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Focal cerebral ischaemia induces CRH mRNA in rat cerebral cortex amygdala
Abstract
Corticotropin-releasing hormone (CRH) antagonism has neuroprotective effects in models of ischemia. We examined CRH mRNA by in situ hybridization in a well-established rat model of focal cerebral ischemia caused by permanent middle cerebral artery occlusion (MCAo). In ischemic cortex CRH mRNA levels were elevated 2.6-fold 60 min after MCAo, compared with sham operated animals. CRH mRNA was also induced in the amygdala, 60 min following ischemia, in a pattern which was qualitatively different from that of sham operated animals. This rapid and profound increase in CRH mRNA levels during focal cerebral ischemia is likely to be associated with neurotoxicity, as CRH antagonism has been reported to cause a significant reduction in neuronal loss during ischemia.
... In addition, CRF has been implicated in ischemia-induced changes in cerebral blood flow (De Michele et al, 2005), blood-brain barrier permeability (Esposito et al, 2003), and cell death (Stevens et al, 2003). Furthermore, CRF levels rise in the amygdala, hypothalamus, hippocampus, and bed nucleus of the stria terminalis within hours of ischemia (Wong et al, 1995). Although CRF is known to change in response to ischemia, the effects of ischemia on CRF receptor binding have not been characterized. ...
... The CA/CPR-induced changes in basal corticosterone concentrations, which precede alterations in negative feedback, may be a manifestation of ischemia-induced changes in CRF neurotransmission. CRF mRNA is rapidly upregulated in the ischemic cortex and amygdala (Wong et al, 1995), and extracellular CRF is elevated in the paraventricular nucleus of the hypothalamus, amygdala, hippocampus, and cortex after cerebral ischemia (Khan et al, 2004). In turn, CRF influences cerebral blood flow, vascular permeability, and cell death (De Michele et al, 2005;Khan et al, 2004;Stevens et al, 2003). ...
... In turn, CRF influences cerebral blood flow, vascular permeability, and cell death (De Michele et al, 2005;Khan et al, 2004;Stevens et al, 2003). Indeed, ischemiainduced neuronal loss is attenuated when CRF antagonists are administered (Charron et al, 2008;Wong et al, 1995). Studies of ischemia in CRF 1 and CRF 2A knockout mice indicate that CRF 1 is an important mediator of ischemic injury (Stevens et al, 2003). ...
Cardiac arrest and cardiopulmonary resuscitation (CA/CPR) increase the risk for affective disorders in human survivors. Postischemic anxiety- and depressive-like behaviors have been documented in animal models of CA/CPR; however, the stability of post-CA/CPR anxiety-like behavior over time and the underlying physiologic mechanisms remain unknown. The hypothalamic-pituitary-adrenal (HPA) axis and the corticotropin releasing factor (CRF) system may mediate the pathophysiology of anxiety and depression; therefore, this study measured CA/CPR-induced changes in CRF receptor binding and HPA axis negative feedback. Mice were exposed to CA/CPR or SHAM surgery and assessed 7 or 21 days later. Consistent with earlier demonstrations of anxiety-like behavior 7 days after CA/CPR, increased anxiety-like behavior in the open field was also present 21 days after CA/CPR. On postoperative day 7, CA/CPR was associated with an increase in basal serum corticosterone concentration relative to SHAM, but this difference resolved by postoperative day 21. The Dexamethasone Suppression Test showed that the CA/CPR group had enhanced negative feedback compared with SHAM controls at postoperative day 21. Furthermore, there was a gradual increase in CRF(1) receptor binding in the paraventricular nucleus of the hypothalamus and bed nucleus of the stria terminalis, as well as a transient decrease of both CRF(1) and CRF(2A) receptors in the dorsal hippocampus. Therefore, sustained changes in activity of the HPA axis and the CRF system after CA/CPR may contribute to the postischemic increase in affective disorders.
... In this context, a role for the 41-amino acid polypeptide corticotropin-releasing hormone (CRH) has been proposed [10,59,61]. CRH has a well-established role in regulating behavioral and neuroendocrine responses to stress [28]. ...
... The piriform cortex has been closely linked to hippocampal-mediated limbic seizures [18], and kainate-induced neurotoxicity increases CRH mRNA expression in this region at a magnitude similar to that produced in the hippocampus [52]. Significant increases in CRH mRNA expression in the amygdala have been reported following both ischemia and traumatic brain injury [55,61]. The central nucleus of the amygdala (CeA) has received particular attention in regard to some excitatory and/or neurotoxic effects of CRH; exogenous administration of CRH in this region has been shown to promote neuronal activation [14], and glutamate induces specific activation of amygdalar CRH release [17]. ...
... Amygdalar increases in CRH release are concordant with the rapid increase in CRH mRNA expression in the CeA reported following exposure to other acute physiological stressors such as hypoxia and ether exposure [50]. Increased CRH mRNA in the amygdala has also been reported 60 min following focal ischemia [61], suggesting increased CRH synthesis at this site. Furthermore, enhanced activation and sensitivity of amygdalar CRH neurons is interesting as CRH-induced limbic seizures appear mediated by the amygdala [3,14], and seizure activity is not uncommon following an ischemic insult. ...
The excitatory actions of corticotropin-releasing hormone (CRH) in the brain and the neuroprotective effects of CRH antagonists in models of ischemia suggest a role for this peptide in the cascade of events leading to cellular damage. The present study aimed to characterize endogenous activation of CRH in discrete brain regions following global ischemia. Time-dependent changes in CRH concentrations were assessed in 10 brain regions including hippocampal, parahippocampal, and hypothalamic regions as well as the amygdala and the frontal cortex at three post-ischemic intervals: 4, 24, and 72 h (Experiment 1). The impact of pretreatment with a neuroprotective dose of the NMDA antagonist (5R,10S)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801; hydrogen maleate) on 24-h ischemia-induced CRH concentrations in the 10 brain regions was also determined (Experiment 2). In vivo microdialysis was used to assess dynamic fluctuations in CRH release at the dorsal hippocampus (CA1 pyramidal layer) and central nucleus of the amygdala (CeA; Experiment 3). Our findings revealed a rapid elevation of CRH concentrations at the piriform cortex (Pir) and hypothalamic nuclei following global ischemia. This was followed by decreased CRH concentrations at the amygdala, the frontal cortex (FC), the CA3, and the hypothalamus 24-h post-ischemia. MK-801 reversed the decreases in the hypothalamic nuclei but not in the other brain regions. Seventy-two hours post-ischemia, CRH levels returned to control values in all regions except the dentate gyrus (DG) where elevated CRH levels were observed. In vivo, a significant increase in CRH release in response to global ischemia was found at the CeA with no alterations at the CA1. These findings support brain region-specific ischemia-induced CRH alterations and suggest that CRH actions to mediate neuronal damage at the hippocampal CA1 layer may be indirect.
... However, increasing evidence supports the involvement of stress regulators in the pathophysiology of cerebral ischemia, which could contribute to behavioral impairments. For example, corticotropin releasing hormone (CRH) release, protein and mRNA expression as well as corticosterone (CORT) levels remain elevated for days in discrete brain regions and in the periphery following cerebral ischemia (Hwang et al., 2006;Khan et al., 2004;Wong et al., 1995). Past studies have also shown that plasma levels of norepinephrine (NE) are up-regulated during (Gustafson et al., 1991), in the hours (Globus et al., 1989), and even weeks (Pich et al., 1993) after brain ischemia. ...
... This observation is consistent with a recent study showing significant losses of CRH-positive neurons at the CeA six weeks following hypoxia-ischemia induced in neonatal rats, a phenomenon associated with hyperactivity in response to novel open field exposure in ischemic rats (Carty et al., 2010). Alterations at longer survival intervals contrast with rapid elevations of CRH release, protein and mRNA levels in the amygdala observed at short time intervals following traumatic brain injury and global or focal ischemia in rats (Khan et al., 2004;Roe et al., 1998;Wong et al., 1995). Although the exact contribution of such changes remains unknown, increase in CRH expression at shorter term intervals likely contributes, above regulatory actions of the stress response, to plastic remodeling affecting amygdalar and hippocampal neurons following various psychogenic and physiological stressors, including cerebral ischemia (Kovalenko et al., 2006;Martone et al., 2000;Ruan et al., 2012). ...
Abnormal function of the neuroendocrine stress system has been implicated in the behavioral impairments observed following brain ischemia. The current study examined long-term changes in stress signals regulation 30 days following global cerebral ischemia. Experiment 1 investigated changes in the expression of corticotropin releasing hormone (CRH) and its subtype 1 receptor (CRHR1), glucocorticoid receptors (GR) in the paraventricular nucleus of the hypothalamus (PVN), the central nucleus of the amygdala (CeA), and the CA1 subfield of the hippocampus. Tyrosine hydroxylase (TH) was determined at the locus coeruleus (LC). Experiment 2 investigated the role of central CRHR1 activation on corticosterone (CORT) secretion at multiple time intervals following global ischemia after exposure to an acute stressor. Findings from Experiment 1 demonstrated a persistent increase in GR, CRH and CRHR1 immunoreactivity (ir) at the PVN, reduced GR and CRHR1 expression in pyramidal CA1 neurons, and increased LC TH expression in ischemic rats displaying working memory errors in the radial arm Maze. Findings from Experiment 2 revealed increased CORT secretion up to 7 days, but no longer present 14 and 21 days post ischemia. However upon an acute restraint stress induced 27 days following reperfusion, ischemic rats had increased plasma CORT secretions compared to sham-operated animals, suggesting HPA axis hypersensitivity. Antalarmin (2μg/2μl) pretreatment significantly attenuated post ischemic elevation of basal and stress-induced CORT secretion. These findings support persistent neuroendocrine dysfunctions following brain ischemia likely to contribute to emotional and cognitive impairments observed in survivors of cardiac arrest and stroke.
