Salvinorin A preserves cerebral pial artery autoregulation after forebrain ischemia via the PI3K/AKT/cGMP pathway

This study aimed to investigate the protective effect of salvinorin A on the cerebral pial artery after forebrain ischemia and explore related mechanisms. Thirty Sprague-Dawley rats received forebrain ischemia for 10 min. The dilation responses of the cerebral pial artery to hypercapnia and hypotension were assessed in rats before and 1 h after ischemia. The ischemia reperfusion (IR) control group received DMSO (1 µL/kg) immediately after ischemia. Two different doses of salvinorin A (10 and 20 µg/kg) were administered following the onset of reperfusion. The 5th, 6th, and 7th groups received salvinorin A (20 µg/kg) and LY294002 (10 µM), L-NAME (10 μM), or norbinaltorphimine (norBIN, 1 μM) after ischemia. The levels of cGMP in the cerebrospinal fluid (CSF) were also measured. The phosphorylation of AKT (p-AKT) was measured in the cerebral cortex by western blot at 24 h post-ischemia. Cell necrosis and apoptosis were examined by hematoxylin-eosin staining (HE) and TUNEL staining, respectively. The motor function of the rats was evaluated at 1, 2, and 5 days post-ischemia. The dilation responses of the cerebral pial artery were significantly impaired after ischemia and were preserved by salvinorin A treatment. In addition, salvinorin A significantly increased the levels of cGMP and p-AKT, suppressed cell necrosis and apoptosis of the cerebral cortex and improved the motor function of the rats. These effects were abolished by LY294002, L-NAME, and norBIN. Salvinorin A preserved cerebral pial artery autoregulation in response to hypercapnia and hypotension via the PI3K/AKT/cGMP pathway.

Neuroprotective effect of the ethanol extract of Artemisia capillaris on transient forebrain ischemia in mice via nicotinic cholinergic receptor / 中国天然药物

Artemisia capillaris Thunberg is a medicinal plant used as a traditional medicine in many cultures. It is an effective remedy for liver problems including hepatitis. Recent pharmacological reports have indicated that Artemisia species can exert various neurological effects. Previously, we reported a memory-enhancing effect of Artemisia species. However, the mechanisms underlying the neuroprotective effect of A. capillaris (AC) are still unknown. In the present study, we investigated the effect of an ethanol extract of AC on ischemic brain injury in a mouse model of transient forebrain ischemia. The mice were treated with AC for seven days, beginning one day before induction of transient forebrain ischemia. Behavioral deficits were investigated using the Y-maze. Nissl and Fluoro-jade B staining were used to indicate the site of injury. To determine the underlying mechanisms for the drug, we measured acetylcholinesterase activity. AC (200 mg·kg) treatment reduced transient forebrain ischemia-induced neuronal cell death in the hippocampal CA1 region. The AC-treated group also showed significant amelioration in the spontaneous alternation of the Y-maze test performance, compared to that in the untreated transient forebrain ischemia group. Moreover, AC treatment showed a concentration-dependent inhibitory effect on acetylcholinesterase activity in vitro. Finally, the effect of AC on forebrain ischemia was blocked by mecamylamine, a nonselective nicotinic acetylcholine receptor antagonist. Our results suggested that in a model of forebrain ischemia, AC protected against neuronal death through the activation of nicotinic acetylcholine receptors.

Neuroprotective effect of the ethanol extract of Artemisia capillaris on transient forebrain ischemia in mice via nicotinic cholinergic receptor / 中国天然药物

Artemisia capillaris Thunberg is a medicinal plant used as a traditional medicine in many cultures. It is an effective remedy for liver problems including hepatitis. Recent pharmacological reports have indicated that Artemisia species can exert various neurological effects. Previously, we reported a memory-enhancing effect of Artemisia species. However, the mechanisms underlying the neuroprotective effect of A. capillaris (AC) are still unknown. In the present study, we investigated the effect of an ethanol extract of AC on ischemic brain injury in a mouse model of transient forebrain ischemia. The mice were treated with AC for seven days, beginning one day before induction of transient forebrain ischemia. Behavioral deficits were investigated using the Y-maze. Nissl and Fluoro-jade B staining were used to indicate the site of injury. To determine the underlying mechanisms for the drug, we measured acetylcholinesterase activity. AC (200 mg·kg) treatment reduced transient forebrain ischemia-induced neuronal cell death in the hippocampal CA1 region. The AC-treated group also showed significant amelioration in the spontaneous alternation of the Y-maze test performance, compared to that in the untreated transient forebrain ischemia group. Moreover, AC treatment showed a concentration-dependent inhibitory effect on acetylcholinesterase activity in vitro. Finally, the effect of AC on forebrain ischemia was blocked by mecamylamine, a nonselective nicotinic acetylcholine receptor antagonist. Our results suggested that in a model of forebrain ischemia, AC protected against neuronal death through the activation of nicotinic acetylcholine receptors.

