Enhanced post-ischemic neurogenesis in aging rats.

Hippocampal neurogenesis persists in adult mammals, but its rate declines dramatically with age. Evidence indicates that experimentally-reduced levels of neurogenesis (e.g., by irradiation) in young rats has profound influence on cognition as determined by learning and memory tests. In the present study we asked whether in middle-aged, 10- to 13-months-old rats, cell production can be restored toward the level present in young rats. To manipulate neurogenesis we induced bilateral carotid occlusion with hypotension. This procedure is known to increase neurogenesis in young rats, presumably in a compensatory manner, but until now, has never been tested in aging rats. Cell production was measured at 10, 35, and 90 days after ischemia. The results indicate that neuronal proliferation and differentiation can be transiently restored in middle-aged rats. Furthermore, the effects are more pronounced in the dorsal as opposed to ventral hippocampus thus restoring the dorso-ventral gradient seen in younger rats. Our results support previous findings showing that some of the essential features of the age-dependent decline in neurogenesis are reversible. Thus, it may be possible to manipulate neurogenesis and improve learning and memory in old age.

Post-ischemic hypothermia attenuates loss of the vascular basement membrane proteins, agrin and SPARC, and the blood-brain barrier disruption after global cerebral ischemia.

Vascular basement membrane (BM) stabilizes brain vessels and inhibits endothelial cell cycle. Cerebral ischemia causes BM breakdown with the loss of structural BM components including collagens and laminins. In this study, the expression changes of the BM proteoglycan agrin, and the non-structural BM constituent SPARC (BM-40, osteonectin), were studied in brain vessels after global cerebral ischemia. A transient 20-min forebrain ischemia followed by 1, 6 or 24 h of reperfusion was induced in adult Sprague-Dawley rats by combined bilateral common carotid artery occlusion and hypotension (42-45 mm Hg). In a separate group of animals, a mild (32 degrees C) post-ischemic hypothermia was induced for 6 h, starting immediately after ischemia. RNA from approximately 500 brain vessels (20-100 microm) extracted by laser-capture microdissection (LCM) microscopy was used to determine the expression of proteoglycans agrin and SPARC mRNAs by quantitative PCR (Q-PCR). Protein expression was determined by immunohistochemistry in adjacent tissue sections. The BBB permeability was assessed using (3)H-sucrose as an in vivo tracer and by examining fibrinogen immunoreactivity in tissue sections. A transient global brain ischemia resulted in a significant (ANOVA, p<0.05; 6 animals/group) reduction in agrin and SPARC mRNAs in LCM-captured brain vessels 24 h after reperfusion. A time-dependent loss of agrin and SPARC from the BM during reperfusion was also observed by immunochemistry. A 6-h post-ischemic hypothermia reduced SPARC and agrin mRNA and protein losses, BBB transfer constant for (3)H-sucrose as well as fibrinogen extravasation 24 h after reperfusion. It is conluded that a transient post-ischemic hypothermia stabilizes brain vessels and reduces BBB disruption in part by preventing proteolytic degradation of regulatory BM constituents, SPARC and agrin.

Graded reversible opening of the rat blood-brain barrier by intracarotid infusion of sodium caprate.

