Preserved neurotransmitter receptor binding following ischemia in preconditioned gerbil brain

Preconditioning the brain with sublethal ischemia induces tolerance to subsequent ischemic insult. Using [3H]quinuclidinyl benzilate (QNB), [3H]MK 801, [3H]cyclohexyladenosine, [3H]muscimol, and [3H]PN200-110, we investigated the alterations in neurotransmitter receptor and calcium channel binding in the gerbil hippocampus following ischemia with or without preconditioning. Two-minute forebrain ischemia, which produced no neuronal damage, resulted in no alterations in binding except for a slight reduction in [3H]QNB binding in the CA1 subfield. Three-minute ischemia destroyed the majority of CA1 pyramidal cells and caused, in CA1, reductions in binding of all ligands used. Preconditioning with 2-min ischemia followed by 4 days of reperfusion protected against CA1 neuronal damage and prevented the reductions in binding although [3H]QNB and [3H]PN200-110 binding transiently decreased in the early reperfusion period, suggesting down-regulation. Thus, preconditioning protects against damage to the neurotransmission system as well as histopathological neuronal death.     

Autoradiographic analysis of second-messenger systems in the gerbil brain

Quantitative in-vitro autoradiographic study was performed to localize two prominent second-messenger systems (the adenylate cyclase and phosphoinositide systems) in the normal gerbil brain. [3H] Forskolin and [3H] phorbol 12, 13-dibutyrate (PDBu) were used to identify the regional distribution of adenylate cyclase and protein kinase C, respectively. The localization of the forskolin binding was not uniform, being particularly concentrated in the striatum, the accumbens nucleus, the olfactory tubercle, the substantia nigra, the CA3 region of the hippocampus and the molecular layer of the cerebellum. On the other hand, the PDBu binding was rather uniform, although the superficial layer of the cerebral neocortices, the strata oriens of the CA1 region of the hippocampus and the molecular layer of the cerebellum showed relatively dense binding. Quantitative autoradiography of the second-messenger systems in the brain is expected to provide important information concerning the role of neurotransmitters in the pathophysiology of various conditions.     

Nuclear binding sites for triiodothyronine in the gerbil brain following ischemia and recirculation

The effect of cerebral ischemia and subsequent recirculation on the nuclear thyroid hormone receptors was investigated. Ischemia was produced by occlusion of the right common carotid artery in the Mongolian gerbil. The thyroid hormone receptors were measured in vitro by a [¹²⁵I]triiodothyronine (T₃) binding assay with isolated nuclei and Scatchard analysis. A rapid increase of the total number of binding sites for T₃ appeared within 30 min of ischemia and reached over 40% by 3 h. During the same 3-h period, the relative binding affinity was reduced by 25%. Upon recirculation after 30 min or 3 h of ischemia, a rapid reversal of measured T₃ binding sites occurred, which progressed to 20–30% below the control value by the recirculation period of 3 h. If the ischemic period was only 30 min, the nuclear T₃ binding capacity recovered toward the control level and the affinity constant returned normal after recirculation for 24 h. When the ischemic period was extended to 3 h, there was progressive loss of receptor sites, and no tendency for recovery of the affinity constant was observed. These results demonstrated a prompt alteration of a specific nuclear regulatory component in cerebral ischemia, which may indicate the importance of such changes within the nuclear regulatory mechanism for reversibility of cerebral function following ischemic insult.     

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.     

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 changes in [H]inositol 1,4,5-triphosphate binding in the gerbil brain following repeated sublethal ischemia

We investigated the regional changes in [³H]inositol 1,4,5-triphosphate (IP₃) binding in the brain following ischemia using in vitro autoradiography. Three 2-min ischemic insults at 1-hr intervals and a 6-min period of ischemia were induced in gerbils and they were killed after 1, 4, and 28 days. Normal animals had high [³H]IP₃ binding in the CA1 subfield of the hippocampus and the striatum. The binding in the CA1 decreased strikingly after both 6-min ischemia and three 2-min ischemic insults. The [³H]IP₃ binding also decreased in the lateral striatum after three 2-min ischemic insults but not after 6 min of ischemia. Histological observations confirmed neuronal damage to these areas of reduced binding. By contrast, we found a marked increase in [³H]IP₃ binding in the ventral thalamus 28 days after three 2-min ischemic insults. Histological observations with Nissl staining revealed an accumulation of fine granular deposits there. Thus, repeated ischemic insults produced more extensive neuronal damage and changes in [³H]IP₃ binding than a single equivalent period of ischemia. The increased [³H]IP₃ binding in the thalamus coincidentally with an accumulation of Nissl-positive granules at the chronic stage after repeated ischemia is of considerable interest.     