Transient forebrain ischemia induces impairment in cognitive performance prior to extensive neuronal cell death in Mongolian gerbil (Meriones unguiculatus)

In Mongolian gerbils, bilateral common carotid artery occlusion (BCCAO) for several minutes induces ischemia, due to an incomplete circle of Willis, resulting in delayed neuronal cell death in the Cornet d'Ammon 1 (CA1) region of the hippocampus. Neuronal cell death in the hippocampus and changes in behavior were examined after BCCAO was performed for 5 min in the gerbils. One day after BCCAO, the pyramidal neurons of the CA1 region of the hippocampus showed degenerative changes (clumped chromatin in nuclei). At 5 and 10 days after BCCAO, extensive neuronal cell death was observed in the hippocampal CA1 region. Cognitive performance was evaluated by using the radial maze and passive avoidance tests. In the radial maze test, which examines win-stay performance, the number of errors was significantly higher in ischemic gerbils than in sham-operated gerbils on days 1 and 2 post-operation. In the passive avoidance test, the latency and freezing times were significantly shorter in ischemic gerbils than in sham-operated gerbils on the days 1, 2, and 4–6 post-operation. These results indicate that transient forebrain ischemia impairs cognitive performance, even immediately after the ischemic insult when there are only subtle signs of neuronal cell death.

The neuroprotective mechanism of ampicillin in a mouse model of transient forebrain ischemia

Ampicillin, a beta-lactam antibiotic, dose-dependently protects neurons against ischemic brain injury. The present study was performed to investigate the neuroprotective mechanism of ampicillin in a mouse model of transient global forebrain ischemia. Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral common carotid artery occlusion for 40 min. Before transient forebrain ischemia, ampicillin (200 mg/kg, intraperitoneally [i.p.]) or penicillin G (6,000 U/kg or 20,000 U/kg, i.p.) was administered daily for 5 days. The pretreatment with ampicillin but not with penicillin G signifi cantly attenuated neuronal damage in the hippocampal CA1 subfield. Mechanistically, the increased activity of matrix metalloproteinases (MMPs) following forebrain ischemia was also attenuated by ampicillin treatment. In addition, the ampicillin treatment reversed increased immunoreactivities to glial fibrillary acidic protein and isolectin B4, markers of astrocytes and microglia, respectively. Furthermore, the ampicillin treatment significantly increased the level of glutamate transporter-1, and dihydrokainic acid (DHK, 10 mg/kg, i.p.), an inhibitor of glutamate transporter-1 (GLT-1), reversed the neuroprotective effect of ampicillin. Taken together, these data indicate that ampicillin provides neuroprotection against ischemia-reperfusion brain injury, possibly through inducing the GLT-1 protein and inhibiting the activity of MMP in the mouse hippocampus.

Effects of endogenous histamine on transient forebrain ischemia-induced neuronal damage at late phase of reperfusion in mice / 中国病理生理杂志

