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Levetiracetam (LEV) is an antiepileptic drug with a unique but as yet not fully resolved mechanism of action. Therefore, by use of a simplified rat-isolated nerve-bouton preparation, we have investigated how LEV modulates glutamatergic transmission from mossy fiber terminals to hippocampal CA3 neurons. Action potential-evoked excitatory postsynaptic currents (eEPSCs) were recorded using a conventional whole-cell patch-clamp recording configuration in voltage-clamp mode. The antiepileptic drug phenytoin decreased glutamatergic eEPSCs in a concentration-dependent fashion by inhibiting voltage-dependent Na⁺ and Ca²⁺ channel currents. In contrast, LEV had no effect on eEPSCs or voltage-dependent Na⁺ or Ca²⁺ channel currents. Activation of presynaptic GABA type A (GABA(A)) receptors by muscimol induced presynaptic inhibition of eEPSCs, resulting from depolarization block. Low concentrations of Zn²⁺, which had no effect on eEPSCs, voltage-dependent Na⁺ or Ca²⁺ channel currents, or glutamate receptor-mediated whole cell currents, reduced the muscimol-induced presynaptic inhibition. LEV applied in the continuous presence of 1 µM muscimol and 1 µM Zn²⁺ reversed this Zn²⁺ modulation on eEPSCs. The antagonizing effect of LEV on Zn²⁺-induced presynaptic GABA(A) receptor inhibition was also observed with the Zn²⁺ chelators Ca-EDTA and RhodZin-3. Our results clearly show that LEV removes the Zn²⁺-induced suppression of GABA(A)-mediated presynaptic inhibition, resulting in a presynaptic decrease in glutamate-mediated excitatory transmission. Our results provide a novel mechanism by which LEV may inhibit neuronal activity.

Three-dimensional (3D) time-of-flight (TOF) is now commonly used in routine magnetic resonance angiography (MRA) studies of the head and neck. However, there are limits to its diagnostic abilities in the clinical field and, in some instances, a more invasive supplementary examination may be required. We incidentally discovered a patient with an aberrant vessel of the head and neck that ran alongside the left carotid artery and contained a constant, slowly pulsating efferent blood flow. 3D-TOF and carotid ultrasonography could not determine the nature and origin of this vessel. Additional studies using flow-spoiled fresh blood imaging (flow-spoiled FBI) and time spatial labeling inversion pulse (time-SLIP) methods were effective in determining that the vessel was the left jugular vein, and that the continuous venous reflux was a result of a venous steal by the right jugular vein. We show that by combining different MRA techniques we can effectively achieve diagnosis without resorting to more invasive examinations. J. Magn. Reson. Imaging 2010;32:429–433. © 2010 Wiley-Liss, Inc.

Preconditioning of gerbil brain with a sublethal forebrain ischemia is known to protect hippocampal CA1 neurons following a subsequent lethal ischemia (the second ischemia) which usually damages neurons (ischemic tolerance). Present report using a confocal laser scanning microscope demonstrated that the hippocampal cells of sham operation gerbils contained immunofluorescent NGF and BDNF and their high-affinity receptors (TrkA and TrkB). A 2-min ischemia caused little change of these proteins (ANOVA test, P0.05). After the second lethal ischemia, in the CA1 area with ischemic preconditioning (2-min ischemia), only BDNF but not NGF and their high-affinity receptors showed a transient reduction at 4 h (ANOVA test, P0.01) and improved from 1 day (ANOVA test, P0.05). In the CA1 area without ischemic preconditioning (sham operation), NGF and its high-affinity TrkA receptor showed a consistent reduction from 4 h to 7 days (ANOVA test, P0.05); BDNF and TrkB decreased transiently from 4 h to 1 day (ANOVA test, P0.05) but were recovered in the surviving neurons from 3 days. At 3 and 7 days after the second lethal ischemia, apoptotic cell injury could be seen in the CA1 area without ischemic preconditioning but was sparsely noted in the CA1 area with ischemic preconditioning. In the ischemia-resistant CA3 and dentate gyrus areas, only BDNF decreased significantly at 7 days in the CA3 area without ischemic preconditioning (ANOVA test, P0.01). However, no significant change occurred in NGF, TrkA and TrkB immunofluorescence from 4 h to 7 days after the second lethal ischemia in the CA3 and dentate gyrus areas with and without ischemic preconditioning. Western blot study showed that in the hippocampal formation with ischemic preconditioning, preconditioning prevented the decline of these protein levels from 1 day to 7 days after the second lethal ischemia (ANOVA test, P0.05). Results of this study demonstrate that ischemic preconditioning recovers the initial decline in NGF and BDNF and their corresponding receptors in the vulnerable CA1 neurons after the second lethal ischemia, suggesting that growth factors might play a role in the protective mechanism of ischemic preconditioning.

