Induction of tolerance to ischemia: Alterations in second-messenger systems in the gerbil hippocampus

Preconditioning the brain with sublethal ischemia protects against neuronal damage following subsequent ischemic insult. Using [3H]inositol 1,4,5-triphosphate (IP3), [3H]phorbol 12,13-dibutyrate (PDBu), [3H]cyclic adenosine monophosphate (cAMP) and [3H]rolipram, we performed quantitative autoradiography to determine postischemic alterations in second-messenger systems in the gerbil hippocampus following preconditioning the brain with sublethal ischemia. At 7 days of reperfusion, no alterations were observed in brains subjected to 2 min of forebrain ischemia which produced no neuronal damage. However, 3-min ischemia caused a 75% reduction in [3H]IP3 binding (p < 0.01 vs. control) and 15-25% reductions in [3H]forskolin (p < 0.01 vs. control), [3H]cAMP (p < 0.05 vs. control), and [3H]rolipram (p < 0.01 vs. control) binding in the CA1 subfield coincident with histopathological CA1 pyramidal cell destruction, but no significant alterations in [3H]PDBu binding. Preconditioning the brain with 2 min of ischemia followed by 4 days of reperfusion prevented both histopathological cell death and the reductions in binding following subsequent 3 min of ischemia. Interestingly, [3H]IP3 and [3H]rolipram binding in CA1 showed a transient reduction, by 30% and 20% (both p < 0.01 vs. control), respectively, in the early reperfusion period. This downregulation of the IP3 system may play a role in the protection against cell death.     

Sequential changes in muscarinic acetylcholine, adenosine A1 and calcium antagonist binding sites in the gerbil hippocampus following repeated brief ischemia.

We performed quantitative autoradiography to determine sequential alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of an L-type calcium channel blocker in the gerbil hippocampus following repeated brief ischemic insults. [3H]Quinuclidinyl benzilate (QNB). [3H]cyclohexyladenosine (CHA) and [3H]PN200-110 were used to label muscarinic and adenosine A1 receptors and L-type calcium channels, respectively. Changes at 1 h, 6 h, 1 day, 4 days and 1 month after three 2-min ischemic insults were compared with changes after single 2- or 6-min ischemia. Two-minute ischemia, which causes no histopathological neuronal damage, produced no persistent alterations in binding sites. We observed a transient and mild increase in binding activities, especially in [3H]CHA binding, at 1 h of recirculation. Following 6-min ischemia and three 2-min ischemic insults. [3H]QNB and [3H]PN200-110 binding decreased by more than 50% in the CA1 subfield by 1 month, but [3H]CHA binding decreased transiently by 20-30% at 4 days when delayed neuronal death of hippocampal CA1 pyramidal cells took place. Reductions in binding, especially in [3H]QNB binding, following three 2-min ischemic insults were greater and appeared earlier than those after 6-min ischemia. Furthermore, alterations extended to the CA3 subfield and the dentate gyrus following repeated insults. Thus, alterations in receptor binding after repeated ischemic insults were greater than those after equivalent single period of ischemia.     

Postischemic alteration of muscarinic acetylcholine, adenosine A1 and calcium antagonist binding sites in selectively vulnerable areas: an autoradiographic study of gerbil brain.

We performed receptor autoradiography to determine sequential alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of a voltage dependent L-type calcium channel blocker 1 h-1 month after transient cerebral ischemia in the gerbil brain. [3H]Quinuclidinyl benzilate (QNB), [3H]cyclohexyladenosine (CHA) and [3H]PN200-110 were used to label muscarinic and adenosine A1 receptors and L-type calcium channels, respectively. Transient ischemia was induced for 10 min. [3H]QNB and [3H]CHA binding showed no significant alteration in selectively vulnerable areas at an early stage (1-24 h) of recirculation. However, the dentate molecular layer which was resistant to ischemia revealed a significant decrease in the [3H]CHA binding sites 24 h after ischemia. Thereafter, the [3H]QNB and [3H]CHA binding showed significant reduction in most of selectively vulnerable areas. Marked reduction was especially found in the dorsolateral part of striatum and the hippocampal CA1 sector which was the most vulnerable to ischemia. In contrast, [3H]PN200-110 binding showed a transient elevation in the hippocampal CA1 sector, the dentate molecular layer and the thalamus 1 h of recirculation. However, the striatum and neocortex revealed no alteration in the [3H]PN200-110 binding. Thereafter, the reduction in the [3H]PN200-110 binding was seen only in the dorsolateral part of the striatum and the hippocampal CA1 sector. The results suggest that transient cerebral ischemia can cause the alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of L-type calcium channel blocker in most of selectively vulnerable areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium antagonist, adenosine A1, and muscarinic bindings in rat hippocampus after transient ischemia