AIM: To determine the effect of endogenous histamine on transient forebrain ischemia-induced neuronal injury at the late phase of reperfusion using histidine decarboxylase knockout ( HDC-KO) mice.METHODS:Wild-type (WT) and HDC-KO mice were subjected to bilateral common carotid artery occlusion for 30 min followed by 3 d or 15 d of reperfusion.At different time points after reperfusion, the body weight, mortality rate, learning and memory in fear conditioning test and hippocampal CA 1 neuronal density were evaluated .RESULTS: At 1 d after reperfusion , the body weight loss was observed in both WT and HDC-KO mice.At 4 d, 5 d, 6 d and 7 d after reperfusion, the increment in the body weight of the HDC-KO mice was significantly smaller than that of the WT mice .During the period between 8 d and 14 d after reperfusion, the mortality rate of the HDC-KO mice was higher than that of the WT mice (P<0.05).At 14 d after reperfusion , the HDC-KO mice exhibited more aggravated deficits in contextual and cue memory compared with the WT mice.Correspondingly , a more severe CA1 neuronal injury in the HDC-KO mice than that in the WT mice was ob-served at 15 d but not at 3 d after reperfusion (P<0.05).CONCLUSION:Endogenous histamine may attenuate learn-ing/memory deficits and neuronal injury at the late phase of ischemia /reperfusion.However, the involved mechanisms need to be further investigated .

Effects of ultrasound-combined microbubbles on hippocampal AchE fibers in rats

OBJECTIVE@#To investigate the protective effect of ultrasound-combined microbubbles on hippocampal acetylcholinesterase (AchE) fibers in rats.@*METHODS@#According to random digits table, 60 SD rats were divided into two groups, marrow stromal cells (MSCs) intracranial transplantation group and MSCs intracranial transplantation + ultrasonic microbubbles group. Marrow stromal cells were cultivated and isolated in vitro; 12 weeks after transplantation, spatial learning and memorizing abilities of rats were assessed by Morris water maze; AchE staining method was used to observe changes in density and appearance of AchE staining positive fibers in hippocampal CA1 region.@*RESULTS@#There was a significant increase in spatial learning and memorizing abilities of rats in MSCs intracranial transplantation + ultrasonic microbubbles group. Hippocampal AchE staining suggested an increase in the density of AchE staining positive fibers in MSCs intracranial transplantation group; the fibers were regular, intact and dense. Density of hippocampal AchE positive fibers was negatively correlated with the escape latent period and was positively correlated with percentage of the time needed to cross each platform quadrant.@*CONCLUSIONS@#Better promotion of spatial learning and memorizing abilities of rats in MSCs intracranial transplantation + ultrasonic microbubbles group may be related with the protective effect of ultrasound-combined microbubbles on hippocampal acetylcholine fibers.

Characterization of nestin expression in astrocytes in the rat hippocampal CA1 region following transient forebrain ischemia / 대한해부학회지

Recent studies have suggested that nestin facilitates cellular structural remodeling in vasculature-associated cells in response to ischemic injury. The current study was designed to investigate the potential role of post-ischemic nestin expression in parenchymal astrocytes. With this aim, we characterized ischemia-induced nestin expression in the CA1 hippocampal region, an area that undergoes a delayed neuronal death, followed by a lack of neuronal generation after transient forebrain ischemia. Virtually all of the nestin-positive cells in the ischemic CA1 hippocampus were reactive astrocytes. However, induction of nestin expression did not correlate simply with astrogliosis, but rather showed characteristic time- and strata-dependent expression patterns. Nestin induction in astrocytes of the pyramidal cell layer was rapid and transient, while a long-lasting induction of nestin was observed in astrocytes located in the CA1 dendritic subfields, such as the stratum oriens and radiatum, until at least day 28 after ischemia. There was no detectable expression in the stratum lacunosum moleculare despite the evident astroglial reaction. Almost all of the nestin-positive cells also expressed a transcription factor for neural/glial progenitors, i.e., Sox-2 or Sox-9, and some cells were also positive for Ki-67. However, all of the nestin-positive astrocytes expressed the calcium-binding protein S100beta, which is known to be expressed in a distinct, post-mitotic astrocyte population. Thus, our data indicate that in the ischemic CA1 hippocampus, nestin expression was induced in astroglia that were becoming reactive, but not in a progenitor/stem cell population, suggesting that nestin may allow for the structural remodeling of these cells in response to ischemic injury.

