4-vessel Occlusion Research Articles

Neuroprotective Effects of Nicorandil in Chronic Cerebral Hypoperfusion-Induced Vascular Dementia

Ischemia-induced chronic cerebral hypoperfusion (CCH) is associated with reduced cerebral blood flow and vascular dementia (VaD). Brain mitochondrial potassium (adenosine triphosphate-sensitive potassium [KATP]) channels have a beneficial role in various brain conditions. The utility of KATP channels in CCH-induced VaD is still unknown. The aim of this study is to investigate the role of nicorandil, a selective KATP channel opener, in CCH-induced VaD. The method of 2-vessel occlusion (2VO) was used to induce CCH in mice. Cognitive impairment was assessed using Morris water maze. Serum nitrosative stress (nitrite/nitrate), brain cholinergic dysfunction (acetylcholinesterase [AChE] activity), brain oxidative stress (thiobarbituric acid reactive substances, glutathione [GSH], catalase [CAT], and superoxide dismutase [SOD]), inflammation (myeloperoxidase [MPO]), and infarct size (2,3,5-triphenyltetrazolium chloride staining) were assessed. 2-vessels-occluded animals have shown significant cognitive impairment, serum nitrosative stress (reduced nitrite/nitrate), cholinergic dysfunction (increased brain AChE activity), and increased brain oxidative stress (reduction in GSH content and SOD and CAT activities with a significant increase in lipid peroxidation), along with a significant increase in MPO activity and infarct size. However, nicorandil treatment has significantly attenuated various CCH-induced behavioral and biochemical impairments. It may be said that 2VO provoked CCH leading to VaD, which was attenuated by the treatment of nicorandil. So, modulation of KATP channels may provide benefits in CCH-induced VaD.

Intranasal Delivery of a Caspase-1 Inhibitor in the Treatment of Global Cerebral Ischemia.

Caspase-1 is an enzyme implicated in neuroinflammation, a critical component of many diseases that affect neuronal degeneration. However, it is unknown whether a caspase-1 inhibitor can modify apoptotic neuronal damage incurred during transient global cerebral ischemia (GCI) and whether intranasal administration of a caspase-1 inhibitor is an effective treatment following GCI. The present study was conducted to examine the potential efficiency of post-ischemic intranasal administration of the caspase-1 inhibitor Boc-D-CMK in a 4-vessel occlusion model of GCI in the rat. Herein, we show that intranasal Boc-D-CMK readily penetrated the central nervous system, subsequently inhibiting caspase-1 activity, decreasing mitochondrial dysfunction, and attenuating caspase-3-dependent apoptotic pathway in ischemia-vulnerable hippocampal CA1 region. Further investigation regarding the mechanisms underlying Boc-D-CMK's neuroprotective effects revealed marked inhibition of reactive gliosis, as well as reduction of the neuroinflammatory response via inhibition of the downstream pro-inflammatory cytokine production. Intranasal Boc-D-CMK post-treatment also significantly enhanced the numbers of NeuN-positive cells while simultaneously decreasing the numbers of TUNEL-positive and PARP1-positive cells in hippocampal CA1. Correspondingly, behavioral tests showed that deteriorations in spatial learning and memory performance, and long-term recognition memory following GCI were significantly improved in the Boc-D-CMK post-treated animals. In summary, the current study demonstrates that the caspase-1 inhibitor Boc-D-CMK coordinates anti-inflammatory and anti-apoptotic actions to attenuate neuronal death in the hippocampal CA1 region following GCI. Furthermore, our data suggest that pharmacological inhibition of caspase-1 is a promising neuroprotective strategy to target ischemic neuronal injury and functional deficits following transient GCI.

Neuroprotection of Ro25-6981 Against Ischemia/Reperfusion-Induced Brain Injury via Inhibition of Autophagy.