The protective roles of Ca2+ channel blockers against ischemic hippocampal damage are still debated. We used autoradiography to study postischemic L-type Ca2+ channels (1,4-dihydropyridine Ca2+ channel blocker binding), adenosine A1 receptors, and muscarinic cholinergic receptors in the rat hippocampus using [3H]PN200-110 (PN), [3H]cyclohexyladenosine (CHA), and [3H]quinuclidinyl benzilate (QNB), respectively, in 49 rats subjected to 20 minutes of forebrain ischemia. The rats were decapitated after 1 (n = 7), 3 (n = 7), 6 (n = 8), 12 (n = 7), 24 (n = 6), 48 (n = 6), or 168 (n = 8) hours of recirculation; eight control rats were sham-operated but experienced no cerebral ischemia. Reduced receptor binding preceding the delayed death of CA1 pyramidal cells was first observed in the stratum oriens of the CA1 subfield. Significant reductions in [3H]PN, [3H]CHA, and [3H]QNB bindings of this stratum compared with control were noticed after 3 (35%, p less than 0.01), 12 (31%, p less than 0.01), and 1 (10%, p less than 0.05) hours of recirculation, respectively. By 168 hours after ischemia (when the populations of CA1 pyramidal cells were depleted) all strata in the CA1 subfield had lost most of their receptor sites, and [3H]PN, [3H]CHA, and [3H]QNB bindings in the stratum oriens were decreased to 23%, 30%, and 63% of control (p less than 0.01). Although [3H]PN binding in the CA3 subfield did not change significantly during 168 hours after ischemia, the histologically intact dentate gyrus exhibited a 31% loss of binding sites compared with control (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic analysis of second messenger and neurotransmitter system receptors in the gerbil hippocampus following transient forebrain ischemia.

Changes in second messenger and neurotransmitter system receptor ligand binding induced by transient forebrain ischemia were studied in the gerbil hippocampus. The animals were allowed variable periods of recovery ranging from 2 h to 7 days after 5-min bilateral carotid artery occlusion. The binding of second messenger systems ([3H]inositol 1,4,5-trisphosphate ([3H]IP3)to inositol 1,4,5-triphosphate, [3H]forskolin to adenylate cyclase and [3H]phorbol 12,13-dibutylate to protein kinase C) and neurotransmitter receptor systems ([3H]PN200-110 to L-type calcium channels. [3H]N6-cyclohexyl-adenosine to adenosine A1 and [3H]quinuclidinyl benzilate to muscarinic cholinergic receptor) were assayed using quantitative autoradiography. In the CA1 subfield, 2 h after ischemia, [3H]IP3, [3H]forskolin, and [3H]quinuclidinyl benzilate binding activities significantly decreased by 25, 17 and 13%, respectively, though no morphological abnormalities were obvious. Six hours after ischemia, the [3H]phorbol 12,13-dibutylate binding activity in the stratum oriens of the CA1 subfield increased by 15%. One day after ischemia, [3H]PN200-110 binding activity in this subfield decreased by 26%, and 7 days after ischemia, [3H]phorbol 12,13-dibutylate and [3H]N6-cyclohexyl-adenosine receptor binding activities decreased in this subfield. In particular, at 7 days after ischemia, [3H]IP3 binding activity in the CA1 subfield showed a complete decline. In the CA3 subfield, [3H]PN200-110 binding activity decreased 2 days after ischemia, and [3H]IP3 and [3H]N6-cyclohexyl-adenosine binding activities decreased 7 days after ischemia. In the dentate gyrus, the structure of which remained histologically intact after ischemic insult, [3H]IP3 and [3H]forskolin binding activities decreased 7 days after ischemia. In contrast, the [3H]phorbol 12,13-dibutylate binding activity increased in the molecular layer of the dentate gyrus 7 days after ischemia. These results indicate that marked alteration of intracellular signal transduction precedes neuronal damage in the hippocampal CA1 subfield and that the histologically intact CA3 and dentate gyrus also shows modulated neuronal transmission after ischemia.     