Gerbil forebrains were frozen in situ to inactivate the tissues, and 1,2-diacylglycerols were first measured quantitatively by HPLC. Although 1,2-diacylglycerols were completely recovered from the HPLC column, the control amount of 1,2-diacylglycerol in gerbil forebrain was only 79.6 nmol/g wet weight, which is about one-fourth of that previously reported for gerbil brain inactivated by liquid N2 after decapitation instead of in situ freezing. The fatty acid composition of 1,2-diacylglycerols in gerbil forebrain was first reported and the control 1,2-diacylglycerols were richer in palmitic acid than in stearic acid or arachidonic acid, which is rather different from the data previously reported for mouse or rat brain obtained by decapitation and analyzed by traditional TLC methods. The amount of 1,2-diacylglycerol increased by 82.9% in gerbil forebrain during 5 min of ischemia induced by bilateral carotid ligation. Arachidonic acid and stearic acid were abundant in the 1,2-diacylglycerols produced by 5 min of ischemia. Thus we were able to obtain accurate values of the amount and the fatty acid composition of 1,2-diacylglycerols in gerbil forebrains using HPLC and in situ freezing technique.

Immunohistochemistry was used to study herpes simplex virus type 1–induced central nervous system demyelination in the trigminal root entry zone of mice inoculated with herpes simplex virus type 1 by the corneal route. There was no change in peripheral nervous system myelin as shown by immunostaining for Po glycoprotein. Double immunoperoxidase staining for herpes simplex virus type 1 antigens and glial fibrillary acidic protein showed that most of the infected cells were astrocytes. Glial fibrillary acidic protein immunostaining was completely lost in the inferior medial portion of the trigeminal root entry zone at 6 days after herpes simplex virus type 1 inoculation, a time when central nervous system myelin was preserved as indicated by immunostaining for myelin basic protein. The pattern of glial fibrillary acidic protein staining did not change and herpes simplex virus type 1 antigens were no longer detected after day 8. There was a progressive loss of myelin basic protein staining within the area unstained by glial fibrillary acidic protein antisera on days 8 to 14. This pattern of astrocyte loss before central nervous system demyelination is strikingly different from the reactive astrocytosis seen in other demyelinating lesions, such as acute experimental allergic encephalomyelitis, progressive multifocal leukoencephalopathy, or acute multiple sclerosis. Herpes simplex virus type 1 infection in mice provides an unusual model of acute central nervous system demyelination preceded by a loss of astrocytes.

Due to two developments that have taken place within the past few years, the treatment for stroke is about to change immensely. First, it has become possible to significantly inhibit the spread of hypoperfusive tissue damage by initiating antioxidant therapy within 24 hours of a stroke. As a result of this new therapy, many physicians now feel that the prognosis for stroke patients has improved considerably. Second, elucidation of the brain function of post-stroke rehabilitation has made headway as advanced functional brain imaging techniques have provided reliable data, thereby substantiating new rationales.

White matter low attenuation (WMLA) on computed tomography (CT) and the central conduction time (CCT) measured by median nerve-somato-sensory evoked potentials were examined in 12 multi-infarct dementia (MID) patients, 10 patients with multiple infarcts (MI) without dementia and 11 age-matched controls. Patients with deep vascular lesions without cortical infarcts on CT were included. In MID, prolongation of CCT and moderate to severe WMLA were observed. The CCT and WMLA in MI patients were intermediate. The CCT was longer when the WMLA was more extensive. The result suggests that white matter disease in MID is concerned to the occurrence of dementia by disturbing axonal conduction.

