were studied within less than 6 h from the onset of symptoms by means of a detailed protocol including clinical evaluation, cerebral computed tomography, digital angiography and ultrasound transcranial Doppler sonography. Early angiography revealed a complete arterial occlusion in 76% of cases, the majority of which were intracranial (66%). Seventy percent of the occlusions that were retested were removed within 1 week. Potential embolic sources were found in more than 80% of cases. Patients with documented intracranial occlusion and scarce or absent collateral filling at early angiography, had the worst clinical outcome (P < 0.05), based on mortality data and the Canadian Neurological Scale. The 30-day mortality rate was 25%. Survival was significantly better (P < 0.01) in patients with a Canadian Neurological Score on entry of ≥ 6.5 than in patients with a < 6.5 value. Our data indicate that early pathophysiological studies augment the clinical information and should be taken into account in the design and analysis of therapeutic trials of acute ischemic stroke.
L-Arginine (> or = 30 mg kg-1, i.v.), but not D-arginine (300 mg kg-1) administered 5 min after unilateral common carotid/middle cerebral artery occlusion increased regional cerebral blood flow (rCBF) within the dorsolateral ischaemic cortex in spontaneously hypertensive rats. L-Arginine (300 mg kg-1) increased rCBF from 22 +/- 2.7 to 33 +/- 4% of baseline as measured by laser-Doppler flowmetry. This increase may explain the ability of L-arginine to reduce infarct size following focal cerebral ischaemia, as reported previously. The mechanism appears to be mediated by nitric oxide since topical L-NAME (1 microM), a nitric oxide synthase inhibitor, decreased pial arteriole calibre from 115 +/- 2.2 to 106 +/- 0.9% of baseline following L-arginine infusion (300 mg kg-1).
Two different techniques were utilized to identify the infiltration of polymorphonuclear leukocytes (PMN) into cerebral tissue following focal ischemia: histologic analysis and a modified myeloperoxidase (MPO) activity assay. Twenty-four hours after producing permanent cortical ischemia by occluding and severing the middle cerebral artery of male spontaneously hypertensive rats, contralateral hemiparalysis and sensory-motor deficits were observed due to cerebral infarction of the frontal and parietal cortex. In hematoxylin-and-eosin-stained histologic sections, PMN, predominantly neutrophils, were identified at various stages of diapedesis from deep cerebral and meningeal vessels at the periphery of the infarct, into brain parenchyma. When MPO activity in normal brain tissue was studied initially, it could not be demonstrated in normal tissues extracted from non-washed homogenates. However, if tissue was homogenized in phosphate buffer (i.e., washed), MPO activity was expressed upon extraction. Utilizing this modified assay, MPO activity was significantly increased only in the infarcted cortex compared to other normal areas of the brain. This was observed in non-perfused animals and after perfusion with isotonic saline to remove blood constituents from the vasculature prior to brain removal. The increased PMN infiltration and MPO activity were not observed in forebrain tissue of sham-operated control rats. Also, MPO activity was not increased in the ischemic cortex of MCAO rats perfused immediately after middle cerebral artery occlusion, indicating that blood was not trapped in the ischemic area. By using a leukocyte histochemical staining assay, activity of peroxidases was identified within vascular-adhering/infiltrating PMN in the infarcted cortex 24 hr after focal ischemia. An evaluation of several blood components indicated that increased MPO activity was selective for PMN. The observed increase of approximately 0.3 U MPO/g wet weight ischemic tissue vs. nonischemic cerebral tissues probably reflects the increased vascular adherance/infiltration of approximately 600,000 PMN/g wet weight infarcted cortex 24 hr after focal ischemia. This combined biochemical and histological study strongly suggests that PMN adhere within blood vessels and infiltrate into brain tissue injured by focal ischemia and that the associated inflammatory response might contribute to delayed progressive tissue damage in focal stroke. This modified MPO assay is a useful, quantitative index of PMN that can be utilized to elucidate the potential deleterious consequences of neutrophils infiltrating into the central nervous system after cerebral ischemia, trauma, or other pro-inflammatory stimuli.