... Several years ago it was reported that a CRF receptor antagonist reduced the damage caused by global ischaemia in the gerbil (Lyons et al. 1991). We have now shown that CRF mRNA is increased by cerebral ischaemia or traumatic brain damage in the rat cortex associated with the primary injury, but more specifically in the amygdala unilateral to the insult, where no neuronal loss occurs in response to damage in the rat (Wong et al. 1995). Injection of a CRF receptor antagonist (i.c.v.) significantly reduces subsequent damage in the cortex (Wong et al. 1995;Roe et al. 1998) (Fig. 11). ...
... We have now shown that CRF mRNA is increased by cerebral ischaemia or traumatic brain damage in the rat cortex associated with the primary injury, but more specifically in the amygdala unilateral to the insult, where no neuronal loss occurs in response to damage in the rat (Wong et al. 1995). Injection of a CRF receptor antagonist (i.c.v.) significantly reduces subsequent damage in the cortex (Wong et al. 1995;Roe et al. 1998) (Fig. 11). Our preliminary data suggest that unilateral lesions of the amygdala reduce subsequent cortical injury (S. ...
The first report that inhibition of IL-1 reduces brain damage in vivo (Relton & Rothwell, 1992) demonstrated that i.c.v. injection of IL-1ra at the time of induction of permanent focal cerebral ischaemia (middle cerebral artery occlusion (MCAo)) in the rat inhibited subsequent brain damage (infarct volume) by almost 70 % (Fig. 3). Numerous studies have subsequently verified and extended this observation (e.g. Betz et al. 1995, 1996; Garcia et al. 1995; Loddick & Rothwell, 1996; Relton et al. 1996; Rothwell et al. 1996a, 1997; Stroemer & Rothwell, 1997). It is now known that even systemic injection of IL-1ra, at considerably higher doses (50–100 mg kg−1), also inhibits ischaemic brain damage (Garcia et al. 1995; Relton et al. 1996), and indeed both IL-1 and IL-1ra are actively transported into the brain from the circulation (Gutierrez et al. 1994). IL-1ra is neuroprotective when administered 30–60 min after focal cerebral ischaemia and reduces not only infarct volume but also oedema, glial activation and neuronal loss, and largely reverses neurological impairment caused by MCAo (Garcia et al. 1995; Relton et al. 1996). In addition to these effects on MCAo in the adult rat or mouse, IL-1ra also markedly reduces brain damage caused by hypoxia/ischaemia in neonatal rats (Martin et al. 1995), global cerebral ischaemia in gerbils (Martin et al. 1996), lateral, cortical fluid percussion injury in the rat (Toulmond & Rothwell, 1995a) and heat stroke damage in rabbits (Lin et al. 1995), and reduces the clinical symptoms of experimental allergic encephalomyelitis (EAE, a rodent model of multiple sclerosis) (Martin & Near, 1995). In several of these paradigms, IL-1ra is effective when administered up to 4 h after the insult, and these actions of IL-1ra are not associated with any changes in body temperature or cardiovascular parameters in normal or brain-damaged rats.
... However, CRF has been implicated in both processes. Experimental focal cerebral ischaemia or brain trauma causes induction of CRF mRNA at the site of injury and in the armygdala (Wong et al., 1995; Roe, McGowan & Rothwell, unpublished data), and administration of CRF receptor antagonists inhibits ischaemic, excitotoxic and traumatic brain injury in the rat (Figure 7; Lyons et al., 1991; Strijbos et al., 1994; Wong et al., 1995; Roe & Rothwell, unpublished data). It is not yet known if the expression or actions of CRF in neurodegeneration are related directly to IL- 1, though this is an attractive hypothesis given the relationship between these molecules in other responses to disease. ...
... However, CRF has been implicated in both processes. Experimental focal cerebral ischaemia or brain trauma causes induction of CRF mRNA at the site of injury and in the armygdala (Wong et al., 1995; Roe, McGowan & Rothwell, unpublished data), and administration of CRF receptor antagonists inhibits ischaemic, excitotoxic and traumatic brain injury in the rat (Figure 7; Lyons et al., 1991; Strijbos et al., 1994; Wong et al., 1995; Roe & Rothwell, unpublished data). It is not yet known if the expression or actions of CRF in neurodegeneration are related directly to IL- 1, though this is an attractive hypothesis given the relationship between these molecules in other responses to disease. ...
... Thus, Caruana et al. [14] reported epileptiform EEG activity in the CA1/subicular region, the perirhinal cortex, and the prefrontal cortex following 15 min global ischemia in rats lasting several hours post reperfusion. Moreover, increased amygdalar CRH mRNA expression and release has been demonstrated following global and focal ischemia in rats [37,82], while significant losses of CRH-positive neurons have recently been demonstrated in the same brain region 6 weeks following hypoxia–ischemia [13], a phenomenon associated with hyperactivity in response to novel open field exposure in ischemic rats. Reduced amygdalar *Fig. 1. ...
... Hence, neither cued nor contextual fear conditioning can completely eliminate the role of the hippocampus. Different studies have reported post ischemic changes in the amygdala associated to increased amygdalar CRH mRNA and release in the hours or days following focal and global ischemia [37,82]. The sensitivity of this brain area to kainite-induced seizures and excitotoxicity [1,2,16,73] also suggests that the cellular integrity and/or functioning of this brain region could be altered following ischemia. ...
The current study characterizes fear conditioning responses following global ischemia and evaluates neuronal damage affecting discrete extra-hippocampal areas susceptible to contribute to post ischemic emotional and memory impairments. Conditioned emotional response, Barnes Maze and object recognition tests were used to assess emotional, spatial and recognition memory, respectively. Behavioural testing was initiated in middle-aged animals (10-12 month old) 1 week following sham (n=16) or 4VO occlusion (n=18). Post-mortem cellular assessment was performed in the hippocampal CA1 layer, the perirhinal cortex and basolateral amygdala. Middle-aged ischemic animals showed impaired spatial memory in the initial three testing days in the Barnes Maze and deficit in recognition memory. Of interest, ischemic rats demonstrated a significant reduction of freezing and increased locomotion during the contextual fear testing period, suggesting reduced fear in these animals. Assessment of neuronal density 40 days following global ischemia revealed that CA1 neuronal injury was accompanied by 20-25% neuronal loss in the basolateral nucleus of the amygdala and perirhinal cortex in middle-aged ischemic compared to sham-operated animals. This study represents the first demonstration of altered conditioned fear responses following ischemia. Our findings also indicate a vulnerability of extra-hippocampal neurons to ischemic injury, possibly contributing to discrete emotional and/or memory impairments post ischemia.
... Evidence indicates that at high levels, in the context of acute events, IL-1 is neurotoxic. Recent data in models of acute brain ischemia suggest that local increases of CRH contribute to acute neurodegenerative processes, 42 while IL-1ra synthesis is involved in the process of neuroprotection; 43 therefore, it is likely that the actions of IL-1 are also mediated through CRH in extrahypothalamic sites. ...
... CRH mRNA was also induced ispilaterally in the amygdala, 60 min following ischemia, in a pattern which was qualitatively different from that of shamoperated animals. 42 This finding, when interpreted in the light of previous studies showing neuroprotection by CRH antagonists in animal models of acute ischemia, 58,59 led to the concept that high levels of endogenous CRH may contribute to neurotoxicity. Strijbos et al 59 indicate that neuroprotection caused by CRH antagonism is not secondary to decreased cortisol levels, as focal cerebral ischemia is not affected by RU-486, a type II glucocorticoid receptor antagonist, or by dexamethasone. ...
Depression represents a major public health problem. It is estimated that 13-20% of the population has some depressive symptoms at any given time and about 5% of the population is assumed to suffer from major depression. Known pathological processes include ischemia, neoplasia, necrosis, apoptosis, infection, and inflammation. Of those, inflammation is the most compatible with the waxing and waning course of depression, and could explain the biology of this disorder that has a fluctuating course with severe episodes that can be followed by partial or complete remission. Over the years a body of evidence has been accumulated suggesting that major depression is associated with dysfunction of inflammatory mediators. Major depression commonly co-occurs with ischemic heart disease and decreased bone mineral density. Depressive symptoms are known to have a negative impact on cardiovascular prognosis, increasing the mortality rate of coronary artery disease. Several lines of evidence indicate that brain cytokines, principally interleukin-1beta (IL-1beta) and IL-1 receptor antagonist may have a role in the biology of major depression, and that they might additionally be involved in the pathophysiology and somatic consequences of depression as well as in the effects of antidepressant treatment. A particularly unique and novel aspect of the studies and views discussed here is their potential to lead to interventions which may reduce the morbidity and mortality risks for osteoporosis, cardiovascular disease, and behavioral symptoms in patients with major depression. We also discuss the emerging concept of peripheral and central cytokine compartments: their integration and differential regulation is a key element for the optimal functioning of the immune and nervous systems.
... There is increasing evidence to support the involvement of CRF in the pathogenesis of ischemic brain damage. Up-regulation of CRF mRNA has been reported in the cerebral parenchyma of rats after permanent middle cerebral artery occlusion (MCAO) (Wong et al., 1995) and traumatic brain injury (Roe et al., 1998). Moreover, CRF antagonists are beneficial in experimental models of neurodegeneration in vivo. ...