Iron mediates endothelial cell damage and blood-brain barrier opening in the hippocampus after transient forebrain ischemia in rats

Blood cells are transported into the brain and are thought to participate in neurodegenerative processes following hypoxic ischemic injury. We examined the possibility that transient forebrain ischemia (TFI) causes the blood-brain barrier (BBB) to become permeable to blood cells, possibly via dysfunction and degeneration of endothelial cells in rats. Extravasation of Evans blue and immunoglobulin G (IgG) was observed in the hippocampal CA1-2 areas within 8 h after TFI, and peaked at 48 h. This extravasation was accompanied by loss of tight junction proteins, occludin, and zonula occludens-1, and degeneration of endothelial cells in the CA1-2 areas. Iron overload and mitochondrial free radical production were evident in the microvessel endothelium of the hippocampus before endothelial cell damage occurred. Administration of deferoxamine (DFO), an iron chelator, or Neu2000, an antioxidant, blocked free radical production and endothelial cell degeneration. Our findings suggest that iron overload and iron-mediated free radical production cause loss of tight junction proteins and degeneration of endothelial cells, opening of the BBB after TFI.

Pre-ischemic Treatment with Ampicillin Reduces Neuronal Damage in the Mouse Hippocampus and Neostriatum after Transient Forebrain Ischemia

Ampicillin, a beta-lactam antibiotic, has been reported to induce astrocytic glutamate transporter-1 which plays a crucial role in protecting neurons against glutamate excitotoxicity. We investigated the effect of ampicillin on neuronal damage in the mouse hippocampus and neostriatum following transient global forebrain ischemia. Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral occlusion of the common carotid artery for 40 min. Ampicillin was administered post-ischemically (for 3 days) and/or pre-ischemically (for 3~5 days until one day before the onset of ischemia). Pre- and post-ischemic treatment with ampicillin (50 mg/kg/day or 200 mg/kg/day) prevented ischemic neuronal death in the medial CA1 area of the hippocampus as well as the neostriatum in a dose-dependent manner. In addition, ischemic neuronal damage was reduced by pre-ischemic treatment with ampicillin (200 mg/kg/day). In summary, our results suggest that ampicillin plays a functional role as a chemical preconditioning agent that protects hippocampal neurons from ischemic insult.

Effects of alpha-Lipoic Acid on Apoptotic Cell Death in Rat Hippocampus Following Transient Forebrain Ischemia-reperfusion Injury

PURPOSE: The purpose of this study was to evaluate effect of alpha-lipoic acid on apoptotic cell death in rat hippocampal neuron following transient forebrain ischemia-reperfusion (I/R) injury. METHODS: The four-vessel occlusion method was used to induce transient I/R injury in the forebrain of Sprague-Dawley rats. In the treatment group, alpha-Lipoic acid (LA) was administered subcutaneously at 50 mg/kg/day for 7 days before induction of I/R injury. RESULTS: Pretreatment with LA significantly reduced the number of TUNEL-positive neurons in the pyramidal cells layer of the hipocampal CA1 region 5 days after the ischemia, suggesting a marked reduction of apoptotic cell death. Pretreatment with LA also resulted in marked suppression at the transcript level of mRNA for caspase-3 at 24 hours, and decreased concentration of the active form of caspase-3 protein in the hippocampus at 1, 3, and 5 days after I/R injury. Furthermore, as indicated by western blot analysis, the concentration of apoptosis-inducing factor (AIF) in the hippocampus was reduced at 1 and 3 days after a transient I/R injury by pretreatment with LA. CONCLUSION: The results of this study suggest that LA has the potential to prevent neuronal cell death in the hippocampus by inhibiting intracellular signaling pathways responsible for apoptosis following transient I/R injury.

The Effect of Fluoxetine on Behaviors in Transient Forebrain Ischemic Gerbil / 대한정신약물학회지

OBJECTIVE: This study aims to explore the effect of fluoxetine on memory, locomotor and depressive behavior in transient forebrain ischemic model of gerbil. METHODS: Doses of fluoxetine (10, 40 mg/kg) or vehicle were intraperitoneally administered once 30 min before ischemic surgery in gerbil. Novel object recognition test, spontaneous motor activity, learned helplessness test were performed 4 days, 8 days, or 9 days, respectively, after sham or ischemic surgery. RESULTS: Fluoxetine treatment (40 mg/kg) significantly reduced recognition memory in sham operated gerbil. However, fluoxetine (10, 40 mg/kg) did not affect ischemia-induced impairment in recognition memory. The treatment of fluoxetine (10, 40 mg/kg) significantly inhibited locomotor hyperactivity induced by transient ischemia even though fluoxetine (40 mg/kg) did not affect spontaneous motor activity in the sham operated gerbils. Fluoxetine did not affect depressive behavior in sham and ischemic gerbils. CONCLUSION: The treatment of fluoxetine inhibited ischemia-induced hyperactivity, but did not affect memory and depressive behavior in transient forebrain ischemic gerbils.

