Ischemia-induced neuronal cell death, calcium accumulation, and glial response in the hippocampus of the Mongolian gerbil and protection by propentofylline (HWA 285)

The localization and timing of cellular calcium loading and glial cell reaction in relation to selective death of hippocampal neurons was studied in Mongolian gerbils following transient forebrain ischemia. Two days after a 5-min period of ischemia, heavy calcium staining was histochemically demonstrated in circumscribed groups of nerve cells, located in the transition zone between the CA1 and CA3 areas. This preceded complete neuronal cell death that was quantitatively assessed by measuring the intensity of Nissl staining. After a 12-min period of ischemia, extensive calcium loading was observed in conjunction with severe neuronal damage throughout the CA1 region as well in the dorsal nuclei of the thalamus. The extent of calcium staining decreased with time and was not seen at stages later than 7 days. Already at 2 days after a 5-min period of ischemia, a strong increase of glial fibrillary acidic protein immunoreactivity was seen. This indicates a marked and early hypertrophy of astrocytes that was not accompanied by an obvious proliferation. Neither the astrocytic response nor the neuronal calcium accumulation were observed in gerbils pretreated with propentofylline, HWA 285 (10 mg/kg, i.p.) 15 min before bilateral carotid artery occlusion. Also, the decrease of Nissl staining in the CA1 area after 5 and 12 min of ischemia was considerably less pronounced and did not significantly differ from sham-operated controls.     

Low dose of barbiturates for prevention of hippocampal lesions after brief ischemic episodes

Summary Male Mongolian gerbils were subjected to bilateral carotid occlusion for 5 and 10 min, followed by 7 days of recirculation. After this interval, serial sections were made of the posterior region of the dorsal hippocampus, and the number of surviving neurons was determined per mm length of CA1 sector. In halothane-anesthetized animals only 21.1% of CA1 neurons survived 5-min ischemia, but this percentage could be raised to 78.6% when animals were pretreated with 25 mg/kg pentobarbital before ischemia. Pretreatment with 50 mg/kg pentobarbital before 5-min ischemia or pretreatment with 25 mg/kg pentobarbital before 10-min ischemia did not reduce CA1 lesions. It is concluded that a non-anesthetic dose of barbiturates is able to prevent selective vulnerability of CA1 sector, but that this effect is limited to the initial 5 min of ischemia.     

Ischemic preconditioning in 18- to 20-month-old gerbils: long-term survival with functional outcome measures.

BACKGROUND AND PURPOSE: In young animals, ischemic preconditioning protects CA1 hippocampal neurons against global ischemia. However, cerebral ischemia occurs most frequently in individuals aged >/=65 years. This study examined the protection provided by ischemic preconditioning in a population of aged (18- to 20-month-old) gerbils. METHODS: One group of animals was exposed to two 1.5-minute episodes of global ischemia separated by 24 hours and followed 72 hours later by a 5-minute occlusion of both carotid arteries. A second group was given 2 episodes of preconditioning only. Two other groups were exposed to 5 minutes of ischemia or sham surgery. The animals survived 10, 30, or 60 days. Functional and histological assessments were used to determine the extent of protection. RESULTS: Ten days after ischemia there was >80% protection of CA1 neurons in ischemic preconditioned animals compared with 6% in ischemic gerbils. Nevertheless, these preconditioned animals were impaired in open-field tests of habituation. In addition, CA1 dendritic field potentials were smaller in amplitude compared with those in sham animals. While there was a complete loss of staining for CA1 microtubule-associated protein-2 in ischemic animals, staining in ischemic preconditioned animals was normal. This suggests that dendritic abnormalities per se were not responsible for the observed functional deficits. CA1 cell survival declined to approximately 75% of sham values (P<0.05) at 60 days after ischemia. CONCLUSIONS: Ischemic preconditioning provided substantial neuroprotection in aged gerbils. Nonetheless, the striking dissociation between histological and functional protection provided by ischemic preconditioning in aged animals emphasizes the need to use functional end points and long-term survival when assessing neuroprotection. Although functional recovery was evident with increasing survival time, CA1 cell death continued, thereby raising the possibility that the level of neuroprotection attained was not permanent.     