1. We review the biochemical and molecular changes in brain with developing cerebral infarction, based on recent findings in experimental focal cerebral ischemia. 2. Occlusion of a cerebral artery produces focal ischemia with a gradual decline of blood flow, differentiating a severely ischemic core where infarct develops rapidly and an area peripheral to the core where the blood flow reduction is moderate (called penumbra). Neuronal injury in the penumbra is essentially reversible but only for several hours. The penumbra area tolerates a longer duration of ischemia than the core and may be salvageable by pharmacological agents such as glutamate antagonists or prompt reperfusion. 3. Upon reperfusion, brain cells alter their genomic properties so that protein synthesis becomes restricted to a small number of proteins such as stress proteins. Induction of the stress response is considered to be a rescue program to help to mitigate neuronal injury and to endow the cells with resistance to subsequent ischemic stress. The challenge now is to determine how the neuroprotection conferred by prior sublethal ischemia is achieved so that rational strategies can be developed to detect and manipulate gene expression in brain cells vulnerable to ischemia. 4. Expansion of infarction may be caused by an apoptotic mechanism. Investigation of apoptosis may also help in designing novel molecular strategies to prevent ischemic cell death. 5. Ischemia/reperfusion injury is accompanied by inflammatory reactions induced by neutrophils and monocytes/macrophages infiltrated and accumulated in ischemic areas. When the role of the inflammatory/immune systems in ischemic brain injury is revealed, new therapeutic targets and agents will emerge to complement and synergize with pharmacological intervention directed against glutamate and Ca2+ neurotoxicity.

Background and Purpose —In vitro studies have shown that nerve growth factor (NGF) is protective to cortical neurons against various insults. However, the role of NGF in relation to its high-affinity trkA receptor in the cortical neurons has not been well discussed. In this experiment, we studied the possible involvement of the NGF/receptor system in the ischemic injury of cortical neurons after focal cerebral ischemia in rats. Methods —Male Wistar rats received right middle cerebral artery occlusion of 90 minutes’ duration. The rats were decapitated at different reperfusion time points: hour 4 and days 1, 3, 7, and 14 of recirculation. Brain sections at the level of striatum were immunostained against NGF, trkA, glial fibrillary acidic protein (GFAP), and stress protein HSP70. Double immunostaining against NGF and GFAP was also performed. Optical density of NGF immunoreactivity in the ischemic and nonischemic cortexes was compared between sham-control and ischemic animals. Results —In the sham-control rats, NGF immunoreactivity was present in the cortical and striatal neurons. However, beginning at hour 4 after recirculation, there was a significant decrease of NGF in the ischemic cortex and striatum. Beginning at day 1, NGF was absent completely in the infarcted striatum and cortex. However, in the peri-infarct penumbra area, despite a decrease in NGF at hour 4 and day 1, NGF recovered beginning at day 3 and returned almost to the sham-control level at day 14. In the nonischemic cortex, NGF increased beginning at hour 4, peaked at day 7, and returned almost to the sham-control level at day 14. The trkA and HSP70 immunoreactivities were not present in the sham-control cortex. However, trkA was induced at hour 4 in the ischemic cortex and at days 1 and 3 in the peri-infarct penumbra cortex. The HSP70 was induced at days 1 and 3 in the peri-infarct penumbra area. Double immunostaining showed that the number of GFAP-positive cells increased gradually, and NGF immunoreactivity in the GFAP-positive cells became gradually intense after ischemia. Conclusions —Our study demonstrated a temporal profile of NGF and trkA in the ischemic cortex and NGF expression by reactive astrocytes. Our data suggest that the NGF/receptor system may play a role in the astrocyte/neuron interaction under certain pathological conditions, such as focal cerebral ischemia.

In the immunohistochemical staining of nerve growth factor, it has been reported that fixation-dependent lability of nerve growth factor hampers its localization. In the present study, we used two different polyclonal antibodies to immunostain nerve growth factor in rat brain tissue. We found that in paraformaldehyde-fixed (immersion- or perfusion-fixed) brains, nerve growth factor-like immunoreactivity was located primarily in the cytoplasmic membrane and fiber tract of hippocampal neurons and was sparse in cortical neurons. When fresh frozen brain sections were fixed in paraformaldehyde solution, nerve-growth factor-like immunoreactivity was distributed evenly in the cell body. However, when fresh frozen brain sections were fixed in acetone, immunoreactivity to nerve growth factor was present as discrete or confluent dense particles in the cell body, especially in the nuclear region. Also, when paraformaldehyde-perfusion-fixed brain sections were heat treated in salt solution before immunostaining, nerve growth factor-like immunoreactivity could be retrieved in the cytoplasmic and nuclear regions. The hippocampal formation, cerebral cortex and basal forebrain expressed nerve growth factor-like immunoreactivity. Double immunostaining in fresh frozen brains showed that the low-affinity nerve growth factor receptor (p75) co-expressed with nerve growth factor and trkA proto-oncogene in basal forebrain neurons. Our study shows that formaldehyde fixation can mask nerve growth factor antigen, and special treatment, such as heating, is needed to retrieve nerve growth factor antigen to permit immunohistochemical detection. For immunohistochemical study of nerve growth factor in rat brain tissue, successful immunostaining can be obtained by using fresh frozen brains to prevent the masking effect of fixatives or by using paraformaldehyde-fixed brains with heat treatment. It is likely that nerve growth factor is synthesized and accumulated mainly in the cell body but not in the fiber tracts, which is similar to the distribution of its messenger RNA. The co-existence of p75 with nerve growth factor and trkA in basal forebrain neurons suggests the role of low- and high-affinity receptors in regulating the trophic effect of nerve growth factor.