In this study, we investigated the neuroprotective effect of Ro25-6981 against cerebral ischemia/reperfusion injury. Ro25-6981 alone or in combination with rapamycin was intracerebroventricularly administered to rats which suffered transient forebrain ischemia inducing by 4-vessel occlusion and reperfusion. Nissl staining was used to determine the survival of CA1 pyramidal cells of the hippocampus, while immunohistochemistry was performed to measure neuron-specific enolase (NSE) expression. The expression of autophagy-related proteins, such as microtubule-associated protein l light chain 3 (LC3), Beclin 1, and sequestosome 1 (p62), was assessed by immunoblotting. Nissl staining showed that neuronal damage was reduced in the hippocampal CA1 pyramidal layer in rats that received Ro25-6981. The protective effect of Ro25-6981 was dose-dependent, with a significant effect in the middle-dose range. The expression of NSE increased after Ro25-6981 treatment. Ro25-6981 significantly decreased LC3II (which is membrane bound) and Beclin 1, and increased p62. In addition, Ro25-6981 decreased rapamycin-induced neuronal damage and excessive activation of autophagy after I/R. Taken together, the results suggest that Ro25-6981 could suppress ischemic brain injury by regulating autophagy-related proteins during ischemia/reperfusion.

Gradual cerebral hypoperfusion in spontaneously hypertensive rats induces slowly evolving white matter abnormalities and impairs working memory

Rats subjected to bilateral common carotid arteries (CCAs) occlusion or 2-vessel occlusion (2VO) have been used as animal models of subcortical ischemic vascular dementia (SIVD). However, these models possess an inherent limitation in that cerebral blood flow (CBF) drops sharply and substantially after ligation of CCAs without vascular risk factors and causative small vessel changes. We previously reported a novel rat model of 2-vessel gradual occlusion (2VGO) in which ameroid constrictors (ACs) were placed bilaterally in the CCAs of Wistar-Kyoto rats. To simulate SIVD pathology more closely, we applied ACs in spontaneously hypertensive rats (SHRs), which naturally develop small vessel pathology, and compared their phenotypes with SHR-2VO and sham-operated rats. The mortality rate of the SHR-2VGO was 0% while that of the SHR-2VO was 56.5%. The CBF of the SHR-2VO dropped to 50% of the baseline level at 3 h, whereas the SHR-2VGO showed a gradual CBF reduction reaching only 68% of the baseline level at seven days. The SHR-2VGO showed slowly evolving white matter abnormalities and subsequent spatial working memory impairments of a similar magnitude to the remaining SHR-2VO at 28 days. We suggest the SHR-2VGO robustly replicates selective aspects of the pathophysiology of SIVD with low mortality rate.

Activation of G protein-coupled estrogen receptor 1 (GPER-1) ameliorates blood-brain barrier permeability after global cerebral ischemia in ovariectomized rats

G protein-coupled estrogen receptor 1 (GPER-1) plays important roles in estrogen-mediated neuroprotection. However, protective effects of GPER-1 on blood-brain barrier (BBB) after ischemic stroke have not been determined. The aim of present study was to determine whether GPER-1 activation ameliorates BBB permeability in ovariectomized rats with induced global cerebral ischemia (GCI). GCI was induced by 4-vessel occlusion for 20 min followed by 24 h reperfusion period. The GPER-1 agonist (G1) was bilaterally administered immediately upon reperfusion by intracerebroventricular (icv) injection. We found that the GPER-1 agonist could significantly decrease immunoglobulin G (IgG) extravasation and increase the levels of tight junctions (occludin and claudin-5) in the CA1 at 24 h of reperfusion after GCI. Further, protein levels of vascular endothelial growth factor A (VEGF-A) was significantly decreased in the ischemic CA1 by G1. Our results suggest that GPER-1 activation reduce tight junctions disruption via inhibition of VEGF-A expression after ischemic injury.

