Mineralocorticoid and glucocorticoid receptor expressions in astrocytes and microglia in the gerbil hippocampal CA1 region after ischemic insult

In the present study, we observed expression and changes of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the gerbil hippocampal CA1 region, but not in the CA2/3 region, after 5 min of transient forebrain ischemia. In blood, corticosterone levels were increased biphasically at 30 min and 12 h after ischemia/reperfusion, and thereafter its levels were decreased. In the sham-operated group, MR and GR immunoreactivities were weakly detected in the CA1 region. By 3 days after ischemia, MR and GR were not significantly altered in the CA1 region: at 12 h after ischemia, GR was expressed in a few neurons in the CA1 region, whereas MR was not expressed in any neurons after ischemic insult. From 4 days after ischemia, MR and GR immunoreactivities were detected in astrocytes and microglia in the CA1 region, and at 7 days after ischemia, MR and GR immunoreactivities peaked in the hippocampal CA1 region. At this time, 55% of astrocytes and 30% of microglia showed MR immunoreactivity, and 20% of astrocytes and 40% of microglia showed GR immunoreactivity. Western blot analyses showed that the pattern of changes in MR and GR protein levels was similar to the immunohistochemical changes observed after transient forebrain ischemia. From 4 days after ischemia, MR and GR protein levels were increased time-dependently after ischemia. In conclusion, enhanced MR and GR expressions in astrocytes and microglia were detected in the hippocampal CA1 region 4-7 days after ischemia/reperfusion. At this time, GR immunoreactivity was abundant in microglia, whereas MR immunoreactivity was prominent in astrocytes. The specific distribution of corticosteroid receptors in the astrocytes and microglia may be associated with the differences of MR and GR functions against ischemic damage.     

Behavioral evaluation of ischemic damage to CA1 hippocampal neurons: effects of preconditioning

In Mongolian gerbils, global forebrain ischemia induces enhanced locomotor activity and the disruption of nest building immediately after the insult, followed by damage to hippocampal neurons developing 3 days later. Preconditioning by a brief episode of sublethal ischemia induces the protection of CA1 hippocampal neurons against a lethal ischemic insult. We examined how preconditioning with 2-min ischemia affects disturbances in the nest building behavior and locomotor activity induced by the injurious 3-min ischemia. Morphological examination confirmed that preconditioning significantly reduced neuronal damage in CA1 evoked by injurious ischemia. Behavioral studies demonstrated that preconditioning reduced the locomotor hyperactivity and latency in nest building after test ischemia, in comparison to sham or naive animals. The results indicate that the nest building test and measurement of locomotor activity may be used for an early in vivo prediction of the extent of ischemic brain damage and tolerance induced by ischemic preconditioning.     

Neural grafting to ischemic lesions of the adult rat hippocampus

Summary The purpose of this study was to examine the structural and connective integration of developing hippocampal neurons grafted to ischemic lesions of the adult rat hippocampus. The 4-vessel occlusion model was used to cause transient cerebral ischemia which damages CA1 pyramidal cells in the dorsal hippocampus, but spares nonpyramidal neurons and afferents in the area. One week later, cell suspensions were made from the CA1 region of fetal (E18-20) rats and injected stereotaxically into the lesion. The recipient brains were examined 6 weeks to 6 months later for survival, morphology, and intrinsic and extrinsic connections of the grafts. The methods used included cell stains, histochemical staining for acetylcholinesterease (AChE), immunocytochemical staining for neuropeptides (cholelecystokinin (CCK), somatostatin (SS), enkephalin (Enk) and an astrocytic marker, glial fibrillary acidic protein (GFAP), as well as tracing by retrograde axonal transport of fluorochromes and light and electron microscopy of anterograde axonal degeneration. The grafts survived well (80%) and were often quite large. They were well integrated in the lesioned host brain area, contained both pyramidal cells and neuropeptidergic neurons and displayed a near normal GFAP immunoreactivity for astrocytes. The latter contrasted the dense gliosis of the host ischemic lesion. Judged by the AChE staining the grafts were innervated by cholinergic host septohippocampal fibers. Ingrowth of host hippocampal commissural fibers was demonstrated by Fink-Heimer staining for degenerating nerve terminals following acute lesions of the hippocampal commissures. At the ultrastructural level degenerating, electron dense terminals of host commissural origin were found even deep inside the graft neuropil in synaptic contact with mainly dendritic spines. A transplant efferent connection to the host brain was demonstrated by retrograde fluorochrome tracing and consisted of a homotypic projection to more posterior levels of the ipsilateral host CA1 and subiculum. Minor abnormal, efferent projections to the host dentate molecular layer were shown in Timm staining. We conclude that fetal CA1 neurons grafted to one week old ischemic lesions of the dorsal CA1 in adult rats become structurally well incorporated and can establish nerve connections with the host brain.     