Protection of rat hippocampus against ischemic neuronal damage by pretreatment with sublethal ischemia

We examined whether preconditioning with sublethal ischemia protects against neuronal damage following subsequent lethal ischemic insults. Forebrain ischemia for 3 min in Wistar rats increased heat shock protein-70 immunoreactivity in the hippocampal CA1 subfield but produced no neuronal damage. Preconditioning with 3 min of ischemia followed by 3 days of reperfusion protected against hippocampal CA1 neuronal damage following 6 and 8 min of ischemia but not damage after 10 min of ischemia. The result strongly suggests that stress response induced by sublethal ischemia protects against ischemic brain damage.     

Autoradiographic analysis of second-messenger systems in the gerbil hippocampus following repeated brief ischemic insults.

Using [3H]inositol 1,4,5-triphosphate (IP3), [3H]phorbol 12,13-dibutyrate (PDBu) and [3H]forskolin, we performed quantitative autoradiography to determine sequential alterations in second-messenger systems in the gerbil hippocampus following repeated brief ischemic insults. Changes following three 2-min ischemic insults were compared with those following single 2- or 6-min ischemia. [3H]IP3 binding was extremely sensitive to ischemic insult, and more than 80% of the binding sites were lost after destruction of CA1 pyramidal cells following 6-min ischemia and three 2-min ischemic insults. Furthermore, a 30% reduction was observed after 2-min ischemia which leads to no neuronal loss. [3H]PDBu binding in the CA1 subfield decreased by 1 day after three 2-min ischemic insults and by 4 days after 6-min ischemia, and 40-50% reductions were observed at 1 month. In contrast, [3H]forskolin binding was relatively preserved. [3H]PDBu and [3H]forskolin binding transiently increased early in the reperfusion period. We also observed a difference in the pattern and severity of alterations between repeated ischemic insults and single ischemia.     

Regional variations in particulate cyclic AMP dependent-protein kinase binding activity in the gerbil hippocampus following transient forebrain ischemia by [3H]cyclic AMP binding.

Changes in the binding of [3H]cyclic AMP as an indicator of particulate cyclic AMP-dependent protein kinase (AMP-DPK) binding activity following transient forebrain ischemia were studied in the gerbil using in vitro autoradiography. [3H]Cyclic AMP binding in the strata pyramidale and lacunosum-moleculare of the hippocampal CA1, the stratum pyramidale of the CA3, and the dentate gyrus decreased transiently in the early postischemic phase but then recovered. However, [3H]cyclic AMP binding in the strata pyramidale and radiatum of the CA1, the granular layer of the dentate gyrus, and the upper layer of the cortex decreased again 7 days after ischemia. In the CA4 subfield and the lower layer of the cortex, the binding showed no significant alterations after ischemia. Administration of pentobarbital prior to the induction of ischemia prevented the decrease in [3H]cyclic AMP binding in the CA1 subfield 6 h and 7 days after ischemia, and showed protective effects against neuronal death of the CA1 pyramidal cells 7 days after ischemia. These results indicate that marked alteration of intracellular signal transduction precedes neuronal damage in the hippocampal CA1 subfield. Furthermore, postischemic reduction of [3H]cyclic AMP binding in the histologically intact cerebral cortex, CA3, and dentate gyrus may be the reflection of cellular dysfunction after energy failure.     