Reduced expression of IA channels is associated with post-ischemic seizures

PurposePost-stroke seizures are considered as a major cause of epilepsy in adults. The pathophysiologic mechanisms resulting in post-stroke seizures are not fully understood. The present study attempted to reveal a new mechanism underlying neuronal hyperexcitability responsible to the seizure development after ischemic stroke. MethodsTransient global ischemia was produced in adult Wistar rats using the 4-vessel occlusion (4-VO) method. The spontaneous behavioral seizures were defined by the Racine scale III–V. The neuronal death in the brain was determined by hematoxylin-eosin staining. The expression levels of A-type potassium channels were analyzed by immunohistochemical staining and western blotting. ResultsWe found that the incidence of spontaneous behavioral seizures increased according to the severity of ischemia with 0% after 15-min ischemia and ∼50% after 25-min ischemia. All behavioral seizures occurred with 48h after ischemia. Morphological analysis indicated that brain damage was not correlated with behavioral seizures. Immunohistochemical staining showed that the expression levels of the A-type potassium channel subunit Kv4.2 was significantly reduced in ischemic brains with behavioral seizures, but not in ischemic brains without seizures. In addition, rats failing to develop spontaneous behavioral seizures within 2days after ischemia were more sensitive to bicuculline-induced seizures at 2 months after ischemia than control rats. Meanwhile, Kv4.2 expression was decreased in brain at 2 months after ischemia. ConclusionOur results demonstrated the reduction of Kv4.2 expression might contribute to the development of post-ischemic seizures and long-term increased seizure susceptibility after ischemia. The mechanisms underlying post-stroke seizures and epilepsy is unknown so far. The down-regulation of IA channels may explained the abnormal neuronal hyperexcitability responsible for the seizure development after ischemic stroke.

One-stage apertura thoracis superior approach for four-vessel occlusion in rats

ObjectiveThere are a great number of modified models based on the four-vessel occlusion (4VO) model of Pulsinelli and Brierley which has been used worldwide for brain ischemia research. However, up to now the problems of collateral circulations of 4VO and the difficulty in arranging a surgery to occlude the basilar artery in other models are not satisfactorily solved yet. In this study, an improved 4-vessel occlusion (I4VO) rat model which is easy to handle and able to decrease the effect of collateral circulation is reported. MethodsThe common carotid arteries and the beginning of the subclavical arteries of rats were occluded for different time by one-stage apertura thoracis superior approach. Neurological deficit scores defined by the modified Garcia scoring system and histopathological method were used to evaluate the effects of this model up to 7 days after reperfusion. ResultsThe neurological scores in the 15-min and 25-min groups decreased significantly at 24, 48 and 72 hours after reperfusion (PO.05), and the histopathologic study showed that there were stable, symmetrical changes of lesions in bilateral hippocampus in all the ischemia samples from two ischemia groups compared with sham operated group (P<0.05). ConclusionThis modified model is safe, easy, reliable, stable, mini-invasive as well as time-saving in making bilateral hemispheric ischemia, which can effectively decrease collateral circulations and meanwhile lead to stable lesions in hippocampus and cortex.

Ischemic preconditioning protects the brain against injury via inhibiting CaMKII–nNOS signaling pathway

Although studies have shown that cerebral ischemic preconditioning (IPC) can ameliorate ischemia/reperfusion (I/R) induced brain damage, but its precise mechanisms remain unknown. Therefore, the aim of this study was to investigate the neuroprotective mechanisms of IPC against ischemic brain damage induced by cerebral I/R and to explore whether the Calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway contributed to the protection provided by IPC. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with 3min of IPC alone or KN62 (selective antagonist of CaMKII) treatment before IPC, after reperfusion for 3 days, 6min ischemia was induced. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting was performed to measure the phosphorylation of CaMKII, nNOS, c-Jun and the expression of FasL. Immunoprecipitation was used to examine the binding between PSD95 and nNOS. The results showed that IPC could significantly protect neurons against cerebral I/R injury, furthermore, the combination of PSD95 and nNOS was increased, coinstantaneously the phosphorylation of CaMKII and nNOS (ser847) were up-regulated, however the activation of c-Jun and FasL were reduced. Conversely, KN62 treatment before IPC reversed all these effects of IPC. Taken together, the results suggest that IPC could diminish ischemic brain injury through CaMKII-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway.

