Increased neuronal damage in apolipoprotein E-deficient mice following global ischaemia

There is accumulating evidence that apolipoprotein E (apoE) plays a role in regulating the response to and outcome following brain injury. The present study compared the histological outcome and recovery following an episode of global ischaemia in apoE-deficient mice and wild-type littermates (12-week-old males, n = 8 per group). Transient global ischaemia was induced for a period of 17 min and the animals were allowed to recover for 72 h. Transient global ischaemia induced selective neuronal degeneration in several brain regions in wild-type mice. There was statistically significant increased ischaemic neuronal damage in apoE-deficient mice compared with wild-type mice in six of the seven regions examined (hippocampal regions CA1, CA3/CA4 and dentate gyrus; thalamus; cortex and caudate nucleus; P < 0.05). The data substantiate a role for apoE in modifying the response of the CNS to acute injury.     

Increased neuronal damage and apoE immunoreactivity in human apolipoprotein E, E4 isoform-specific, transgenic mice after global cerebral ischaemia

Apolipoprotein E (apoE, protein; APOE, gene) is expressed as three isoforms in humans (E2, E3, E4). The APOE-epsilon4 allele is associated with a poor outcome in patients after head injury of which ischaemic brain damage is a contributor of mortality and morbidity. The aim of the study was to determine whether mice expressing human APOE-epsilon4 displayed more extensive ischaemic neuronal damage 72 h after transient global ischaemia compared with mice which express human APOE-epsilon3. APOE-epsilon3 and -epsilon4 transgenic mice, under the control of a human promoter, were used which express human APOE in neurons and glia. Ischaemic neuronal damage in the CA1 pyramidal cell layer in the APOE-epsilon4 transgenic mice was significantly greater than in the APOE-epsilon3 mice after global ischaemia (36.4+/-8.9%, 18.2+/-7.3%; P<0.05). This was associated with more extensive neuronal apoE immunoreactivity in the CA1 pyramidal cell layer in the APOE-epsilon4 transgenic mice compared with APOE-epsilon3 transgenic mice. In contrast, in the caudate nucleus, there were similar levels of ischaemic neuronal damage in the APOE-epsilon3 and -epsilon4 transgenic mice (39.2 +/-10.1%; 44.6+/-8.4%, P = 0.32). In the caudate, similar numbers of neurons were immunostained for apoE in the APOE-epsilon3 and -epsilon4 transgenic mice. The present study demonstrated that the APOE-epsilon4 allele is associated with an increased vulnerability of a specific brain region to the effects of global ischaemia, which is closely associated with an increase in neuronal apoE. The data extend previous work and are consistent with an association of the APOE-epsilon4 allele with a poor outcome after acute brain injury in humans.     

Marked alterations in the cellular localisation and levels of apolipoprotein E following acute subdural haematoma in rat

Apolipoprotein E (apoE) plays a role in the response to acute brain injury, the mechanisms as yet remain unknown. In the present study, alterations in the immunohistochemical localisation of apoE in rat cortex were examined at 30 min. 2 h or 4 h following production of an acute subdural haematoma. Levels of apoE were determined in cortex by immunoblotting at 30 min and 4 h post-haematoma. Extensive areas of ischaemic cell damage were observed in the cortex underlying the haematoma with minimal damage observed in shams. In sham animals, apoE immunoreactivity was confined to astrocytes and their processes. Following the haematoma induction, apoE immunoreactivity was dramatically altered. At 30 min post-haematoma, intense apoE staining was observed in clusters of neuronal perikarya and the neuropil throughout the cortical layers underlying the haematoma and this persisted at 2 h and 4 h post-haematoma. Additionally, at 4 h post-haematoma marked apoE staining of discrete foci within the neuropil closely associated with capillaries was consistently observed in the ipsilateral cortex. Immunoblotting indicated there were no significant alterations in the cortical levels of apoE at 30 min post-haematoma but, at 4 h post-haematoma, there was a significant elevation (27%, P < 0.001) in the levels of apoE in cortex underlying the haematoma compared to control levels. The results indicate that following acute subdural haematoma, a rapid cellular redistribution of apoE occurs and precedes a significant elevation in the levels of apoE. These alterations in apoE may occur, at least initially, as part of the brain's protective response to injury.     

