Hippocampal CA1 Sector Research Articles

Neuroprotective Effect of Post Ischemic Treatment of Acetylsalicylic Acid on CA1 Hippocampus Neuron and Spatial Learning in Transient MCA Occlusion in Rat

The present study was designed to examine the neuroprotective effects of post-ischemic treatment with ASA on pyramidal neurons of the hippocampal CA1 sector and spatial learning and memory. Animals were allocated to ASA (20, 40 and 80 mg kg-1) and vehicle groups. Ischemia was induced by 20 min middle cerebral artery occlusion. ASA solution was administered intraperitoneal at 30 min, 6 and 24 h after induction of ischemia. Four days after ischemia, animals were subjected to 5 days of training in the Morris water maze (MWM); 4 days with the invisible platform to test spatial learning and the 5th day without platform to test spatial memory. At the end of behavioral test rats were sacrificed and pyramidal neurons in the CA1 sector were stained with Hematoxylin and Eosin. We demonstrated that repeated injections of 20 mg kg-1 ASA (p<0.05) but not 40 and 80 mg kg-1 ASA (p = 0.989) increased percentage of spent time in the target zone across the four days post stroke test period significantly in the MWM as compared to vehicle group. But repeated injections of 20, 40 and 80 mg kg-1 of ASA after ischemia not reduced the prolongation of the escape latency significantly as compared to vehicle group (p = 0.556). In treated groups there was no significant effect on spatial memory (p = 0.987) as compared to vehicle group. Histological verification of CA1 area showed that repeated injections of 20, 40 and 80 mg kg-1 ASA at 30 min, 6 and 24 h after ischemia reduced the number of dead hippocampal neuronal cells significantly (p<0.001). Study findings show that repeated ASA injections at 30 min, 6 and 24 h after stroke onset decreases neuronal injuries following ischemia and attenuates cerebral ischemia-induced learning dysfunctions.

Age-related changes of NGF, BDNF, parvalbumin and neuronal nitric oxide synthase immunoreactivity in the mouse hippocampal CA1 sector

We investigated the age-related alterations in nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), parvalbumin and neuronal nitric oxide synthase (nNOS) immunoreactivity of the mouse hippocampal CA1 sector. NGF and BDNF immunoreactivity was unchanged in the hippocampal CA1 pyramidal neurons from 2 to 50-59 weeks of birth. In contrast, a significant increase in the NGF and BDNF immunoreactivity was observed in glial cells of the hippocampal CA1 sector from 40-42 to 50-59 weeks of birth. On the other hand, the number of parvalbumin- and nNOS-positive interneurons was unchanged in the hippocampal CA1 sector during aging processes, except for a significant decrease of nNOS-positive interneurons 2 weeks of birth. Our results indicate that NGF and BDNF immunoreactivity was unaltered in the hippocampal CA1 pyramidal neurons during aging processes. In contrast, a significant increase in the NGF and BDNF immunoreactivity was observed in glial cells of the hippocampal CA1 sector during aging processes. The present study also shows that the number of parvalbumin- and nNOS-positive interneurons was unchanged in the hippocampal CA1 sector during aging processes, except for a significant decrease of nNOS-positive interneurons 2 weeks of birth. These results demonstrate that the expression of glial NGF and BDNF may play a key role for helping survival and maintenance of pyramidal neurons and neuronal functions in the hippocampal CA1 sector during aging processes. Furthermore, our findings suggest that parvalbumin- and nNOS-positive interneurons in the hippocampal CA1 sector are resistant to aging processes. Moreover, our findings suggest that nitric oxide synthesized by the nNOS may play some role for neuronal growth during postnatal development.

Spontaneous temperature changes in the 2-vessel occlusion model of cerebral ischemia in rats

Transient global ischemia (ISC) in rats and humans causes selective and delayed neuronal death in the hippocampal CA1 sector. It is clear from rodent studies that hyperthermia aggravates, whereas hypothermia lessens, this injury. In this study we sought to relate core (Tc) and brain (Tb) temperature, measured via telemetry probes, after ISC produced in rats by bilateral common carotid artery occlusion combined with systemic hypotension (2-VO model). We also tested whether spontaneous postischemic temperature fluctuations occurred and whether they were related to cell death as previous studies indicate. We report that Tc and Tb readings are similar and are highly correlated before and after 10 min of 2-VO ISC. In the second experiment, rats were subjected to 8, 9, or 10 min of 2-VO ISC. Despite a range in CA1 injury among these animals, there was no evidence of post-ISC hyperthermia, contrary to earlier work, and neither temperature nor the physiological variables measured during ISC (e.g., glucose) predicted injury. Our findings suggest that, under the present conditions, 2-VO rats do not experience postoperative hyperthermia, which can be adequately measured with Tc telemetry probes.