We examined the change of regional cerebral blood flow (rCBF) and local cerebral glucose utilization (LCGU) in the middle cerebral artery (MCA) occlusion or recirculation model of rats, and tested anti-ischemic effects of a free radical scavenger, 3-methyl-1-phenyl-pyrazolon-5-one (MCI-186). A remarkable increase in LCGU was observed in the cortex supplied by the anterior cerebral artery after recirculation. This hypermetabolism of glucose was at least partly caused by the postischemic oxidative injury, since MCI-186 ameliorated the high LCGU in this area. These results suggested the usefulness of this type of free radical scavenger for inhibiting the postischemic injury.

Earlier we indicated that neutrophilic invasion into cerebral parenchyma is an important step in rat cerebral ischemia-reperfusion injury and the production of chemotactic factors, cytokine-induced neutrophil chemoattractant (CINC) precede the neutrophilic invasion. The aim of the present study was to evaluate the role of CINC production and the therapeutic possibility of blocking CINC activity in the transient ischemic brain damage in rats. Focal transient ischemia was produced by intraluminal occlusion of the right middle cerebral artery for 60 min. An enzyme immunoassay was used to measure the brain concentration of CINC and myeloperoxidase activity in ischemic areas was measured as a marker of neutrophilic accumulation. An immunohistochemical staining technique was used to detect the immunopositive cells for anti-CINC antibody. Further, application of anti-CINC antibody or anti-neutrophil antibody to rats was used to evaluate the role of CINC production. In ischemic areas, CINC production was detected and peaked 12 h after reperfusion, which followed 60 min of ischemia. Intraperitoneal injection of anti-neutrophil antibody 24 h before and immediately after reperfusion significantly reduced the brain water content and partially reduced the CINC production in ischemic areas. Further, immunohistochemical staining showed that anti-CINC antibody was found on the endothelial surface of venules and on parts of neutrophils that had invaded the ischemic area 6 to 24 h after reperfusion. Also, treatment with anti-CINC antibody reduced ischemic edema formation 24 h after reperfusion and the size of infarction areas 7 days after reperfusion. It thus appears that CINC, mainly produced by endothelium activated by factors released from neutrophils, plays an important role in ischemic brain damage. Furthermore, the blocking of CINC activity with antibody suggests an immuno-therapeutic approach to the treatment of stroke patients.

Levetiracetam (LEV) is an antiepileptic drug with a unique but as yet not fully resolved mechanism of action. Therefore, by use of a simplified rat-isolated nerve-bouton preparation, we have investigated how LEV modulates glutamatergic transmission from mossy fiber terminals to hippocampal CA3 neurons. Action potential-evoked excitatory postsynaptic currents (eEPSCs) were recorded using a conventional whole-cell patch-clamp recording configuration in voltage-clamp mode. The antiepileptic drug phenytoin decreased glutamatergic eEPSCs in a concentration-dependent fashion by inhibiting voltage-dependent Na⁺ and Ca²⁺ channel currents. In contrast, LEV had no effect on eEPSCs or voltage-dependent Na⁺ or Ca²⁺ channel currents. Activation of presynaptic GABA type A (GABA(A)) receptors by muscimol induced presynaptic inhibition of eEPSCs, resulting from depolarization block. Low concentrations of Zn²⁺, which had no effect on eEPSCs, voltage-dependent Na⁺ or Ca²⁺ channel currents, or glutamate receptor-mediated whole cell currents, reduced the muscimol-induced presynaptic inhibition. LEV applied in the continuous presence of 1 µM muscimol and 1 µM Zn²⁺ reversed this Zn²⁺ modulation on eEPSCs. The antagonizing effect of LEV on Zn²⁺-induced presynaptic GABA(A) receptor inhibition was also observed with the Zn²⁺ chelators Ca-EDTA and RhodZin-3. Our results clearly show that LEV removes the Zn²⁺-induced suppression of GABA(A)-mediated presynaptic inhibition, resulting in a presynaptic decrease in glutamate-mediated excitatory transmission. Our results provide a novel mechanism by which LEV may inhibit neuronal activity.

