Chronic Effects of Pyridoxine in the Gerbil Hippocampal CA1 Region after Transient Forebrain Ischemia

In a previous study, we reported that the administration of pyridoxine (vitamin B₆) to mice for 3 weeks significantly increased cell proliferation and neuroblast differentiation in the dentate gyrus without any neuronal damage. In the present study, we investigated the restorative potentials of pyridoxine on ischemic damage in the hippocampal CA1 region of Mongolian gerbils. Gerbils were subjected to 5 min of transient ischemia, and surgical operation success was assessed by ophthalmoscope during occlusion of common carotid arteries and spontaneous motor activity at 1 day after ischemia/reperfusion. Pyridoxine (350 mg/kg) or its vehicle (physiological saline) was intraperineally administered to ischemic gerbils twice a day starting 4 days after ischemia/reperfusion for 30 or 60 days. The repeated administration of pyridoxine for 30 and 60 days significantly increased doublecortin-immunoreactive neuroblasts in the dentate gyrus and increased NeuN-immunoreactive mature neurons and βIII-tubulin-immunoreactive dendrites in the hippocampal CA1 region. Furthermore, brain-derived neurotrophic factor (BDNF) protein levels were significantly increased in pyridoxine-treated groups compared to those in the vehicle-treated groups. These results suggest that chronic administration of pyridoxine enhances neuroblast differentiation in the dentate gyrus and induces new mature neurons in the hippocampal CA1 region by up-regulating BDNF expression in hippocampal homogenates.     

Ischemia-Induced Changes of PRAS40 and p-PRAS40 Immunoreactivities in the Gerbil Hippocampal CA1 Region After Transient Cerebral Ischemia

Proline-rich Akt substrate of 40-kDa (PRAS40) is one of the important interactive linkers between Akt and mTOR signaling pathways. The increase of PRAS40 is related with the reduction of brain damage induced by cerebral ischemia. In the present study, we investigated time-dependent changes in PRAS40 and phospho-PRAS40 (p-PRAS40) immunoreactivities in the hippocampal CA1 region of the gerbil after 5 min of transient cerebral ischemia. PRAS40 immunoreactivity in the CA1 region was decreased in pyramidal neurons from 12 h after ischemic insult in a time-dependent manner, and, at 5 days post-ischemia, PRAS40 immunoreactivity was newly expressed in astrocytes. p-PRAS40 immunoreactivity in the CA1 pyramidal neurons was hardly found 12 h and apparently detected again 1 and 2 days after ischemic insult. At 5 days post-ischemia, p-PRAS40 immunoreactivity in the CA1 pyramidal neurons was not found. These results indicate that ischemia-induced changes in PRAS40 and p-PRAS40 immunoreactivities in CA1 pyramidal neurons and astrocytes may be closely associated with delayed neuronal death in the hippocampal CA1 region following transient cerebral ischemia.     

Comparison of Trophic Factors Changes in the Hippocampal CA1 Region Between the Young and Adult Gerbil Induced by Transient Cerebral Ischemia

In the present study, we investigated neuronal death/damage in the gerbil hippocampal CA1 region (CA1) and compared changes in some trophic factors, such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), in the CA1 between the adult and young gerbils after 5?min of transient cerebral ischemia. Most of pyramidal neurons (89?%) were damaged 4?days after ischemia?Creperfusion (I?CR) in the adult; however, in the young, about 59?% of pyramidal neurons were damaged 7?days after I?CR. The immunoreactivity and levels of BDNF and VEGF, not GDNF, in the CA1 of the normal young were lower than those in the normal adult. Four days after I?CR in the adult group, the immunoreactivity and levels of BDNF and VEGF were distinctively decreased, and the immunoreactivity and level of GDNF were increased. However, in the young group, all of their immunoreactivities and levels were much higher than those in the normal young group. From 7?days after I?CR, all the immunoreactivities and levels were apparently decreased compared to those of the normal adult and young. In brief, we confirmed our recent finding: more delayed and less neuronal death occurred in the young following I?CR, and we newly found that the immunoreactivities of trophic factors, such as BDNF, GDNF, and VEGF, in the stratum pyramidale of the CA1 in the young gerbil were much higher than those in the adult gerbil 4?days after transient cerebral ischemia.     