Effects of ischemia on regional ligand binding to adrenoceptors in the rat brain.

Changes in ligand binding to adrenoceptors ([3H]prazosin to alpha 1-receptors, [3H]idazoxan to alpha 2-receptors and [125I]cyanopindolol to beta-receptors) following transient cerebral ischemia were investigated using autoradiographic methods. The binding was quantified in brain sections from control rats, rats subjected to 15 min of 2-vessel occlusion ischemia, and rats with recirculation times of 1 h, 1 week or 4 weeks after ischemia. No significant change in alpha 1-receptor binding was observed during and immediately following ischemia, but a decrease was noted in the vulnerable hippocampal CA1 region following 1 week's survival. In the parietal cortex, the ligand binding to alpha 1-receptors increased at 4 weeks. A reduced [3H]idazoxan binding was observed 1 h after ischemia in the temporal cortex and amygdala. No change in ligand binding to beta-receptors was seen in the early phase postischemia, but a marked increase had occurred in the hippocampal CA1 region at 1 and 4 weeks after ischemia (+163% and +142%, respectively), presumably due to accumulation of macrophages expressing beta-receptors. The early postischemic changes in receptor binding may represent downregulation of the adrenoceptors by processes activated during ischemia, while neuronal degeneration, compensatory mechanisms in surviving neurons and proliferation of non-neuronal cells may account for the subsequent changes.     

Blood–brain barrier damage in reperfusion following ischemia in the hippocampus of the Mongolian gerbil brain

Vascular permeability to intravenously injected horseradish peroxidase (HRP) was qualitatively examined in the hippocampus of ischemic Mongolian gerbil brains by light and electron microscopy. After 30 min of right common carotid artery occlusion followed by 90 min of reperfusion, the animal was perfused with a fixative and killed. Before the perfusion of the fixative, HRP was injected into the femoral vein. HRP was visualized with tetramethyl benzidine (TMB) and diamino-benzidine (DAB) for light and electron microscopy, respectively. Staining reaction with TMB for HRP appeared in medial or dorsal portions of the operated side of the hippocampus, especially around some vessels along the hippocampal fissure. Ultrastructural examination in the vessels along hippocampal fissure revealed that the endothelial cytoplasm contained HRP-filled vesicles or vacuoles in close proximity to the basal lamina, and seemed to be slightly electron-dense. Swollen pericytes, swollen astrocytic foot processes and perivascular cells with HRP-filled cytoplasm were also observed in that area. In this study, it was clearly demonstrated that intravascular macromolecules leaked transendothelially, through vessel walls in the hippocampal fissure, from the blood stream in the medial portions of the hippocampus during reperfusion following ischemia. These findings suggest that the blood–brain barrier in some vessels along the hippocampal fissure in the medial parts of the hippocampus is more vulnerable to ischemic insults than those in other brain areas.     

Alteration of muscarinic binding in specific brain areas following estrogen treatment

Systemic administration of estradiol benzoate (EB) to ovariectomized female rats significantly altered cholinergic muscarinic binding of [³H]quinuclidinyl benzilate in discrete brain regions. Specifically, EB increased muscarinic binding in the medial basal hypothalamus (MBH) and decreased muscarinic binding in the medial preoptic area (POA). These alterations were dose-dependent and appeared to reflect changes in the number of muscarinic binding sites. Treatment with EB failed to significantly affect binding in several control areas in females or in the MBH and POA of males.     

Effects of felbamate on brain polyamine changes following transient cerebral ischemia in the Mongolian gerbil

We sought to determine whether treatment with felbamate was capable to reduce the accumulation of putrescine induced by transient forebrain ischemia in the Mongolian gerbil. Gerbils underwent 10 min ligation of common carotid arteries followed by recirculation. Immediately after the release of the arterial occlusion, felbamate (75 and 150 mg kg(-1) i.p.) was administered. Putrescine and polyamine levels were measured in hippocampus and striatum at 1, 8, 24 and 48 h after recirculation. Putrescine levels appeared enhanced already 8 h after the release of the arterial occlusion and kept increasing up to 48 h in the hippocampus and striatum. No significant changes in spermidine levels during recirculation were detected. Conversely, spermine appeared to decrease in the hippocampus while it did not show changes in the striatum. Felbamate significantly reduced the ischemia induced changes in putrescine brain content only at the dose of 150 mg kg(-1) i.p.     