The behavioral effects of transient cerebral ischemia in adult Wistar rats were studied. In Experiment 1, rats were subjected to 90-min occlusion of the unilateral, left or right, middle cerebral artery (MCA) followed by recirculation. The locomotor activity had not changed 3 and 30 days after the occlusion, except that the number of rearing was significantly decreased by left MCA occlusion. Rats were examined in a radial maze system for learning and memory ability during 4 weeks from the 3rd day after ischemia (the 3rd day was counted as day 1 of the experiment). Maze performance was slightly disturbed due to focal brain damage by MCA occlusion, but the disturbance was statistically significant only on days 6, 11, and 15 in the right occlusion. In Experiment 2, rats were trained to master a radial maze task completely for 4 weeks, and then subjected to transient unilateral (right) ischemia as described above. These rats showed an increase in incorrect entry in the radial maze task from day 4 to day 14. However, on day 21, the number of incorrect entry decreased to the control level of the sham-operated group. The numbers of correct choice were inversely related with those of incorrect entry, though slightly blunted. Coincidentally, the time required to solve the maze task was also prolonged from day 4 to day 14, but returned to the control time on day 21. These results suggest that unilateral ischemia transiently suppresses both acquiring radial maze performance and maintenance of learned performance and that it is a good model for studying human focal cerebral ischemia.

We studied the temporal profile of nerve growth factor-like immunoreactivity (NGF-LI[ in the rat brains following 30 min of middle ] cerebral artery occlusion. The rats were decapitated at 4 h, 1, 3, 7, and I4 days of recirculation. Brain sections at the level of striatum were immunostained against NGF as well as a stress protein, HSP70. Also, double immunostaining of NGF and glial fibrillary acidic protein was performed. In the sham-control rats, NGF-LI was normally present in the cortical and striatal neurons. However, at 4 h of recirculation, there was a significant decrease of NGF-LI in the ischemic cortex and striatum. From I day, NGF-LI was absent completely in the ischemic striatum. However, in the ischemic cortex, NGF-LI decreased to the lowest level at I day, but it recovered gradually from 3 days and increased significantly to above sham-control level at 7 days. At 14 days of recirculation, NGF-LI returned to a near sham-control level. In the non-ischemic cortex, NGF-LI increased gradually from 4 h with a peak at 7 days, and returned to the sham-control level at I4 days of recirculation. A HSP70 was induced in the ischemic cortex at 1 and 3 days, when there was a significant reduction of NGF-LI. The number of reactive astrocytes increased gradually and NGF-LI in the reactive astrocytes became gradually intense after ischemia. The present finding showing that NGF-LI can be recovered in the stressed cortical neurons suggests a possible involvement of NGF in the process of neuronal survival after focal cerebral ischemia. The expression of NGF in reactive astrocytes indicates that astrocyte may also play a role in supporting neuronal survival after ischemia.

Heme oxygenase (HO) is a rate-limiting enzyme in heme catabolism, the end products of which include iron, carbon monoxide and bilirubin. We investigated the changes in expression of an inducible form, heme oxygenase-1 (HO-1), and a constitutive form, HO-2, in rat brain following 20 min of forebrain ischemia, using specific antisera for HO-1 and HO-2. HO-1 protein was remarkably induced in brain following ischemia, while the level of HO-2 protein was not noticeably affected. The level of HO-1 protein expression was maximal at 12 h, which is in good agreement with the time course of the HO-1 mRNA induction. In the cortical mantle, most of the cells expressing increased HO-1 protein were identified as pyramidal neurons and astrocytes by their shapes and locations. In hippocampal CA-2 and CA-3 subfields, prominent induction was observed in astrocytes rather than in neuronal cells. By contrast, the HO-1 protein was not detected in the CA1 subfield following the insult, although the increased level of transcripts was evident in neurons and glial cells. These results suggest that not only in neuronal cells but also in astrocytes within the CA1 subfield, there may be an impairment of protein metabolism, preceding the delayed CA1 pyramidal cell losses.