Electroacupuncture Ameliorates Learning and Memory and Improves Synaptic Plasticity via Activation of the PKA/CREB Signaling Pathway in Cerebral Hypoperfusion.

Electroacupuncture (EA) has shown protective effects on cognitive decline. However, the underlying molecular mechanisms are ill-understood. The present study was undertaken to determine whether the cognitive function was ameliorated in cerebral hypoperfusion rats following EA and to investigate the role of PKA/CREB pathway. We used a rat 2-vessel occlusion (2VO) model and delivered EA at Baihui (GV20) and Dazhui (GV14) acupoints. Morris water maze (MWM) task, electrophysiological recording, Golgi silver stain, Nissl stain, Western blot, and real-time PCR were employed. EA significantly (1) ameliorated the spatial learning and memory deficits, (2) alleviated long-term potentiation (LTP) impairment and the reduction of dendritic spine density, (3) suppressed the decline of phospho-CREB (pCREB) protein, brain-derived neurotrophic factor (BDNF) protein, and microRNA132 (miR132), and (4) reduced the increase of p250GAP protein of 2VO rats. These changes were partially blocked by a selective protein kinase A (PKA) inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H89), suggesting that the PKA/CREB pathway is potentially involved in the effects of EA. Moreover, any significant damage to the pyramidal cell layer of CA1 subregion was absent. These results demonstrated that EA could ameliorate learning and memory deficits and alleviate hippocampal synaptic plasticity impairment of cerebral hypoperfusion rats, potentially mediated by PKA/CREB signaling pathway.

Abstract 17177: Crosstalk and Synergism Between Local Brain-derived and Exogenous 17β-Estradiol in Regulating Rapid, Pro-survival Signaling in the Hippocampus Following Global Cerebral Ischemia

Cardiac arrest-induced global cerebral ischemia (GCI) can lead to death of hippocampal CA1 neurons and cognitive impairment. Previous work has shown that the steroid hormone, 17β-estradiol (E2) is strongly neuroprotective against GCI, an effect that involves, in part, rapid signaling via the membrane estrogen receptor, GPR30. To gain greater insight into E2 actions, the current study examined: 1) whether GPR30 expression in the hippocampus changes after CGI, and 2) the role and potential crosstalk/synergism between local (brain-derived) and exogenous E2 in regulation of GPR30 and rapid prosurvival signaling after GCI. GCI was induced in adult ovariectomized female rats by a 4-vessel occlusion model (12-min occlusion). Vehicle or low diestrous levels of E2 were administered chronically by minipump beginning at the time of ovariectomy. The results showed that GPR30 expression in the hippocampal CA1 was unchanged at 3h, but significantly decreased at 1d and 3d after GCI. In contrast, aromatase expression showed an initial decrease at 3h, a rebound at 1day, and a robust elevation at 3 days after CGI. Administration of exogenous E2 increased both GPR30 and aromatase expression at 3h, 1d and 3d after GCI. Interestingly, central administration of the aromatase inhibitor, letrozole or aromatase antisense (AS) oligonucleotides abolished exogenous E2 enhancement of GPR30 and aromatase at 3d after GCI. Furthermore, letrozole and aromatase-AS treatment attenuated exogenous E2-induced phosphorylation/activation of prosurvival ERK1/2 and CREB following GCI, as well as exogenous E2-induced enhanced expression of BDNF, PSD95 and synaptophsin. As a whole, the results suggest that local-derived E2 may help mediate beneficial protective effects of exogenous E2 after GCI to enhance GPR30 expression, increase activation of ERK1/2-CREB-BDNF pro-survival signaling, and improve synaptic function.

Targeting type-2 metabotropic glutamate receptors to protect vulnerable hippocampal neurons against ischemic damage.