A Simple Method for Predicting Hippocampal Neurodegeneration in a Mouse Model of Transient Global Forebrain Ischemia

In the present study, we developed a simple method to predict the neuronal cell death in the mouse hippocampus and striatum following transient global forebrain ischemia by evaluating both cerebral blood flow and the plasticity of the posterior communicating artery (PcomA). Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral occlusion of the common carotid artery (BCCAO) for 30 min. The regional cerebral blood flow (rCBF) was measured by laser Doppler flowmetry. The plasticity of PcomA was visualized by intravascular perfusion of India ink solution. When animals had the residual cortical microperfusion less than 15% as well as the smaller PcomA whose diameter was less than one third compared with that of basilar artery, neuronal damage in the hippocampal subfields including CA1, CA2, and CA4, and in the striatum was consistently observed. Especially, when mice met these two criteria, marked neuronal damage was observed in CA2 subfield of the hippocampus. In contrast, after transient BCCAO, neuronal damage was consistently produced in the striatum, dependent more on the degree of rCBF reduction than on the plasticity of PcomA. The present study provided simple and highly reproducible criteria to induce the neuronal cell death in the vulnerable mice brain areas including the hippocampus and striatum after transient global forebrain ischemia.

Mechanism of anti-apoptotic action of dipfluzine on neuronal damage of the rat hippocampal CA1 region subjected to transient forebrain ischemia / 药学学报

Aim To explore the relations between anti-apoptotic role of dipfluzine (DIP) and the death signaling transduction pathway initiated by CD95 molecules, and the transcription factor involved in the transcription regulation of CD95 molecules in the hippocampal CA1 region after transient forebrain ischemia. Methods The rat forebrain transient ischemia model was established through 15 min ischemia followed by 3 days reperfusion by using the four-vessel method. The rats were divided randomly into five groups: sham control group, ischemia / reperfusion (I/R) group, DIP treated groups (20, 40 and 80 mg·kg-1 body weight, ig, separately). Western blotting and RT-PCR were performed to detect the expression changes of Fas, FasL, caspase 10 p20, caspase 8, I-κB-α, and p-I-κB-α molecules in protein and mRNA levels, separately, and immunohistochemistry for molecular localization of Fas and FasL in rat hippocampus. Results The expression of Fas, FasL, and caspase 10 p20 in protein and mRNA levels increased after I/R, which was inhibited significantly after treatment with 20 and 40 mg·kg-1 of DIP (P<0.01). In 80 mg·kg-1 of DIP group, the expression of Fas and FasL protein was not significantly different from that of I/R group (P>0.05). The expression of caspase 8 and I-κB-α showed no significant differences in all groups (P>0.05), and no gene expression was observed for p-I-κB-α protein in the study. DIP significantly affected molecular distribution of Fas and FasL protein in CA1 subregion of hippocampus. Conclusion DIP inhibits the death signaling transduction pathway initiated by CD95 molecules in rat hippocampal CA1 subregion, and NF-κB transcription factor may not be involved in the transcription regulation of CD95 molecules after transient forebrain ischemia.

Postischemic Treatment with Aminoguanidine Inhibits Peroxynitrite Production in the Rat Hippocampus Following Transient Forebrain Ischemia