Oxygen radicals produced by activated neutrophils have been involved in brain injury during ischemia-reperfusion. Platelet-activating factor (PAF) is a candidate as one of the mediators of neutrophil activation during cerebral ischemia-reperfusion. Recent evidence indicates that PAF-induced neutrophil activation is mediated by thromboxane A2 (TXA2). To study the role of PAF and TXA2 in radical production during cerebral ischemia-reperfusion, we evaluated the effects of a PAF antagonist, Y-24180, and a TXA2 antagonist, S-1452, on radical formation in rats with 1 h middle cerebral artery (MCA) occlusion. In the present study, we employed a new electron spin resonance (ESR) method coupled with brain microdialysis. The method uses the endogenous ascorbyl radical (AR) concentration as a marker of oxygen radicals and requires no spin-trapping agents. In the vehicle controls, extracellular AR from the ischemic brain cortex decreased during MCA occlusion. Following reperfusion, AR significantly increased at 30 mm and 1 h, returned to near the basal levels at 2 h, and increased again at 24 h after reperfusion. In the rats treated with S-1452 or Y-24180, AR decreased during MCA occlusion to the same extent as in the vehicle control. However, pretreatment with Y-24180 or S-1452 significantly attenuated the increase in extracellular AR after reperfusion, while it exerted no effect on the changes in extracellular ascorbate or tissue pO2 throughout the experimental period. In conclusion, PAF and TXA2 might contribute to cerebral ischemia-reperfusion injury by increasing the generation of oxygen radicals.

Cytokines which promote emigration of leukocytes from the vascular lumen into the injured brain tissue are produced at the site of incipient cerebral infarction. The blood-borne invaders then accelerate the decomposition of brain cells by their toxic by-products, phagocytic action, and by the immune reaction. Recently accumulated data in our laboratories and other research facilities show that depleting the amount of circulating leukocytes or administering anti-inflammatory chemicals such as cytokine blocking agents, anti-adhesion molecule antibodies, and immunosuppressants effectively minimize the size of ischemia induced cerebral infarction. Based on the fact that leukocyte invasion of the affected brain tissue occurs 6 to 24 hours after onset of ischemia, administration of an anti-inflammatory therapy may widen the therapeutic window against stroke.

The contribution of cytokines in an inflammatory cascade on cerebral reperfusion injury are characterized as typical phases; leukocytes invision, microglial activation, and remodeling. Within 1-2 days, IL-1 (interleukin-1) and TNF (tumour necrosis factor) induce the expression of adhesion molecules that cause leukocytes adhere to endothelial cells. IL-8 (CINC; cytokine-induced neutrophil chemoattractant) is a well-known chemokine that promotes invasion of these leukocytes into brain parenchyma. The activation of proteases and free radical formation by invading neutrophils induces lipid peroxidation and subsequently neuronal damage. From 2 to 7 days, microglia is activated mainly in the "reactive zone" at the boundary of the infarct, and secrete IL-1 and TNF. These cytokines induce astroglial proliferation and production of trophic factors by astroglia to limit the neuronal damage. However, excess astrogliosis exert a negative effect on neuroregeneration. From 7 to 30 days, phagocytic macrophages are observed in the core of infarction sites. The macrophages release a number of cytophylactic agents including proteases and superoxide anions to degrade the damaged areas. TGF-beta and basic FGF (fibroblast growth factor) from glial cells and macrophages induce angiogenesis to discard the debris for subsequent remodeling. These complicated cascade after cerebral reperfusion injury are indeed controlled by cytokines: IL-1 and TNF are considered to be primary mediators that work in concert with IL-8 and growth factors to initiate and regulate the local inflammation in the brain.

A growing body of experimental data indicate that oxygen radicals may mediate the brain injury during ischemia–reperfusion. One potential source of oxygen radicals is activated neutrophils. To study the role of neutrophils in radical production during cerebral ischemia–reperfusion, we evaluated the effects of depletion of circulating neutrophils by administration of an anti-neutrophil monoclonal antibody (RP3) on radical formation in rats with 1-h middle cerebral artery (MCA) occlusion. In the present study, we employed a new electron spin resonance method coupled with brain microdialysis. The method uses the endogenous ascorbyl radical (AR) concentration as a marker of oxygen radicals and requires no spin-trapping agents. In the vehicle controls, extracellular AR decreased during MCA occlusion. After reperfusion, AR significantly increased at 30 min and 1 h, returned to near basal level until 2 h, and increased again at 24 h after reperfusion. In the rats treated with RP3, AR decreased during MCA occlusion to the same extent as in the vehicle control. However, RP3 treatment completely inhibited the increase in extracellular AR after reperfusion. RP3 treatment exerted no effect on the changes in extracellular ascorbate or tissue Po2 throughout the experimental period. In conclusion, neutrophils are a major source of oxygen radicals during reperfusion after focal cerebral ischemia.