Quantitative autoradiographic analysis of muscarinic cholinergic and adenosine A1 binding sites after transient forebrain ischemia in the gerbil

The influence of transient forebrain ischemia on adenosine A₁ and muscarinic cholinergic receptors in the gerbil brain 1–27 days after recirculation was studied. The topographical distribution and the alteration in the adenosine A₁ and muscarinic receptor sites were analyzed by means of quantitative receptor autoradiography using [³H]cyclohexyladenosine ([³H]CHA) and [³H]quinuclidinyl benzilate ([³H]QNB), respectively. In most regions examined, the temporal profiles of the alteration of the receptor density were in accordance with the histopathological findings. [³H]CHA binding activity decreased suddenly after neuronal damage, while [³H]QNB grain density showed a gradual decrease in the dorsolateral caudate-putamen and in the CA₁ subfield of the hippocampus. In the caudate-putamen, [³H]CHA and [³H]QNB binding activity in the dorsal aspect was markedly reduced 1–27 days after ischemia. [³H]CHA binding activity in the ventromedial region of the caudate-putamen also decreased 1–3 days after ischemia, though neuronal damage was restricted to the dorsolateral aspect. Neuronal death in CA₁ was preceded by the decrease in [³H]QNB binding activity in the stratum radiatum 1 and 2 days after ischemia. Marked decrease in [³H]QNB and [³H]CHA binding activity was noted in the CA₁ subfield 3–27 days after recirculation. Three to 27 days after ischemia, the A₁ binding activities in the CA₃ subfield of the hippocampus and int he dentate gyrus were reduced despite the normal appearance of these areas throughout the reperfusion period. Muscarinic binding sites in the CA₃ subfield were also reduced 27 days after ischemia. Despite minimal neuronal damage in the lateral septal nucleus and in the substantia nigra, the A₁ binding activity in these regions was reduced by 70% and 50%, respectively. These results provide further evidence that the muscarinic receptors in the dorsolateral region of the caudate-putamen are localized postsynaptically on small and medium-sized neurons and that those in the CA₁ subfield of the hippocampus are localized on the CA₁ pyramidal cells.     

Alteration of voltage-dependent calcium channels in canine brain during global ischemia and reperfusion.

Elevated intracellular calcium (iCa2+) plays an important role in the pathophysiology of ischemic brain damage. The mechanisms by which iCa2+ increases are uncertain. Recent evidence implicates the voltage-dependent calcium channel (VDCC) as a likely site for the alteration in Ca2+ homeostasis during ischemia. The purpose of this study was to determine whether VDCCs are altered by global ischemia and reperfusion in a canine cardiac arrest, resuscitation model. We employed the radioligand, [3H]PN200-110, to quantitate the equilibrium binding characteristics of the VDCCs in the cerebral cortex. Twenty-five adult beagles were separated into four experimental groups: (a) nonischemic controls, (b) those undergoing 10-min ventricular fibrillation and apnea, (c) those undergoing 10-min ventricular fibrillation and apnea followed by spontaneous circulation and controlled respiration for 2 and (d) 24 h. Brain cortex samples were taken prior to killing of the animal, frozen immediately in liquid nitrogen, and crude synaptosomal membranes isolated by differential centrifugation/filtration. After 10 min of ischemia the maximal binding (Bmax) of [3H]PN200-110 increased to greater than 250% of control values (control Bmax 11.16 +/- 0.98; ischemic 28.35 +/- 2.78 fmol/mg protein; p less than 0.05). Bmax returned to near control values after 2 h of reperfusion but remained significantly greater than the control at 24 h. Although the affinity constant (Kd) (control = 0.12 +/- 0.03 nM) appeared to increase with ischemia and normalize with reperfusion, the changes were not statistically significant. We conclude that the binding of [3H]PN200-110 to L-type VDCCs is increased after 10 min of global ischemia/anoxia produced by ventricular fibrillation and apnea in the dog.(ABSTRACT TRUNCATED AT 250 WORDS)     

Facilitated recovery from postischemic suppression of protein synthesis in the gerbil brain with ischemic tolerance

Preconditioning of the gerbil brain with a 2-min period of sublethal ischemia followed by 4 days of reperfusion protects against neuronal damage following a subsequent 3-min period of ischemia, which normally destroys pyramidal neurons in the CAI region of the hippocampus. To clarify the role of protein synthesis in this ischemic tolerance phenomenon, we performed an autoradiographic analysis with [¹⁴C]leucine at 4 h, 24 h, and 48 h after 3 min of ischemia with and without preconditioning. General protein synthesis in the CAI region was severely suppressed after 4 h in both groups. The protein synthesis in CAI partially recovered after 24 h and fully recovered after 48 h in animals with preconditioning, but never recovered in animals without preconditioning. Protein synthesis in the neocortex and the striatum was suppressed in the early reperfusion periods only in animals without preconditioning. The results show that the ischemic tolerance is closely related to the facilitated recovery from suppressed protein synthesis in the brain after ischemia.     