BackgroundTo examine whether metabotropic glutamate (mGlu) receptors have any role in mechanisms that shape neuronal vulnerability to ischemic damage, we used the 4-vessel occlusion (4-VO) model of transient global ischemia in rats. 4-VO in rats causes a selective death of pyramidal neurons in the hippocampal CA1 region, leaving neurons of the CA3 region relatively spared. We wondered whether changes in the expression of individual mGlu receptor subtypes selectively occur in the vulnerable CA1 region during the development of ischemic damage, and whether post-ischemic treatment with drugs targeting the selected receptor(s) affords neuroprotection.ResultsWe found that 4-VO caused significantly reduction in the transcript of mGlu2 receptors in the CA1 region at times that preceded the anatomical evidence of neuronal death. Down-regulation of mGlu2 receptors was associated with reduced H3 histone acetylation at the Grm2 promoter. The transcripts of other mGlu receptor subtypes were unchanged in the CA1 region of 4-VO rats. Ischemia did not cause changes in mGlu2 receptor mRNA levels in the resistant CA3 region, which, interestingly, were lower than in the CA1 region. Targeting the mGlu2 receptors with selective pharmacologic ligands had profound effects on ishemic neuronal damage. Post-ischemic oral treatment with the selective mGlu2 receptor NAM (negative allosteric modulator), ADX92639 (30 mg/kg), was highly protective against ischemic neuronal death. In contrast, s.c. administration of the mGlu2 receptor enhancer, LY487379 (30 mg/kg), amplified neuronal damage in the CA1 region and extended the damage to the CA3 region.ConclusionThese findings suggest that the mGlu2 receptor is an important player in mechanisms regulating neuronal vulnerability to ischemic damage, and that mGlu2 receptor NAMs are potential candidates in the experimental treatments of disorders characterized by brain hypoperfusion, such as hypovolemic shock and cardiac arrest.

Mild Hypothermia Combined with Hydrogen Sulfide Treatment During Resuscitation Reduces Hippocampal Neuron Apoptosis Via NR2A, NR2B, and PI3K-Akt Signaling in a Rat Model of Cerebral Ischemia-Reperfusion Injury.

We investigated whether mild hypothermia combined with sodium hydrosulfide treatment during resuscitation improves neuron survival following cerebral ischemia-reperfusion injury beyond that observed for the individual treatments. Male Sprague-Dawley rats were divided into seven groups (n = 20 for each group). All rats underwent Pulsinelli 4-vessel occlusion. Ischemia was induced for 15min using ligatures around the common carotid arteries, except for the sham group. Immediately after initiating reperfusion, the mild hypothermia (MH), sodium hydrosulfide (NaHS), hydroxylamine (HA), MH + NaHS, MH + HA, and ischemia-reperfusion (I/R) control groups received an intraperitoneal injection of saline, sodium hydrosulfide, hydroxylamine, sodium hydrosulfide, hydroxylamine, and saline, respectively, and mild hypothermia (32 to 33°C) was induced in the MH, MH + NaHS, and MH + HA groups for 6h. The levels of NR2A, NR2B, p-Akt, and p-Gsk-3β in the hippocampus of the MH, NaHS, and MH + NaHS groups were higher than those in the I/R control group, with the highest levels observed in the MH + NaHS group (P < 0.05). Treatment with hydroxylamine reduced the levels of these proteins in the HA and MH + HA groups, compared with the I/R control and MH groups, respectively. The apoptotic index of the CA1 region of the hippocampus was 45.2, 66.5, 63.5, and 84.8% in the MH + NaHS, MH, NaHS, and I/R control groups, respectively (P < 0.05), indicating that the combination treatment shifted the NR2A/NR2B balance in favor of synaptic neuron stimulation and phosphatidylinositol 3'-kinase (PI3K)/Akt signaling. The combination of mild hypothermia and sodium hydrosulfide treatment for resuscitation following ischemia-reperfusion injury was more beneficial for reducing hippocampal apoptosis and pathology than that of mild hypothermia or hydrogen sulfide treatment alone.