The fatty acid salt, sodium caprate (C10) is a well recognized drug absorption enhancer in intestine because of its ability to widen tight junctions in the epithelial cell lining. Caprate's potential usefulness to similarly alter the blood-brain barrier (BBB) tight junctions of brain vasculature and enhance CNS drug delivery has undergone little investigation. Adult SD rats were anesthetized and C10 was infused into the left internal carotid artery (dosing parameters: 10-30 mM, 1 or 2 ml min(-1), for 0.5-1.5 min). Beginning 5 or 60 min after infusion an i.v. bolus of [3H]mannitol was allowed to circulate for 30 min and degree of BBB leakiness measured as magnitude of the transfer constant (Ki, nl g(-1)s(-1)) for blood to brain mannitol permeation determined from brain and plasma samples. In initial experiments identical C10 infusions caused dramatic BBB opening in some rats, e.g., 10-fold increase in Ki, but not in others. Higher dosing produced consistent opening measured 5-35 or 60-90 min post-infusion but was also toxic as shown by severe brain edema and cardio-respiratory failure. The variable effect of moderate doses was attributed to the fact that arterial blood pressure markedly increased during C10 infusion and may have altered the flow dynamics of cerebrovascular caprate distribution from rat to rat. We modified the procedure by temporarily withdrawing blood to produce hypovolemia and systemic arterial hypotension during C10 infusion. Caprate infusions of 15-25 mM, 2 ml min(-1) for 1 min, produced reliable dose-related openings that lasted as much as an hour, were reversible, and accompanied by little or moderate edema, depending on dose. These findings confirm an earlier report showing that intracarotid caprate infusion opens the BBB but also show that control of the temporary hypertensive response produced by intracarotid caprate infusion is key to tailoring the dosage to consistently achieve graded, reversible BBB opening.

Synaptic activity and triphenyltetrazolium chloride metabolism are correlated after oxygen-glucose deprivation in acute, but not cultured, hippocampal slices.

The importance of the hippocampus to learning and memory has attracted significant attention to how the structure responds to damage. Although many studies have used either the acute hippocampal slice preparation or organotypic hippocampal slice cultures, little work has been done to determine if the choice of model is an important variable. The present study examined whether differences exist in how each model responds to a commonly studied ischemic-like paradigm, oxygen-glucose deprivation. Following the insult, synaptic activity was examined by recording orthodromically evoked CA1 subfield responses, while mitochondrial activity was assessed by spectrophotometric measurement of formazan produced by metabolism of 2,3,5-triphenyltetrazolium chloride. The insult significantly decreased both synaptic and mitochondrial activity within acutely prepared slices, but a disparity existed between these measures in cultured slices. While evoked activity was greatly reduced by an insult of moderate duration, a much longer period was required to cause a comparable decrease in formazan production. Quantitative immunoblotting revealed that one possible explanation for the discrepancy was an elevated expression of astrocytes, which display resistance to hypoxia-aglycemia. Our data indicate that acutely prepared and cultured slices respond differently to ischemic-like challenge; therefore, assays examining viability in these models must consider their innate differences.

Cerebral ischemia induces neuronal expression of novel VL30 mouse retrotransposons bound to polyribosomes.

Mammalian genomes are burdened with a large heterogeneous group of endogenous replication defective retroviruses (retrotransposons). Previously, we identified a transcript resembling a virus-like 30S (VL30) retrotransposon increasing in mouse brain following transient cerebral ischemia. Paradoxically, this non-coding RNA was found bound to polyribosomes. Further analysis revealed that multiple retrotransposon species (BVL-1-like and mVL30-1-like) were bound to polyribosomes and induced by ischemia. These VL30 transcripts remained associated with polyribosomes in the presence of 0.5 M KCl, indicating that VL30 mRNA was tightly associated with ribosomal subunits. Furthermore, the profile of BVL-1 distribution on polyribosomal profiles was distinct from those of translated and translationally repressed mRNA. Consistent with expectations, 5.0 kb VL30 transcripts were detected in ischemic brain with a temporal pattern of expression that was distinct from c-fos. Expression of VL30 was localized in neurons using a combination of in situ hybridization and immunocytochemistry. 3'-RACE-PCR experiments yielded two unique sequences (VL30x-1 and VL30x-2) that were homologous to known VL30 genes. Phylogenetic analysis of VL30 promoter sequence (U3 region) resulted in the identification of two large VL30 subgroups. VL30x-1 and VL30x-2 were closely related and classified in a group that was distinct from the well-characterized VL30 genes BVL-1 and mVL30-1. The promoter regions of VL30x-1 and VL30x-2 did not possess the consensus sequences for either hypoxia or anoxia response elements, suggesting an alternative mechanism for induction. This is the first report that demonstrates ischemia-induced, neuronal expression of unique VL30 retrotransposons in mouse brain.

Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia.

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-D-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.

Electrophysiological correlates of neural plasticity compensating for ischemia-induced damage in the hippocampus.

Injury to the brain often results in loss of synapses or cell death in the damaged area. Subsequent to the injury, the areas that are not directly affected often exhibit enhanced neuronal plasticity. Although there are many reports of morphological changes resulting from such plasticity, their functional consequences are poorly understood. In this study we examined electrophysiological changes associated with ischemia-induced neurogenesis in the hippocampus, a brain region that is particularly vulnerable but also exceptionally plastic. Transient global ischemia was induced in Sprague-Dawley rats by occlusion of both carotid arteries and a reduction in blood pressure for 12 min. The procedure resulted in delayed cell death in the CA1 field of the hippocampus while the dentate gyrus (DG) was spared. To assess neurogenesis and synaptic changes in parallel we used both hemispheres from each animal. One side was used for immunohistochemistry and the other for in vitro electrophysiological experiments in brain slices. Synaptic field responses and synaptic plasticity (LTP) in perforant path within the DG were reduced by 50% at 10 days after the ischemic injury but recovered at 35 days. Synaptic responses in non-neurogenic CA1 were abolished in parallel with cell death and did not recover. Gamma irradiation applied focally to the head selectively prevented neurogenesis and the synaptic recovery in the DG. These experiments reveal electrophysiological changes associated with reactive neural plasticity in the hippocampus.

A two-hour window for hypothermic modulation of early events that impact delayed opening of the rat blood-brain barrier after ischemia.

Opening of the blood-brain barrier (BBB) and consequent edema are known to intensify 24-72 h after ischemic stroke, and research on potential ameliorative therapies in animal models may lead to improved clinical treatments to prevent brain swelling and the secondary damage it causes. In this study, post-ischemic hypothermia treatment, which is an established neuroprotective strategy, was examined for its ability to prevent delayed BBB opening in a rat model of global ischemia. Anesthetized, normothermic SD rats (340-380 g) underwent 20 min of two-vessel (carotid) occlusion plus hypotension (2VO ischemia, between 0900-1100 h). Marked cortical BBB leakiness, which developed overnight, was indicated at sacrifice 24 h post-2VO by an average six- to eightfold increase above baseline in transfer constant values (K(i) ) for rate of blood to brain diffusion of intravenously delivered [(3)H]sucrose. A post-2VO treatment involving whole body cooling to 31.5 degrees-32.5 degrees C, maintenance for 6 h and rewarming to normothermia, significantly reduced BBB leakiness at 24 h, whether cooling was initiated immediately after reperfusion, or after a 1-h delay, but not after 2-h delay. Immediate hypothermia treatment reduced overall tissue injury at 24 h as evidenced by an assay of mitochondrial succinate dehydrogenase activity, and also reduced brain edema. By contrast, treatment of rats with the anti-inflammatory drugs cyclosporine A or minocycline offered no protection of BBB or mitochondria. It is concluded that hypothermic alteration of critical events during the first 2 h after prolonged ischemia powerfully mitigates the BBB damage and associated events that normally develop 24 h later.

Translation-state analysis of gene expression in mouse brain after focal ischemia.