Using an in situ nick translation procedure, DNA single-strand breaks (SSB) in postischemic gerbil hippocampus were investigated after 15-min forebrain ischemia followed by 0–4 h of recirculation. In the control group, increased SSB were noticed in the ependymal cell layer and the dentate gyrus. After 15-min ischemia without recirculation, no remarkable changes in SSB were observed. However, after 1 h of recirculation, a marked increase in SSB was recognized throughout the hippocampus, especially in the cells in CAI subfield and the dentate gyrus. After 4 h of recirculation, SSB decreased to a level near that of the control group. The results of the present study indicate that ischemic insults may injure intranuclear DNA during postischemic recirculation periods. Although many factors may be involved, activated endonuclease due to an intracellular Cat+ rise, free radicals, and postischemic hyperthermia appear to be involved in this phenomenon.

Preconditioning of the gerbil brain with a 2-min period of sublethal ischemia protects against neuronal damage following a subsequent 3-min period of ischemia which normally damages CA1 neurons of the hippocampus (ischemic tolerance). In this study, we investigated the role of a small stress protein, heat shock protein-27, in the induction of ischemic tolerance. For this purpose, we used immunohistochemistry with an antibody against heat shock protein-27. Normal hippocampus contained very low levels of heat shock protein-27. The preconditioning ischemia for 2 min caused little changes in the heat shock protein-27 immunostaining in CA1 neurons but an increase in heat shock protein-27 immunostaining in a small number of astrocytes in the CA3 region and in many astrocytes in the dentate hilus. The second ischemia for 3 min caused no specific changes in heat shock protein-27 immunostaining in CA1 neurons both with and without tolerance in early reperfusion periods. After seven days, destruction of CA1 neurons occurred in animals without preconditioning and reactive astrocytes were intensely immunostained for heat shock protein-27. An intense heat shock protein-27 iimmunostaining was also seen in astrocytes in the dentate hilus after the second ischemia in both groups. Thus, we observed no temporal correlation between the induction of heat shock protein-27 and the manifestation of ischemic tolerance in the CA1 neurons. Most intense heat shock protein-27 immunostaining was observed in reactive astrocytes that accumulated in the damaged CA1 region and dentate hilus. Therefore, heat shock protein-27 is unlikely to have a direct role in the mechanism of ischemic tolerance in the gerbil, but may rather be a component of glial reaction to injury.

Calcium/calmodulin-dependent protein kinase II (kinase II) and protein phosphatase 2B (calcineurin) immunoreactivity in the rat hippocampus was studied 100 days after ischemic damage to hippocampal CA1 pyramidal neurons. One-hundred days after ischemia, only a few CA1 pyramidal neurons survived and they exhibited enhanced kinase II and calcineurin immunoreactivity in their basal and apical dendrites. The stratum lucidum of the CA3 (mossy fiber terminal area) had enhanced kinase II and calcineurin immunoreactivity. These results suggest activity-dependent regulation and redistribution of kinase II and calcineurin after intervention in neuronal circuitry.

Background and Purpose We have indicated that neutrophils play an important role in cerebral ischemia-reperfusion injury. Neutrophils are also known to adhere to the endothelial wall through adhesion molecules and to infiltrate into the tissue, and this neutrophilic invasion correlates with the concentration gradient of chemotactic factors. The aim of the present study was to evaluate the role of cytokine-induced neutrophil chemoattractant (CINC) on brain damage in rats from transient ischemia. Methods The brain water content was measured to evaluate postischemic brain injury in rats with 60 minutes of middle cerebral artery occlusion with reperfusion. An enzyme-linked immunosorbent assay was used to evaluate the blood and brain concentrations of CINC, and enzymatic and histological techniques were used to measure the neutrophilic infiltration into the brain. Results The increase of water content was first observed at 6 hours after reperfusion, after which this increase was gradual, with brain edema peaking from 24 to 48 hours after reperfusion. Neutrophilic infiltration into the parenchyma and myeloperoxidase activity were first noted 12 hours after reperfusion, after which a marked increase occurred from 24 to 48 hours after reperfusion. In the ischemic brain areas, CINC was first detected at 3 hours after reperfusion. The CINC level peaked at 12 hours after reperfusion (9.15±0.45 ng/g wet wt, n=5) and then gradually reduced from 24 to 48 hours after reperfusion (5.35±0.95 ng/g wet wt, n=5, and 1.25±0.10 ng/g wet wt, n=5, respectively). Interestingly, the serum CINC concentration was transiently elevated from 3 to 6 hours after reperfusion. No CINC production was detected in the brain of rats subjected to 60 minutes of ischemia without reperfusion. Conclusions A marked increase in CINC concentration was detected in brain and serum during early reperfusion. This suggests that the time course of CINC production precedes brain edema formation and neutrophilic infiltration. It thus appears that CINC may play an important role in neutrophilic infiltration in ischemic lesion and in brain edema formation after ischemia-reperfusion injury.