Effects of naftidrofuryl oxalate, a 5HT2 antagonist, on neurotransmission and transduction systems in the gerbil hippocampus

We investigated the effects of age and naftidrofuryl oxalate (Naftidrofuryl), a 5-HT₂ antagonist, on neurotransmission and transduction systems in the gerbil hippocampus using quantitative autoradiography. [³H]Quinuclidinyl benzilate (QNB), [³H]cyclohexyl-adenosine (CHA), [³H]MK-801, and [³H]muscimol were used to label muscarinic acetylcholine, adenosine A1, N-methyl-d-aspartate (NMDA), and γ-aminobutyric acid-A (GABA_A) receptors, respectively. [³H]PN200-110 labeled L-type Ca²⁺ channels. [³H]Forskolin, [³H]cyclic adenosine monophosphate (cAMP), [³H]phorbol 12,13-dibutyrate (PDBu), and [³H]inositol 1,4,5-triphosphate (IP₃) were used to label adenylate cyclase, cAMP-dependent protein kinase, protein kinase C (PKC), and IP₃ receptors, respectively. Approximately 20% reductions in [³H]QNB, [³H]forskolin, and [³H]PDBu binding were observed in the hippocampus of 9-month-old gerbils in comparison with 5-week-old gerbils. Treatment with Naftidrofuryl (10 mg/kg, i.p., once a day for 7 days) ameliorated these reductions. No changes were found in [³H]CHA, [³H]MK-801, [³H]muscimol, [³H]PN200-110, [³H]cAMP, and [³H]IP₃ binding. The results suggest that Naftidrofuryl may have beneficial effects on the age-related alterations in signal transmission and transduction systems in the brain. Because the acetylcholine system, adenylate cyclase, and PKC are considered to be involved in learning and memory processes, the result may have clinical implications.     

Cerebral ischemic preconditioning induces lasting effects on CA1 neuronal survival, prevents memory impairments but not ischemia-induced hyperactivity

In ischemic preconditioning, prior exposure to a short 3-min global ischemia provides substantial protection against the deleterious effects of a subsequent prolonged ischemic insult in rats. The objective of the present study was to determine if the neuronal protection induced by ischemic preconditioning influence functional recovery following a 6-min ischemic insult in rats. Animals received either sham-operation, a 3-min ischemia, a preconditioning 3-min global ischemia followed 3 days later by a 6-min global ischemia or a single 6-min global ischemia. Open field habituation, memory performance in the 8-arm radial maze and object recognition were assessed at different intervals following ischemia. Our findings revealed that preconditioning reversed ischemia-induced spatial memory deficits in the 8-arm radial maze, as suggested by significant reduction of working memory errors in preconditioned as compared to ischemic animals. Preconditioning also attenuated ischemia-induced object recognition deficits at short-term intervals. Nonetheless, preconditioning failed to alter ischemia-induced hyperactivity as demonstrated by enhanced behavioral activity in the open field in both preconditioned and ischemic animals compared to 3-min ischemic and sham-operated rats. CA1 cell counts revealed significant neuronal sparing in preconditioned animals that was observed 6-month following reperfusion. Together, these findings suggest that neuronal survival in preconditioned rats is associated with significant improvements of hippocampal-dependent memory functions and, further support that ischemia-induced hyperactivity may not solely depend on selective neuronal damage to hippocampal neurons.     