Comparison of the Immunoreactivity of Trx2/Prx3 Redox System in the Hippocampal CA1 Region Between the Young and Adult Gerbil Induced by Transient Cerebral Ischemia

In the present study, we compared the immunoreactivities and levels of Trx/prx redox system, thioredoxin 2 (Trx2), thioredoxin reductase 2 (TrxR2) and peroxiredoxin 3 (Prx3), as well as neuronal death in the hippocampal CA1 region between the adult and young gerbil after 5 min of transient cerebral ischemia. At 4 days post-ischemia, pyramidal neurons (about 90%) in the adult stratum pyramidale of the CA1 region showed "delayed neuronal death (DND)"; however, at this time point, few pyramidal neurons showed DND in the young stratum pyramidale. At 7 days post-ischemia, about 56% of pyramidal neurons showed DND in the young stratum pyramidale. The immunoreactivities of all the antioxidants in the young sham-group were similar to those in the adult sham-group. At 4 days post-ischemia, the immunoreactivity of TrxR2, not Trx2 and Prx3 in the adult ischemia-group was dramatically decreased in CA1 pyramidal neurons. At this time point, the immunoreactivities of all the antioxidants in the young ischemia-group were apparently increased compared to the adult ischemia-group. From 7 days pots-ischemia, non-pyramidal cells showed the immunoreactivities of all the antioxidants in the ischemic CA1 region; however, in the young ischemia-groups, the immunoreactivities were much lower than those in the adult ischemia-groups. In brief, our results showed that the immunoreactivities of Trx2, TrxR2 and Prx3 were dramatically increased in CA1 pyramidal neurons of the young ischemia-groups at 4 days post-ischemia compared to those in the adult ischemia-groups induced by transient cerebral ischemia.     

Comparison of Glial Activation in the Hippocampal CA1 Region Between The Young and Adult Gerbils After Transient Cerebral Ischemia

It has been reported that young animals are less vulnerable to brain ischemia. In the present study, we compared gliosis in the hippocampal CA1 region of the young gerbil with those in the adult gerbil induced by 5?min of transient cerebral ischemia by immunohistochemistry and western blot for glial cells. We used male gerbils of postnatal month 1 (PM 1) as the young and PM 6 as the adult. Neuronal death in CA1 pyramidal neurons in the adult gerbil occurred at 4?days posti-schemia; the neuronal death in the young gerbil occurred at 7?days post-ischemia. The findings of glial changes in the young gerbil after ischemic damage were distinctively different from those in the adult gerbil. Glial fibrillary acidic protein-immunoreactive astrocytes, ionized calcium-binding adapter molecule (Iba-1), and isolectin B4-immunoreactive microglia in the ischemic CA1 region were activated much later in the young gerbil than in the adult gerbil. In brief, very less gliosis occurred in the hippocampal CA1 region of the young gerbil than in the adult gerbil after transient cerebral ischemia.     

Ionized Calcium-binding Adapter Molecule 1 Immunoreactive Cells Change in the Gerbil Hippocampal CA1 Region after Ischemia/Reperfusion

Ionized calcium-binding adapter molecule 1 (iba-1) is specifically expressed in microglia and plays an important role in the regulation of the function of microglia. We observed chronological changes of iba-1-immunoreactive cells and iba-1 level in the gerbil hippocampal CA1 region after transient ischemia. Transient forebrain ischemia in gerbils was induced by the occlusion of bilateral common carotid arteries for 5 min. Immunohistochemical and Western blot analysis of iba-1 were performed in the gerbil ischemic hippocampus. In the sham-operated group, iba-1-immunoreactive cells were detected in the CA1 region. Thirty minutes after ischemia/reperfusion, iba-1 immunoreactivity significantly increased, and its immunoreactive cells were well ramified. Three hours after ischemia/reperfusion, iba-1 immunoreactivity and level decreased, and thereafter they increased again with time after ischemia/reperfusion. Three days after ischemia/reperfusion, iba-1-immunoreactive cells had well-ramified processes, which projected to the stratum pyramidale of the CA1 region. Seven days after ischemia/reperfusion, iba-1 immunoreactivity and level were highest in the CA1 region, whereas they significantly decreased in the CA1 region 10 days after ischemia/reperfusion. Iba-1-immunoreactive cells in the ischemic CA1 region were co-localized with OX-42, a microglia marker. In brief, iba-1-immunoreactive cells change morphologically and iba-1 immunoreactivity alters in the CA1 region with time after ischemia/reperfusion. These may be associated with the delayed neuronal death of CA1 pyramidal cells in the gerbil ischemic hippocampus.     