Abstract 42: Effects Of Intracerebroventricular Application Of Granulocyte Colony-stimulating Factor On Cerebral Recovery After Cardiac Arrest In Rats

Introduction: After transient global cerebral ischemia, selective vulnerable brain areas show delayed neuronal degeneration with characteristic signs of apoptosis. Recent data demonstrated potent neuroprotective effects of the application of granulocyte colony-stimulating factor (G-CSF) after focal cerebral ischemia. In order to assess possible effects of intracerebroventricular application of G-CSF on cerebral recovery after global cerebral ischemia due to cardiac arrest (CA) neurological testing according to a tape removal test as well as histological analysis of the hippocampal CA-1 sector were performed. Methods: Global cerebral ischemia was initiated by ventricular fibrillation in rats during general anesthesia. After 6 min of CA cardio pulmonary resuscitation (CPR) was initiated. After restoration of spontaneous circulation (ROSC) the animals were divided into two groups (G-CSF vs. placebo; n=16 per group). G-CSF (4μl=3.84μg) or placebo was applied in single shot technique 30 min after ROSC. During CA, CPR and for the first 24 h after ROSC all animals were kept normothermic using a feedback control system with intraperitoneal implanted telemetric probes. The tape removal test was applied pre-CA, 24 h, 3 d, 7 d, 10 d and 14 d after ROSC. After 14 days histological analysis was done by counting TUNEL positive neurons in the hippocampal CA-1 sector. All experiments were performed in a randomized and blinded setting. Results: All animals were kept normothermic within the first 24 h after ROSC (G-CSF: 37.3 ± 0.3 °C; placebo: 37.5 ± 0.5 °C). Pre-CA all animals removed the adhesive tapes promptly. After ROSC in both treatment groups a clear neurological damage was shown that improved within the first 14 days, without reaching baseline values. No statistically significant differences between the group could be seen. With regard to TUNEL-positive neurons in the hippocampal CA-1 sector no significant differences could be observed between the groups (G-CSF: 47 ± 22; placebo: 51 ± 24). Conclusions: Regarding neurological recovery and neurohistopathological outcome 14 d after global cerebral ischemia, the present study was not able to show any beneficial effects of G-CSF, despite the well known effects of G-CSF in non global cerebral ischemia models.

Abstract 2837: Delayed Calpain Inhibition Is Neuroprotective After Transient Global Brain Ischemia

Introduction/Hypothesis: The delay in neurodegeneration after transient global brain ischemia offers a potentially broad therapeutic window for inhibiting molecular injury mechanisms. One such mechanism, calpain-mediated proteolysis, peaks between 24 and 72 hours after transient forebrain ischemia (TFI) in rats. This study tests the hypothesis that calpain inhibition is neuroprotective when initiated 22 hours after TFI. Methods: Male Long Evans rats (400 – 450 g) were anesthetized (halothane), mechanically ventilated, and instrumented for temperature and hemodynamic monitoring. Each underwent 10 minutes of TFI induced by reversible bilateral carotid occlusion and hypovolemic hypotension (MAP 30 mm Hg). Twenty-two hours after TFI, rats were block randomized (n=6/group) to receive either calpain inhibitor (CEP-3453 (Cephalon), 60 mg/kg IV bolus and 30 mg/kg IV infusion for 50 hours) or vehicle (normal saline IV bolus and infusion for 50 hours). Sham operated rats served as controls. Rats were euthanized 72 hours after injury and brains were processed for immunohistochemistry. Hippocampal CA1 sector calpain activity was analyzed by immunofluorescence using primary antibody that specifically detects calpain-cleaved alpha-spectrin (Ab38). Neurodegeneration was quantified by counting normal appearing Hoechst-stained neuronal nuclei in the hippocampal CA1 pyramidal layer. Ab38 immunofluorescence intensity and normal nuclei counts were compared between vehicle and CEP-3453 treatment groups using a two-tailed Student’s t-test (alpha error 0.05). Results: Baseline and post-injury hemodynamic parameters and temperature were not significantly different between vehicle and CEP-3453 treatment groups. Relative to sham operated controls, mean CA1 sector Ab38 immunofluorescence increased by 263 ± 281% in vehicle treated rats and 68 ± 147% in CEP-3453 treated rats (p=0.17). Normal CA1 pyramidal layer nuclei averaged 10 ± 12% of control in vehicle treated rats and 50 ± 42% of control in CEP-3453 treated rats (p=0.047). Conclusion: These results suggest a causal role for calpains in delayed post-ischemic neurodegeneration, and demonstrate a broad therapeutic window for calpain inhibition in this model of transient global brain ischemia.