Ischemia-related changes of glial-derived neurotrophic factor and phosphatidylinositol 3-kinase in the hippocampus: their possible correlation in astrocytes

In the present study, we observed the changes of endogenous expression of glial-cell-line-derived neurotrophic factor (GDNF) and phosphatidylinositol 3-kinase (PI-3 kinase) in the gerbil hippocampus after transient forebrain ischemia and investigated the correlation between GDNF and PI-3 kinase in the ischemic hippocampus. In the sham-operated group, GDNF and PI-3 kinase immunoreactivity was not found in any cells in the hippocampal CA1 region. GDNF, not PI-3 kinase, immunoreactivity was expressed in non-pyramidal cells in the CA1 region at 6 h after ischemic insult. At 12-24 h after ischemia, GDNF and PI-3 kinase immunoreactivity in the CA1 region was similar to that of the sham-operated group. From 2 days after ischemic insult, GDNF- and PI-3-kinase-immunoreactive astrocytes were detected in the CA1 region, and GDNF and PI-3 kinase immunoreactivity in astrocytes was highest in the CA1 region 4 days after ischemic insult. Moreover, at this time point, GDNF and PI-3 kinase were co-localized in some astrocytes. Western blotting showed that ischemia-related changes of GDNF and PI-3 kinase protein levels were similar to the immunohistochemical changes after ischemia. These results suggest that GDNF and PI-3 kinase may be related to delayed neuronal death and that GDNF and PI-3 kinase may be involved in activation of astrocytes.     

Alz-50 and ubiquitin immunoreactivity is induced by permanent focal cerebral ischaemia in the cat

The effects of permanent focal cerebral ischaemia on Alz-50 and ubiquitin antibody immunohistochemical staining were investigated in vivo in the cat. Alz-50 and ubiquitin antibody staining was compared to the distribution of ischaemic cell damage. Six hours following permanent occlusion of one middle cerebral artery, Alz-50 immunoreactivity was present in neurones in the ipsilateral ischaemic cerebral cortex and caudate nucleus but not in any region of the contralateral hemisphere or in sham-operated cats. Only a proportion of neurones were stained with Alz-50 and these did not have the shrunken, pyknotic appearance characteristic of irreversible ischaemic cell damage. Ubiquitin immunoreactivity was also increased in the ischaemic hemisphere, again only a proportion of neurones were stained. The Alz-50 antibody recognises the microtubule-associated protein tau and stains neurofibrillary tangles as well as neurones vulnerable to neurofibrillary change in tissue sections of Alzheimer brain. The results indicate that there are changes in tau protein in response to an ischaemic insult, but only in some neurones, which may reflect an early stage of the degenerative process. Increased ubiquitin immunoreactivity may be a response to the presence of abnormal proteins, including tau, which are induced by an ischaemic challenge.Supported by the Wellcome Trust     

Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA‐074: a novel strategy for neuroprotection based on 'calpain–cathepsin hypothesis'

Although Cornu Ammonis (CA) 1 neurons of the hippocampus are known to be vulnerable to transient ischaemia, the mechanism of ischaemic neuronal death is still unknown, and there are very few strategies to prevent neuronal death at present. In a previous report we demonstrated μ-calpain activation at the disrupted lysosomal membrane of postischaemic CA1 neurons in the monkey undergoing a complete 20 min whole brain ischaemia. Using the same experimental paradigm, we observed that the enzyme activity of the lysosomal protease cathepsin B increased throughout the hippocampus on days 3–5 after the transient ischaemia. Furthermore, by immunocytochemistry cathepsin B showed presence of extralysosomal immunoreactivity with specific localization to the cytoplasm of CA1 neurons and the neuropil of the vulnerable CA1 sector. When a specific inhibitor of cathepsin B, the epoxysuccinyl peptide CA-074 (C₁₈H₂₉N₃O₆) was intravenously administered immediately after the ischaemic insult, ≈ 67% of CA1 neurons were saved from delayed neuronal death on day 5 in eight monkeys undergoing 20 min brain ischaemia: the extent of inhibition was excellent in three of eight and good in five of eight monkeys. The surviving neurons rescued by blockade of lysosomal activity, showed mild central chromatolysis and were associated with the decreased immunoreactivity for cathepsin B. These observations indicate that calpain-induced cathepsin B release is crucial for the development of the ischaemic neuronal death, and that a specific inhibitor of cathepsin B is of potential therapeutic utility in ischaemic injuries to the human CNS.     