... Corticotropin-releasing factor has been reported to elevate both heart rate and blood pressure by stimulating noradrenergic sympathetic outflow (Fisher et al., 1983) and to cause vasodilation (Lei et al., 1993). During focal cerebral ischemia in the rat brain, mRNA for CRF is increased in the occluded hemisphere (Wong et al., 1995;Yatsushiro et al., 1997c) and striking elevations are also observed in pial and parenchymal arterioles (Yatsushiro et al., 1997c). The relevance of the elevated CRF in blood vessels during ischemia is less clear. ...
The neuroprotective effects of a systemically active, highly selective, corticotropin-releasing factor-1 (CRF1) receptor antagonist, R121920 ((7-(dipropylamino)-2,5-dimethyl-3- [2-(dimethylamino)-5-pyridyl] pyrazolo [1,5-a] pyrimidine), was assessed in two rat models of permanent focal cerebral ischemia, where the middle cerebral artery (MCA) was occluded either through the subtemporal approach or using the intraluminal suture technique. R121920 rapidly crossed the blood-brain barrier after intravenous (IV) bolus administration (10 mg/kg), with peak brain concentrations at 5 minutes (2.26 +/- 0.40 microg/mL), which were approximately 2-fold greater than those in plasma (0.98 +/- 0.24 microg/mL). Treatment with R121920 (10 mg/kg IV followed by 5 mg/kg subcutaneously at hourly intervals for 4 hours) significantly (P < 0.001) reduced total (by 40%) and cortical (by 37%) infarct volume at 24 hours after subtemporal MCA occlusion (MCAO). In the intraluminal suture MCAO model, IV administration of R121920 (10 mg/kg) at the time of ischemia onset (and at multiple times thereafter) reduced both hemispheric infarct volume (by 34%, P < 0.001) and brain swelling (by 50%, P < 0.001) when assessed at 24 hours. In this model of focal ischemia, significant reduction (P < 0.05) in both outcome measures was obtained when R121920 administration was delayed up to 1 hour after MCAO. These results further define the antiischemic properties of selective CRF 1 antagonists in two experimental models of permanent focal cerebral ischemia.
... Moderate to extensive loss of hippocampal CA1 pyramidal cells has been shown to induce severe anterograde amnesia in humans [62], and a variety of functional deficits, including locomotor hyperactivity as well as working and spatial memory impairments in rodents [5,12,13,28]. Among the molecular targets suggested to play a role in excitotoxic cellular damage and cerebral ischemia is the 41-amino acid polypeptide corticotrophin-releasing hormone (CRH) [37,56,61]. CRH-like immunoreactive cells, fibres and receptors have been identified in different hypothalamic, brain stem and limbic structures including the amygdala and hip- pocampus [7,39,51,58,59]. ...
... Similarly, within 4 h, CRH concentrations increased by approximately 50–100% at the piriform cortex, as well as the lateral, ventromedial and paraventricular nucleus of the hypothalamus in response to the ischemic stress [32] . Similar increases in amygdalar CRH mRNA expression were observed following focal ischemia [61]. These structures are not associated with neuronal degeneration, suggesting that high increases in CRH in response to a global ischemic insult are not necessarily associated with concomitant cell death. ...
Corticotropin-releasing hormone (CRH) has been implicated in ischemia-induced neurotoxicity, due in part to excitatory effects at the hippocampus, and the demonstrated neuroprotective effects of centrally administered, non-specific CRH antagonists. However, a number of issues remain to be clarified from these studies, including the relative contribution of CRH receptor subtypes, and the efficacy of these compounds to alter ischemia-induced behavioral impairments. In the current study, a highly selective, systemically administered CRH1 antagonist (CP154,526) failed to reverse global ischemia-induced cell death in hippocampal CA1 neurons or spatial memory impairments as assessed in the radial arm maze. Similarly, central administration of alpha-helical CRH failed to confer protection against ischemic damage. Interestingly, CRH1 antagonism reversed ischemia-induced hyperactivity in a novel open field, suggesting that modulation of this behavior is independent of effects on hippocampal CA1 cell loss. Failure of the current study to demonstrate neuroprotective effects of either the selective or non-selective CRH antagonists tested challenges the proposed neurotoxic role of CRH in global ischemia. These findings are discussed in relationship to recent findings reconsidering the participation of CRH in excitotoxic-mediated cellular damage.
... cJun phosphorylation is closely associated with neuronal ischemic stress [23]. CRF was chosen bibliographically because although this gene is known to be constitutively expressed in retinal amacrine neurons [24], it is upregulated in neurons after ischemia [25]. We confirmed expression in RGCs using Brn3a colocalization ( Figure 5). ...
Nonarteritic anterior ischemic optic neuropathy (NAION) is the most common cause of sudden optic nerve (ON)-related vision loss in humans. Study of this disease has been limited by the lack of available tissue and difficulties in evaluating both treatments and the window of effectiveness after symptom onset. The rodent nonarteritic anterior ischemic optic neuropathy model (rNAION) closely resembles clinical NAION in its pathophysiological changes and physiological responses. The rNAION model enables analysis of the specific responses to sudden ischemic axonopathy and effectiveness of potential treatments. However, there are anatomic and genetic differences between human and rodent ON, and the inducing factors for the disease and the model are different. These variables can result in marked differences in lesion development between the two species, as well as in the possible responses to various treatments. These caveats are discussed in the current article, as well as some of the species-associated differences that may be related to ischemic lesion severity and responses.
... In rodents, CRH concentrations increase by approximately 50-100% at the piriform cortex, the central nucleus of the amygdala, as well as the lateral, ventromedial and paraventricular nucleus of the hypothalamus within 4 h of global cerebral ischemia (Khan et al., 2004). Similarly, elevated CRH mRNA levels in the amygdala and cerebral cortex are observed within hours of focal ischemia (Wong et al., 1995). Increased levels of CRH, norepinephrine and CRHR1 are observed in the rat PVN and pituitary after hypoxia, cold, or restraint stress Wang et al., 2004). ...
Although it is well accepted that changes in the regulation of the hypothalamic-pituitary adrenal (HPA) axis may increase susceptibility to affective disorders in the general population, this link has been less examined in stroke patients. Yet, the bidirectional association between depression and cardiovascular disease is strong, and stress increases vulnerability to stroke. Corticotropin-releasing hormone (CRH) is the central stress hormone of the HPA axis pathway and acts by binding to CRH receptors (CRHR) 1 and 2, which are located in several stress-related brain regions. Evidence from clinical and animal studies suggests a role for CRH in the neurobiological basis of depression and ischemic brain injury. Given its importance in the regulation of the neuroendocrine, autonomic, and behavioral correlates of adaptation and maladaptation to stress, CRH is likely associated in the pathophysiology of post stroke emotional impairments. The goals of this review article are to examine the clinical and experimental data describing (1) that CRH regulates the molecular signaling brain circuit underlying anxiety- and depression-like behaviors, (2) the influence of CRH and other stress markers in the pathophysiology of post stroke emotional and cognitive impairments, and (3) context and site specific interactions of CRH and BDNF as a basis for the development of novel therapeutic targets. This review addresses how the production and release of the neuropeptide CRH within the various regions of the mesocorticolimbic system influences emotional and cognitive behaviors with a look into its role in psychiatric disorders post stroke.
... In this study the central administration of α-helical-CRH (9-41) lowered the severity of cerebral infarction, and alleviated cerebral infarction-related gastrointestinal barrier dysfunction. Studies suggest that CRH is involved in stroke injury: CRH-mRNA increases rapidly after stroke [33,34] and the central use of CRH receptor antagonist, α-helical-CRH (9-41) after stroke can reduce ischemic injury. [35][36][37] Its mechanism however remains unclear. ...
Background:
Corticotrophin releasing hormone (CRH) is believed to mediate stress-induced behaviors, implying a broader, integrative role for the hormone in the psychological stress response, and studies on CRH in physical stress are few. This study was undertaken to investigate whether CRH plays an important role in cerebral infarction-related gastrointestinal barrier dysfunction.
Methods:
Thirty male Wistar rats were randomly divided into a pseudo-operation group (group C, n=10), a cerebral infarction group (group I, n=10), and a cerebral infarction + ic α-helical-CRH (9-41) group (group Aic, n=10). Urine samples were collected to determine the levels of epinephrine, norepinephrine, cortisol, and sucrose. At 24 hours after establishment of the models, blood samples were taken to determine the activity of diamine oxidase (DAO) and the concentration of D-lactic acid (D-lac). The stomach was taken to determine gastric Guth score, and the hypothalamus was also taken to determine tissue CRH protein expression using Western blotting.
Results:
The hypothalamus CRH protein, the indicators of stress, the plasma DAO activity and plasma D-lac, urine sucrose exertion and gastric Guth score in group I were higher than those in groups Aic and C.
Conclusions:
After cerebral infarction, CRH in the hypothalamus was increased, the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system were activated, gastrointestinal permeability was increased, and gastrointestinal barrier function was destroyed. CRH receptor antagonist alleviated the gastrointestinal barrier function.
... 17 target genes, listed in Table 3, amongst which some have reported neuroprotective functions e.g., AID genes (Atf3, Btg2, Gadd45g), Nr4a1 [48,49], Fos [50], Cited2 [51], Dusp1 [52,53], Klf4 [54], and Nfil3 [55,56] whereas others are associated with neuronal apoptosis e.g. Nos3 [57], Crh [58,59], Crem [60], Homer1 [61]. Others still (Ccl3 and TNF) are inflammatory markers [62], since inflammation is a known brain response to nerve agents. ...