A reversible type of neuronal injury following ischemia in the gerbil hippocampus

The Mongolian gerbil is known to develop delayed neuronal death in the hippocampus following brief forebrain ischemia (Brain Res 239: 57-69, 1982). The effect of pentobarbital on this slow process of neuronal damage was examined. Immediately following 5 min of bilateral carotid occlusion, pentobarbital (10, 20, or 40 mg/kg) was injected. The control animals received saline injection. Seven days following ischemic insult, animals were perfusion-fixed and the neuronal density in the hippocampal CA1 subfield was counted. Most of the neurons in the CA1 sector survived ischemic insult when pentobarbital was given, whereas most of control group neurons were lost without the treatment. The average neuronal density of 20 mg/kg group was 168.2 +/- 12.3 (SEM) per 1 mm linear length of the CA1 subfield. The density in 40 mg/kg group was 181.1 +/- 14.9. The neuronal density in the whole control group was 34.3 +/- 5.1. The density of unoperated normal gerbils was 212.3 +/- 3.9. This result indicates that the neuronal damage of "delayed neuronal death" is reversible. On the other hand, when pentobarbital was injected 1 hr following ischemia, it showed no effect. The cell change in the CA1 sector, reversible at the initial stage, seems to rapidly become irreversible, while neurons still remain intact morphologically.     

Role for gamma-aminobutyric acid in selective vulnerability in gerbils

We tested the efficacy of various putative neuroprotective agents in the gerbil model of delayed neuronal death. The selective loss of anterior CA1 neurons of the hippocampus 4 days after 5 minutes of bilateral ischemia was complete in greater than 90% of the gerbils examined. We tested 11 agents for their ability to protect against neuronal loss. Only those agents that were associated with the GABAergic system exhibited protection and only when administered before the ischemic insult. The possibility that delayed neuronal death is the result of a primary defect in inhibitory neurotransmission is considered.     

Calpain inhibitor entrapped in liposome rescues ischemic neuronal damage

Transient forebrain ischemia induces activation of calpain and proteolysis of a neuronal cytoskeleton, fodrin, in gerbil hippocampus. This phenomenon precedes delayed neuronal death in hippocampal CA1 neurons. We examined effects of a calpain inhibitor on delayed neuronal death after transient forebrain ischemia. In gerbils, a selective calpain inhibitor entrapped in liposome was given transvenously and 30 min later, 5-min forebrain ischemia was produced by occlusion of both common carotid arteries. On day 7, CA1 neuronal damage was examined in the hippocampal slices stained with cresyl violet. Calpain-induced proteolysis of fodrin was also examined by immunohistochemistry and immunoblot. Additionally, to assure entrapment of the inhibitor by CA1 neurons, the inhibitor-liposome complex was labeled with FITC and given to gerbils. Fluorescence in the hippocampal slices was examined by confocal laser scanning microscope. Selective CA1 neuronal damage induced by forebrain ischemia was prevented by administration of the inhibitor in a dose-dependent manner. Calpain-induced proteolysis of fodrin was also extinguished by the calpain inhibitor in a dose-dependent manner. Bright fluorescence of the FITC-labeled inhibitor was observed in the CA1 neurons. The data show an important role of calpain in the development of the ischemic delayed neuronal death. Calpain seems to produce neuronal damage by degrading neuronal cytoskeleton. Our data also show a palliative effect of the calpain inhibitor on the neurotoxic damage, which offers a new and potent treatment of transient forebrain cerebral ischemia.     

Regional neuroprotective effects of pentobarbital on ischemia-induced brain damage

We investigated the neuroprotective effect of pentobarbital, a GABAA receptor-effector, on ischemic neuronal damage in the gerbils. The animals were allowed to survive for 7 days after 10-min ischemia induced by bilateral occlusion of the common carotid arteries. Morphological changes and abnormal calcium accumulation were evaluated in selectively vulnerable areas after ischemia. Pentobarbital (40 mg/kg, IP), administered 30 min prior to ischemia, significantly reduced neuronal cell loss in the neocortex, the striatum, and the hippocampal CA3 sector. However, pentobarbital failed to prevent the damage to the hippocampal CA1 sector and the thalamus. 45Ca autoradiographic study also revealed that a marked calcium accumulation was found in the selectively vulnerable regions after ischemia, which was consistent with the extent of histological neuronal damage. The abnormal calcium accumulation was reduced in the sites corresponding to most of the regions in which the protective effect of pentobarbital was found. The results suggest that ischemia-induced neuronal damage may be partly caused by an imbalance between excitatory and inhibitory input.     