The alteration of corticotropin-releasing factor (CRF) receptor immunoreactivity in the gerbil hippocampus and neocortex following ischemic insults

Recently, we suggested that the ectopic expression of corticotropin-releasing factor (CRF) is associated with processes linked to neuronal injury and/or degeneration in response to an ischemic insult. However, little experimental data currently links the CRF receptor directly to neuronal death induced by ischemia. Therefore, in the present study, we investigated the temporal and spatial changes in CRF receptor immunoreactivity in the hippocampus and the neocortex after transient ischemia. CRF receptor immunoreactivity in the hippocampus was reduced up to 24h after ischemia insult, as compared to the sham. Interestingly, CRF receptor immunoreactivity disappeared in the CA1 region of the hippocampus at 4 days in the post-ischemic group. The other regions of hippocampus maintained their immunoreactivities at this time point. On the other hand, in the neocortex, 3h after transient ischemia, the CRF receptor immunoreactivity was elevated in regions vulnerable to ischemia. At 12h post-ischemia, its immunoreactivity had decreased versus the sham operated animals. These results suggest that the selectively ectopic expression of CRF following ischemia, which we reported previously, may regulate inflammatory responses. In addition, these findings also suggest that the mechanisms of neuronal death as mediated by CRF receptor differ in the hippocampus and the neocortex.     

Consequences of Transient Focal Cerebral Ischaemia for Second Messenger and Neurotransmitter Binding in the Rat: Quantitative Autoradiographic Analysis of Forskolin, Dopamine D1 Receptor Binding and Cerebral Blood Flow Changes

In order to study the consequences of reperfusion for ischaemic brain injury, quantitative ligand binding autoradiography was carried out in a model of reversible focal cerebral ischaemia. Endothelin-1 applied to the abluminal surface of the middle cerebral artery in anaesthetized Sprague-Dawley rats induced severe focal ischaemia and subsequent reperfusion (assessed by blood flow tracers [^99mTc]HMPAO and [¹⁴C]iodoantipyrine respectively) by 2 h after insult. Ligand binding autoradiography on consecutive sections demonstrated these blood flow changes to be associated with a significant reduction in forskolin binding throughout the middle cerebral artery territory (e.g. 25% in parietal cortex, 11% in dorsolateral caudate nucleus). The most marked losses in forskolin binding were in areas where ischaemia was severe and reperfusion was poor. However, the same changes in cerebral blood flow had no significant effect on D₁ dopamine receptor binding (e.g. >2% reduction in the caudate nucleus). These data demonstrate that ligand binding characteristics are significantly affected as early as 2 h after insult, with evidence of differential sensitivity for forskolin and D₁ dopamine binding. With regard to the consequences of reperfusion, comparison with our previous study of 2 h maintained ischaemia demonstrates reperfusion-related salvage of dopamine and forskolin binding in the caudate nucleus but possible exacerbation of forskolin binding loss in the cortex.     

Influence of apolipoprotein E genotype on neuronal damage and apoE immunoreactivity in human hippocampus following global ischemia

Apolipoprotein E (apoE) influences the response to and outcome from brain injury possibly through alterations in neuronal repair mechanisms. This study aimed to determine alterations in neuronal and glial apoE after brain injury in patients and sought to determine whether possession of an apoE-epsilon4 allele influences the degree of apoE immunoreactivity or the degree of neuronal damage following brain injury. ApoE immunoreactivity and neuronal damage were semiquantitatively assessed in the temporal lobe of a group of controls (n = 44) and in a group of patients who had an episode of global ischemia and subsequently died (n = 58, survival ranged from 1 hour to 3 months). There was a significant degree of neuronal damage in all hippocampal sectors and in the neocortex of the global ischemia group compared with controls (p < 0.0001). Glial apoE immunoreactivity was significantly increased in hippocampal sectors (CA1, CA2, CA3/CA4, dentate fascia) in the global ischemia group compared with controls (p < 0.01). Neuronal apoE immunoreactivity was significantly increased in all hippocampal sectors (CA1, CA2, CA3/CA4, dentate fascia) and in the neocortex of the global ischemia group compared with controls (p < 0.0001). There was a significant and positive association between the degree of neuronal apoE immunoreactivity and the degree of neuronal damage in the global ischemia cases (r2 = 0.691, p < 0.001) and there was not an association in the control group. Possession of an apoE-epsilon4 allele did not influence the degree of neuronal or glial apoE immunoreactivity or the degree of neuronal damage in the global ischemia cases or the controls. The data indicate apoE is markedly increased in neurons and glia following brain injury. In this study, apoE genotype did not appear to influence neuronal damage, glial apoE or intraneuronal apoE following injury     