Abstract
Organophosphorus (OP) nerve agents are deadly chemical weapons that pose an alarming threat to military and civilian
populations. The irreversible inhibition of the critical cholinergic degradative enzyme acetylcholinesterase (AChE) by OP
nerve agents leads to cholinergic crisis. Resulting excessive synaptic acetylcholine levels leads to status epilepticus that, in
turn, results in brain damage. Current countermeasures are only modestly effective in protecting against OP-induced brain
damage, supporting interest for evaluation of new ones. (-)-Phenserine is a reversible AChE inhibitor possessing
neuroprotective and amyloid precursor protein lowering actions that reached Phase III clinical trials for Alzheimer’s Disease
where it exhibited a wide safety margin. This compound preferentially enters the CNS and has potential to impede soman
binding to the active site of AChE to, thereby, serve in a protective capacity. Herein, we demonstrate that (-)-phenserine
protects neurons against soman-induced neuronal cell death in rats when administered either as a pretreatment or posttreatment
paradigm, improves motoric movement in soman-exposed animals and reduces mortality when given as a
pretreatment. Gene expression analysis, undertaken to elucidate mechanism, showed that (-)-phenserine pretreatment
increased select neuroprotective genes and reversed a Homer1expression elevation induced by soman exposure. These
studies suggest that (-)-phenserine warrants further evaluation as an OP nerve agent protective strategy.
... 17 target genes, listed in Table 3, amongst which some have reported neuroprotective functions e.g., AID genes (Atf3, Btg2, Gadd45g), Nr4a1 [48,49], Fos [50], Cited2 [51], Dusp1 [52,53], Klf4 [54], and Nfil3 [55,56] whereas others are associated with neuronal apoptosis e.g. Nos3 [57], Crh [58,59], Crem [60], Homer1 [61]. Others still (Ccl3 and TNF) are inflammatory markers [62], since inflammation is a known brain response to nerve agents. ...
Organophosphorus (OP) nerve agents are deadly chemical weapons that pose an alarming threat to military and civilian populations. The irreversible inhibition of the critical cholinergic degradative enzyme acetylcholinesterase (AChE) by OP nerve agents leads to cholinergic crisis. Resulting excessive synaptic acetylcholine levels leads to status epilepticus that, in turn, results in brain damage. Current countermeasures are only modestly effective in protecting against OP-induced brain damage, supporting interest for evaluation of new ones. (-)-Phenserine is a reversible AChE inhibitor possessing neuroprotective and amyloid precursor protein lowering actions that reached Phase III clinical trials for Alzheimer's Disease where it exhibited a wide safety margin. This compound preferentially enters the CNS and has potential to impede soman binding to the active site of AChE to, thereby, serve in a protective capacity. Herein, we demonstrate that (-)-phenserine protects neurons against soman-induced neuronal cell death in rats when administered either as a pretreatment or post-treatment paradigm, improves motoric movement in soman-exposed animals and reduces mortality when given as a pretreatment. Gene expression analysis, undertaken to elucidate mechanism, showed that (-)-phenserine pretreatment increased select neuroprotective genes and reversed a Homer1expression elevation induced by soman exposure. These studies suggest that (-)-phenserine warrants further evaluation as an OP nerve agent protective strategy.
... CRH not only activates the hypothalamic-pituitary-adrenal axis, but also the sympathetic nervous system, as well as exerting important proinflammatory effects, reversible by the administration of the CRH antagonist, antalarmin. In the brain, CRH can be neurotoxic, an effect ameliorated by antagonizing of the CRH receptor 1. 37 Moreover, CRH could also contribute to some phenotypic changes characteristic of melancholic depression. Administration of CRH centrally leads to fear-related behaviors 38 and decreases in appetite, 39 sleep 40 and sexual activity. ...
Major depression and bipolar disorder are heterogeneous conditions in which there can be dysregulation of (1) the stress system response, (2) its capacity for counterregulation after danger has passed and (3) the phase in which damaging molecules generated by the stress response are effectively neutralized. The response to stress and depressed mood share common circuitries and mediators, and each sets into motion not only similar affective and cognitive changes, but also similar systemic manifestations. We focus here on two highly interrelated processes, parainflammation and endoplasmic reticulum (ER) stress, each of which can potentially interfere with all phases of a normal stress response in affective illness, including adaptive neuroplastic changes and the ability to generate neural stem cells. Parainflammation is an adaptive response of the innate immune system that occurs in the context of stressors to which we were not exposed during our early evolution, including overfeeding, underactivity, aging, artificial lighting and novel foodstuffs and drugs. We postulate that humans were not exposed through evolution to the current level of acute or chronic social stressors, and hence, that major depressive illness is associated with a parainflammatory state. ER stress refers to a complex program set into motion when the ER is challenged by the production or persistence of more proteins than it can effectively fold. If the ER response is overwhelmed, substantial amounts of calcium are released into the cytoplasm, leading to apoptosis. Parainflammation and ER stress generally occur simultaneously. We discuss three highly interrelated mediators that can effectively decrease parainflammation and ER stress, namely the central insulin, klotho and peroxisome proliferator-activated receptor-γ (PPAR-γ) systems and propose that these systems may represent conceptually novel therapeutic targets for the amelioration of the affective, cognitive and systemic manifestations of major depressive disorder.Molecular Psychiatry advance online publication, 27 November 2012; doi:10.1038/mp.2012.167.
... Different studies have showed potent effects of cerebral ischemia on stress hormones and associated regulatory systems. For example, corticotropin-releasing hormone (CRH) and corticosterone (CORT) levels remain elevated for days in discrete brain regions and in the periphery following cerebral ischemia (Chen et al., 1998;Hwang et al., 2006;Khan et al., 2004;Wong et al., 1995) and upregulation of norepinephrine secretions has been documented in brain tissue during (Bentue-Ferrer et al., 1986;Gustafson et al., 1991) and for weeks after ischemia (Pich et al., 1993), indicating that it represents a potent physiological stressor. ...
This study investigates the association of ischemia-induced spatial memory impairment to alterations of the HPA axis and noradrenergic activation post insult. Experiment 1 characterized the effects of 10 min forebrain ischemia on corticosterone (CORT) secretion following ischemia and in response to spatial memory assessment in the Barnes maze, as well as the impact of pre-ischemia treatment with the glucocorticoid inhibitor metyrapone (175 mg/kg; s.c.). The results showed that cerebral ischemia represents a significant physiological stressor that upregulated CORT secretion 1, 24 and 72 h post-ischemia but not at 7 days. In response to testing in the Barnes maze ischemic animals showed elevated CORT secretion simultaneously with spatial memory deficits. The single dose of metyrapone attenuated the ischemia-induced adrenocortical hyper-responsiveness and subsequent memory deficits despite not providing neuroprotection in the hippocampal CA1 pyramidal cells. To complement these findings, we examined whether norepinephrine which provides positive feedback to the HPA axis and is upregulated following brain ischemia could influence memory performance at delayed intervals after ischemia. Experiment 2 demonstrated that pre-testing administration of the alpha2-adrenoceptor agonist clonidine (.04 mg/kg, s.c.) attenuated ischemia-induced working memory impairments in a radial maze while opposite effects were obtained with the antagonist yohimbine (.3 mg/kg, s.c.). Post-testing administration of clonidine produced spatial reference memory impairments in ischemic rats. The findings from the current study demonstrate increased sensitization and responsiveness of systems regulating stress hormones at long intervals post ischemia. Importantly, we demonstrate that these effects contribute to post ischemic cognitive impairments which can be attenuated pharmacologically even in the presence of hippocampal degeneration at time of testing.
... In work conducted to show the role of CRH in acute neurodegeneration, it was shown that CRH mRNA levels were elevated 2.6-fold within 1 h after middle cerebral artery occlusion, when compared to shamoperated animals. Furthermore, the induction of CRH mRNA ipsilaterally in the amygdala, following ischemia, was qualitatively different from that of sham-operated animals (37). These findings, when interpreted in the light of previous studies showing neuroprotection by CRH antagonists in animal models of acute ischemia (38,39), led to the concept that high levels of endogenous CRH may contribute to neurotoxicity. ...
Cytokines are molecules that were initially discovered in the immune system as mediators of communication between various types of immune cells. However, it soon became evident that cytokines exert profound effects on key functions of the central nervous system, such as food intake, fever, neuroendocrine regulation, long-term potentiation, and behavior. In the 80's and 90's our group and others discovered that the genes encoding various cytokines and their receptors are expressed in vascular, glial, and neuronal structures of the adult brain. Most cytokines act through cell surface receptors that have one transmembrane domain and which transduce a signal through the JAK/STAT pathway. Of particular physiological and pathophysiological relevance is the fact that cytokines are potent regulators of hypothalamic neuropeptidergic systems that maintain neuroendocrine homeostasis and which regulate the body's response to stress. The mechanisms by which cytokine signaling affects the function of stress-related neuroendocrine systems are reviewed in this article.
... After washing at 408C, the sections were exposed to a b-max Hyper®lm (Amersham, Braunschweig, Germany) at a room temperature for 14 days. After hybridization, washing out and autoradiography of the brain slices, the distribution and density of signal in the ®lm was analysed; it was found that patterns of hybridization fully agreed with previously described distribution of CRF mRNA (Hsu et al., 1998;Palkovits et al., 1998;Wong et al., 1995) and of NPY mRNA (Morris, 1989;Obuchowicz and Turchan, 1998;S Â miaøowska et al., 1997). A speci®city test with RNAase or with the excess of an unlabelled probe, completely eliminated the hybridization signal. ...