Effects of the application of erythropoietin on cerebral recovery after cardiac arrest in rats

After transient global cerebral ischaemia, selectively vulnerable areas of the brain show delayed neuronal degeneration. Recent data have demonstrated potent neuroprotective effects of the application of growth hormones like erythropoietin (EPO) after focal cerebral ischaemia. In order to assess possible effects of the application of EPO on cerebral recovery after cardiac arrest in rats, the vulnerable hippocampal CA-1 sector was investigated. Thirty male Wistar rats were randomised into two groups (EPO versus placebo; n=15 per group). Cardiac arrest was induced by ventricular fibrillation during general anaesthesia. After 6 min of global cerebral ischaemia, animals were resuscitated by external chest compressions combined with defibrillation. Investigator blinded bolus application of EPO (5000 IE/kg bodyweight) and placebo was performed at three different time points, respectively: 5 min before cardiac arrest (i.v.; intravenously), 24h (i.p.; intraperitoneally) and 72 h (i.p.) after ischaemia. At 24h, 72 h and 7 days, animals were tested according to a neurological deficit score. After of reperfusion, coronal brain sections were analysed by TUNEL- and Nissl-staining. A caspase activity assay was done to determine antiapoptotic properties of EPO. Statistical analyses were done using ANOVA. Neurological deficit scoring did not show differences between the groups. However, in all groups typical delayed neuronal degeneration could be found in the CA-1 sector. There was no difference in neuronal survival between the groups (viable neurons EPO (median [interquartile range]): 15.5 [10.1-21.3]; placebo: 16.8 [7.7-26.3]). Results from TUNEL-staining revealed no differences in the amount of apoptotic cell death between the groups (EPO: 71.2 [58.1-81.8]; placebo: 73.4 [67.8-78.2]). Caspase activity assays demonstrated a strong expression of general caspase activity as well as caspase-3 activity in the CA-1 sector and the nucleus reticularis thalami, without any differences between both groups. Despite the well known neuroprotective properties of EPO in ischaemia induced neuronal degeneration, this study could not reveal any beneficial effects of EPO after global cerebral ischaemia due to cardiac arrest in rats.

Age-related changes of astorocytes, oligodendrocytes and microglia in the mouse hippocampal CA1 sector

We investigated the age-related alterations of astorocyte, oligodendrocyte and microglia in the mouse hippocampal CA1 sector under the same conditions using immunohistochemistry. Glial fibrillary acidic protein (GFAP), 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and isolectin B 4 immunoreactivity was measured in 2-, 8-, 18-, 40–42- and 50–59-week-old mice. Total number of GFAP-positive cells was unchanged in the hippocampal CA1 sector up to 40–42 weeks of birth. In 50–59-week-old mice, however, a significant increase in the number of GFAP-positive cells was observed in the hippocampal CA1 sector, exhibiting the morphology of reactive astrocytes. In contrast, the fibers of CNPase immunoreactivity were unchanged in the hippocampal CA1 sector up to 18 weeks of birth. In 40–42- and 50–59-week-old mice, however, a significant decrease in the densities of CNPase-positive fibers was observed in the hippocampal CA1 sector. On the other hand, total number of isolectin B 4-positive cells was unchanged in the hippocampal CA1 sector up to 40–42 weeks of birth. In 50–59-week-old mice, however, a significant decrease in the number of isolectin B 4-positive cells was observed in the hippocampal CA1 sector. Our results show that astrocytes proliferate and are activated in the hippocampal CA1 sector with advancing age. Furthermore, the present study demonstrates that the fibers of oligodendrocytes and total number of microglial cells in the hippocampal CA1 sector are decreased during ageing processes. These results suggest that age-related changes of astorocytes, oligodendrocytes and microglia had occurred in the mouse hippocampal CA1 sector.