Changes in immunoreactivity of HSP60 and its neuroprotective effects in the gerbil hippocampal CA1 region induced by transient ischemia

Heat shock proteins act as molecular chaperones and are involved in protein folding, refolding, transport, and translocation. In the present study, we observed changes in heat shock protein 60 (HSP60) immunoreactivity and protein level in the gerbil hippocampal CA1 region after 5 min of transient forebrain ischemia and its neuroprotective effect against ischemic damage. HSP60 immunoreactivity in the CA1 region began to increase in the stratum pyramidale at 30 min after ischemia/reperfusion, and peaked 24 h after ischemia/reperfusion. Thereafter, HSP60 immunoreactivity was decreased in the CA1 region with time. Seven days after ischemia/reperfusion, HSP60 immunoreactivity was increased again in the CA1 region: at this time point after ischemia/reperfusion, HSP60 immunoreactivity was expressed in glial cells in the ischemic CA1 region. HSP60 immunoreactive glial cells were astrocytes containing glial fibrillar acidic protein. In contrast, change in HSP60 immunoreactivity in the ischemic CA2/3 region was not significant compared with that in the ischemic CA1 region. In Western blot study, HSP60 protein level in the CA1 region was increased after ischemia/reperfusion and highest 24 h after ischemia/reperfusion. Animals treated with recombinant adenoviruses expressing Hsp60 (Ad-Hsp60) showed the neuroprotection of CA1 pyramidal neurons from ischemic damage. These results suggest that HSP60 may be associated with delayed neuronal death of CA1 pyramidal neurons after transient ischemia, and the induction of HSP60 protects the neurons from ischemic damage.     

Time interval after ischaemic preconditioning affects neuroprotection and gliosis in the gerbil hippocampal CA1 region induced by transient cerebral ischaemia

Objectives: Ischaemic preconditioning (IPC) can increase ischaemic tolerance of the central nervous system (CNS) to a subsequent longer or lethal period of transient ischaemia. In this study, we examined neuroprotective effects of time intervals after IPC against ischaemic insult in the hippocampus.

Methods: Animals were randomly assigned to six groups; sham-operated-group, ischaemia-operated-group, and three IPC (12?hours, 1- and 2-day intervals after IPC) plus ischaemia-groups (IPC-12?hour, 1 and 2-day interval-ischaemia-operated-groups). For neuroprotection, we carried out cresyl violet (CV) staining neuronal nuclei (NeuN) immunohistochemistry and Fluoro-Jade B histofluorescence staining. In addition, we examined gliosis using immunohistochemistry for GFAP (a marker for astrocytes) and Iba-1 (a marker for microglia).

Results: A significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischaemia-operated-group and IPC-12?hours interval-ischaemia-operated-groups. In the IPC-1?day interval-ischaemia-operated-group, CA1 pyramidal neurons were well protected from ischaemic insult; the neuroprotective effect in the IPC-2?day interval-ischaemia-operated-group was less than that in the IPC-1?day interval-ischaemia-operated-group. On the other hand, we observed changes in glial cells (astrocytes and microglia) in the CA1 of all groups. The distribution pattern of glial cells only in the IPC-1?day interval-ischaemia-operated-group was similar to that in the sham-group.

Conclusion: In brief, our findings indicate that 1?day after IPC displays a mighty neuroprotection and shows an inhibition of glial activation in the CA1 induced by transient ischaemic insult.     