Protein disulfide isomerase immunoreactivity and protein level changes in neurons and astrocytes in the gerbil hippocampal CA1 region following transient ischemia

We investigated the temporal and spatial alterations of protein disulfide isomerase (PDI) immunoreactivity and protein level in the hippocampus proper after 5 min transient forebrain ischemia in gerbils. PDI immunoreactivity was significantly altered in the hippocampal CA1 region. PDI immunoreactivity in the sham-operated animals was found in non-pyramidal cells. At 30 min after ischemia, PDI immunoreactivity was shown in the pyramidal cells of the stratum pyramidale (SP): the PDI immunoreactivity in the pyramidal cells was increased up to 12 h after ischemia. Thereafter PDI immunoreactivity was decreased, and the PDI immunoreactivity was shown in non-pyramidal cells 2 days after ischemia. Four to 5 days after ischemia, almost pyramidal cells in the CA1 region were lost because the delayed neuronal death occurred. At this time period, PDI immunoreactivity was expressed in some astrocytes as well as some neurons. The results of the Western blot analysis were consistent with the immunohistochemical data. These findings suggest that increase of PDI in pyramidal cells may play a critical role in resistance to ischemic damage at early time after ischemic insult, and that expression of this protein in astrocytes at late time after ischemic insult is partly implicated in the acquisition of tolerance against ischemic stress.     

Very delayed neuronal loss occurs in the glomerular layer of the main olfactory bulb following transient ischemia in gerbils

Olfactory dysfunction could happen following various insults such as ischemic-hypoxic state. Neurons of the main olfactory bulb (MOB) are resistant to ischemic damage. In the present study, we investigated the ischemia-related changes of neurons and glial cells in the glomerular layer (GL) of the gerbil MOB after transient ischemia. The number of NeuN-immunoreactive neurons became to decrease from 10 days after ischemic insult. Fifteen days after ischemic insult, astrocytes and microglia were increased in number. By 60 days after ischemia, NeuN-immunoreactive neurons were significantly decreased by 42% per glomerulus. At this time period, astrocytes and microglia were pronouncedly increased. This result indicates that neuronal loss must be much delayed in the GL following transient ischemia.     

大鼠脑缺血长期存活后海马可塑性的研究

本实验试图进一步探讨经长期存活后，脑缺血及不全脑缺血大鼠海马的可塑性变化，并验证它们之间的关系。脑缺血模型采用pulsinelli四血管结扎及其改变法，苔藓纤维显示采用Timm染色法．结果显示，大鼠脑缺血20muin、再存活90d后，海马CAI区细胞几乎全部丧失，CA2／CA3也出现严重的细胞消亡，CAI区明显萎缩，多数伴有苔藓纤维的侧枝抽芽，同期不全脑缺血大鼠海马未见或仅见局限于CAI的部分细胞丢失，齿状回或CA3区也伴有苔藓纤维抽芽．提示脑缺血经长期存活后引起的苔藓纤维的侧枝抽芽并不依赖于CAI区的细胞死亡．     

DNA cleavage and proteolysis of microtubule-associated protein 2 after cerebral ischemia of different severity.

We report temporal profiles of cytoplasmic proteolysis and genomic DNA cleavage after cerebral ischemia of different severity in gerbils. Global forebrain ischemia by bilateral common carotid artery occlusion for 5 min with reperfusion, severe unilateral hemispheric ischemia by unilateral common carotid artery occlusion for 30 min with reperfusion, and complete ischemia by decapitation were used. The hippocampus was examined for proteolysis by using immunohistochemistry for microtubule-associated protein 2, DNA cleavage by using in situ nick-end labelling, and nuclear morphology by Hematoxylin staining. During evolution of delayed neuronal death after transient forebrain ischemia, loss of the immunoreaction for microtubule-associated protein 2 occurred almost in parallel with DNA cleavage in the CA1 region. In contrast, disappearance of the immunoreaction for microtubule-associated protein 2 was much faster than genomic DNA cleavage after unilateral hemispheric ischemia and reperfusion. The microtubule-associated protein 2 immunoreactivity was completely lost before development of changes in nuclear morphology or DNA cleavage after complete ischemia. The present study demonstrated the differences between necrosis and delayed neuronal death, but the nuclear morphology in the latter was not exactly the same as seen in apoptosis. Some elements of both necrotic and apoptotic machineries may work following transient ischemia, and the degree of ischemic insult may determine the character of cell death process.     