Our earlier findings concerning the 6-OHDA lesion suggested dopaminergic regulation of neuropeptide Y (NPY) and corticotropin releasing factor (CRF) synthesis and expression in amygdala neurons. On the other hand, some other studies indicated that not only dopamine, but also other monoamines may modulate peptidergic neurons. Therefore the present study examined the effect of pharmacological deprivation of monoaminergic influences on NPY and CRF neurons in rat brain amygdala by means of in situ hybridization and immunohistochemical methods. It was found that NPY mRNA expression in the amygdala decreased after 24h blockade of dopaminergic D1 and D2 receptors, by haloperidol or SCH23390. At the same time the NPY-peptide expression measured immunohistochemically was not significantly changed. A prolonged, 14-day, blockade of dopaminergic receptors by haloperidol induced an opposite effect, an increase in NPY mRNA expression. Impairment of the serotonergic transmission by blockade of 5-HT synthesis using p-chlorophenylalanine, as well as attenuation of the noradrenergic transmission by NA depletion from terminals by DSP4, did not significantly change NPY mRNA expression or the mean number of NPY-immunoreactive neurons in the amygdala. Only a decrease in the staining intensity observed as a decreased number of darkly stained neurons was found after both compounds. Neither the dopamine receptor blockade nor the impairment of serotonergic or noradrenergic transmission changed CRF mRNA or the peptide expression in the amygdala. The obtained results indicate that in rat brain amygdala, of all the monoamines, dopamine seems to be the most important modulator of NPY biosynthesis and expression. The effect of blockade of dopaminergic receptors is biphasic: first it induces a decrease and then - after prolonged treatment an increase in NPY mRNA. Serotonergic and noradrenergic systems in the amygdala seem to be connected with regulation of NPY release rather than the biosynthesis.
... In addition to its effects on hormone regulation, CRH has been implicated in pathological and pathophysiological responses in various neurodegenerative disorders, including brain traumatic, ischemic and excitotoxic injuries. These neurodegenerative disorders are accompanied by an increase in brain CRH and its receptors (Wong et al. 1995;Greenwood et al. 1997;Roe et al. 1998;Wang et al. 2000). Several lines of evidence suggest that CRH contributes to neuronal loss in vitro and in vivo while some studies show a protective role of CRH. ...
We have previously demonstrated that corticotropin-releasing hormone (CRH) receptor 1 (CRH-R1) is functionally expressed in rat microglia. In the present study, we show that CRH, acting on CRH-R1, promoted cell proliferation and tumour necrosis factor-alpha (TNF-alpha) release in cultured rat microglia. Exogenous CRH resulted in an increase in BrdU incorporation compared with control cells, which was observed in a range of concentrations of CRH between 10 and 500 nm, with a maximal response at 50 nm. The effect of CRH on BrdU incorporation was inhibited by a CRH antagonist astressin but not by a cAMP-dependent protein kinase inhibitor H89. Exposure of microglial cells to CRH resulted in a transient and rapid increase in TNF-alpha release in a dose-dependent manner. In the presence of astressin, the effects of CRH on TNF-alpha release were attenuated. CRH effects on TNF-alpha release were also inhibited by specific inhibitors of MEK, the upstream kinase of the extracellular signal-regulated protein kinase (ERK) (PD98059) or p38 mitogen-activated protein kinase (SB203580), but not by H89. Furthermore, CRH induced rapid phosphorylation of ERK and p38 kinases. Astressin, PD98059, and SB230580 were able to inhibit CRH-induced kinase phosphorylation. These results suggest that CRH induces cell proliferation and TNF-alpha release in cultured microglia via MAP kinase signalling pathways, thereby providing insight into the interactions between CRH and inflammatory mediators.
... For instance, Fos expression is elevated in the CeA after a coronary artery occlusion (Xie et al, 2000). Focal cerebral ischaemia has been associated with acutely elevated corticotrophin-releasing hormone (CRH) release and mRNA in the amygdala (Wong et al, 1995;Khan et al, 2004) with a corresponding decrease in intracellular CRH (Khan et al, 2004), as well as changes in other neurotrans-mitters (Allen et al, 1995). Also, expression of voltage-gated K þ channels is increased in the rat amygdala after a transient focal ischaemia (Chung et al, 2001), providing a possible molecular basis for altered neuronal excitability in the region. ...
Infection, inflammation, and hyperthermia associated with cerebral ischaemia are known to contribute to enhanced neuronal cell loss and more severe behavioural deficits. Because neonatal exposure to an immune challenge has been shown to alter the severity of inflammatory and febrile responses to a further immune challenge experienced in adulthood, we hypothesised that this could also alter temperature responses and neuronal survival after ischaemia. Thus, male Sprague-Dawley rats were treated at postnatal day 14 with a single injection of the bacterial endotoxin lipopolysaccharide (LPS) and were examined as adults for temperature changes, behavioural deficits, and neuronal cell loss associated with global cerebral ischaemia after a two-vessel occlusion (2VO). Neonatally LPS-treated rats showed behavioural differences in a novel object exploration paradigm, as well as altered temperature responses to the 2VO compared with neonatally saline-treated controls. Interestingly, these neonatally LPS-treated rats also showed increased cell loss in the central nucleus of the amygdala, a region that is important in the processing of emotional responses, but that is not usually examined in animal models of cerebral ischaemia. No differences were seen in the CA1, CA3, or dentate gyrus regions of the hippocampus. This work shows the importance of examining brain regions other than the hippocampus in association with global ischaemia. We also highlight the importance of the early period of development in programming an animal's ability to deal with injury such as cerebral ischaemia in adulthood.
... CRH has been shown previously to exert either neurotoxic or neuroprotective effects. The level of CRH is increased after several forms of brain injury such as trauma ( Roe et al. 1998), hypoxia/ischemia ( Wong et al. 1995;Wang et al. 2000) and seizures (Maecker et al. 1997). Administration of CRH provoked progressive loss of hippocampal CA3 neurons independently of glucocorticoids (Brunson et al. 2001). ...
Neuropeptides are short-chain peptides found in brain tissue, some of which function as neurotransmitters and others as hormones. Neuropeptides may directly or indirectly modulate glial functions in the CNS. In the present study, effects of various neuropeptides on the viability and inflammatory activation of cultured microglia were investigated. Vasoactive intestinal peptide, substance P, cholecystokinin and neuropeptide Y did not affect microglial cell viability, whereas corticotropin-releasing hormone (CRH) induced a classical apoptosis of mouse microglia in culture as shown by nuclear condensation and fragmentation, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and cleavage of caspase 3 and poly(ADP-ribose) polymerase protein. CRH, however, did not influence nitric oxide production or expression of inflammatory genes including those encoding cytokines and chemokines, indicating that CRH did not affect the inflammatory activation of microglia. The CRH-induced microglial apoptosis appeared to involve a mitochondrial pathway and reactive oxygen species, based on the mitochondrial membrane potential change, caspase 9 activation and sensitivity to antioxidants. Taken together, our results indicate that the stress neuropeptide CRH may regulate neuroinflammation by inducing the apoptosis of microglia, the major cellular source of inflammatory mediators in the CNS.
Ischaemic neurovascular stroke represents a leading cause of death in the developed world. Preclinical and human epidemiological evidence implicates the corticotropin‐releasing factor (CRF) family of neuropeptides as mediators of acute neurovascular injury pathology. Preclinical investigations of the role of CRF, CRF receptors and CRF‐dependent activation of the hypothalamic–pituitary–adrenal (HPA) axis have pointed toward a tissue‐specific and temporal relationship between activation of these pathways and physiological outcomes. Based on the literature, the major phases of ischaemic stroke aetiology may be separated into an acute phase in which CRF and anti‐inflammatory stress signalling are beneficial and a chronic phase in which these contribute to neural degeneration, toxicity and apoptotic signalling. Significant gaps in knowledge remain regarding the pathway, temporality and systemic impact of CRF signalling and stress biology in neurovascular injury progression. Heterogeneity among experimental designs poses a challenge to defining the apparent reciprocal relationship between neurological injury and stress metabolism. Despite these challenges, it is our opinion that the elucidated temporality may be best matched with an antibody against CRF with a half‐life of days to weeks as opposed to minutes to hours as with small‐molecule CRF receptor antagonists. This state‐of‐the‐art review will take a multipronged approach to explore the expected potential benefit of a CRF antibody by modulating CRF and corticotropin‐releasing factor receptor 1 signalling, glucocorticoids and autonomic nervous system activity. Additionally, this review compares the modulation of CRF and HPA axis activity in neuropsychiatric diseases and their counterpart outcomes post‐stroke and assess lessons learned from antibody therapies in neurodegenerative diseases.
Zinc toxicity is one of the key factors responsible for the neuronal injuries associated with various neurological conditions. Zinc accumulation in some cells is accompanied by the increase of blood stress hormone levels, which might indicate a functional connection between stress and zinc toxicity. However, the cellular mechanism for the effect of stress on zinc toxicity is not known. Recently, it was reported that the zinc permeable transient receptor potential melastatin 7 (TRPM7) channel may represent a novel target for neurological disorders where zinc toxicity plays an important role. To investigate the effect of stress hormone on zinc-induced cell death, neuroblastoma SH-SY5Y cells were pretreated with urocortin, a corticotropin releasing factor (CRF)-related peptide. Urocortin potentiated zinc-induced cell death at μM range of extracellular zinc concentrations. It significantly increased TRPM7 channel expression, and zinc influx into cytosol. Moreover, application of TRPM7 channel blockers and RNA interference of TRPM7 channel expression attenuated the zinc-induced cell death in urocortin-pretreated cells, indicating that TRPM7 channel may serve as a zinc influx pathway. These results suggest that TRPM7 channel may play a critical role for zinc toxicity associated with stress.