The immunoreactivity and activity of adenylate cyclase type I are changed in the hippocampal CA1 region after transient forebrain ischemia in gerbils

Adenylate cyclase (AC) has a specific sensitivity to Ca²⁺/calmodulin. AC-I, one of the mediator of learning and memory, plays an important role in signal transduction underlying learning and memory function. In the present study, we found ischemia-related changes of AC-I in the hippocampal CA1 region, but not in the CA2/3 region, after 5 min of transient forebrain ischemia in gerbils. In the sham-operated group, AC-I immunoreactive neurons were detected in pyramidal and non-pyramidal cells in the hippocampus proper. AC-I immunoreactivity was significantly increased at 3 h in the CA1 region after ischemic insult. Thereafter, AC-I immunoreactivity was gradually decreased. Four days after ischemic insult, AC-I-immunoreactive CA1 pyramidal cells in the stratum pyramidale were very few due to delayed neuronal death. The results of Western blot analysis showed that changes of AC-I protein contents were similar to immunohistochemical data after ischemic insult. Gpp(NH)p-dependent AC-I activity in hippocampal CA1 region was not changed in all groups, while Ca²⁺/calmodulin-dependent AC-I activity in hippocampal CA1 region was significantly decreased 24 h after ischemia–reperfusion. These results suggest that the decrease of AC-I activity may be associated with impairment of neurodevelopment and neuroplasticity including learning and memory although the AC-I immunoreactivity was maintained 24 h postischemic group compared to that of the sham-operated group.     

Expression and changes of Ca2+-ATPase in neurons and astrocytes in the gerbil hippocampus after transient forebrain ischemia

Ca2+-ATPase is one of the most powerful modulators of intracellular calcium levels. In this study, we focused on chronological changes in the immunoreactivity and protein levels of Ca2+-ATPase in the hippocampus after 5 min of transient forebrain ischemia. Ca2+-ATPase immunoreactivity was significantly altered in the hippocampal CA1 region and in the dentate gyrus, but not in the CA2/3 region after ischemic insult. In the sham-operated group, Ca2+-ATPase immunoreactivity was detected in the hippocampus. Ca2+-ATPase immunoreactivity in the CA1 region and in the dentate gyrus, and its protein levels peaked 3 h after ischemic insult. At this time, CA1 pyramidal cells and dentate polymorphic cells showed strong Ca2+-ATPase immunoreactivity. Thereafter, Ca2+-ATPase immunoreactivity reduced in the CA1 region and in the dentate gyrus. One day after ischemic insult, Ca2+-ATPase immunoreactivity was observed in some CA1 non-pyramidal cells, and 4 days after ischemic insult, Ca2+-ATPase immunoreactivity was detected in astrocytes throughout the CA1 region, but Ca2+-ATPase immunoreactivity in the dentate gyrus had nearly disappeared. Our results suggest that Ca2+-ATPase changes may be associated with a response to ischemic damage in hippocampal CA1 pyramidal cells, and that increased Ca2+-ATPase immunoreactivity in the reactive astrocytes may be associated with the maintenance of intracellular calcium levels.     

Antioxidant-like protein 1 is altered in non-pyramidal cells and expressed in astrocytes in the gerbil hippocampal CA1 region after transient forebrain ischemia

In the present study, we observed chronological changes of antioxidant-like protein 1 (AOP-1) in the gerbil hippocampal CA1 region after 5 min of transient forebrain ischemia using immunohistochemistry and western blot. AOP-1 was significantly altered in the CA1 region after transient ischemia. In the sham-operated group, AOP-1 immunoreactivity was detected in pyramidal and non-pyramidal cells of the CA1 region. At 30 min after ischemic insult, AOP-1 immunoreactivity and protein level was decreased in the CA1 region. At 12 h after ischemic insult, AOP-1 immunoreactivity and protein level was highest in this region. At this time, after ischemia, AOP-1 immunoreactivity in non-pyramidal cells was high compared to the sham-operated group. Based on double immunofluorescence study, AOP-1-immunoreactive neurons were identified as GABAergic, which were stained with GAD or parvalbumin. Thereafter, AOP-1 immunoreactivity and protein levels were decreased time-dependently. From 4 days after ischemic insult, AOP 1 immunoreactivity was generally expressed in astrocytes. Five days after ischemic insult, AOP-1 immunoreactivity and protein level was increased again to 1.4 folds compared to that of the sham-operated group. In brief, AOP-1 immunoreactivity was increased in GABAergic non-pyramidal cells in the hippocampal CA1 region at early time after ischemic insult and was expressed in astrocytes at late time after ischemia. This result suggests that AOP-1 may be important role in homeostasis of GABAergic neurons because these neurons are resistant to ischemic damage.     