Differential regulation of cyclooxygenase-2 in the rat hippocampus after cerebral ischemia and ischemic tolerance

We investigated the temporal changes and cellular localization of cyclooxygenase-2 (COX-2) in the rat hippocampus during the induction of acquired ischemic tolerance by sublethal ischemia, and compared these changes with those occurring following transient forebrain ischemia. Adult male Sprague Dawley rats were subjected to either 10 min of lethal global ischemia with or without 3 min of sublethal ischemic preconditioning, or 3 min of ischemia only. A short (3 min) cerebral ischemia as well as lethal ischemia with preconditioning substantially and significantly upregulated COX-2 expression in dentate granule cells, as confirmed by immunoblot analysis. This became evident by 4 h, peaked at 1-3 days, and returned to the basal level around 7 days. COX-2 expression was also increased in CA2 and CA3 neurons, although with weaker staining intensity, but in CA1 neurons very weak immunoreactivity was transiently observed. In the ischemic hippocampus, however, in agreement with previous reports, COX-2 expression was induced strongly in vulnerable CA1 and hilar neurons as well as in resistant CA3 and dentate granule cells. These data demonstrated that COX-2 expression is upregulated in neuronal subpopulations destined to survive, i.e., in CA3 and dentate granule cells after ischemia and ischemia-tolerance induction, as well as in ischemia-vulnerable neurons, i.e., in CA1 neurons after lethal ischemia, suggesting that hippocampal neuronal subpopulations have differential sensitivity to COX-2 upregulation.     

Immunohistochemical expression of phospholipase C in global and focal ischemic encephalopathy in gerbil: relationship with morphological changes.

Phospholipase C (PLC) and related enzymes in signal transduction system are closely linked to cellular damage in ischemic encephalopathy. This study was undertaken to elucidate the time sequential changes of PLC isoenzymes (beta and gamma) in vulnerable areas of hippocampus in global ischemia and infarcted area in focal infarction. Mongolian gerbils were used because of their susceptibility to ischemic encephalopathy and divided into the following groups: the bilateral ischemia with various reperfusion periods group, unilateral progressive ischemia group, and focal ischemia group induced by infusion of iron particles through the femoral artery. The changes of PLC isoenzymes were observed immunohistochemically and matched with morphological changes. In the global ischemia with reperfusion group, the changes were most significant in hippocampus. Sequential changes of neurons such as red neurons at an early stage progressed to pknotic neurons at a later stage were noted with typical delayed neuronal damage in the corns ammonis (CA) 1 subfield of hippocampus. Red neurons and pyknotic neurons as well as intracytoplasmic inclusion in 3 to 24 hours of reperfusion showed loss of PLC isoenzymes as well as tubulin. The changes of PLC expression were corresponding to the degeneration of neurons with no discernible time sequential changes in remaining neurons. In the unilateral hemispheric progressive ischemia group, ischemic damage was far more marked and extensive with no selective injury pattern according to time and location. At 1 day, there was diffuse vacuolization and necrosis of neuropil with a loss of neuron. Admixed surviving neurons and vacuolated neuropil showed increased reaction to anti-PLC antibodies, which could be either an evidence of protein synthesis responding to ischemic insult or an artifactual change. Focal ischemia group showed time sequential changes of blood vessels and white blood cells with necrosis of surrounding tissue. Degenerating hippocampal neurons in infarction also showed a strong positive reaction to anti-PLC antibody, which was most likely due to condensation of cytoplasm rather than increased synthesis. This study showed different changes of PLC expression in global ischemic encephalopathy with reperfusion, progressive ischemia, and focal infarction, which suggested different pathophysiologic mechanism between these conditions.     

Expression of estrogen receptor-beta 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) alpha and beta in the hippocampal CA1 sector of postischemic adult macaque monkeys. ERbeta was present in control CA1 pyramidal neurons, decreasing on day 4 after ischemia. In contrast, ERbeta immunoreactivity increased remarkably in the radiate and molecular layers of CA1, where it was present in astrocytes and microglia. ERalpha was negligible in both control and postischemic monkeys. These results indicate that ERbeta is the major receptor responsible for the direct estrogen actions on the monkey hippocampus, regulating glial response after ischemia.     