Quantitative analysis of effects of kappa-opioid agonists on postischemic hippocampal CA1 neuronal necrosis in gerbils

The ability of the kappa-opioid receptor agonists U50488H and U62066E (spiradoline mesylate) compared with the non-kappa close structural analogue U54494A to affect postischemic necrosis of the selectively vulnerable hippocampal CA1 neurons was examined in male Mongolian gerbils. The gerbils were treated with either saline vehicle or 10 mg/kg i.p. of one of the test drugs 30 minutes before and again 2 hours after a 10-minute period of bilateral carotid artery occlusion or sham occlusion under light methoxyflurane anesthesia. Seven days after ischemia and reperfusion the brains were perfusion-fixed, and hippocampal CA1 cells were counted in a blind fashion. In ischemic gerbils that received only vehicle, there was a 78.9% loss of CA1 neurons compared with sham-occluded gerbils. In contrast, in U50488H-treated gerbils, mean cell loss was reduced to 33.9% (p less than 0.01 vs. vehicle-treated group). U62066E was even more effective in reducing postischemic CA1 degeneration to only 20.7% (p less than 0.0001 vs. vehicle-treated group). However, treatment with the non-kappa analogue U54494A did not cause any apparent protection; the gerbils in this group showed an 80.7% loss of CA1 neurons. Our results are consistent with the hypothesis that kappa-receptor stimulation is associated with improved postischemic neuronal preservation.     

The highly selective cyclooxygenase-2 inhibitor DFU is neuroprotective when given several hours after transient cerebral ischemia in gerbils

Several studies suggest that cyclooxygenase-2 contributes to the delayed progression of ischemic brain damage. In this study we examined whether the highly selective cyclooxygenase-2 inhibitor DFU reduces neuronal damage when administered several hours after 5 min of transient forebrain ischemia in gerbils. The extent of ischemic injury was assessed behaviorally by measuring the increases in locomotor activity and by histopathological evaluation of the extent of CA1 hippocampal pyramidal cell injury 7 days after ischemia. DFU treatment (10 mg/kg, p.o.) significantly reduced hippocampal neuronal damage even if the treatment is delayed until 12 h after ischemia. These results suggest that selective cyclooxygenase-2 inhibitors may be a valuable therapeutic strategy for ischemic brain injury.     

Protective effects of human recombinant superoxide dismutase on transient ischemic injury of CA1 neurons in gerbils.

BACKGROUND AND PURPOSE: It has been postulated that oxygen-derived free radicals are produced in significant quantities upon reperfusion of ischemic brain and that the free radicals play a pivotal role in triggering the ischemic neuronal damage causing delayed neuronal death. This study was undertaken to examine the effects of human recombinant superoxide dismutase on the delayed neuronal death of CA1 neurons and on the change in the expression of messenger ribonucleic acid for endogenous copper-zinc superoxide dismutase after transient ischemia. METHODS: Human recombinant superoxide dismutase (8 x 10(5) units/kg) or apo-superoxide dismutase was administered intravenously 1 minute before bilateral carotid artery occlusion in gerbils divided among four experimental groups. Endogenous copper-zinc superoxide dismutase messenger ribonucleic acid was analyzed by in situ hybridization histochemistry using a sulfur-35-labeled oligonucleotide probe. Immunohistochemical localizations of administered human recombinant superoxide dismutase were investigated. RESULTS: All gerbils receiving apo-superoxide dismutase exhibited almost complete destruction of CA1 neurons 7 days after 5 minutes of ischemia. The gerbils treated with human recombinant superoxide dismutase showed mild lesions (p less than 0.01). Discrete localizations were observed for endogenous copper-zinc superoxide dismutase messenger ribonucleic acid. Transient ischemia increased labeling throughout the hippocampus after 30 minutes and 24 hours of reperfusion. This increase was abolished by treatment with human recombinant superoxide dismutase. This phenomenon was confirmed by Northern blot analysis. The interneurons in CA3 and cells in the hilus were mainly stained against administered superoxide dismutase at 5 and 30 minutes, and these reactions had disappeared at 20 hours after the administration. CONCLUSIONS: Our data demonstrate protective effects of human recombinant superoxide dismutase against ischemic neuronal damage and support the hypothesis that the generated free radicals induce a vicious cycle leading to delayed neuronal death.     