Free radicals generated by a partial reduction of O2 pose a serious threat to tissues and vital organs and cells. The major site of interaction between the lung and inhaled oxidants is the epithelium. We have examined the effect of pyrogallol, an O2- generator, on the ability of human epithelial cells to produce active DNA binding proteins and inducible nitric oxide synthase (iNOS) mRNA in cultured A549 epithelial cells. NF kappa B binding in the nuclei of these cells was determined by electrophoretic mobility shift assays. iNOS mRNA was measured using reverse transcription of PCR. There was a time- and concentration-dependent induction of NF kappa B binding, followed by a time and dose dependent increase in iNOS mRNA levels. These results suggest that in airways the initial response to oxidative stress may be to induce NF kappa B-responsive genes, such as iNOS, which may play an important role in defending the airway against oxidative stress.
We previously showed that interferon (IFN)-gamma inhibited the proliferation of rat vascular smooth muscle cells (VSMC) by generation of nitric oxide (NO) through the induction of an NO synthase (NOS) and cloned the rat inducible NOS cDNA in VSMC (VSM-NOS). To study the regulation of human inducible NOS (hiNOS) transcription in VSMC, we now cloned and sequenced a 2.9-kb fragment for the hiNOS gene containing a putative promoter, exon 1 and exon 2. The 5'-flanking region contains several consensus sequences for the binding of transcription factors involved in the inducibility of other genes by cytokines. These include IFN-gamma responsive element and NF-IL6 and NF-kappa B binding consensus sequences. Interestingly, hiNOS gene contains a shear-stress responsive element (GAGACC) which was also found to exist in human endothelial-type NOS but not in murine inducible NOS.
The proposal that nitric oxide (NO) or its reactant products mediate toxicity in brain remains controversial in part because
of the use of nonselective agents that block NO formation in neuronal, glial, and vascular compartments. In mutant mice deficient
in neuronal NO synthase (NOS) activity, infarct volumes decreased significantly 24 and 72 hours after middle cerebral artery
occlusion, and the neurological deficits were less than those in normal mice. This result could not be accounted for by differences
in blood flow or vascular anatomy. However, infarct size in the mutant became larger after endothelial NOS inhibition by nitro-L-arginine
administration. Hence, neuronal NO production appears to exacerbate acute ischemic injury, whereas vascular NO protects after
middle cerebral artery occlusion. The data emphasize the importance of developing selective inhibitors of the neuronal isoform.
We sought to determine whether expression of the inducible, calcium-independent isoform of nitric oxide synthase (iNOS) contributes to the tissue damage produced by focal cerebral ischemia. The middle cerebral artery was occluded in halothane-anesthetized spontaneously hypertensive rats. Twenty-four hours later rats received intraperitoneal injections of the iNOS inhibitor aminoguanidine (100 mg/kg twice per day; n = 10) or of aminoguanidine + L-arginine (300 mg/kg four times per day; n = 7), aminoguanidine + D-arginine (n = 7), arginine alone (n = 6), or vehicle (n = 9). Drugs were administered for 3 consecutive days. Infarct volume was determined by image analysis in thionin-stained brain sections 4 days after induction of ischemia. Administration of aminoguanidine reduced infarct volume by 33 +/- 4% (P < 0.05 from vehicle; analysis of variance and Tukey's test), a reduction that was antagonized by coadministration of L- but not D-arginine. Administration of L-arginine alone did not affect infarct size (P > 0.05 vs. vehicle). In separate rats (n = 10), aminoguanidine attenuated calcium-independent NOS activity in the infarct (P < 0.05 vs. vehicle) without affecting calcium-dependent activity (P > 0.05). Aminoguanidine did not affect resting cerebral blood flow or the cerebrovascular vasodilation elicited by hypercapnia, as determined by laser-Doppler flowmetry (n = 4). We conclude that aminoguanidine selectively inhibits iNOS activity in the area of infarction and reduces the volume of the infarct produced by middle cerebral artery occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)
We evaluated the ability of monoclonal antibodies directed against leukocyte adhesion molecules (intercellular adhesion molecule-1 [ICAM-1], CD18) to enhance the efficacy of thrombolysis in a rabbit cerebral embolism stroke model. Both tissue-type plasminogen activator (tPA) and anti-CD18 (alpha-CD18) monoclonal antibody administered 5 minutes after embolization increased the quantity of clots required to produce neurologic damage, although the combination was no more effective than either substance alone. Neither alpha-CD18 nor anti-ICAM-1 (alpha-ICAM-1) improved neurologic outcome at postischemic delays of 15 or 30 minutes. However, the combination of alpha-ICAM-1 (15 minutes after embolization) and tPA (2 hours after embolization) significantly improved neurologic outcome even though neither substance was effective alone at these postembolization delays. These findings suggest that prevention of leukocyte adhesion increases the postischemic duration at which thrombolytic therapy remains effective.