Quantitative morphology of human hippocampus early neuron development

Previous findings in adults revealed significant hemispheric asymmetry in the size of neuronal somata in hippocampal subfield CA2 (the resistant sector) with no age‐related changes. A paucity of quantitative data on the developmental status of these protected neurons has led to the investigation of their morphology in comparison to neurons in adjacent subfield CA3, bilaterally. Bilateral coronal sections from postmortem hippocampus, 24 to 76 weeks postmenstrual age (gestational age plus postnatal age), were studied. The neurons were digitized and measured on a computer. Soma size correlated positively and significantly with age in CA2 and CA3, bilaterally. CA2 somata were significantly larger (left, 34%; right, 32%) than adjacent CA3 somata. Variability in soma form or size increased appreciably with age, in both subfields, bilaterally, while variability in soma orientation was weakly correlated with brain growth. The results suggest that in early development there are similarities in hemispheric growth patterns in CA2 and CA3. Large CA2 soma size implies axonal connectivity to distantly located targets very early in development. The results have functional implications, including memory, to brain development. Anat Rec 254:87–91, 1999. © 1999 Wiley‐Liss, Inc.     

Selective neuronal death after transient forebrain ischemia in the Mongolian gerbil: a silver impregnation study

An important feature of ischemic brain damage is the exceptional vulnerability of specific neuronal populations and the relative resistance of others. Silver impregnation was used to delineate the extent and time-course of neuronal degeneration produced by 5 min of complete forebrain ischemia in the Mongolian gerbil. Lesions were confined to four brain regions: (1) hippocampal areas CA1, CA2-CA3a and CA4; (2) the dorsomedial portion of the lateral septal nucleus; (3) the dorsolateral portion of the striatum; and (4) the somatosensory neocortex. The ischemic lesion evolved with time in all four regions, but at different rates. Somatic argyrophilia developed rapidly in the striatum and hippocampal area CA4 (maximal in 24 h or less), at intermediate rates in the somatosensory neocortex, hippocampal areas CA1a and CA2-CA3a and the lateral septal nucleus (maximal in 2 days), and slowly in hippocampal area CA1b (maximal in 3 days). These results emphasize that the extent and rate of neuronal degeneration can vary even within a presumably homogeneous neuronal population, as evidenced by the different results in areas CA1a and CA1b. Similar results were obtained from analysis of brain sections stained with Cresyl Violet, hematoxylin-eosin or hematoxylin-eosin/Luxol Fast Blue. Terminal-like silver granules were observed in the projection fields of degenerated neurons. They also appeared, however, in the perforant path terminal zone of the hippocampal dentate molecular layer 1-2 days after transient ischemia and in stratum oriens and stratum radiatum of area CA1b prior to somatic degeneration. These granular deposits could not be clearly related to the degeneration of neuronal somata. Novel findings of this study include the degeneration of some dentate basket cells and lateral septal neurons and the appearance of terminal-like argyrophilia in the hippocampal formation without any obvious relation to somatic degeneration. Some of our results lend support to the hypothesis that ischemic neuronal cell death constitutes an excitotoxic process. Other results, however, suggest that the selective vulnerability of neurons to transient ischemia must involve factors beyond excitotoxicity.     

MK-801, but not anisomycin, inhibits the induction of tolerance to ischemia in the gerbil hippocampus.

We examined whether MK-801, an N-methyl-D-aspartate (NMDA)-receptor antagonist, or anisomycin, a reversible protein synthesis inhibitor, inhibits the induction of ischemic tolerance following preconditioning with sublethal ischemia in gerbil hippocampus. Preconditioning with 2 min of ischemia, which induced heat shock protein-72 immunoreactivity, prevented hippocampal CA1 neuronal damage following 3 min of ischemia produced 3 days later. MK-801, but not anisomycin, inhibited the induction of tolerance although the heat shock protein synthesis was reduced in both groups. The present result suggests that NMDA receptor activation, causing stress response, induces the ischemic tolerance.     

Significance of nerve growth factor content levels after transient forebrain ischemia in gerbils.

Involvement of nerve growth factor (NGF) in the pathogenesis of delayed neuronal death (DND) of CA1 neurons in the hippocampus has been suggested. We measured regional changes in the content of tissue NGF of the hippocampus in the Mongolian gerbil after 5 min forebrain ischemia. The NGF content was found to decrease significantly in the CA3 and dentate regions by 32% two days after ischemia. By contrast in the CA1 region, the level of NGF became significantly elevated by 50% two weeks after ischemia or later. The early reduction of NGF content in the afferent area projecting to the CA1 sector might be primarily linked to the pathogenesis of DND, whereas the delayed increase within the CA1 sector might be a secondary local response mainly of reactive astroglia.     