Hyperoxidized Peroxiredoxins and Glyceraldehyde-3-Phosphate Dehydrogenase Immunoreactivity and Protein Levels are Changed in the Gerbil Hippocampal CA1 Region After Transient Forebrain Ischemia

Oxidative stress is a major pathogenic event occurring in several brain disorders and is a major cause of brain damage due to ischemia/reperfusion. Thiol proteins are easily oxidized in cells exposed to reactive oxygen species (ROS). In the present study, we investigated transient ischemia-induced chronological changes in hyperoxidized peroxiredoxins (Prx-SO₃) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH-SO₃) immunoreactivity and protein levels in the gerbil hippocampus induced by 5 min of transient forebrain ischemia. Weak Prx-SO₃ immunoreactivity is detected in the hippocampal CA1 region of the sham-operated group. Prx-SO₃ immunoreactivity was significantly increased 12 h and 1 day after ischemia/reperfusion, and the immunoreactivity was decreased to the level of the sham-operated group 2 days after ischemia/reperfusion. Prx-SO₃ immunoreactivity in the 4 days post-ischemia group was increased again, and the immunoreactivity was expressed in glial components for 5 days after ischemia/reperfusion. GAPDH-SO₃ immunoreactivity was highest in the CA1 region 1 day after ischemia/reperfusion, the immunoreactivity was decreased 2 days after ischemia/reperfusion. Four days after ischemia/reperfusion, GAPDH-SO₃ immunoreactivity increased again, and the immunoreactivity began to be expressed in glial components from 5 days after ischemia/reperfusion. Prx-SO₃ and GAPDH-SO₃ protein levels in the ischemic CA1 region were also very high 12 h and 1 day after ischemia/reperfusion and returned to the level of the sham-operated group 3 days after ischemia/reperfusion. Their protein levels were increased again 5 days after ischemia/reperfusion. In conclusion, Prx-SO₃ and GAPDH-SO₃ immunoreactivity and protein levels in the gerbil hippocampal CA1 region are significantly increased 12 h-24 h after ischemia/reperfusion and their immunoreactivity begins to be expressed in glial components from 4 or 5 days after ischemia/reperfusion.     

Clioquinol Inhibits Zinc-Triggered Caspase Activation in the Hippocampal CA1 Region of a Global Ischemic Gerbil Model

Background

Excessive release of chelatable zinc from excitatory synaptic vesicles is involved in the pathogenesis of selective neuronal cell death following transient forebrain ischemia. The present study was designed to examine the neuroprotective effect of a membrane-permeable zinc chelator, clioquinol (CQ), in the CA1 region of the gerbil hippocampus after transient global ischemia.

Methodology/Principal Findings

The common carotid arteries were occluded bilaterally, and CQ (10 mg/kg, i.p.) was injected into gerbils once a day. The zinc chelating effect of CQ was examined with TSQ fluorescence and autometallography. Neuronal death, the expression levels of caspases and apoptosis inducing factor (AIF) were evaluated using TUNEL, in situ hybridization and Western blotting, respectively. We were able to show for the first time that CQ treatment attenuates the ischemia-induced zinc accumulation in the CA1 pyramidal neurons, accompanied by less neuronal loss in the CA1 field of the hippocampus after ischemia. Furthermore, the expression levels of caspase-3, -9, and AIF were significantly decreased in the hippocampus of CQ-treated gerbils.

Conclusions/Significance

The present study indicates that the neuroprotective effect of CQ is related to downregulation of zinc-triggered caspase activation in the hippocampal CA1 region of gerbils with global ischemia.     