Immunohistochemical changes related to ageing in the mouse hippocampus and subventricular zone

We investigated mainly immunohistochemical changes of nestin (a marker of neuroepithelial stem cells) and Ki-67 (a marker of proliferating cells) proteins related to ageing in the mouse hippocampus and subventricular zone (SVZ) using young adult (8 weeks old) and middle-aged (40 weeks old) mice. In the present study, no significant changes in neurons and astrocytes of the hippocampal CA1 sector were found in a middle-aged male ICR mice without severe senile weakness, as compared with young adult animals. In contrast, a significant change in the number of microglia was found in the hippocampal CA1 sector of the middle-aged mice. Furthermore, no significant changes in the number of nestin- and Ki-67-positive cells were observed in the hippocampal CA1 sector of the middle-aged mice. On the other hand, decreases in the number of nestin- and Ki-67-immunopositive cells were observed in the SVZ of the middle-aged mice. Furthermore, a migration of nestin- and Ki-67-immunoreactive cells in the corpus callosum was not observed in the SVZ of the middle-aged mice. In the dentate gyrus, significant decreases in the number of Ki-67-immunopositive cells were observed in the middle-aged mice. Our study also showed that nestin immunoreactivity was observed in both Ki-67-postive cells and astrocytes in the SVZ of young adult mice. These findings emphasize the need to recognize ageing as important factors in studies of microglia, which may help to clarify the role of glial cell structure and function during ageing processes. Furthermore, the present findings suggest that ageing processes may decrease neurogenesis in the corpus callosum, SVZ and dentate gyrus. Thus our present findings provide valuable information for the neurogenesis during ageing processes.

Immunohistochemical Study on Distribution of NF-κB and p53 in Gerbil Hippocampus after Transient Cerebral Ischemia: Effect of Pitavastatin

We investigated the immunohistochemical alterations of the transcription nuclear factor kappa-B (NF-kappaB) and transcription factor p53 in the hippocampus after transient cerebral ischemia in gerbils. We also examined the effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor pitavastatin against the alterations of NF-kappaB, p53 and neuronal nuclei in the hippocampus after ischemia. Severe neuronal damage was observed in the hippocampal CA1 neurons 5 and 14 days after ischemia. In the present study, the increase of NF-kappaB immunoreactivity in glial cells and p53 immunoreactivity in neurons preceded neuronal damage in the hippocampal CA1 sector after ischemia. Thereafter, NF-kappaB immunoreactivity was induced highly in reactive astrocytes and microglia of the hippocampal CA1 sector where severe neuronal damage was observed. Our immunohistochemical study showed that pitavastatin prevented the alterations of NF-kappaB and p53 in the hippocampal CA1 sector 5 days after transient ischemia. Furthermore, our results with neuronal nuclei immunostaining indicate that pitavastatin dose-dependently prevented the neuronal cell death in the hippocampal CA1 sector 5 days after transient cerebral ischemia. These results suggest that the up-regulations of NF-kappaB in glia and p53 in neurons can cause neuronal cell death after ischemia. Our findings also support the hypothesis that NF-kappaB- and/or p53-mediated neuronal cell death is prevented through decreasing oxidative stress by pitavastatin. Thus, NF-kappaB and p53 may provide an attractive target for the development of novel therapeutic approaches for brain stroke.

Vasopressors are essential during cardiopulmonary resuscitation in rats: Is vasopressin superior to adrenaline?