Ca3 neuronal activities of dorsal and ventral hippocampus are differentially altered in rats after prolonged post-ischemic survival

The aim of the present study is to explore the potential hyper-excitability of hippocampal CA3 neurons in rats after prolonged post-ischemic survival. We conducted 15-min four-vessel-occlusion ischemic episodes in rats, allowed these animals to survive for approximately 8 months and then examined the basic morphological features and population synaptic activities of CA3 neurons. In fixed tissue sections obtained from dorsal hippocampi of post-ischemic rats, we observed a complete loss of the CA1 neurons together with a shrunken CA1 sector. Extracellular recordings in slices revealed that the overall synaptic activities of dorsal hippocampal CA3 neurons were decreased in post-ischemic rats compared with sham-operated controls. Both sham control and post-ischemic ventral hippocampal neurons were capable of exhibiting intermittent spontaneous field potentials in slices. These spontaneous field potentials spread from the CA3 to the CA1 area and their generation relied on the activity of glutamate alpha-amino-3-hydroxy-5-methyl-4 isoxazole proprionic acid (AMPA) receptors. The propensity for displaying these spontaneous field potentials appeared to be greater in post-ischemic slices than sham control slices. Our data suggest that the hyper-excitability of the post-ischemic hippocampus, if it occurs, may preferentially take place in the ventral CA3 circuitry.     

Postischemic alterations in ultrastructural cytochemistry of neuronal Golgi apparatus

Functional activity of the Golgi apparatus in postischemic neurons was evaluated by using thiamine pyrophosphatase (TPPase) activity as an histochemical marker for the trans cisternae. Ischemia was produced in rats by permanent occlusion of vertebral arteries and 30-minute occlusion of the carotid arteries. This insult produces irreversible ischemic injury to neurons in the striatum and CA1 zone of hippocampus but only reversible injury to neurons in the paramedian cortex and CA3 hippocampus. The number of neurons with TPPase activity in controls correlated in part with neuronal size and was found in greater than 90% of neurons in cortex and CA3 hippocampus, 70% in CA1 hippocampus, and 40% in striatum. Ischemia plus recirculation for 30 minutes resulted in a decrease in the number of neurons with TPPase activity by 50% in CA1 hippocampus and by 80% in the three other areas. Resistant neurons in cortex and CA3 hippocampus showed partial recovery of TPPase activity by 2 hours after ischemia although the number of neurons was still less than that in controls (55% and 72%, respectively; p less than 0.01). At 24 and 48 hours, TPPase activity in cortical and CA3 neurons was similar to controls. In contrast, irreversibly injured neurons in striatum and CA1 hippocampus showed a persistent loss of TPPase activity during the entire postischemic period. Furthermore, TPPase activity remained significantly decreased in CA1 hippocampus even though previous studies in our laboratory indicated partial recovery of Golgi cisternae before subsequent cell death at 48 to 72 hours. Since TPPase activity has been correlated with functional activity within the Golgi apparatus these results suggest that glycosylation of glycoproteins and glycolipids may be markedly impaired in neurons after cerebral ischemia. The persistent abnormalities in Golgi function may contribute to the development of irreversible injury by interfering with the normal maintenance of plasma membranes and axonal transport.     

Extrapyramidal motor symptoms versus striatal infarction volume after focal ischemia in mongolian gerbils

Few behavioral tests are available to evaluate extrapyramidal dysfunctions after focal cerebral ischemia in rodents, although extrapyramidal motor dysfunctions are often observed clinically in patients with cerebral infarction. We evaluated the methamphetamine (MP)-induced rotation test for the detection and quantification of extrapyramidal motor dysfunction induced by striatal infarction in gerbils after focal cerebral ischemia. Mongolian gerbils (n=79) underwent the left common carotid artery occlusion (CCAO) for 10, 15, or 20 min. Spontaneous and MP-induced rotation tests were repeated postischemia, and the results compared with the extent of ischemic tissue injury. The density of dopaminergic neurons immunostained with a tyrosine hydroxylase antibody in the substantia nigra pars compacta (SNpc) also was measured. Histological examination revealed selective neuronal death of the hippocampal cornu ammonis 1 (CA1) sector in 10-min CCAO animals, infarction confined to the striatum and hippocampal neuronal death in 15-min CCAO animals, and widespread hemispheric infarction in 20-min CCAO animals. Dopaminergic neurons in the SNpc were preserved in 10- and 15-min CCAO animals but were significantly reduced in 20-min CCAO animals. In MP-induced rotation tests, 15-min CCAO animals showed biased rotation ipsilateral to the lesioned side. Biased rotation persisted 4 weeks postischemia, and the number of rotations significantly correlated with the regional infarction volume of the striatum. Twenty-minute CCAO animals showed biased rotation contralateral to the lesioned side; rotation number was not correlated with the infarction volume. Our results show that biased rotation behavior is a sensitive parameter of the extent of striatal injury after focal cerebral ischemia provided the lesion is not extended to the ipsilateral cortex. MP-induced rotation in rodents probably coordinates with the extrapyramidal motor dysfunction after striatal infarction in patients with vascular Parkinsonism.     