Autoradiographic analysis on second-messenger systems and local cerebral blood flow in ischemic gerbil brain

Alterations of the second-messenger systems, adenylate cyclase (AC) and protein kinase C (PKC), and local cerebral blood flow (lCBF) were evaluated during experimental cerebral ischemia in gerbils employing a quantitative autoradiographic method, which permitted these three parameters to be measured in the same brain. Ischemia was induced by occlusion of the right common carotid artery for 6 h. Animals attaining more than 5 in their ischemic scores were utilized for further experiments. At the end of ischemia, lCBF was measured by the [14C]iodoantipyrine method. The AC and PKC activities were estimated by the autoradiographic technique developed in our laboratory using [3H]forskolin (FK) and [3H]phorbol-12,13-dibutyrate (PDBu), respectively. The lCBF fell below 10 ml/100 g/min in most cerebral regions on the ligated side. The greatest reduction in FK binding was noted in the olfactory tubercle, caudate-putamen, and globus pallidus, followed by the hippocampus and cerebral cortices. The FK binding tended to be low at lCBF less than 20 ml/100 g/min in the cerebral cortices. However, the PDBu binding was relatively well preserved in each cerebral structure, and no significant correlation between lCBF and PDBu binding was noted in the cerebral cortices. The AC system may thus be vulnerable to ischemic insult over extensive brain regions, while the PKC system may be relatively resistant to ischemia.     

Neuronal damage following repeated brief ischemia in the gerbil

The effect of repetition of brief ischemia, which causes no morphological brain damage when given as a single insult, was studied. Two-minute forebrain ischmia was induced in gerbils singly and 3 or 5b times at 60-min intervals. Although 2-min ischemia induced no neuronal damage, 3 or 5 repeated ischemic insults caused neuronal damage in the selectively vulnerable regions, the severity being dependent on the number of episodes.     

Adenylate cyclase and histopathological changes in the gerbil brain following prolonged unilateral ischemia and recirculation

This study was designed to correlate histopathological changes in gerbil brain following unilateral primary and secondary ischemia to enzymatic-adenylate cyclase damage. At three hrs permanent occlusion of the right common carotid artery only minimal histological changes were evident in cerebrum, hippocampus, striatum and olfactory tubercle while the enzyme responses were unremarkable. Severe histological and enzymatic alterations were present at one hour of recirculation subsequent to 3 hrs of unilateral occlusion. Similar damage was evident at 6 and 24 hrs permanent occlusion. Principal enzyme damage was directed toward basal activity, as well as stimulation of the catalytic (forskolin-sensitive) sites on the enzyme complex. For the most part the transducer (GTP-sensitive) site was unaffected by ischemia until 24 hr ligation. These changes were observed in only those gerbils developing severe symptoms of stroke.     

A flavonoid inhibitor of 5-lipoxygenase inhibits leukotriene production following ischemia in gerbil brain

Leukotrienes C4 and D4 are arachidonic acid metabolites that constrict blood vessels and enhance vascular permeability; their biosynthesis is initiated by the reaction of arachidonic acid with 5-lipoxygenase enzyme. After bilateral carotid artery occlusion for 15 minutes and reperfusion of the gerbil brain for 15 minutes, we determined the brain tissue concentrations of leukotrienes C4 and D4 by radioimmunoassay; they had increased from a baseline concentration of less than 1 to a mean +/- SEM concentration of 12.8 +/- 3.9 pmol/g brain. We also studied the effect of a flavonoid 5-lipoxygenase inhibitor on leukotriene production in the reperfused gerbil brain. A water-soluble flavonoid (5-hexyloxy-3',4'-dihydroxy-6,7-dimethoxyflavone 4'-disodium phosphate) was administered intravenously at a dose of 200 mg/kg body wt; 15 minutes later, both carotid arteries were occluded. The enhanced production of leukotrienes C4 and D4 in the reperfused brain was reduced by approximately 80% (from a mean +/- SEM of 12.8 +/- 3.9 to 2.2 +/- 1.3 pmol/g brain) in the presence of the 5-lipoxygenase inhibitor. The flavonoid did not affect the production of prostaglandin D2, the concentration of which also increased in the reperfused ischemic brain.     

Selective vulnerability in the gerbil hippocampus following transient ischemia

Summary Following brief ischemia, the Mongolian gerbil is reported to develop unusual hippocampal cell injury (Brain Res 239:57–69, 1982). To further clarify this hippocampal vulnerability, gerbils were subjected to ischemia for 3, 5, 10, 20, and 30 min by bilateral occlusion of the common carotid arteries. They were perfusion-fixed after varying intervals of survival time ranging from 3 h up to 7 days. Following brief ischemia (5–10min), about 90% of the animals developed typical hippocampal damage. The lesion was present throughout the extent of the dorsal hippocampus, whereas damage outside the hippocampus was not observed. Each sector of the hippocampus showed different types of cell reaction to ischemia. Ischemic cell change was seen in scattered CA4 neurons, and reactive change was found in CA2, whereas CA1 pyramidal cells developed a strikingly slow cell death process. Ischemia for 3 min did not produce hippocampal lesion in most cases. Following prolonged ischemia (20–30min), brain injury had a wide variety in its extent and distribution. These results revealed that the gerbil brief ischemia model can serve as an excellent, reliable model to study the long-known hippocampal selective vulnerability to ischemia. Delayed neuronal death in CA1 pyramidal cells was confirmed after varying degrees of ischemic insult. These findings demonstrated that the pathology of neuronal injury following brief ischemia was by no means uniform nor simple.     