Ischaemic pre-conditioning in organotypic hippocampal slice cultures is inversely correlated to the induction of the 72 kDa heat shock protein (HSP72)

In vivo, preconditioning with a sublethal insult can confer resistance to normally lethal episodes of cerebral ischaemia. This phenomenon has been linked with the induction of the 72 kDa heat shock protein (HSP72), but this has not been clearly demonstrated in vitro. We have used organotypic hippocampal slice cultures to investigate whether tolerance to lethal ischaemia is dependent on HSP72. Cultures were maintained in vitro for 14 days, and neuronal damage assessed using propidium iodide fluorescence. Prolonged neuronal HSP72 upregulation occurred following exposure to 30 min ischaemia, 45 min hypoxia and 1 microM kainate, but not 1 microM NMDA or 20 min ischaemia, all sublethal insults. Preconditioning with ischaemia, kainate or hypoxia 24 h prior to lethal ischaemia (45 min) was not protective, and when the delay was increased to 48 h, damage in the CA3 pyramidal cell region was significantly increased compared to cultures exposed to 45 min ischaemia alone. Preconditioning with 20 min ischaemia had no effect on the severity of ischaemic damage. Preconditioning with 1 microM NMDA significantly reduced neuronal damage produced by either 45 or 60 min ischaemia when the delay between insults was 48 h. NMDA pre-treatment also prevented neurotoxicity produced by glutamate (5-10 mM) but not NMDA (10-30 microM). These data suggest that in vitro, the increased expression of HSP72 following some sublethal insults should be considered as a marker of cell stress prejudicial to the survival of neurones subsequently exposed to ischaemia, while tolerance can be produced through mechanisms independent of HSP72 induction.     

In vivo 31phosphorus spectroscopy during transient cerebral ischaemia in the gerbil

The depletion of the high energy phosphates; phosphocreatine and ATP, during cerebral ischaemia disrupts normal cellular function and can lead to cerebral infarction. Using in vivo nuclear magnetic resonance spectroscopy; the metabolic effects of the gerbil model of transient bilateral carotid artery occlusion were quantified. By examining the changes in the inorganic phosphate (Pi), phosphocreatine (PCr) and μ-ATP peaks, the PCr/Pi ratio, the PCr/μ-ATP ratio and intracellular pH (pHi) before, during and after an ischaemic insult were calculated. Preischaemic values for these parameters were: PCr/Pi = 2.466 ± 0.130, PCr/μ-ATP = 1.691 ± 0.053, pHi = 7.112 ± 0.021. By the end of 20 min of global ischaemia, the PCr and μ-ATP peaks fell to levels similar to background in most animals. Calculated values were: PCr/Pi = 0.488 ± 0.126, PCr/μ-ATP = 1.833 ± 0.179, pHi = 6.551 ± 0.258. With reperfusion, PCr/Pi increased rapidly back towards preischaemic levels but pHi improvement was delayed 10 min after that of PCr/Pi. By 1 h of reperfusion, both PCr/Pi and pHi were statistically equivalent to preischaemic values. During ischaemia, ATP was lost more rapidly than the storage form, PCr, but recovery of both was parallel. This suggested an intact ability to store such energy. These data indicate that the gerbil brain recovers normal high energy phosphate levels within an hour following a 20 min ischaemic insult, but that initial reperfusion does not immediately correct intracellular acidosis. Such a delay may prove a useful marker of those animals with more severe ischaemic injury.     

Calpain activation and inhibition in organotypic rat hippocampal slice cultures deprived of oxygen and glucose.

It has been suggested that, after ischaemia, activation of proteases such as calpains could be involved in cytoskeletal degradation leading to neuronal cell death. In vivo, calpain inhibitors at high doses have been shown to reduce ischaemic damage and traumatic brain injury, however, the relationship between calpain activation and cell death remains unclear. We have investigated the role of calpain activation in a model of ischaemia based on organotypic hippocampal slice cultures using the appearance of spectrin breakdown products (BDPs) as a measure of calpain I activation. Calpain I activity was detected on Western blot immediately after a 1-h exposure to ischaemia. Up to 4 h post ischaemia, BDPs were found mainly in the CA1 region and appeared before uptake of the vital dye propidium iodide (PI). 24 h after the insult, BDPs were detected extensively in CA1 and CA3 pyramidal cells, all of which was PI-positive. However, there were many more PI-positive cells that did not have BDPs, indicating that the appearance of BDPs does not necessarily accompany ischaemic cell death. Inhibition of BDP formation by the broad-spectrum protease inhibitor leupeptin was not accompanied by any neuroprotective effects. The more specific and more cell-permeant calpain inhibitor MDL 28170 had a clear neuroprotective effect when added after the ischaemic insult. In contrast, when MDL 28170 was present throughout the entire pre- and post-incubation phases, PI labelling actually increased, indicating a toxic effect. These results suggest that calpain activation is not always associated with cell death and that, while inhibition of calpains can be neuroprotective under some conditions, it may not always lead to beneficial outcomes in ischaemia.     