Vasopressors are recommended for cardiopulmonary resuscitation (CPR) after cardiac arrest. In order to assess possible benefits regarding neurological recovery, vasopressin versus adrenaline and the combination of both was tested against placebo in a cardiac arrest model in rats. Under anaesthesia with halothane and N2O, cardiac arrest was initiated via transoesophageal electrical fibrillation. After 7 min of global ischaemia, CPR was performed by external chest compression combined with defibrillation. Animals were randomly assigned to three groups receiving adrenaline, vasopressin and a combination of both (n = 15 per group) versus placebo (n = 8). At 1, 3 and 7 days animals were tested according to a neurological deficit score (NDS). After 7 days of reperfusion, coronal brain sections were analysed by Nissl- and TUNEL-staining. Viable as well as TUNEL-positive neurons were counted in the hippocampal CA-1 sector. For statistical analysis, the log rank and the Kruskal-Wallis ANOVA test were used. All data are given as mean+/-S.D.; a p-value <0.05 was considered significant. Mean arterial blood pressure (MAP) measured in the aorta did not differ between the vasopressor groups, whereas placebo animals had significantly lower levels. Survival to 7 days revealed significant differences between the placebo (n = 0/8) and all vasopressor groups (adrenaline, 10/15; adrenaline/vasopressin, 8/15; vasopressin, 12/15). Histological deficit scoring by quantitative analysis of the Nissl- and TUNEL-staining showed no difference in the amount of viable and apoptotic neurons in the vasopressin group (viable: 33+/-18; apoptotic: 63+/-23) versus the adrenaline group (viable: 21+/-12; apoptotic: 67+/-17) and the adrenaline/vasopressin group (viable: 31+/-26; apoptotic: 61+/-27). Neurological deficit scoring did not show any differences between the vasopressor groups. Administration of arginine-vasopressin during CPR does not improve behavioural and cerebral histopathological outcome, compared to the use of adrenaline or the combination of both vasopressors, after cardiac arrest in rats.

Postischemic alterations of BDNF, NGF, HSP 70 and ubiquitin immunoreactivity in the gerbil hippocampus: pharmacological approach.

1. We investigated the immunohistochemical alterations of BDNF, NGF, HSP 70 and ubiquitin in the hippocampus 1 h to 14 days after transient cerebral ischemia in gerbils. We also examined the effect of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor pitavastatin against the changes of BDNF, NGF, HSP 70 and ubiquitin in the hippocampus after cerebral ischemia in the hippocampus after ischemia. 2. The transient cerebral ischemia was carried out by clamping the carotid arteries with aneurismal clips for 5 min. 3. In the present study, the alteration of HSP 70 and ubiquitin immunoreactivity in the hippocampal CA1 sector was more pronounced than that of BDNF and NGF immunoreactivity after transient cerebral ischemia. In double-labeled immunostainings, BDNF, NGF and ubiquitin immunostaining was observed both in GFAP-positive astrocytes and MRF-1-positive microglia in the hippocampal CA1 sector after ischemia. Furthermore, prophylactic treatment with pitavastatin prevented the damage of neurons with neurotrophic factor and stress proteins in the hippocampal CA1 sector after ischemia. 4. These findings suggest that the expression of stress protein including HSP 70 and ubiquitin may play a key role in the protection against the hippocampal CA1 neuronal damage after transient cerebral ischemia in comparison with the expression of neurotrophic factor such as BDNF and NGF. The present findings also suggest that the glial BDNF, NGF and ubiquitin may play some role for helping surviving neurons after ischemia. Furthermore, our present study indicates that prophylactic treatment with pitavastatin can prevent the damage of neurons with neurotrophic factor and stress proteins in the hippocampal CA1 sector after transient cerebral ischemia. Thus our study provides further valuable information for the pathogenesis after transient cerebral ischemia.

The Antibiotic Erythromycin Induces Tolerance against Transient Global Cerebral Ischemia in Rats (Pharmacologic Preconditioning)

Cerebral ischemic tolerance can be induced by a variety of noxious stimuli, but no clinically applicable regimen for preconditioning has been described. Therefore, the authors tested the ability of a pharmacologic preconditioning strategy using the well-known macrolide antibiotic erythromycin to induce tolerance against transient global cerebral ischemia in vivo. They also investigated whether tolerance induction by erythromycin involves transcriptional and translational changes of cerebral B-cell leukemia/lymphoma-2 (bcl-2) expression. Male Wistar rats were treated with erythromycin (25 mg/kg intramuscularly) or vehicle and subjected to 15 min of transient global cerebral ischemia 6, 12, or 24 h after pretreatment. Neurologic deficit was evaluated once daily, and neuronal cell survival was assessed after 7 days of reperfusion. Additional animals were similarly pretreated, and cerebral bcl-2 messenger RNA (mRNA) and protein expression was analyzed 6 and 24 h later. Erythromycin improved postischemic neuronal survival in hippocampal CA1 and CA3 sectors and reduced functional deficit, with 12 h being the most efficient pretreatment interval. Bcl-2 mRNA in hippocampus was transiently up-regulated 6 h after erythromycin, but neuronal Bcl-2 protein remained unchanged. Erythromycin can induce cerebral ischemic tolerance in vivo (pharmacologic preconditioning), suggesting a potential clinical strategy of preemptive neuroprotection. Changes in bcl-2 expression after erythromycin were small and transient. The induction of bcl-2-related pathways, although important for other preconditioning regimens, may therefore be less relevant for the neuroprotective effects of pharmacologic preconditioning using erythromycin.