Intercellular adhesion molecule-1 (ICAM-1) is a glycoprotein expressed on endothelial cells that facilitates leukocyte adhesion. To test the hypothesis that reduction of leukocytes in an ischemic lesion reduces ischemic brain damage, we measured the effect of administration of an anti-ICAM-1 monoclonal antibody on ischemic brain damage after transient middle cerebral artery occlusion in the rat. ICAM-1 expression increased in the ischemic lesion, and the lesion volume was significantly reduced by 41% in the anti-ICAM-1 antibody group compared with the control group (p < 0.05). Numbers of polymorphonuclear leukocytes (PMNs) were significantly reduced in the cortices of the anti-ICAM-1 antibody group compared with the control animals (p < 0.05). Our data indicate that administration of anti-ICAM-1 antibody results in a significant reduction of ischemic brain damage concomitant with a reduction of PMNs in the lesion after transient focal cerebral ischemia in the rat.
The present experiments were designed to study under what circumstances middle cerebral artery (MCA) occlusion by an intraluminal filament technique leads to hyperthermia and what the mechanisms are. We found that permanent MCA occlusion by this technique lead to a rise in body (core) temperature to 39.0-39.5 degrees C during the first 2-4 h, and to sustained hyperthermia thereafter (38.5-39.0 degrees C). After 2 h of transient MCA occlusion hyperthermia could only be avoided if anesthesia (with control of temperature) was maintained for 2 h of ischemia and 1 h of recirculation or, in unanesthetized animals, if external cooling was maintained for 2 h of ischemia and 2 h of recirculation. Control of temperature only during ischemia did not prevent a postischemic rise in temperature. One hour of MCA occlusion had less effect on body temperature. Results are presented which suggest that the hyperthermia observed is due to an interference, by the intraluminal filament, of circulation to hypothalamic centers regulating body temperature. It is speculated that the hyperthermia induced may blunt or obliterate the effect of drugs, normally considered to ameliorate brain damage due to focal ischemia.
We investigated the temporal profile of ischemic tissue damage, neutrophil response, and vascular occlusion after permanent and transient middle cerebral artery occlusion in the rat. Focal cerebral ischemia was induced by advancing a nylon monofilament to occlude middle cerebral artery (MCA). Two groups of rats were investigated: (1) those with permanent MCA occlusion (n = 29), and (2) and those having the arterial occlusion released after 2 h (n = 34). Experiments were terminated at 6, 12, 24, 48, 72, 96 and 168 h after the onset of ischemia, and brain sections were stained with hematoxylin and eosin for histological evaluation. Initially, the cortical lesion was smaller in rats subjected to transient MCA occlusion than in rats subjected to permanent MCA occlusion (p < 0.02). The surface area of the lesion was identical in both groups at 48 h after the onset of ischemia. Neutrophil infiltration into tissue and the time of peak neutrophil infiltration occurred earlier after transient MCA occlusion than after permanent MCA occlusion (6 h, 48 h in transient; 12 h, 72 h in permanent). Within the lesions, the number of occluded vessels was significantly lower in the transient ischemia group than in the permanent ischemia group during the time interval between 12-48 h (p < 0.01). Our data suggest that the temporal evolution of the lesion, the pattern of neutrophil infiltration and the chronology of microvascular occlusion differs depending on whether the MCA occlusion is transient (2 h) or permanent; however, significant differences in the size of the brain lesion disappeared 48 h after onset of ischemia.