Changes in Corticosteroid Hormone Receptors in the Ischemic Gerbil Hippocampal CA1 Region Following Repeated Restraint Stress

Restraint stress produces physiological changes including suppression of long-term potentiation in the brain. We observed the effects of repeated stress on ischemic damage associated with corticosteroid hormone receptors in gerbils. Animals were placed into restrainers for 5 h (between 09:30 h and 14:30 h) for 21 consecutive days prior to induction of transient cerebral ischemia. The animals were divided into 4 groups; (1) sham-operated-control-group (sham-group), (2) ischemia-operated-control-group (ischemia-group), (3) sham-operated-stress-group (stressed-sham-group), and (4) ischemia-operated-stress-group (stressed-ischemia-group). We found that serum corticosterone level in the ischemia-group was highest (374% of the sham-group) 12 h after ischemia/reperfusion and its level in the stressed-ischemia-group was significantly lower than the ischemia-group. Locomotor activity in the ischemia-group was significantly increased (295% of the sham-group) at 1 day post-ischemia; however, the locomotor activity in the stressed-ischemia-group was less increased compared to the ischemia-group. Cresyl violet positive (CV⁺) cells were significantly decreased in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) of the 4 days post-ischemia-group, while 79.4% of CV⁺ cells were detected in the CA1 of the stressed-ischemia-group. Also, a few NeuN (neuron-specific soluble nuclear antigen)⁺ cells were detected in the SP of the 4 days post-ischemia-group; however, in the 4 days stressed-post-ischemia-group, 77.2% of NeuN⁺ neurons were found in the SP. Glial fibrillary acidic protein⁺ astrocytes in the CA1 in the stressed-ischemia-groups were similar to those in the ischemia-groups; however, ionized calcium-binding adapter molecule 1⁺ microglia in the stressed-ischemia-groups were less activated compared to the ischemia-groups. Mineralocorticoid receptor (MCR) and glucocorticoid receptor (GR) immunoreactivity in the SP of the stressed-ischemia-group were higher than the ischemia-group; at 4 days post-ischemia, MCR and GR immunoreactivity were expressed in non-pyramidal cells. In brief, our results indicate that repeated restraint stress significantly increase levels of corticosteroid hormone receptors and attenuates neuronal damage in the ischemic hippocampal CA1 region.     

蒙古沙鼠脑缺血后大脑皮质Calretinin(CR)的表达变化

目的:观察蒙古沙鼠前脑短暂缺血后前额皮质Calretinin(CR)的表达变化,为进一步研究其功能及治疗提供形态学依据。方法:24只健康雄性长爪鼠随机分为正常组对照组(6只)和缺血组(18只),夹闭蒙古沙鼠双测颈总动脉10 min诱导前脑缺血后,动物分别存活1天、3天或7天。用免疫组织化学方法检测了前额皮质中CR的表达。结果:与正常组相比,各缺血组中前额皮质中CR表达均上调。缺血再灌1天时,CR表达最强(P＜0．01);3天时CR表达开始恢复(P＜0．05);7天时CR表达进一步恢复,但仍高于正常组(P＜0．01)。结论:前脑短暂缺血后可造成蒙古沙鼠前额皮质CR的表达在短时间内急剧上调;但随着时间的CR的表达会恢复正常。     

Isoform-Specific Redistribution of Calcineurin Aα and Aβ in the Hippocampal CA1 Region of Gerbils After Transient Ischemia

Abstract: To investigate isoform-specific roles of Ca²⁺/calmodulin-dependent phosphatase [calcineurin (CaN)] in ischemia-induced cell death, we raised antibodies specific to CaN Aα and CaN Aβ and localized the CaN isoforms in the hippocampal CA1 region of Mongolian gerbils subjected to a 5-min occlusion of carotid arteries. In the nonischemic gerbil, immunoreactions of both isoforms were highly enriched in CA1 regions, especially in the cytoplasm and apical dendrites of CA1 pyramidal neurons. At 4–7 days after the induced ischemia, immunoreactivities of the CaN Aα isoform in CA1 pyramidal cells were markedly reduced, whereas they were enhanced in the CA1 radiatum and oriens layers. In contrast, CaN Aβ immunoreactivities were reduced in all layers of the ischemic CA1 region, whereas they were enhanced in activated astrocytes, colocalizing with glial fibrillary acidic protein. These findings suggest that up-regulation of CaN Aα in afferent fibers in CA1 and up-regulation of CaN Aβ in reactive astrocytes may be involved in neuronal reorganization after ischemic injury.     