Cytokines are established mediators and modulators of inflammation and damage in peripheral tissues. However, the realization that they are also involved in degeneration and damage in the nervous system has emerged only recently, but is now an intensely active and exciting field of research.
Stroke is a common and devastating neurological disorder (Scatton, 1994) which is the third leading cause of death in major industrialized countries and also a major cause of long-lasting disability. Cerebral ischemia is always vascular origin and can be divided into haemorrhagic and thromboembolic, with the latter accounting for approximately 90% of strokes (Scatton, 1994) and results from embolic or thrombotic occlusion of the major cerebral arteries, most often the middle cerebral artery.
Jemula802 is a simulator developed by Disney Research for the standard IEEE 802.11, more specifically 802.11a/g. Jemula802 is used for simulations on scenarios related with Theme Park deployments. This paper describes the progress of the design and implementation of the IEEE 802.15.4 over Jemula. Jemula802.15.4-adaptation transforms Jemula802 (simulator of IEEE 802.11 standard) into the IEEE 802.15.4 standard simulator. IEEE 802.15.4, standard for low-rate wireless personal area networks (LR-WPANs) that specifies the physical layer and medium access control that is the base for upper layer protocols like Zigbee, MiWi and WirelessHART. Thereby, Jemula is offering a simulator and emulation framework to evaluate and compare deployments based on IEEE 802.11 (WiFi) and IEEE 802.15.4 (Wireless Sensor Networks). In addition, Jemula offers the visualization libraries for theme parks that can be also applied for smart cities. Therefore, Jemula can be considered as a evaluation framework for IEEE 802.15.4 and IEEE 802.11 for Smart Cities.
Interleukins (ILs) are cytokines, best known for their actions on immune cells and as mediators of systemic inflammation and responses to systemic tissue damage. The IL family includes peptides of molecular size 8–25 kDa but with diverse actions. Interleukin 1 (IL-1) was first proposed as a modulator of neuronal damage several years ago, but early studies implied that it might promote repair and regeneration. IL-1 exerts a number of potentially beneficial actions, including synthesis of nerve growth factor (NGF), which is neuroprotective, and neurovascularization. In pure neuronal cultures, IL-1 and interleukin 6 (IL-6) inhibit excitotoxic damage. Studies relating to the role of other cytokines in cerebral ischemia are largely circumstantial as few studies have investigated the effects of blocking endogenous brain cytokines. Preliminary data indicate that the central administration of physiological relevant doses of recombinant IL-6 inhibit ischemic brain damage.
There is increasing evidence for structural brain changes associated with unipolar re current major depression. Many depressed patients have comorbid physical illnesses, producing a high rate of subcortical white matter changes and brain damage to key structures involved in the modulation of emotion. This is especially true in the case of late-onset depression, which typically occurs in the setting of age-related illnesses, such as Parkinson's disease, Alzheimer's disease, poststroke syndromes, and myocardial in farction. In addition, there is now evidence for brain changes associated with early-onset major depression. Volume decreases have been reported in the hippocampus, amygdala, caudate, putamen, and frontal cortex. These structures are extensively interconnected and are part of a neuroanatomical circuit that has been termed the limbic-cortical-striatal pallidal-thalamic tract. Possible mechanisms for tissue loss include neuronal loss through exposure to repeated episodes of hypercortisolemia or glial cell loss, resulting in in creased vulnerability to glutamate neurotoxicity. Studies combining the anatomical and morphological information of MRI studies with functional studies have the potential to localize abnormalities in blood flow, metabolism, and neurotransmitter receptors and provide a better integrated model of depression. NEUROSCIENTIST 4:331-334, 1998
Depression is highly co-morbid with a number of neurodegenerative conditions, including Parkinson’s disease (PD), Alzheimer’s
disease (AD), stroke and multiple sclerosis. Although psychosocial stress and impairment play a substantial role, accumulating
evidence suggests that co-morbid depressive illness may emerge from alterations in processes related to the primary neurodegenerative
condition. For instance, depression in PD may occur long before motor disability and in many cases is likely related to early
degeneration in brainstem or limbic regions. We posit that pro-inflammatory cytokines and their associated inflammatory signaling
pathways (e.g., JAK-STAT, NFκB, and MAP kinases), as well as other immuno-inflammatory factors such as the microglial inducible
enzyme, cyclooxygenase-2 (COX-2), may play a primary role in modulating the emergence of co-morbid depression. In this regard,
neuroprotective/neurotrophic anti-inflammatory factors may have important antidepressant properties. The present review will
cover the evidence concerning the mechanisms through which depression might emerge in PD and other neurodegenerative disorders.
Secondly, we will focus on the important cytokines, inflammatory co-factors and intracellular signaling proteins that could
be targeted to potentially provide therapeutic benefit for depression as well as the primary neurodegenerative condition.
KeywordsParkinson’s disease-Depression-Co-morbidity-Inflammation-Cytokine-Cyclooxygenase
Antalarmin is a pyrrolopyrimidine compound that antagonizes corticotropin-releasing hormone (CRH) type 1 receptors (CRHR1). In order to assess the effects of antalarmin treatment on hypothalamic-pituitary-adrenal (HPA) function we measured the plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone in animals treated with either antalarmin or vehicle for 1 week or for 8 weeks. We found that antalarmin treatment for 1 week did not affect basal concentrations of ACTH or corticosterone. In contrast, treatment for 8 weeks significantly lowered basal ACTH and corticosterone concentrations and also significantly decreased the basal corticosterone to ACTH ratio, indicating decreased basal adrenocortical responsiveness to ACTH. However, immobilization stress resulted in ACTH and corticosterone concentrations that were the same in animals treated with vehicle or antalarmin for either 1 or 8 weeks. We conclude that even though 8-week antagonism of CRHR1 by the non-peptide antalarmin blunts basal concentrations of ACTH and corticosterone, and affects the adrenal responsiveness to ACTH, it does not blunt the HPA response to acute stress, and it does not appear to cause stress-induced adrenal insufficiency.
Several studies have demonstrated that antagonists of the corticotrophin releasing factor (CRF) receptor markedly inhibit experimentally induced excitotoxic, ischaemic and traumatic brain injury in the rat, and that CRF expression is elevated in response to experimentally induced stroke or traumatic brain injury. CRF is also induced by the pro-inflammatory cytokine interleukin 1 (IL-1), which participates in various forms of neurodegeneration. The aim of this study was to test the hypothesis that CRF is toxic directly in vivo or in vitro. In primary cultures of rat cortical neurons, exposure to CRF (10 pM–100 nM) for 24 h failed to cause cell death directly, or to modify the neurotoxic effects of N-methyl-d-aspartate (NMDA). Similarly, infusion of CRF (0.3–5 μg) into specific brain regions of the rat did not induce cell death and did not significantly alter the neuronal damage produced by infusion of excitatory amino acids. These data demonstrate that CRF is not directly neurotoxic, and suggest that either CRF mediates neuronal damage by indirect actions (e.g. on the vasculature) and/or that CRF is not the endogenous ligand which contributes to neurodegeneration through activation of CRF receptors.
We previously reported that mice over-expressing the human amyloid precursor protein gene with the double Swedish mutation of familial Alzheimer's disease (mtAPP), which exhibit progressive deposition of amyloid β-peptide in hippocampal and cortical brain regions, have an impaired ability to maintain a sustained glucocorticoid response to stress. Corticotropin releasing hormone (CRH), which initiates neuroendocrine responses to stress by activating the hypothalamic—pituitary—adrenal (HPA) axis, is expressed in brain regions prone to degeneration in Alzheimer's disease. We therefore tested the hypothesis that CRH can modify neuronal vulnerability to amyloid β-peptide toxicity. In primary neuronal culture, CRH was protective against cell death caused by an amyloid-β peptide, an effect that was blocked by a CRH receptor antagonist and by an inhibitor of cyclic AMP-dependent protein kinase. The increased resistance of CRH-treated neurons to amyloid toxicity was associated with stabilization of cellular calcium homeostasis. Moreover, CRH protected neurons against death caused by lipid peroxidation and the excitotoxic neurotransmitter glutamate. The level of mRNA encoding CRH was unchanged in mtAPP mouse brain, whereas the levels of mRNAs encoding glucocorticoid and mineralocorticoid receptors were subtly altered. Our results suggest that disturbances in HPA axis function can occur independently of alterations in CRH mRNA levels in Alzheimer's disease brain and further suggest an additional role for CRH in protecting neurons against cell death.