Changes of pyridoxal kinase expression and activity in the gerbil hippocampus following transient forebrain ischemia

In the previous study, we observed chronological alterations of glutamic acid decarboxylase (GAD), which is the enzyme converting glutamate into GABA. GAD isoforms (GAD65 and GAD67) differ substantially in their interactions with cofactor pyridoxal 5'-phosphate, which is catalyzed by pyridoxal kinase (PLK). In the present study, we examined the chronological changes of PLK expression and activity in the hippocampus after 5 min transient forebrain ischemia in gerbils. PLK immunoreactivity in the sham-operated group was detected weakly in the hippocampus. Ischemia-related change of PLK immunoreactivity in the hippocampus was significant in the hippocampal cornu ammonis (CA1)region, not in the hippocampal CA2/3 region and dentate gyrus. PLK immunoreactivity was observed in non-pyramidal GABAergic neurons at 30 min to 3 h after ischemic insult. At 12 h after ischemic insult, PLK immunoreactivity was shown in many CA1 pyramidal cells as well as some non-pyramidal cells. At this time point, PLK immunoreactivity and protein content was highest after ischemia. Thereafter, PLK immunoreactivity and protein content is decreased time-dependently by 4 days after ischemic insult. Four days after ischemia, some astrocytes expressed PLK in the CA1 region. The specific PLK activity was not altered following ischemic insult up to 2 days after ischemic insult. Thereafter, the specific PLK activity decreased time-dependently. However, total activity of PLK was significantly increased 12-24 h after ischemic insult, and thereafter total activity of PLK decreased. Therefore, we suggest that the over-expression of PLK in the CA1 pyramidal cells at 12 h after ischemia may induce increase of GAD in the CA1 pyramidal cells, which plays an important role in delayed neuronal death via the increase of GABA or enhancement of GABA shunt pathway.     

Environmental enrichment enhances recovery of function but exacerbates ischemic cell death.

Prior exposure to brief 'conditioning' episodes of ischemia protects hippocampal CA1 neurons against a subsequent more severe ischemic insult. However, protected cells exhibit abnormal function and as survival times are extended this ischemic tolerance dissipates and cells begin to die. In this study, we sought to determine whether environmental enrichment could alter the above pattern of delayed cell death and functional impairment in a gerbil model of ischemic tolerance. Gerbils received either ischemic preconditioning, 5 min of ischemia without preconditioning or sham surgery. Three days after ischemia, gerbils were placed in either an enriched environment or standard laboratory housing. Open field habituation was assessed 3, 7, 10, 30 and 60 days after ischemia. Subsequently, animals were trained in two versions (win-shift and win-stay) of a T-maze task. Following behavioral testing, extracellular CA1 field potential amplitudes and CA1 cell counts were determined. Initial open field activity was significantly higher in all experimental groups compared to sham animals (P<0.001). By 60 days, enriched ischemic preconditioned and enriched ischemic gerbils were not different than shams whereas non-enriched, ischemic preconditioned and ischemic gerbils continued to have higher activity scores (P<0.05). Preconditioned and enriched ischemic animals learned the win-shift T-maze problem as quickly as shams while non-enriched ischemic gerbils were severely impaired compared with all other groups (P<0.001). Only the sham and enriched preconditioned groups readily acquired the win-stay paradigm. CA1 field potential amplitudes were lower (P<0.05) in ischemic than sham gerbils irrespective of treatment. Surprisingly, CA1 cell counts were significantly lower (P<0.01) in enriched versus non-enriched ischemic preconditioned animals.These data demonstrate that early, intensive intervention after ischemia can improve functional outcome but that this is accompanied by increased brain damage. Careful consideration needs to be given to the timing of rehabilitation after stroke and related types of brain injury.