A modified bilateral carotid artery stenosis procedure to develop a chronic cerebral hypoperfusion rat model with an increased survival rate

BackgroundBilateral carotid artery occlusion (2-vessel occlusion, 2VO) in rats is a classic and frequently used approach to develop an animal model of chronic cerebral hypoperfusion. However, this method results in substantial mortality in rats. New methodWe investigated whether a modified 2VO procedure, which induces bilateral carotid artery stenosis via ligation of each bilateral common carotid artery (CCA) with a 32 gauge (G) needle followed by needle removal, could produce a chronic cerebral hypoperfusion rat model with an increased survival rate. Sprague-Dawley (SD) rats were treated with the standard or modified 2VO procedure, and changes in cerebral blood flow (CBF) and survival rates were determined. On day 28, cognitive function was assessed with the Morris Water Maze (MWM) test, and neuronal survival and degeneration within the hippocampal CA1 area were measured. Damage to the white matter (WM) within the corpus striatum was assessed via Luxol fast blue (LFB) staining and analyses analyzing the levels of the myelin basic protein (MBP) protein levels. ResultsThe modified 2VO procedure induced similar cognitive impairments, hippocampal lesions and WM damage compared with the standard 2VO procedure in rats; however, it had an increased survival rate.Comparison with existing methods This novel method can be used to quickly and effectively establish a chronic cerebral hypoperfusion rat model with common materials and an improved survival rate. ConclusionBilateral carotid artery stenosis using a 32G needle is a useful and reliable method to develop a rat model of chronic cerebral hypoperfusion with increased survival.

Investigation of redox status in chronic cerebral hypoperfusion-induced neurodegeneration in rats

Aging related reduction in cerebral blood flow (CBF) has been linked with neurodegenerative disorders including Alzheimer's disease and dementia. Experimentally, a condition of chronic cerebral hypoperfusion due to reduced CBF can be induced by permanent bilateral occlusion of common carotid arteries (2-vessel occlusion, 2VO) in rats. Since oxidative stress, leading to neuronal apoptosis and death, is one of the mechanisms, which is thought to play a significant role in chronic degenerative neurological disorders, the present study was planned to assess the ROS status by measuring the levels of anti-oxidant enzymes that might occur during chronic cerebral hypoperfusion. Antioxidant enzymes namely glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase were measured in the brain tissue at eight weeks of 2VO induction in rats. Results show significantly elevated levels of GPx, SOD, and catalase enzymes as compared with the control group. It is possible that compensatory rise in antioxidant enzymes occurs in response to increased oxidative stress following ischemic insult.

Comparative analysis of autophagy and tauopathy related markers in cerebral ischemia and Alzheimer's disease animal models.

Alzheimer’s disease (AD) and cerebral ischemia (CI) are neuropathologies that are characterized by aggregates of tau protein, a hallmark of cognitive disorder and dementia. Protein accumulation can be induced by autophagic failure. Autophagy is a metabolic pathway involved in the homeostatic recycling of cellular components. However, the role of autophagy in those tauopathies remains unclear. In this study, we performed a comparative analysis to identify autophagy related markers in tauopathy generated by AD and CI during short-term, intermediate, and long-term progression using the 3xTg-AD mouse model (aged 6,12, and 18 months) and the global CI 2-VO (2-Vessel Occlusion) rat model (1,15, and 30 days post-ischemia). Our findings confirmed neuronal loss and hyperphosphorylated tau aggregation in the somatosensory cortex (SS-Cx) of the 3xTg-AD mice in the late stage (aged 18 months), which was supported by a failure in autophagy. These results were in contrast to those obtained in the SS-Cx of the CI rats, in which we detected neuronal loss and tauopathy at 1 and 15 days post-ischemia, and this phenomenon was reversed at 30 days. We proposed that this phenomenon was associated with autophagy induction in the late stage, since the data showed a decrease in p-mTOR activity, an association of Beclin-1 and Vps34, a progressive reduction in PHF-1, an increase in LC3B puncta and autophago-lysosomes formation were observed. Furthermore, the survival pathways remained unaffected. Together, our comparative study suggest that autophagy could ameliorates tauopathy in CI but not in AD, suggesting a differential temporal approach to the induction of neuroprotection and the prevention of neurodegeneration.