Prevention of delayed neuronal death in gerbil hippocampus by a novel vinca alkaloid derivative (Vinconate)

We investigated the effect of vinconate, a novel vinca alkaloid derivative, on delayed neuronal death using Mongolian gerbils. The animals were allowed to survive for 7 d after 3 or 5 min of forebrain ischemia induced by bilateral occlusion of the common carotid arteries. Morphological changes and calcium (⁴⁵Ca) accumulation were evaluated in the CA1 sector of the hippocampus after ischemia. Vinconate (50, 100, and 300 mg/kg) showed protective effects against neuronal death in a dose-dependent manner when administered intraperitoneally (ip) 10 min before 5 min of ischemia. However, the administration of vinconate (100 and 300 mg/kg, ip) immediately after 5 min of ischemia showed no therapeutic effect, whereas a marked therapeutic effect of vinconate (50 and 100 mg/kg, ip) was observed when administered immediately after 3 min of ischemia. An anesthetic dose of pentobarbital (40 mg/kg, ip) also produced significant protection against neuronal death. Furthermore, a⁴⁵Ca autoradiographic study indicated that a marked calcium accumulation was found in the CA1 sector at 7 d after 5 min of ischemia, which was consistent with the extent of histological neuronal damage. When vinconate (100 and 300 mg/kg, ip) was administered 10 min before 5 min of ischemia, the abnormal calcium accumulation was not detected in the CA1 sector. These data indicate that suppression of abnormal neuronal activity may be owing to the antagonistic action of vinconate on calcium accumulation.     

Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investi-gated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal tran-sient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining cal-bindin D28k immunoreactivity.     

Delayed pentobarbital administration limits ischemic brain damage in gerbils.

The capacity of delayed barbiturate administration to limit brain damage after unilateralcerebral ischemia was examined histologically in gerbils. The right common carotid artery was occluded in 50 animals under brief (3-minute) halothane anesthesia; 18 animals (36%) developed motor abnormalities consistent with stroke. The arterial clasps were removed after 1 hour and the abnormal animals were divided into treatment and placebo groups. Treated gerbils received sodium pentobarbital (70 mg/kg) intarperitoneally 1 hour after clasp removal and a smaller dose (50 mg/kg) 2 hours later; these animals lost corneal reflexes but retained spontaneous respiration and were kept normothermic. Animals in the placebo group received equivalent volumes of normal saline. Except for the period of anesthesia, both groups had similar postischemic motor behavior. Neuropathological examination of animals killed by perfusion-fixation after 24 hours revealed fewer pentobarbital-treated animals with shift of midline structures and with ipsilateral ischemic damage (including infarction). Compared with the placebo group, there was less extensive neuronal ischemic cell change in five regions of the ipsilateral cerebral hemispheres of the pentobarbital-treated animals (p less than 0.05). The results suggest that barbiturates administered as long as 1 hour after the end of an ischemic insult can still limit brain damage.     

Oxypurinol attenuates ischemia-induced hippocampal damage in the gerbil

Oxypurinol, an inhibitor of the enzyme xanthine oxidase, reduced ischemic hippocampal damage and the associated hypermotility in Mongolian gerbils. Cerebral ischemia was induced in unanesthetized gerbils by a bilateral 5-min occlusion of the carotid arteries. Oxypurinol (40 mg/kg, IP), administered 20 min prior to carotid occlusion, prevented the increase in locomotor activity observed in saline-injected ischemic animals and significantly reduced the damage to, and loss of, CA1 hippocampal neurons observed 5 days postischemia. These findings suggest that oxypurinol may be useful for the prevention of cerebral ischemic damage.     

Evaluation of CA1 damage using single-voxel 1H-MRS and un-biased stereology: Can non-invasive measures of N-acetyl-asparate following global ischemia be used as a reliable measure of neuronal damage?

Global brain ischemia provoked by transient occlusion of the carotid arteries (2VO) in gerbils results in a severe loss of neurons in the hippocampal CA1 region. We measured the concentration of the neuron specific N-acetyl-aspartate, [NAA], in the gerbil dorsal hippocampus by proton MR spectroscopy (1H-MRS) in situ, and HPLC, 4 days after global ischemia. The [NAA] was correlated with graded hippocampus damage scoring and stereologically determined neuronal density. A basal hippocampal [NAA] of 8.37+/-0.10 and 9.81+/-0.44 mmol/l were found from HPLC and 1H-MRS, respectively. HPLC measurements of [NAA] obtained from hippocampus 4 days after 2VO showed a 20% reduction in the [NAA] following 4 min of ischemia (P<0.001). 1H-MRS measurements on gerbils subjected to 4 or 8 min of ischemia showed a similar 24% decline in the [NAA] (P<0.05). Thus, there was correlation between the HPLC and 1H-MRS determined NAA decline. There was also a significant correlation between 1H-MRS [NAA] and the corresponding reduction in CA1 neuronal density (P<0.004). In summary our findings show that single voxel 1H-MRS can be used as a supplement to histological evaluation of neuronal injury in studies after global brain ischemia. Accordingly, volume selective spectroscopy has a potential for assessment of neuroprotective therapeutic compounds/strategies with respect to neuronal rescue for delayed ischemic brain damage.     