Time-Course Change of Redd1 Expressions in the Hippocampal CA1 Region Following Chronic Cerebral Hypoperfusion

Redd1, also known as RTP801/Dig2/DDIT4, is a stress-induced protein and marked changes of Redd1 expression occurs in response to hypoxia or cerebral ischemia. In the present study, we examined the time-course changes in Redd1 protein expressions in the rat hippocampal CA1 region following chronic cerebral hypoperfusion (CCH) induced by permanent bilateral common carotid arteries occlusion (2VO). Redd1 immunoreactivity in the pyramidal neurons of the hippocampal CA1 region was increased at 7 days after 2VO surgery, and then the immunoreactivity was decreased with time. Especially, very weak Redd1 immunoreactivity was observed in the hippocampal CA1 region at 28 days after 2VO surgery. Western blot analysis showed that Redd1 level in the hippocampal CA1 region was significantly increased at 7 days following CCH and significantly decreased at 28 days after 2VO surgery, compared with that of the sham-operated group. These results indicate that Redd1 expressions is markedly changed in the hippocampal CA1 region following CCH and that change of Redd1 expression may be associated with the CCH-induced neuronal damage in the hippocampal CA1 region.     

Damage to Neurons and Oligodendrocytes in the Hippocampal CA1 Sector after Transient Focal Ischemia in Rats

Focal brain lesions such as transient focal cerebral ischemia can lead to neuronal damage in remote areas, including the ipsilateral substantia nigra and hippocampus, as well as in the ischemic core. In this study, we investigated acute changes in the ipsilateral hippocampus from 1 up to 7 days after 90 min of transient focal cerebral ischemia in rats, using anti-NeuN (neuronal nuclei), anti-Cu/Zn-superoxide dismutase (Cu/Zn-SOD), anti-Mn-SOD, anti-neuronal nitric oxide synthase (nNOS), anti-inducible NOS (iNOS), anti-glial fibrillary acidic protein (GFAP), anti-ionized calcium-binding adaptor molecule 1(Iba 1) and anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) antibodies. In our western blot and histochemical analyses, present results show that transient focal cerebral ischemia in rats can cause a severe and acute damage of neurons and oligodendrocytes in the ipsilateral hippocampal CA1 sector. The present findings also demonstrate that the expression of iNOS produced by Iba 1-immunopositive microglia precedes the damage of neurons and oligodendrocytes in the ipsilateral hippocampal CA1 sector after transient focal cerebral ischemia. In contrast, our results suggest that increased reactive oxygen species (ROS) production during reperfusion cannot lead to damage of neurons and oligodendrocytes in the ipsilateral hippocampal CA1 sector after transient focal cerebral ischemia, because of an insufficient expression of Cu/Zn-SOD and Mn-SOD. Our double-labeled immunohistochemical study demonstrates that the overexpression of iNOS produced by Iba 1-immunopositive microglia may play a pivotal role in the damage of neurons and oligodendrocytes in the ipsilateral hippocampal CA1 sector at an acute stage after transient focal cerebral ischemia.     

Effect of Transient Cerebral Ischemia on the Expression of Receptor for Advanced Glycation End Products (RAGE) in the Gerbil Hippocampus Proper

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor of the immunoglobulin superfamily that has been implicated in multiple neuronal and inflammatory stress processes. In this study, we examined changes in RAGE immunoreactivity and its protein levels in the gerbil hippocampus (CA1-3 regions) after 5 min of transient global cerebral ischemia. The ischemic hippocampus was stained with cresyl violet, neuronal nuclei (a neuron-specific soluble nuclear antigen) antibody and Fluoro-Jade B (a marker for neuronal degeneration). 5 days after ischemia–reperfusion, delayed neuronal death occurred in the stratum pyramidale of the CA1 region. RAGE immunoreactivity was not detected in any regions of the CA1-3 regions of the sham-group; the immunoreactivity was markedly increased only in the CA1 region from 3 days after ischemia–reperfusion. On the other hand, RAGE immunoreactivity was newly expressed in astrocytes, not in microglia. Western blot analysis showed that RAGE protein level was highest at 5 days post-ischemia. In brief, both the RAGE immunoreactivity and protein level were distinctively increased in astrocytes in the ischemic CA1 region from 3 days after transient cerebral ischemia. These results indicate that the increase of RAGE expression in astrocytes after ischemia–reperfusion may be related to the ischemia-caused activation of astrocytes in the ischemic CA1 region.     