Neuroprotection of Sevoflurane Against Ischemia/Reperfusion-Induced Brain Injury Through Inhibiting JNK3/Caspase-3 by Enhancing Akt Signaling Pathway.

In this study, we investigated the neuroprotective effect of sevoflurane against ischemic brain injury and its underlying molecular mechanisms. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with sevoflurane alone or sevoflurane combined with LY294002/wortmannin (selective inhibitor of PI3K) before ischemia. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting and immunoprecipitation were performed to measure the phosphorylation of Akt1, PRAS40, ASK1, and JNK3 and the expression of cleaved-caspase-3. The results demonstrated that a moderate dose of sevoflurane inhalation of 2% for 2 h had significant neuroprotective effects against ischemia/reperfusion induced hippocampal neuron death. Sevoflurane significantly increased Akt and PRAS40 phosphorylation and decreased the phosphorylation of ASK1 at 6 h after reperfusion and the phosphorylation of JNK3 at 3 days after reperfusion following 15 min of transient global brain ischemia. Conversely, LY294002 and wortmannin significantly inhibited the effects of sevoflurane. Taken together, the results suggest that sevoflurane could suppress ischemic brain injury by downregulating the activation of the ASK1/JNK3 cascade via increasing the phosphorylation of Akt1 during ischemia/reperfusion.

Hypertension-mediated enhancement of JNK activation in association with endoplasmic reticulum stress in rat model hippocampus with cerebral ischemia-reperfusion.

Acute brain ischemia can induce the activation of c-Jun N-terminal kinases (JNKs). Hypertension is a critical etiology for brain ischemia. We identified the effects of hypertension on the activation of JNK as well as its impact on SP600125, a JNK inhibitor, during endoplasmic reticulum stress (ERS) in the hippocampus using a rat model. Transient whole-brain ischemia was induced by 4-vessel occlusion (bilateral vertebral and bilateral common carotid arteries) in normal and spontaneous hypertensive rats. SP600125 (0.05 mg/kg, iv) was administered 30 min before ischemia. Morphological changes in hippocampal nerve cells were observed by cresyl violet staining. Phosphorylation of JNK, and expression levels of CHOP and GPR78, markers for ERS, were detected by western blot at 1, 6, 24, and 48 h, and neurological outcomes were measured using an eight-arm radial maze 48 h after ischemia. Hypertension apparently aggravated impairment of memory function, decreased the density of surviving neurons, increased phosphorylation of JNK, and enhanced CHOP expression, but reduced GPR78 levels in hippocampal tissues following brain ischemia. SP600125 alleviated neurological dysfunction, improved neuron survival, decreased phosphorylation of JNK and levels of CHOP, but increased expression of GPR78 in rats with hypertension during cerebral ischemia by inhibition of ERS.

Acetyl-L-carnitine and oxaloacetate in post-treatment against LTP impairment in a rat ischemia model. An in vitro electrophysiological study.

A high proportion of research relating to cerebral ischemia focuses on neuroprotection. The application of compounds normally present in the organism is popular, because they do not greatly influence the synaptic activity by receptor modulation, and can be administered without serious side effects. Oxaloacetate (OxAc) and acetyl-L-carnitine (ALC) are such favorable endogenous molecules. ALC can exert a protective effect by improving the energy state of the neurons under ischemic conditions. A promising neuroprotective strategy is glutamate scavenging, which can be achieved by the intravenous administration of OxAc. This study involved the possible protective effects of ALC and OxAc in different post-treatment protocols against long-term potentiation (LTP) impairment. Ischemia was induced in rats by 2-vessel occlusion, which led to a decreased LTP relative to the control group. High-dose (200 mg/kg) ALC or OxAc post-treatment resulted in a higher potentiation relative to the 2VO group, but it did not reach the control level, whereas low-dose ALC (100 mg/kg) in combination with OxAc completely restored the LTP function. Many previous studies have concluded that ALC can be protective only as pretreatment. The strategy described here reveals that ALC can also be neuroprotective when utilized as post-treatment against ischemia.