The aim of this study was to investigate the role of the neuropeptide corticotrophin-releasing hormone (CRH) in neurodegeneration induced by traumatic brain injury, using a well characterized model of lateral fluid percussion injury in male, Sprague-Dawley rats. In the first series of experiments, CRH gene expression was assessed by in situ hybridization after traumatic brain injury. A bilateral increase in CRH mRNA in the paraventricular nucleus was observed in rats subjected to traumatic brain injury compared with sham-operated controls. A maximal (40%) increase in hybridization signal was detected 2 h after trauma compared with control rat brains. In addition, marked induction of CRH transcripts was found in the ipsilateral amygdala after trauma, but no increase was detected in the ipsilateral cortex around the area of damage. In a separate experiment, the effects of the CRH antagonist, D-Phe CRH(12-41) (25 microg total dose), or appropriate vehicle injected intracerebroventricularly, was tested on infarct volume caused by brain injury. Repeated intracerebroventricular injection of D-Phe CRH(12-41) significantly reduced, by 45%, the volume of cortical damage in injured rats compared with vehicle-treated, trauma animals. The rapid upregulation of CRH gene expression in the paraventricular nucleus and amygdala following lateral fluid percussion injury and the marked neuroprotection achieved by inhibiting CRH action suggest that CRH is involved directly in the pathogenesis of traumatic brain injury. This observation may have important implications for the development of novel therapeutic strategies for treating the neurological consequences of brain trauma and related conditions.
This study takes advantage of continuing advances in the precision of magnetic resonance imaging (MRI) to quantify hippocampal volumes in a series of human subjects with a history of depression compared with controls. We sought to test the hypothesis that both age and duration of past depression would be inversely and independently correlated with hippocampal volume. A sample of 24 women ranging in age from 23 to 86 years with a history of recurrent major depression, but no medical comorbidity, and 24 case-matched controls underwent MRI scanning. Subjects with a history of depression (post-depressed) had smaller hippocampal volumes bilaterally than controls. Post-depressives also had smaller amygdala core nuclei volumes, and these volumes correlated with hippocampal volumes. In addition, post-depressives scored lower in verbal memory, a neuropsychological measure of hippocampal function, suggesting that the volume loss was related to an aspect of cognitive functioning. In contrast, there was no difference in overall brain size or general intellectual performance. Contrary to our initial hypothesis, there was no significant correlation between hippocampal volume and age in either post-depressive or control subjects, whereas there was a significant correlation with total lifetime duration of depression. This suggests that repeated stress during recurrent depressive episodes may result in cumulative hippocampal injury as reflected in volume loss.
The influence of dopaminergic denervation on neuropeptide Y and corticotropin-releasing factor-containing neurons in the amygdala was investigated in rats by examining the effects of a selective, unilateral 6-hydroxydopamine lesion of mesencephalic dopaminergic neurons in both the substantia nigra and the ventral tegmental area on these peptides and their messenger RNA expression, observed eight to 10 days after the lesion. The studies were conducted by immunocytochemical and in situ hybridization methods. Neuropeptide Y or corticotropin-releasing factor-immunoreactive neurons were counted in sections of the amygdala under a microscope, and the messenger RNA expression was measured as optical density units in autoradiograms. A significant increase in both neuropeptide Y and corticotropin-releasing factor messenger RNA expression was found in the amygdala on the lesioned side in comparison with the contralateral one, as well as with the ipsilateral side of vehicle-injected controls. Immunohistochemical studies showed that the number of neuropeptide Y-immunoreactive neurons increased in the whole amygdala on the lesioned side. At the same time, the number of corticotropin-releasing factor-immunoreactive neurons grouped in the central amygdaloid nucleus declined, and so did the staining intensity. The obtained results indicate that dopaminergic denervation stimulates the synthesis of neuropeptide Y and corticotropin-releasing factor in rat amygdala, but the peptide levels are differently regulated, which points to a diverse release of these peptides.
Ischemia is accompanied by abundant corticosterone secretion, which could potentially exacerbate brain damage via activation of glucocorticoid receptors. We addressed whether manipulating steroid levels during ischemia affects hippocampal synaptic function along with neuronal structure. Moreover, we established whether pretreatment with the glucocorticoid receptor antagonist RU38486 is as effective in preventing deleterious effects after ischemia as is the steroid synthesis inhibitor metyrapone.
Rats underwent 20 minutes of unilateral hypoxia/ischemia (HI). Convulsions were monitored after HI, and 24 hours later, field potentials were recorded in vitro in the hippocampal CA1 area in response to stimulation of the Schaffer collateral/commissural fibers. Morphological alterations were determined in brain slices from the same animals. Data were correlated with steroid treatment before HI.
Metyrapone suppressed plasma corticosteroid levels during HI, whereas corticosterone treatment significantly elevated plasma steroid levels. These treatments affected the incidence of visible seizures after HI: corticosterone treatment resulted in the highest incidence, whereas metyrapone attenuated the occurrence of seizures. Moreover, the HI-induced impairment in synaptic transmission in the CA1 area in vitro was exacerbated by concomitant corticosteroid treatment and alleviated by pretreatment with metyrapone. In parallel, degenerative changes in the hippocampus after HI were most pronounced after corticosterone treatment, whereas metyrapone reduced these alterations. RU38486 was effective only in reducing the incidence of seizures shortly after ischemia.
We tentatively conclude that synaptic function along with cellular integrity is preserved after HI by preventing the ischemia-evoked rise in corticosteroid levels rather than blocking the glucocorticoid receptor.
We analysed the influence of oxygen-glucose deprivation (OGD) on the expression of corticotropin releasing factor (CRF) receptors (CRF-R1 and CRF-R2) in fetal hippocampal neurons in vitro. A 2 h exposure of neurons to OGD resulted in death of 18+/-2.8% cells at 24 h following exposure, which was considered sublethal hypoxia. Expression of both receptors was quantitated by competitive reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Levels of mRNA for CRF-R1 were increased 3.2-fold compared to control neurons, while CRF-R2 mRNA levels remained unchanged. The increase of CRF-R1 mRNA levels was observed at 6 h and peaked at 24 h. CRF-R1 protein levels were also increased by 2.4-fold and 1.7-fold at 24 h and 48 h, respectively. These data suggest that the effects of CRF on neuronal survival are mediated in part through the induction and expression of CRF-R1 following a hypoxic/ischemic insult.
Throughout life organisms are challenged with various physiological and psychological stressors, and the ability to handle these stressors can have profound effects on the overall health of the organism. In mammals, the effects of stressors on the aging process and age-related diseases are complex, involving the nervous, endocrine and immune systems. Certain types of mild stress, such as caloric restriction, may extend lifespan and reduce the risk of diseases, whereas some types of psychosocial stress are clearly detrimental. We now have a basic understanding of the brain regions involved in stress responses, their neuroanatomical connections with neuroendocrine pathways, and the neuropeptides and hor mones involved in controlling responses of different organ systems to stress. Not surprisingly, brain regions involved in learning and memory and emotion play prominent roles in stress responses, and monoaminergic and glutamatergic synapses play particularly important roles in transducing stressful sensory inputs into neuroendocrine responses. Among the neuropeptides involved in stress responses. corticotropin-releasing hormone appears to be a pivotal regulator of fear and anxiety responses. This neuropeptide is responsible for activation of the hypothalamic-pituitary-adrenal (HPA) axis, which is critical for mobilizing energy reserves and immune responses, and improper regulation of the HPA axis mediates many of the adverse effects of chronic physical and psychosocial stress.
Sequencing of the human genome is nearing completion and biologists, molecular biologists, and bioinformatics specialists have teamed up to develop global genomic technologies to help decipher the complex nature of pathophysiologic gene function. This review will focus on differential gene expression in ischemic stroke. It will discuss inheritance in the broader stroke population, how experimental models of spontaneous stroke might be applied to humans to identify chromosomal loci of increased risk and ischemic sensitivity, and also how the gene expression induced by stroke is related to the poststroke processes of brain injury, repair, and recovery. In addition, we discuss and summarise the literature of experimental stroke genomics and compare several approaches of differential gene expression analyzes. These include a comparison of representational difference analysis we have provided using an experimental stroke model that is representative of stroke evolution observed most often in man, and a summary of available data on stroke differential gene expression. Issues regarding validation of potential genes as stroke targets, the verification of message translation to protein products, the relevance of the expression of neuroprotective and neurodestructive genes and their specific timings, and the emerging problems of handling novel genes that may be discovered during differential gene expression analyses will also be addressed.
To evaluate the effect of Radix Acanthopanacis Senticosi in treating acute cerebral infarction and its mechanism.
Clinical study using single-blind, control trial, in which 60-80 ml of Acanthopanax Injection (AI) added to 500 ml of 0.9% normal saline was given by intravenous drip once a day for 14 days in the AI group(34 patients), while 500 ml of dextra-40 was given by the same way in the control group(26 patients). In experimental study, rabbit model of acute incomplete cerebral ischemia was made by ligation of bilateral common carotid artery. 10 ml of AI were given once daily intravenously for 7 days to the AI group and 10 ml of normal saline was given to the control group in the same way. Clinical effects (in clinical study) and effect of AI on cerebral oxygen free radicals (OFR), superoxide dismutase (SOD), and serum concentration of ACTH and cortisone were observed (in experimental study).
Clinical study showed that the total effective rate of AI group (86%) was higher than that of the control group (50%) while nervous functional deficit score was lower in AI group (9.96 +/- 4.66) than that in the control group (13.56 +/- 1.84) significantly. Experimental study showed that the cerebral level of OFR decreased while SOD increased and serum concentration of ACTH and cortisone decreased after treatment in AI group.
AI was effective in treating acute cerebral infarction, the mechanism is probably by ameliorating peroxidation in brain and improving hypothalamic-pituitary-adrenocortical axis function.