Postischemic changes of [3H]nimodipine and [3H]ryanodine binding in the gerbil striatum and hippocampus

Sequential alterations of [³H]nimodipine and [³H]ryanodine binding in gerbils were investigated in selectively vulnerable regions, such as the striatum and hippocampus, 1 h to 7 days after 10 min of transient cerebral ischemia. [³H]Nimodipine binding showed no significant changes in the striatum and hippocampus up to 48 h after ischemia. Seven days after ischemia, however, a severe reduction in [³H]nimodipine binding was observed in the dorsolateral striatum, hippocampal CA1 (stratum oriens, stratum pyramidale and stratum radiatum) and hippocampal CA3 sector. On the other hand, [³H]ryanodine binding showed a significant increase in the hippocampus 1 h after ischemia. Five hours after ischemia, a significant reduction in [³H]ryanodine binding was observed only in the hippocampal CA1 sector. Thereafter, the striatum and hippocampus showed no significant alterations in [³H]ryanodine binding up to 48 h after ischemia. After 7 days, a marked reduction in [³H]ryanodine binding was observed in the striatum and hippocampus which were particularly vulnerable to ischemia. These results demonstrate that postischemic alteration in [³H]nimodipine and [³H]ryanodine binding is produced with different processes in the hippocampus. They also suggest that the mechanism for striatal cell damage caused by transient cerebral ischemia may, at least in part, differ from that for hippocampal neuronal damage. Furthermore, our findings suggest that abnormal calcium release from intracellular stores may play a pivotal role in the development of hippocampal neuronal damage.     

GABA and benzodiazepine receptors in the gerbil brain after transient ischemia: demonstration by quantitative receptor autoradiography

Quantitative receptor autoradiography was used to measure the binding of gamma-aminobutyric acid (GABA) and benzodiazepine receptors after ischemia by means of transient occlusion of bilateral common carotid arteries in the gerbil. [3H]Muscimol was used to label the GABAA receptors and [3H]flunitrazepam to label central type benzodiazepine receptors. In the superolateral convexities of the frontal cortices, [3H]muscimol binding was increased in 60% of the animals killed 3 days after ischemia, and decreased in 67% of the animals killed 27 days after ischemia. Twenty-seven days after ischemia, [3H]flunitrazepam binding in the substantia nigra pars reticulata increased to 252% of the control, though the increase in [3H]muscimol binding was not significant. In the dorsolateral region of the caudate putamen, marked neuronal necrosis and depletion of both [3H]muscimol and [3H]flunitrazepam binding sites were observed 27 days after ischemia, the ventromedial region being left intact. In spite of the depletion of pyramidal cells in the CA1 region of the hippocampus, both [3H]muscimol and [3H]flunitrazepam binding sites were preserved 27 days after ischemia. Since our previous study revealed that adenosine A1 binding sites were depleted in the CA1 subfield of the hippocampus after ischemia correlating with neuronal damage, GABAA and benzodiazepine receptors may not be distributed predominantly on the pyramidal cells in the CA1 region.     

Discrepancy between cell injury and benzodiazepine receptor binding after transient middle cerebral artery occlusion in rats

We investigated postischemic alterations in benzodiazepine receptor, D1 dopamine receptor, and muscarinic acetylcholine receptor binding after transient middle cerebral artery (MCA) occlusion in rats using [3H]-flumazenil, [3H]-SCH23390, and [3H]-N-methyl-4-piperidyl benzilate ([3H]-NMPB), respectively, as radioligand. These ligand bindings were determined at 3 and 24 h and at 3 and 7 days after ischemia/reperfusion of MCA by using autoradiographic methods. Ischemic cell injury was clearly detected from 3 h after ischemia/reperfusion and progressively increased from 3-24 h after ischemia/reperfusion of MCA. The area of cell injury reached maximum at 24 h after ischemia/reperfusion of MCA. [3H]-SCH23390 binding was reduced to 47% of the contralateral side at 3 days after ischemia/reperfusion of MCA. After 7 days, [3H]-SCH23390 binding was further reduced by 20% in the striatum. [3H]-NMPB binding was slightly decreased in both the striatum and cerebral cortex at 3 days after ischemia/reperfusion of MCA, and [3H]-NMPB binding in the striatum and cerebral cortex were reduced to 42 and 62% of the contralateral side at 7 days after ischemia/reperfusion of MCA. [3H]-NMPB was also decreased at 24 h. In contrast, [3H]-flumazenil binding was not decreased in the striatum and cerebral cortex within 7 days after ischemia/reperfusion of MCA. These results suggest that [3H]-SCH23390 and [3H]-NMPB binding do not correlate with cell injury by ischemia/reperfusion, although vulnerability to ischemia/reperfusion was observed with these receptors. In addition, central benzodiazepine receptor imaging might be essentially stable to neuronal cell injury induced by transient focal cerebral ischemia in rats, in contrast to the results of PET studies.     