Protein-energy malnutrition developing after global brain ischemia induces an atypical acute-phase response and hinders expression of GAP-43.

Protein-energy malnutrition (PEM) is a common post-stroke problem. PEM can independently induce a systemic acute-phase response, and pre-existing malnutrition can exacerbate neuroinflammation induced by brain ischemia. In contrast, the effects of PEM developing in the post-ischemic period have not been studied. Since excessive inflammation can impede brain remodeling, we investigated the effects of post-ischemic malnutrition on neuroinflammation, the acute-phase reaction, and neuroplasticity-related proteins. Male, Sprague-Dawley rats were exposed to global forebrain ischemia using the 2-vessel occlusion model or sham surgery. The sham rats were assigned to control diet (18% protein) on day 3 after surgery, whereas the rats exposed to global ischemia were assigned to either control diet or a low protein (PEM, 2% protein) diet. Post-ischemic PEM decreased growth associated protein-43, synaptophysin and synaptosomal-associated protein-25 immunofluorescence within the hippocampal CA3 mossy fiber terminals on day 21, whereas the glial response in the hippocampal CA1 and CA3 subregions was unaltered by PEM. No systemic acute-phase reaction attributable to global ischemia was detected in control diet-fed rats, as reflected by serum concentrations of alpha-2-macroglobulin, alpha-1-acid glycoprotein, haptoglobin, and albumin. Acute exposure to the PEM regimen after global brain ischemia caused an atypical acute-phase response. PEM decreased the serum concentrations of albumin and haptoglobin on day 5, with the decreases sustained to day 21. Serum alpha-2-macroglobulin concentrations were significantly higher in malnourished rats on day 21. This provides the first direct evidence that PEM developing after brain ischemia exerts wide-ranging effects on mechanisms important to stroke recovery.

Toll-like receptor 4 is associated with seizures following ischemia with hyperglycemia

Seizures are a common sequel of cerebral ischemia, and hyperglycemia markedly increases the onset of seizures following an ischemic insult. However, the underlying mechanism of seizures is unclear. The toll-like receptor 4 (TLR4) pathway is known to be involved in temporal lobe epilepsy. The present study investigated the potential involvement of TLR4 in the pathogenesis of seizures following cerebral ischemia with hyperglycemia. Fifteen minutes of global ischemia was produced in adult Wistar rats using a 4-vessel occlusion method. Hyperglycemia was induced via an intraperitoneal injection of glucose 15min prior to ischemia. We determined that 56.7% of the hyperglycemic rats, but none of the normoglycemic rats, developed tonic-clonic seizures within 12h after ischemia. TLR4 was mildly expressed in a few cells in the control hippocampus, primarily in interneurons, and was localized in the cytoplasm. The TLR4-positive cells were significantly increased 3–12h after ischemia. In the hyperglycemic ischemia group, TLR4-positive cells were further increased in quantity and intensity, with a peak at 3h after ischemia relative to the normoglycemic group. There was no difference in the expression of TLR4 between the hyperglycemic ischemia and LPS groups or between the hyperglycemic non-ischemia and control groups. Western blot analysis consistently exhibited an increase in TLR4 protein levels in the CA3 region 3h after hyperglycemic ischemia. High mobility group box 1 (HMGB1) (an endogenous ligand of TLR4) was localized in the nucleus of neuronal cells throughout the hippocampus in the control animals. We observed a dramatic decrease in HMGB1 immunostaining at 3h after hyperglycemic ischemia that gradually returned to control levels. These results suggest that the TLR4 pathway is associated with seizures following global ischemia with hyperglycemia, which provides a new direction for the study of the pathogenesis of seizures that result from hyperglycemic ischemia.