Ischemia-related changes in naive and mutant forms of ubiquitin and neuroprotective effects of ubiquitin in the hippocampus following experimental transient ischemic damage

Ubiquitin binds to short-lived proteins and denatured proteins produced by various forms of injury. The loss of ubiquitin leads to an accumulation of abnormal proteins and may affect cellular structure and function. The aim of the present study is to observe the chronological changes in ubiquitin naive form and its mutant form (ubiquitin⁺¹) in the hippocampal CA1 region (CA1) after transient cerebral ischemia in gerbils. Delayed neuronal death in the CA1 was confirmed 4 days after ischemic insult with NeuN immunohistochemistry. Ubiquitin immunoreactivity and protein level in the CA1 were lowest at 12 h after ischemia/reperfusion; thereafter, they were increased with time. Ubiquitin⁺¹ immunoreactivity and protein levels in the CA1 were slightly decreased at 3 h after ischemia/reperfusion, and they were significantly increased 1 day after ischemia/reperfusion. In addition, ubiquitin and ubiquitin⁺¹ immunoreaction was expressed in astrocytes after delayed neuronal death in the ischemic CA1. To elucidate the protective effect of ubiquitin on ischemic damage, the animals were treated with ubiquitin (1.5 mg/kg body weight) intravenously via the femoral vein. Ubiquitin treatment significantly reduced ischemia-induced locomotor hyperactivity, neuronal death and reactive gliosis such as astrocytes and microglia. In addition, 5 days after ubiquitin treatment in the ischemic group, ubiquitin immunoreactivity was similar to that in the ubiquitin-treated sham group, however, ubiquitin⁺¹ immunoreactivity was higher than that in the ubiquitin-treated sham group. These findings indicate that the depletion of ubiquitin and the accumulation of ubiquitin⁺¹ in CA1 pyramidal neurons after transient cerebral ischemia may inhibit ubiquitin proteolytic pathway and this leads to delayed neuronal death of CA1 pyramidal neurons directly or indirectly after transient cerebral ischemia.     

In vivo 31phosphorus spectroscopy during transient cerebral ischaemia in the gerbil

The depletion of the high energy phosphates; phosphocreatine and ATP, during cerebral ischaemia disrupts normal cellular function and can lead to cerebral infarction. Using in vivo nuclear magnetic resonance spectroscopy, the metabolic effects of the gerbil model of transient bilateral carotid artery occlusion were quantified. By examining the changes in the inorganic phosphate (Pi), phosphocreatine (PCr) and beta-ATP peaks, the PCr/Pi ratio, the PCr/beta-ATP ratio and intracellular pH (pHi) before, during and after an ischaemic insult were calculated. Preischaemic values for these parameters were: PCr/Pi = 2.466 +/- 0.130, PCr/beta-ATP = 1.691 +/- 0.053, pHi = 7.112 +/- 0.021. By the end of 20 min of global ischaemia, the PCr and beta-ATP peaks fell to levels similar to background in most animals. Calculated values were: PCr/Pi = 0.488 +/- 0.126, PCr/beta-ATP = 1.833 +/- 0.179, pHi = 6.551 +/- 0.258. With reperfusion, PCr/Pi increased rapidly back towards preischaemic levels but pHi improvement was delayed 10 min after that of PCr/Pi. By 1 h of reperfusion, both PCr/Pi and pHi were statistically equivalent to preischaemic values. During ischaemia, ATP was lost more rapidly than the storage form, PCr, but recovery of both was parallel. This suggested an intact ability to store such energy. These data indicate that the gerbil brain recovers normal high energy phosphate levels within an hour following a 20 min ischaemic insult, but that initial reperfusion does not immediately correct intracellular acidosis. Such a delay may prove a useful marker of those animals with more severe ischaemic injury.