美满霉素对沙鼠急性脑缺血后脑皮质小白蛋白表达的影响

目的：观察美满霉素对蒙古沙鼠短暂性脑缺血再灌后前额皮质中小白蛋白Parvalbumin（PV）表达的影响,为进一步研究其生理功能变化及治疗提供参考。方法：42只健康雄性蒙古沙鼠随机分为：正常组对照组（6只）、缺血再灌组（18只）,美满霉素治疗组（18只）。夹闭蒙古沙鼠双测颈总动脉10min诱导前脑缺血后,动物分别存活1天、3天或7天。用免疫组化方法检测前额皮质中PV的表达,用图像分析仪测定灰度值。结果：与正常组相比,各缺血再灌组与美满霉素治疗组中前额皮质中PV表达均先减少后回升。缺血再灌1天组,PV表达缓慢减少（P〉0.05）;3天组PV表达减少到低谷（P〈0.05）;7天组PV表达有明显的恢复,但仍低于正常组（P〈0.05）;然而美满霉素治疗各组中的PV表达均强于对应的缺血组（P〈0.05）。结论：美满霉素能抑制蒙古沙鼠脑缺血再灌注后脑皮质中小白蛋白的表达,发挥脑保护作用。     

Rapid Down-Regulation of GABAA Receptors in the Gerbil Hippocampus Following Transient Cerebral Ischemia

Abstract: During transient cerebral ischemia, there is a temporary and robust accumulation of extracellular GABA in the hippocampus. We examined whether the acute exposure of GABA_A/benzodiazepine receptors to high concentrations of GABA early after ischemia results in receptor down-regulation as observed in vitro. Gerbils were killed 30 and 60 min following a 5-min bilateral carotid occlusion, and their brains were prepared for receptor autoradiography. The hydrophilic GABA_A receptor antagonist [³H]SR-95531 and the hydrophobic benzodiazepine agonist [³H]flunitrazepam were used to distinguish between cell surface and internalized receptors. Ischemia significantly decreased [³H]SR-95531 binding in hippocampal areas CA1 and CA3 and in the dentate gyrus 30 min after ischemia. Scatchard analysis in area CA1 revealed that ischemia decreased the B _max as low as 44%. The affinity of the remaining sites was increased substantially (72% decrease in K _D). As expected, there were no changes in the binding of [³H]flunitrazepam to hippocampus in the early postischemic period because the benzodiazepine could bind to both internalized receptors and those on the cell surface. We hypothesize that prolonged exposure (∼30–45 min) of GABA_A receptors to high concentrations of synaptic GABA in vivo causes receptor down-regulation, perhaps via receptor internalization.     

Comparison of Phosphorylated Extracellular Signal-Regulated Kinase 1/2 Immunoreactivity in the Hippocampal Ca1 Region Induced by Transient Cerebral Ischemia Between Adult and Aged Gerbils

In this study, the authors examined the difference of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in the hippocampal CA1 region (CA1) between adult and aged gerbils after 5 min of transient cerebral ischemia. Delayed neuronal death in the CA1 of the aged group was much slower than that in the adult group after ischemia/reperfusion (I/R). pERK1/2 immunoreaction was observed in the CA1 region of the sham-operated adult gerbil. pERK1/2 immunoreactivity and protein levels in the ischemic CA1 region of the adult group were markedly increased 4 days after I/R, and then reduced up to 10 days after I/R. In contrast, pERK1/2 immunoreaction was hardly detected in the CA1 region of sham-operated aged gerbils, and the immunoreactivity increased from 1 day after the ischemic insult, and still observed until 10 days post-ischemia. In addition, pERK1/2-immunoreaction was expressed in astrocytes in the ischemic CA1 region: The expression in the ischemia-operated aged gerbils was later than that in the ischemia-operated adult gerbils. These results indicate that different patterns of ERK1/2 immunoreactivity may be associated with different processes of delayed neuronal death in adult and aged animals.