Immunohistochemical Detection of Apoptosis-Related Proteins in Gerbil Hippocampus Transient Cerebral Ischemia: Neuroprotective Effect of Pitavastatin

Delayed and selective neuronal damage was caused in the CA1 sector of hippocampus following 5 min of transient cerebral ischemia in gerbils. We investigated the immunohistochemical alterations of apoptosis-related proteins such as bcl-2α, bcl-xs/l, bax, cytochrome c, and active caspase 3 and TUNEL staining in the hippocampus at 1 and 5 hr and 1, 2, 5 and 14 days after transient cerebral ischemia in gerbils. We also examined the effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor pitavastatin against the alterations of apoptosis-related proteins and TUNEL staining in the hippocampus after cerebral ischemia. The alterations of apoptosis-related proteins in the hippocampal CA1 sector were more pronounced than the changes of hippocampal CA3 sector and dentate gyrus after cerebral ischemia. The alterations of apoptosis-related proteins in the hippocampal CA1 sector after cerebral ischemia preceded the neuronal damage in this region. Furthermore, the study with TUNEL staining showed that a marked increase of TUNEL-positive nuclei was evident only in the hippocampal CA1 sector 5 days after cerebral ischemia. Our immunohistochemical study also showed that pitavastatin prevented the alterations of apoptosis-related proteins and the increase of TUNEL-positive nuclei in the hippocampal CA1 sector 5 days after cerebral ischemia. The present study indicates that transient cerebral ischemia in gerbils causes the mitochondrial-dependent apoptosis in the hippocampal CA1 sector. Furthermore, our present study demonstrates that pitavastatin can prevent the alterations of apoptosis-related proteins and the increase of TUNEL-positive nuclei in the hippocampal CA1 sector after cerebral ischemia. Thus our study provides novel therapeutic strategies in clinical stroke.

Novel Surrogate Markers for Acute Brain Damage: Cerebrospinal Fluid Levels Corrrelate with Severity of Ischemic Neurodegeneration in the Rat

Previously, we identified proteins released from degenerating cultured cortical neurons as novel cerebrospinal fluid (CSF) markers for acute brain injury in the rat. Here, we investigate relationships between CSF changes in these novel markers and the severity of acute ischemic brain injury. Rats underwent sham surgery or 3,6,8, or 10 mins of transient global forebrain ischemia. At 48 h after insult, CSF levels of 14-3-3beta, 14-3-3zeta, and calpain cleavage products of alpha-spectrin and tau were quantified. Regional acute neurodegeneration was assessed by Fluoro-Jade and silver impregnation staining, and confirmed by immunohistochemical detection of the activation of calpain and caspase, cysteine proteases involved in neurodegenerative signaling. Ischemic neurodegeneration and activation of at least one cysteine protease were observed in the hippocampal CA1 sector, dentate hilus, caudate nucleus, parietal cortex, thalamus, and inferior colliculus. As expected, the total number of degenerating cells increased as a function of ischemia duration. Cerebrospinal fluid levels of the four marker proteins increased markedly after ischemia, and rose in proportion with its duration. Irrespective of the length of ischemia, CSF levels of the neuron-enriched proteins 14-3-3beta and calpain-cleaved tau correlated significantly with the magnitude of acute ischemic neurodegeneration. Additionally, CSF levels of the two proteins correlated with one another. These results show that certain proteins released from degenerating neurons are CSF markers for brain injury in the rat whose levels reflect the severity of acute ischemic neurodegeneration. Measurement of 14-3-3beta and calpain-cleaved tau may be useful for the minimally invasive diagnosis, prognosis, and therapeutic evaluation of acute brain damage.