Confounding any genome-scale analysis of gene expression after cerebral ischemia is massive suppression of protein synthesis. This inefficient translation questions the utility of examining profiles of total transcripts. Our approach to such postischemic gene profiling in the mouse by microarray analysis was to concentrate on those mRNAs bound to polyribosomes. In our proof-of-principle study, polysomally bound and unbound mRNAs were subjected to microarray analysis: of the 1,161 transcripts that we found to increase after ischemia, only 36% were bound to polyribosomes. In addition to the expected increases in heat-shock proteins and metallothioneins, increases in several other bound transcripts involved in the promotion of cell survival or antiinflammatory behavior were noted, such as CD63 (Lamp3), Lcn2 (lipocalin-2), Msn (moesin), and UCP2 (uncoupling protein 2), all of which showed increases in cognate protein by Western blotting. The list of heretofore nonfunctionally annotated transcripts (RIKEN clones/ESTs) that increased appeared to be novel. How some transcripts are selected in ischemic brain for translation into protein, while others are rejected, is not clear. The length of the 5'-UTR in the ischemically induced transcripts that occur in the NCBI RefSeq database did not indicate any general tendency to be more than 200 nt, nor to be longer than the 5'-UTRs of the unbound transcripts. Thus, the presence of a complex 5'-UTR region with internal ribosome entry sites (IRES) or polypyrimidine tracts (TOP) does not appear to be the basis of selection for translation in ischemic brain.

Absence of the transcription factor E2F1 attenuates brain injury and improves behavior after focal ischemia in mice.

Because of observations that cultured neurons from mice deficient in the transcription factor E2F1 exhibit resistance after treatment with a wide variety of cell-death inducers, the authors investigated whether resistance extended to a cerebral ischemic insult. No differences in cerebral blood flow or physiologic parameters were observed in the mutant E2F1 littermates after the focal ligation. After 2 hours of left middle cerebral artery occlusion and 1 day of reperfusion, a 33% smaller infarct (P < 0.05) was observed by 2,3,5-triphenyltetrazolium staining in the brains of E2F1-null mice compared with their E2F1+/+ and +/- littermates. A milder ischemic insult produced by 20 minutes of middle cerebral artery occlusion and 7 days of reperfusion produced a greater difference in the E2F1-null animals with a 71% smaller infarct (P < 0.001) compared to littermate controls. A decrease in neuronal damage after mild ischemia in E2F1-null mice was observed by immunohistochemical monitoring of the loss in neuronal-specific microtubule-associated protein 2 cytoskeletal protein and the appearance of nuclear DNA fragmentation by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling. This decreased brain damage was evidenced by improved behavior in motor function of E2F1 -/- mice compared with their E2F1 +/+ littermates by 7 days of reperfusion. In an effort to address the underlying molecular mechanism of the resistance of E2F1-null mice, the expression of several downstream proapoptotic target genes (p73, Apaf1, Arf) of the E2F1 transcription factor was measured by quantitative polymerase chain reaction. Although an attenuated increase in Hsp68 mRNA was found in E2F1 -/- mice, no changes in the proapoptotic transcripts were found after ischemia, and a mechanistic inference was not possible. The authors conclude that the transcription factor E2F1 does modulate neuronal viability in brain after cerebral ischemia and corroborates the findings with cultured neurons.

Differential passage of [14C]sucrose and [3H]inulin across rat blood-brain barrier after cerebral ischemia.

The biophysical nature of blood-brain barrier (BBB) opening after ischemic or hemorrhagic stroke or traumatic brain injury is unresolved. Ultrastructural (electron micrograph) investigations of experimental BBB injury commonly indicate the abnormal presence of vesicles or tubular structures in cerebrovascular endothelial cells, suggesting the likelihood of convective, fluid-phase transport of blood substances into brain. We measured transfer constants (K(i)s) for the simultaneous passage of two intravenously delivered tracers ([14C]sucrose, mol wt=342; [3H]inulin approximately 5,000) across the intact BBB in the rat, and 24 h after global cerebral ischemia (16-20 min duration) or 24, 48 or 72 h after focal ischemia (2 h duration). In both ischemia models, the upward increment in K(i) (DeltaK(i)) for sucrose, indicating the extra injury-related tracer flux into brain, significantly exceeded that for inulin, as might be expected with faster diffusion of the smaller molecule through injury pores or channels. This inequality of DeltaK(i)s did not suggest a major role for convective, fluid-phase transport by endothelial vesicular or tubular structures and a predominance of diffusional transport was indicated.