Mitochondrial metabolism following bilateral cerebral ischemia in the gerbil

Following periods of bilateral cerebral ischemia in urethane-anesthetized Mongolian gerbils, the metabolism of isolated brain mitochondria was assessed in an in vitro oxygenated medium in the presence of the substrates glutamate-malate. Mitochondria from both control and ischemic brains were well coupled. Urethane-anesthetized controls showed an early mild depression and a subsequent enhancement of State 3 respiration (ADP present) relative to awake controls. Respiratory activity in State 3, calculated per mol of cytochrome oxidase, was first depressed below control values following 60 minutes of ischemia; and a further decline, to 46 to 58% of control values, followed 90 to 120 minutes of ischemia. Mitochondrial calcium ion accumulation was depressed by ischemia of 2 hours in moribund animals. In contrast, a significant degree of cerebral edema was observed with ischemia of only 30 minutes' duration. Respiration of brain mitochondria appears relatively resistant to irreversible impairment following cerebral ischemia.     

Delayed neuronal death in the gerbil hippocampus following ischemia

In the CA1 subfield of the gerbil hippocampus, an unusual series of changes were noticed after ischemia. Mongolian gerbils were subjected to bilateral carotid occlusion for 5 min. Perfusion fixation was performed 3, 6 and 12 h or 1, 2, 4, 7 and 21 days afterwards. Specimens obtained from the dorsal hippocampus were processed for light and electron microscopy. Three different types of changes were observed in the CA4, CA2 and CA1 subfields. In CA4, the change was rapid and corresponded to ischemic cell change. The alteration in CA2 was relatively slow, and identical to what has been called reactive change. On the contrary, the change in the CA1 pyramidal cells was very slow, only becoming apparent by light microscopy 2 days following ischemia. The CA1 subfield was selected for electron microscopic observation. The lamellar alignment of proliferated cisterns of the endoplasmic reticulum was the most conspicuous finding in these cells. Four days following ischemia, almost all of the pyramidal cells in CA1 were destroyed. In the CA1 neuropil, numerous presynaptic terminals remained without being apposed to normal postsynaptic sites. These changes in CA1, called here ‘delayed neuronal death’, may differ from those thought to be typical of ischemic neuronal damage. It was unlikely that the disturbance of local blood vessels was the cause of these changes.     

Sequential alteration of [H]rolipram binding in gerbil brain after transient cerebral ischemia

The time course of rolipram (Ca²⁺/calmodulin independent cyclic adenosine monophosphate inhibitor) binding sites changes following gerbil transient forebrain ischemia was determined using receptor autoradiography. Gerbils subjected to 10-min ischemia revealed a significant reduction in rolipram binding in most selectively vulnerable regions early in the recirculation (1–5 h). Marked reduction in the rolipram binding was seen in the selectively vulnerable areas 48 h or 7 days after ischemia. Thereafter, the rolipram binding in the hippocampal CA1 and CA3 sectors, which were most vulnerable to ischemia, was severely reduced up to 1 month after recirculation. In contrast, the reduction of the rolipram binding activity in other regions recovered to sham-operated level or showed a slight recovery. Interestingly, the dentate gyrus, which was resistant to ischemia, also exhibited a significant reduction of the rolipram binding activity up to I month after ischemia. Eight months after ischemia, the hippocampal CA 1 and CA3 sectors showed severe shrinkage and marked reduction in the rolipram binding. Other regions exhibited no significant reduction in the rolipram binding except for a slight reduction in the thalamus. These results demonstrate that transient cerebral ischemia causes severe reduction in rolipram binding sites in selectively vulnerable areas, and this reduction precedes the neuronal cell loss. These findings may reflect the alteration of an intracellular phosphodiesterase activity after ischemia.     

DNA fragmentation in the CA2 sector of gerbil hippocampus following transient forebrain ischemia

It has been reported that following transient forebrain ischemia in the gerbil, "delayed neuronal death" and "reactive change" occur in hippocampal CA1 and CA2 sectors, respectively. In the present study, using the gerbil transient forebrain ischemia model, we examined brain sections after various recirculation periods and demonstrated, employing the in situ nick-end labeling (TUNEL) method, a nuclear DNA fragmentation in the damaged CA2 neurons.