Null mutation of P53 attenuates hippocampal neuronal death in vivo following global ischemia in mice

Tumor suppressor gene p53 controls cell death after various stresses. However, the role of p53 in neuronal death after brain ischemia has been poorly understood. Here, we evaluated a direct causal link of p53 with neuronal death following global cerebral ischemia using p53 deficient mice. We used p53 mutant mice backcrossed for twelve generations to avoid bias in the genetic background. These mutants, as well as wild-type mice, were subjected to transient global ischemia by 3-vessel occlusion method, in which no difference in severity of ischemia was noted as evidenced by anoxic depolarization and cortical blood flow. The resulting neuronal death in the hippocampal CA1 sector was extensive in p53 wild-type mice: the surviving neuronal count was 9.3 3.0 % of the normal control. However in mutant mice homozygous for p53, marked and significant attenuation of ischemic injury was observed, in which the neuronal count amounted to 61.3 34.0 % (p < 0.0037). In wilt-typ mice, intense p53-like immunoreactivity was observed in hippocampal CA1 neurons at 12 hours after ischemia, and mRNA for Bax, a direct down stream target of p53, was also increased. These results indicated that p53 plays a crucial role in ischemic neuronal death in vivo, and suggest that this molecule could be a therapeutic target in neuronal death following cerebral ischemia.

Alterations of Interneurons of the Gerbil Hippocampus after Transient Cerebral Ischemia: Effect of Pitavastatin

We investigated the immunohistochemical alterations of parvalbumin (PV)-expressing interneurons in the hippocampus after transient cerebral ischemia in gerbils in comparison with neuronal nitric oxide synthase (nNOS)-expressing interneurons. We also examined the effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor pitavastatin against the damage of neurons and interneurons in the hippocampus after cerebral ischemia. Severe neuronal damage was observed in the hippocampal CA1 pyramidal neurons 5 and 14 days after ischemia. The PV immunoreactivity was unchanged up to 2 days after ischemia. At 5 and 14 days after ischemia, in contrast, a conspicuous reduction of PV immunoreactivity was observed in interneurons of the hippocampal CA1 sector. Furthermore, a significant decrease of PV immunoreactivity was found in interneurons of the hippocampal CA3 sector. No damage of nNOS-immunopositive interneurons was detected in the gerbil hippocampus up to 1 day after ischemia. Thereafter, a decrease of nNOS immunoreactive interneurons was found in the hippocampal CA1 sector up to 14 days after ischemia. Pitavastatin significantly prevented the neuronal cell loss in the hippocampal CA1 sector 5 days after ischemia. Our immunohistochemical study also showed that pitavastatin prevented significant decrease of PV- and nNOS-positive interneurons in the hippocampus after ischemia. Double-labeled immunostainings showed that PV immunoreactivity was not found in nNOS-immunopositive interneurons of the brain. The present study demonstrates that cerebral ischemia can cause a loss of both PV- and nNOS-immunoreactive interneurons in the hippocampal CA1 sector. Our findings also show that the damage to nNOS-immunopositive interneurons may precede the neuronal cell loss in the hippocampal CA1 sector after ischemia and nNOS-positive interneurons may play some role in the pathogenesis of cerebral ischemic diseases. Furthermore, our present study indicates that pitavastatin can prevent the damage of interneurons in the hippocampus after cerebral ischemia. Thus, our study provides valuable information for the pathogenesis after cerebral ischemia.

Effects of Chronic Administration with Nilvadipine Against Immunohistochemical Changes Related to Aging in the Mouse Hippocampus

We investigated the effect of Ca2+ antagonist nilvadipine on age-related immunohistochemical alterations in ubiquitin and S100beta protein of the hippocampal CA1 sector in mice using 8-, 18-, 40-, and 59-week-old mice. No significant changes in the number of neuronal cells were observed in the hippocampal CA1 sector up to 59 weeks after birth. The administration of nilvadipine did not affect the number of the hippocampal CA1 cells of 40-week-old mice. Age-dependent increases in ubiquitin immunoreactivity were observed in the hippocampal CA1 neurons up to 59 weeks after birth. The administration of nilvadipine prevented dose-dependently the increases in the number of ubiquitin-immunoreactive neurons in the hippocampal CA1 sector of 40-week-old mice. S100,beta immunoreactivity was unchanged in the hippocampal CA1 sector up to 40 weeks after birth. In 59-week-old mice, the level of staining of S100beta-immunoreactive cells increased significantly in the hippocampal CA1 sector. The administration of nilvadipine decreased dose-dependently the number of S 100beta-immunoreactive cells in the hippocampal CA1 sector of 40-week-old mice. The present study demonstrates that age-related increases in ubiquitin system may play a pivotal role in protecting neuronal cell damage during aging. In contrast, our results suggest that expression of S 100beta protein in the hippocampal CA1 sector may play an exacerbating factor in some neuronal cells damaged by aging. Our results also demonstrate that nilvadipine, a dihydropyridine-type calcium channel blocker, can prevent dose-dependently the increases in the ubiquitin immunoreactive neurons and decrease the number of S100beta immunoreactive cells in the hippocampal CA1 neurons of aged mice. These results suggest that nilvadipine may offer a new approach for the treatment of neuronal dysfunction in aged humans.