A correlative study of calcium channel antagonist binding and local neuropathological features in the hippocampus in Alzheimer''s disease

[3H]PN200-110 binding sites were studied by means of quantitative autoradiography in hippocampal sections of patients with Alzheimer's disease and age-matched control subjects. Choline acetyltransferase activity, plaque, tangle and cell densities were also determined in the same tissue samples used for autoradiographic studies. Quantitative autoradiographic analysis of [3H]PN200-110 binding in control hippocampus revealed a heterogeneous pattern similar to that described in rodents, being particularly high in the dentate gyrus. In Alzheimer's disease, [3H]PN200-110 binding was markedly reduced in the subiculum (control = 9.85 +/- 1.41 pmol/g; Alzheimer = 3.41 +/- 0.54 pmol/g, mean +/- S.E.M., P less than 0.001). In the subiculum there was a disproportionate reduction of [3H]PN200-110 binding in comparison to cell loss in Alzheimer's disease. The activity of choline acetyltransferase in the hippocampus was markedly reduced in Alzheimer's disease (controls 6.9 +/- 1.0; Alzheimer 2.7 +/- 0.9 nmol/h/mg protein, mean +/- S.E.M., P less than 0.01). There was a strong correlation between choline acetyltransferase activity and [3H]PN200-110 binding in the subiculum. [3H]PN200-110 binding did not correlate with plaque density in the subiculum. The discrete reduction and preservation of [3H]PN200-110 binding in the present study is consistent with the pattern of selective cellular vulnerability in the hippocampal region in Alzheimer's disease.     

针刺预处理对短暂性脑缺血再灌注损伤海马神经元的保护

The present study established a model of brain ischemia in aged rats using four-vessel occlusion.We observed hippocampal CA1 neuronal apoptosis and apoptosis-mediated protease caspase-3 expression following preconditioning of electroacupuncture at Baihui(GV 20).Our results showed that the number of hippocampal CA1 normal neurons was decreased,and degenerated neurons were increased 12 hours to 3 days following cerebral ischemia/reperfusion.The number of hippocampal CA1 apoptotic neurons and caspase-3-positive neurons in rats with cerebral ischemia/reperfusion injury was significantly decreased following acupuncture preconditioning.Acupuncture preconditioning protects aged rats against ischemia/reperfusion injury by regulating caspase-3 protein expression.     

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.     

Remifentanil postconditioning improves global cerebral ischemia-induced spatial learning and memory deficit in rats via inhibition of neuronal apoptosis through the PI3K signaling pathway

Global cerebral ischemia followed by reperfusion, which leads to extensive neuronal damage, particularly the neurons in the hippocampal CA1 region. Apoptosis is one of the major mechanisms that lead to neuronal death after cerebral ischemia and reperfusion. The neuroprotective effects of remifentanil preconditioning against cerebral ischemia/reperfusion injury have been recently reported. Here we investigated whether remifentanil postconditioning exerts neuroprotective effects against global cerebral ischemia/reperfusion injury in rats and its potential mechanisms. Global cerebral ischemia was performed via 10 min of four-vessel occlusion. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells and expression of Bcl-2 and Bax in the hippocampal CA1 region were assessed after reperfusion. Morris water maze task was used to quantify spatial learning and memory deficits after reperfusion. We found remifentanil postconditioning markedly improved the spatial learning and memory as well as attenuated neuronal apoptosis in hippocampus caused by cerebral ischemia/reperfusion injury. In addition, remifentanil postconditioning enhanced the expression of anti-apoptotic gene Bcl-2 while suppressed the expression of pro-apoptotic gene Bax in hippocampal CA1 region. However, the neuroprotective effects of remifentanil postconditioning were abolished by pretreatment of the PI3K inhibitor LY294002. The results suggest that remifentanil postconditioning exhibits neuroprotective effects against global cerebral ischemia/reperfusion injury in rats, and its mechanisms might involve inhibition of neuronal apoptosis through the PI3K pathway.