We tested the hypothesis that administration of the antioxidant tirilazad mesylate improves electrophysiological recovery and decreases infarct volume after transient focal cerebral ischemia in cats.
Halothane-anesthetized cats underwent 90 minutes of left middle cerebral artery and bilateral common carotid artery occlusion followed by 180 minutes of reperfusion. Cats were assigned to receive tirilazad (1.5 mg/kg plus 0.2 mg/kg per hour IV infusion) either at the beginning (n = 9) or conclusion (n = 9) of ischemia. Control cats received an equal volume of diluent (citrate buffer, pH 3.0; n = 7) at the beginning and conclusion of ischemia in a blinded fashion. Infarct volume was measured by 2,3,5-triphenyltetrazolium chloride staining.
Blood flow to the left temporoparietal cortex decreased to less than 10 mL/min per 100 g with ischemia but was minimally affected on the right side. Blood flow distribution during ischemia or reperfusion was not different in the tirilazad-treated groups. No group demonstrated postischemic hyperemia or delayed hypoperfusion. Somatosensory evoked potential recorded over the left cortex was ablated during ischemia and recovered to less than 15% of baseline amplitude at 180 minutes of reperfusion in all groups. There were no differences among groups in infarct volume of left hemisphere (pretreatment, 25 +/- 6% [mean +/- SE]; posttreatment, 33 +/- 5%; control, 28 +/- 8% of hemisphere) or caudate nucleus (pretreatment, 46 +/- 7%; posttreatment, 41 +/- 10%; control, 55 +/- 13% of hemisphere).
In an experimental model of focal ischemia involving severe reductions of blood flow followed by reperfusion in cats, administration of tirilazad at the onset of either ischemia or reperfusion does not ameliorate infarct volume assessed during early reperfusion. Our study does not address potential efficacy of tirilazad in the setting of a different dosing strategy or duration of reperfusion.
The cytokine interleukin-1 (IL-1) is synthesised within the brain and acts as a mediator of host defence responses to disease and injury. Several of these central actions of IL-1 are inhibited by an endogenous calcium and phospholipid binding protein, lipocortin-1. Synthesis of IL-1 and lipocortin-1 in the brain is markedly increased by neuronal damage, and inhibition of the actions of endogenous IL-1 by central injection of IL-1 receptor antagonist in the rat significantly inhibits ischaemic and excitotoxic brain damage. Lipocortin-1 appears to act as an endogenous neuroprotective agent that markedly attenuates ischaemic and excitotoxic damage. In contrast, inhibition of the actions of lipocortin-1 by injection of neutralising antiserum exacerbates both forms of neurodegeneration. The mechanisms underlying these effects of IL-1 and lipocortin-1 are largely unknown, but are probably independent of changes in body temperature. Actions of these molecules on corticotrophin releasing factor, arachidonic acid, excitatory amino acids, and nitric oxide, and the possible involvement of these factors in brain damage are discussed.