Urocortin and urocortin II are members of the corticotropin-releasing hormone (CRH) family of neuropeptides that function to regulate stress responses. Two high-affinity G-protein-coupled receptors have been identified that bind CRH and/or urocortin I and II, designated CRHR1 and CRHR2, both of which are present in hippocampal regions of mammalian brain. The hippocampus plays an important role in regulating stress responses and is a brain region in which neurons are vulnerable during disease and stress conditions, including cerebral ischemia, Alzheimer's disease, and anxiety disorders. Here we report that urocortin exerts a potent protective action in cultured rat hippocampal neurons with concentrations in the range of 0.5-5.0 pm, increasing the resistance of the cells to oxidative (amyloid beta-peptide, 4-hydroxynonenal, ferrous sulfate) and excitotoxic (glutamate) insults. We observed that urocortin is 10-fold more potent than CRH in protecting hippocampal neurons from insult, whereas urocortin II is ineffective. RT-PCR and sequencing analyses revealed the presence of both CRHR1 and CRHR2 in the hippocampal cultures, with CRHR1 being expressed at much higher levels than CRHR2. Using subtype-selective CRH receptor antagonists, we provide evidence that the neuroprotective effect of exogenously added urocortin is mediated by CRHR1. Furthermore, we provide evidence that the signaling pathway that mediates the neuroprotective effect of urocortin involves cAMP-dependent protein kinase, protein kinase C, and mitogen-activated protein kinase. This is the first demonstration of a biological activity of urocortin in hippocampal neurons, suggesting a role for the peptide in adaptive responses of hippocampal neurons to potentially lethal oxidative and excitotoxic insults.
Corticotropin releasing hormone (CRH) and its family of related peptides are involved in regulating physiologic responses to multiple stressors, including stroke. Although CRH has been implicated in the exacerbation of injury after stroke, the mechanism remains unclear. After ischemia, both excitotoxic damage and inflammation contribute to the pathology of stroke. CRH is known to potentiate excitotoxic damage in the brain and has been shown to modulate inflammatory responses in the periphery. Here the present authors examine the relative contribution of the two known CRH receptors, CRH-R1 and CRH-R2, to ischemic injury using CRH receptor knockout mice. These results implicate CRH-R1 as the primary mediator of ischemic injury in this mouse model of stroke. In addition, the authors examine a potential role for CRH in inflammatory injury after stroke by identifying functional CRH receptors on astrocytes and microglia, which are cells that are known to be involved in brain inflammation. By single cell PCR, the authors show that microglia and astrocytes express mRNA for both CRH-R1 and CRH-R2. However, CRH-R1 is the primary mediator of cAMP accumulation in response to CRH peptides in these cells. The authors suggest that astrocytes and microglia are cellular targets of CRH, which could serve as a link between CRH and inflammatory responses in ischemic injury via CRH-R1.
While corticotropin-releasing hormone (CRH) has been implicated in a variety of brain disorders such as ischemic injury, the molecular mechanism by which CRH elicits its activities is largely unclear. In the present study, we have determined the effect of CRH on oxygen-glucose deprivation (OGD) induced apoptosis in fetal hippocampal neurons. CRH alone at concentrations of 10-200 nM had no effect on neuronal apoptosis. However, when neurons were co-cultured with microglia, CRH alone at concentrations greater than 100 nM induced neuronal apoptosis and CRH potentiated significant neuronal apoptosis following exposure to OGD. The effect of CRH on neuronal apoptosis was inhibited in the presence of the CRH antagonist astressin. Real-time RT-PCR revealed an increase in mRNA levels of Fas ligand (Fas-L), a membrane protein related to the TNF family, in cultured microglia following OGD exposure. In the presence of CRH, OGD-induced Fas-L expression was significantly increased. The effect of CRH on Fas-L expression was inhibited by specific inhibitors of the extracellular signal-regulated protein kinase (PD98059) and p38 mitogen-activated protein kinase (SB203580). These results suggest that CRH potentiates neuronal apoptosis induced by OGD in the presence of microglia and that this effect may be mediated through the induction of proinflammatory mediators in microglia.
The expression of corticotrophin-releasing factor (CRF) receptors in cerebral arteries and arterioles suggests that CRF may modulate cerebral blood flow (CBF). In the present study, the effects of CRF, CRF-like peptides and the CRF broad spectrum antagonist DPhe-CRF on CBF have been investigated under normal physiologic conditions and in the margins of focal ischaemic insult. The experiments were carried out in anaesthetised and ventilated rats. Changes in CBF after subarachnoid microapplication of CRF and related peptides were assessed with a laser-Doppler flowmetry (LDF) probe. In the ischaemic animals, agents were injected approximately 60 minutes after permanent middle cerebral artery occlusion (MCAo). Microapplication of CRF and related peptides in normal rats into the subarachnoid space produced sustained concentration-dependent increases in CBF. This effect was attenuated by co-application with DPhe-CRF, which did not alter CBF itself. A second microapplication of CRF 30 min after the first failed to produce increases in CBF in normal animals. Microapplication of CRF in the subarachnoid space overlying the ischaemic cortex effected minor increases in CBF whereas D-Phe-CRF had no significant effect on CBF. Activation of the CRF peptidergic system increases CBF in the rat. Repeated activation of CRF receptors results in tachyphylaxis of the vasodilator response. CRF vasodilator response is still present after MCAo in the ischaemic penumbra, suggesting that the CRF peptidergic system may modulate CBF in ischaemic stroke.
To explore the influence of one Chinese herbal extract complex (GETO) on the expression of corticotropin-releasing factor(CRF) and protein kinasec(PKC) proteins of the hippocampus in middle cerebral artery orilusion(MCAO) rats.
All rats were subjected to MCAO by nylon thread, except the sham-group rats. Rats were divied into four groups: sham-group, cerebral ischemia model-group, GETO-group(6. 1 g x kg(-1) x d(-1) )and Duxil-group (7.3 mg x kg(-1) x d(-1)). Using immunohistochemistry technique we measured the expression quantity of CRF and PKC protein in hippocampus of MCAO rats at 2 h,6 h and 24 h after reperfusion, contrasted to Duxil.
CRF: There were lots of positive and deeper dyeing neurons in hippocampus of model-group rats, while there were a few of positive and lighter dyeing neurons in sham-group, GETO-group and Duxil-group. The positive expression areas of CRF protein in hippocampus of model-group was significantly bigger than that of sham-group, GETO -group and Duxil-group respectively( P <0. 01). PKC: There were a great number of denser positive granules in hippocampus of model-group rats, while there were a few of scattered positive granules in sham-group, GETO-group and Duxil-group. The positive expression areas of CRF protein in hippocampus of model-group was significantly bigger than that of sham-group, GETO-group and Duxil-group respectively( P < 0. 01). At the same time there was not significant difference about the expression of CRF and PKC protein between GETO group and Duxil-group.
The high expression of CRF and PKC induced by cerebral ischemia may be one important factor that resulted in the delayed neuronal death in hippocampus. GETO can down-regulate the expression of CRF and PKC induced by cerebral ischemia, which may be one of the mechanisms that the Chinese herbal extract complex, protect cerebral ischemic injury.
Corticotropin-releasing factor (CRF) induces the dilatation of cerebral blood vessels and increases cerebral blood flow (CBF). CRF receptor antagonists reduce ischaemic damage in the rat. In the present study, the expression of CRF around cerebral vessels has been investigated in the rat. No CRF immunoreactivity was identified around pial or intracerebral vessels in the absence of cerebral ischaemia. Four hours after middle cerebral artery occlusion (MCAo), intensely CRF-positive blood vessels were evident on the ischaemic cortical surface and in the peri-infarct and infarct zone. Increased CRF immunoreactivity was also detected in swollen axons in subcortical white matter, caudate nucleus and lateral olfactory tract of the ipsilateral hemisphere, consistent with the failure of axonal transport. These data provide morphologic support for a role of CRF in the pathophysiology of cerebral ischaemia.
Persistent hypothalamo-pituitary adrenal axis dysregulation occurs in up to 40% of patients who have suffered a stroke. The degree of hypercortisolemia is partly determined by the size and site of the vascular lesion. Adrenocortical hyperactivity begins almost immediately after a cerebrovasacular infarct but is persistent in an important subgroup of patients. In the early poststroke period (1 day to 1 month) high corticosteroid levels correlate with the presence of an acute confusional state. In the medium term (1 month to 1 year) hypercortisolemia is associated with the development of a major depressive episode and also relates to functional outcome and survival. Neuroanatomical deficits (particularly in the frontal or medial temporal lobes), age of onset, cognitive impairment, and reduced functional status may act as maintaining factors in both the poststroke depression and the adrenocortical hyperactivity. Patients with persisting hypercortisolemia, with or without depression or cognitive impairment, have a worse prognosis with an increased mortality rate. The mechanism for this effect may involve induced hyperglycemia or direct glucocorticoid neurotoxicity, which impairs the brain's capacity for recovery. It is suggested that the cautions use of antiglucocorticoid strategies may be of value in the medical management of the neuropsychiatric complications that follow cerebrovascular accidents.
The method of choice for studying the distribution and density of a particular mRNA in small anatomical and heterogeneous tissues is in situ hybridization histochemistry (ISHH). The brain is anatomically complex and the adjacent brain regions can express many different genes; therefore, the ISHH approach is a powerful tool for the localization of interleukin mRNA in the brain because single-cell resolution can easily be obtained. Single-cell resolution is not possible with solution or Northern blot hybridization. In ISHH, labeled cDNA or cRNA probes form hybrids with a specific cellular mRNA, and the hybrids are visualized by autoradiography. High sensitivity and unique resolution are the advantages of the ISHH technique. Three main classes of probe in present use are (1) cDNA, (2) RNA probes (riboprobes or cRNA), and (3) oligonucleotide probes. Probes may be synthesized by vector systems, chemically, or with a combination of synthetic and enzymatic approaches.