Immunohistochemical investigation of ischemic and postischemic damage after bilateral carotid occlusion in gerbils

We investigated progression and recovery of neuronal damage during and after global cerebral ischemia in gerbils after bilateral occlusion of the common carotid arteries, using the immunohistochemical method (reaction for tubulin and creatine kinase BB-isoenzyme). The earliest, but reversible, ischemic lesions occurred after 3 minutes' ischemia in the subiculum-CA1 and CA2 regions of the hippocampus. The lesions became irreversible after 4 minutes' ischemia. The ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen were partially or completely reversible if the ischemic period was 5 minutes, whereas delayed degeneration occurred in the pyramidal cells of the medial CA1 region after reperfusion for 48 hours (delayed neuronal death). After 10 minutes' ischemia and subsequent reperfusion, delayed neuronal death extended from the medial to the lateral CA1 region; the ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen also expanded during reperfusion. Our investigation demonstrates that selective vulnerability existed in global cerebral ischemia as in incomplete or regional ischemia and suggests that neurons in many areas of the brain possessed the potential for recovery, progressive deterioration, and even delayed neuronal death depending on the severity and duration of cerebral ischemia.     

Selective vulnerability of hippocampus and disturbances of memory storage after mild unilateral ischemia of gerbil brain

The effect of selective injury of hippocampal neurons on the consolidation of memory traces was studied in gerbils (meriones unguiculatus) after production of mild cerebral ischemia. The right carotid artery was permanently ligated, and animals without gross neurological deficits ("symptom-negative" gerbils) were selected. Eight days and eight weeks after vascular ligation, cell counts of hippocampal neurons were carried out and correlated with regional blood flow and the acquisition of operant behaviour. Eight days after carotid artery occlusion, learning behaviour was significantly impaired although the number of hippocampal neurons had not changed and blood flow had even increased above normal. After eight weeks, learning behaviour and blood flow were normal but now a significant loss of pyramidal neurons was present in the CA1 and CA2 sectors of the hippocampus. Our observations demonstrate that it is possible to detect subtle functional disturbances by appropriate behavioural investigation before manifestation of selective injury of the hippocampus. Recovery of integrative function, despite persistent cellular damage, provides further evidence for central nervous plasticity.     

Effects of delayed administration of (-)-epigallocatechin gallate,a green tea polyphenol on the changes in polyamine levels and neuronal damage after transient forebrain ischemia in gerbils

(-)-Epigallocatechin gallate has a potent antioxidant property and can reduce free radical-induced lipid peroxidation as a green tea polyphenol. In previous study, systemic administration of (-)-epigallocatechin gallate immediately after ischemia has been shown to inhibit the hippocampal neuronal damage in the gerbil model of global ischemia. Polyamines are thought to be important in the generation of brain edema and neuronal cell damage associated with various types of excitatory neurotoxicity. We examined the effects of delayed administration of (-)-epigallocatechin gallate on the changes in polyamine levels and neuronal damage after transient global ischemia in gerbils. To produce transient global ischemia, both common carotid arteries were occluded for 3 min with micro-clips. The gerbils were treated with (-)-epigallocatechin gallate (50 mg/kg, i.p.) at 1 or 3 h after ischemia. The polyamines; putrescine, spermidine, and spermine levels were examined using high performance liquid chromatography in the cerebral cortex and hippocampus 24 h after ischemia. Putrescine levels in the cerebral cortex and hippocampus were increased significantly after ischemia and the delayed administrations of (-)-epigallocatechin gallate (1 or 3 h after ischemia) attenuated the increases. Only minor changes were noted in the spermidine and spermine levels after ischemia. In histology, neuronal injuries in the hippocampal CA1 regions were evaluated quantitatively 5 days after ischemia. (-)-Epigallocatechin gallate administered 1 h or 3 after ischemia significantly reduced hippocampal neuronal damage. The present results show that the delayed administrations of (-)-epigallocatechin gallate inhibit the transient global ischemia-induced increase of putrescine levels in the cerebral cortex and hippocampus. (-)-Epigallocatechin gallate is neuroprotective against neuronal damage even when administered up to 3 h after global ischemia. These findings suggest that (-)-epigallocatechin gallate may be promising in the acute treatment of stroke.     

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1–3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.