The results of several experimental studies of focal ischemia and anecdotal observations suggest that leukocytes may contribute to the injury initiated by an arterial occlusion. The timing and the nature of leukocyte responses in evolving brain infarcts (either human or experimental) are incompletely characterized. This is a study of experimental brain lesions in 96 Wistar rats that underwent occlusion of a large intracranial artery for variable intervals ranging between 30 minutes and 7 days. The experimental model, based on the occlusion of a middle cerebral artery ostium via the insertion of a nylon monofilament through the external carotid artery, does not require opening the skull; therefore, the inflammatory response is not influenced by the effects of craniotomy and changes in intracranial pressure are only those induced by the ischemic lesion. All 96 animals having the same type of arterial occlusion developed an ischemic brain lesion (limited to the territory of the corresponding artery) that evolved into an area of extensive neuronal necrosis over a period of 6 to 12 hours followed by pan-necrosis (infarct) approximately 60 hours later. In this study, leukocytes (in particular polymorphonuclear cells) were detected in the microvessels (capillaries and venules) of the ischemic hemisphere as early as 30 minutes after the arterial occlusion. Numbers of intravascular neutrophils peaked at 12 hours, whereas intraparenchymal granulocytes were most numerous at 24 hours; a few granulocytes were visible in the brain infarct as late as day 7. Circulating monocytes were first detected within the capillaries/venules of the ischemic area after 4 to 6 hours. Platelet aggregates were more abundant in the arterial than the venous side of the circulation, and luminal obstruction of arteries by platelet aggregates became noticeable only 48 hours after the arterial occlusion. Fibrin thrombi were conspicuous for their absence. These observations provide the background for studies that will attempt to unravel the relationship between the biological responses of leukocytes and neuronal necrosis secondary to focal ischemia.
Development and resolution of the lesion produced by occlusion of the middle cerebral artery (MCAO) was studied through quantitative planimetry and histologic/immunohistochemical techniques. MCAO, performed in spontaneously hypertensive rats (SHR), initially (1-3 days) produced large, consistent cerebral cortical infarctions and an increase in ipsilateral hemispheric size (i.e., swelling) quantitated by planimetry on 2,3,5-triphenyltetrazolium chloride (TTC)-stained gross tissue sections. These initial changes correlated well with changes identified from 2 h to 3 days using hematoxylin and eosin stained histologic tissue sections and immunohistochemical techniques including: the progressive development of a cortical area of pan necrosis, infiltration of neutrophils into infarcted tissues, and activation of astroglia. During the initial 2 days following MCAO, glial fibrillary acidic protein immunoreactive cells increased in number and became larger and more intensely fluorescent medial to the cortical infarct. At 5 to 15 days, both the infarct and the ipsilateral hemisphere decreased in size. These changes correlated with the presence of abundant macrophages, and cavitation of the lesion along its medial border. Also during this period, a loose connective tissue matrix formed along the superficial aspect of the infarct. This connective tissue contained fibroblasts, extracellular matrix immunoreactive for laminin and collagen, capillary buds indicating neovascularization, and abundant macrophages. By the final timepoint (30 days), necrotic tissue could no longer be detected in either gross or histologic tissue sections, the inflammatory infiltrate had resolved, and the connective tissue was removed.(ABSTRACT TRUNCATED AT 250 WORDS)
This chapter discusses the experimental strategies for studying the role of nitric oxide (NO) in cerebral ischemic damage and focuses on the strength and weaknesses of each approach. NO is involved in the mechanisms of cerebral ischemic damage. Studies in animal models of cerebral ischemia have provided evidence that modulation of NO production can influence the outcome of the ischemic brain tissue. The roles of NO in the mechanisms of cerebral ischemia are multifaceted and depend on the stage of evolution of the tissue damage. The role of NO in ischemic damage has utilized pharmacological inhibitors of NO synthase (NOS). The purification and molecular cloning of the different isoforms of NOS has allowed the development of monoclonal and polyclonal antibodies directed against the different isoforms of NOS. The level of NOS protein produced in experimental animals or cultures may be determined by Western blot analysis using standard protocols. A more sensitive and quantitative measure of messenger RNA (mRNA) is provided by RNase protection assays.
Hemodynamics of postischemic reperfusion of the brain
- K-A Hossmann
Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat
- J H Garcia
- Y Yoshida
- H Chen
- Y Li
- Z G Zhang
- J Lian
- S Chen
- M Chopp