Effects of age on immunohistochemical changes in the mouse hippocampus

We investigated the age-related changes in neuronal cell death and synaptophysin of the hippocampal CA1 sector in mice using immunohistochemistry. Microtubule-associated protein 2a, b (MAP2) and synaptophysin immunoreactivity was measured in 2-, 8-, 18-, 42- and 59-week-old mice. MAP2 immunoreactivity was unchanged in the hippocampal CA1 sector up to 42 weeks after birth. In 59-week-old mice, however, a significant decrease in MAP2 immunoreactivity was observed in the hippocampal CA1 sector. Total number of synaptophysin-positive boutons was also unchanged in the hippocampal CA1 sector up to 42 weeks of birth. In 59-week-old mice, however, a significant increase in synaptophysin-positive boutons was observed in the hippocampal CA1 sector. These results demonstrate that dendrites and axons in the hippocampal CA1 neurons are particularly susceptible to ageing processes. In contrast, a marked increase in synaptophysin-positive boutons was found in the hippocampal CA1 sector of aged mice. These findings suggest that increase in synaptophysin-positive boutons may play a role in the maintenance of the structural components in the hippocampal CA1 sector of aged mice although most postsynaptic CA1 pyramidal neurons are generated. Thus, our findings provide further valuable information on age-related neurodegeneration and deficits in hippocampus-dependent memory and synaptic plasticity.

Induction of Neuronal Tolerance by Electroconvulsive Shock in Rats Subjected to Forebrain Ischemia

We have examined ischemic tolerance induced by electroconvulsive shock before exposure to forebrain ischemia. Subjects were 40 rats, which were randomly allocated to control, single ECS (sECS), repeated ECS (rECS) or sham group. sECS group and rECS group received ECS only once 2 days before the subsequent 8-min forebrain ischemia and once a day for 9 consecutive days until 2 days before the exposure to ischemia, respectively. Forebrain ischemia was produced by modified bilateral carotid artery occlusion technique. Control group underwent brain ischemia without ECS pretreatment. Sham group received ECS without following exposure to ischemia. Pyramidal cell injury of the hippocampal CA1 sector was microscopically examined on the 7th day after the ischemic exposure or the sham operation. Damage of the pyramidal cells was assessed by the injury ratio, which was ratio of non-viable pyramidal cells to the whole pyramidal cells. The injury ratios of CA1 pyramidal cells in sECS, rECS and control groups were 30.5 +/- 10.8 (n=10), 42.3 +/- 18.4% (n=10) and 90.4 +/- 2.9% (n=9), respectively. The injury ratios in sECS and rECS groups were lower than the ratio in control group (p<0.01), while the ratios of sECS and rECS groups were not different. The pyramidal cells in sham group were intact. Our results indicate that both preconditionings of sECS and rECS have a potency to induce delayed tolerance against temporary forebrain ischemia, though the potency was not different between sECS and rECS. Electroconvulsive shock may be added to the list of preconditioning stimuli to protect brain against ischemic neuronal damage.

Expression of estrogen receptor-β in the postischemic monkey hippocampus

The molecular basis of estrogen-mediated neuroprotection against brain ischemia remains obscure. Here, we studied by immunohistochemistry the expression of estrogen receptor (ER) α and β in the hippocampal CA1 sector of postischemic adult macaque monkeys. ERβ was present in control CA1 pyramidal neurons, decreasing on day 4 after ischemia. In contrast, ERβ immunoreactivity increased remarkably in the radiate and molecular layers of CA1, where it was present in astrocytes and microglia. ERα was negligible in both control and postischemic monkeys. These results indicate that ERβ is the major receptor responsible for the direct estrogen actions on the monkey hippocampus, regulating glial response after ischemia.