Transient forebrain ischemia results in the delayed neuronal death in the CA1 area of the hippocampus. The present study was performed to determine effects of aminoguanidine, a selective iNOS inhibitor, on the generation of peroxynitrite and delayed neuronal death occurring in the hippocampus following transient forebrain ischemia. Transient forebrain ischemia was produced in the conscious rats by four-vessel occlusion for 10 min. Treatment with aminoguanidine (100 mg/kg or 200 mg/kg, i.p.) or saline (0.4 ml/100 g, i.p.) was started 30 min following ischemia-reperfusion and the animals were then injected twice daily until 12 h before sacrifice. Immunohistochemical method was used to detect 3-nitrotyrosine, a marker of peroxynitrite production. Posttreatment of aminoguanidine (200 mg/kg) significantly attenuated the neuronal death in the hippocampal CA1 area 3 days, but not 7 days, after ischemia-reperfusion. 3-Nitrotyrosine immunoreactivity was enhanced in the hippocampal CA1 area 3 days after reperfusion, which was prevented by the treatment of aminoguanidine (100 mg/kg and 200 mg/kg). Our findings showed that (1) the generation of peroxynitrite in the hippocampal CA1 area 3 days after ischemia-reperfusion was dependent on the iNOS activity; (2) the postischemic delayed neuronal death was attenuated in the early phase through the prevention of peroxynitrite generation by an iNOS inhibitor.

Morphological Analysis of Short and Long Term Changes after Ligation of Unilateral Common Carotid Artery in Gerbils

BACKGROUND: The ligation of the unilateral common carotid artery (CCA) in the gerbil has been known as an ischemic animal model showing various changes including selective neuronal necrosis as well as infarction. This study was performed to analyze the short and long term morphological changes of transient unilateral forebrain ischemia with special attention to astroglial proliferation. METHODS: 67 mongolian gerbils were subjected to 2 hr, 3 hr, 4 hr, or 5 hr of forebrain ischemia by the unilateral CCA ligation method. Each of the ischemic groups were examined after a 1 day, 3 day, or 7 day period of reperfusion. Long term reperfusion groups consisted of 2, 3, and 4 weeks of reperfusion after 5hr of unilateral CCA ligation. Morphological changes were analyzed by H-E staining and an immunohistochemical reaction with GFAP antibody. RESULTS: The ligation of the unilateral CCA, induced unilateral hemispheric infarction in 14 gerbils, selective neuronal necrosis (SNN) involving caudate in 1 gerbil, and delayed neuronal necrosis (DND) of the hippocampal CA1 neurons in 2 gerbils. Infarction was most frequent in 1 day reperfusion groups and did not show any differences according to the duration of ischemia. The GFAP reaction was strongly positive in the center of infarction at a 1 day period and negative at a 3 & 7 day period. The surrounding brain parenchyme progressively revealed increased positive reactions. Gerbils with SNN and DND showed moderately or markedly increased GFAP positive reactions in the unilateral caudate, thalamus, and hippocampus, whereas no apparent changes were shown by a H-E stain. CONCLUSIONS: Reactive astrogliosis is a stereotyped reaction of ischemic brain injury and is a more sensitive parameter than neuronal changes.

Behamioral Change and Memory Inpairment Following Transient Forebrain Ischemia in Rats / 대한마취과학회지

This study examined the behavioral change and memory impairment following transient forebrain ischemia in 20 male rats: ten rats were subjected to sham operation. Inchemia was induced by a combination of bilateral common carotid artery occlusion and induced hypotension. During the recovery period, behavioral change was evaluated by open-field method. Histopathological examination was performed 7 days after ischemic insult. The results were as follows 1) Hippocampus CA 1 neuron was the most vulnerable to 10-minute forebrain ischemia. 2) There was increase in locomotor activity and rearing during early post-ischemic period. 3) Ischemia resulted in suppressed preening and grooming. From the above mentioned results, authors suggested that transient forebrain ischemia produced severe neuronal damage in CA l sector of hippocampus, therefore produced behavioral change and memory impairment.

Experimental Study of Cerebral Protective Action with Dexamethasone in a Rat`s Forebrain Ischemic Model / 대한마취과학회지

This study examined the protective effect of dexamethansone following bilateral forebrain ischemia in a rat model of two-vessel occlusion. Twenty rats were subjected to dexamethasone (1 mg/kg intravenously) before bilateral common carotid artery occlusion for 10 minutes. Carotid artery blood flow was restored and the rats were terminated by a perfusion-fixation method after 7 days. Ten minutes of two-vessel occlusion produced cellular ischemic change in the cerebral hemispheres of most rats. This region was studied mainly with light microscope, and it was noticed that the CA 1 neuron in the hippocampus is the most vulnerable to ischemia. Ischemic neuronal damage was graded in accordance with conventional neuropathological criteria. The grade had no statistical significance.