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 [ 14 C]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.

Changes of immunoreactivities for microtubule based motor proteins, kinesin and cytoplasmic dynein, and non-motor protein, microtubule associated protein (MAP) 2 were investigated in gerbil hippocampus after transient ischemia. The immunoreactivities for kinesin showed a progressive decrease in hippocampal CA1 cells from 8 h after transient 5 or 15 min of ischemia that is lethal to the CA1 cells, while it showed no change after 2 min of ischemia that is non-lethal to the cells. The immunoreactivities for cytoplasmic dynein showed a decrease from 3 or 1 h of reperfusion in the CA1 cells after 5 or 15 min of ischemia, respectively. In contrast, the immunoreactivity for MAP2 remained normal until 2 days in the CA1 cells after 5 min of ischemia. These results showed an early changes of microtubule based motor proteins, such as kinesin and cytoplasmic dynein in vulnerable CA1 neurons. These changes may affect the mitochondrial shuttle system between neuronal cell body and the peripheries such as axon terminal and dendrites. This early disturbance may cause a failure to obtain newly synthesized nuclear encoded mitochondrial protein, and result in mitochoncrial dysfunctions and the subsequent cell death.

We examined the effects of rolipram, a cyclic AMP-selective phosphodiesterase inhibitor, on cerebral ischemia-induced neuronal damage in Mongolian gerbils. Transient forebrain ischemia was induced by 3-min occulusion of bilateral common carotid arteries. Rolipram, at a dose of 0.3 or 3 mg/kg, was injected i.p. 30 min before ischemia. Histopathological observations showed that neuronal damage to the hippocampal CA1 subfield, which was seen 7 days after ischemia in vehicle-treated animals, was reduced in animals treated with the higher dose of rolipram.

We investigated postischemic changes of non-pyramidal neurons in the gerbil hippocampus 1 h - 7 days after 10 min of cerebral ischemia, with parvalbumin and microtubule-associated protein 2 (MAP2)-immunohistochemistry. Parvalbumin-immunoreactive interneurons in the hippocampus were unaffected up to 24 h after ischemia. A slight reduction of the immunoreactivity in neuronal processes was seen in the hippocampal CA1 sector 48 h after ischemia. Seven days after ischemia, a marked loss of parvalbumin-immunoreactive interneurons was observed in the hippocampal CA1 and CA3 sectors. Furthermore, reduced staining in the dentate granular and molecular layers was observed. MAP2-immunoreactive pyramidal neurons in the hippocampus were unchanged up to 48 h after ischemia. Seven days after ischemia, a severe loss of MAP2 immunoreactivity was found in the hippocampal CA1 and CA3 neurons and dentate hilar neurons. However, scattered CA1 neurons, most likely interneurons, preserved MAP2 immunoreactivity. The results demonstrate that transient cerebral ischemia can cause a loss of parvalbumin-immunoreactive interneurons in the hippocampus. Furthermore, some interneurons seem to lose parvalbumin synthesis. Although dentate granule cells are resistant to ischemia, considerable reductions of afferent input was suggested by parvalbumin staining.

Immunohistochemical changes of striatal interneurons in the gerbil were investigated 1 h–7 days after 10 min cerebral ischemia. Marked reduction of parvalbumin-immunoreactive interneurons was seen in the striatum from 24 h after ischemia. MAP2 (microtubule-associated protein 2) immunoreactivity markedly decreased in striatal neurons 5 h after ischemia but was unaffected in interneurons. Thereafter, a severe loss of MAP2 immunoreactivity in the interneurons was found 48 h and 7 days after ischemia. The results demonstrate that transient cerebral ischemia can cause a loss of parvalbumin and MAP2 immunoreactivity in interneurons in the dorsolateral striatum in a delayed fashion as compared with a rapid loss of striatal neurons.

Neutrophils have been implicated in the pathogenesis of ischemia-reperfusion injury. The aim of the present study was to evaluate the correlation between neutrophil infiltration into ischemic tissues and brain injury after transient focal ischemia. We evaluated the effects of depletion of circulating neutrophils by administration of an antineutrophil monoclonal antibody (RP3) on brain edema formation, infarct size, and neutrophil infiltration (myeloperoxidase [MPO]-quantified) in rats with 1 hour of middle cerebral artery (MCA) occlusion. In the cerebral cortex perfused by the anterior cerebral artery (ACA area), there was a significant increase in MPO activity only 24 hours (P < .05) after reperfusion. In the cerebral cortex perfused by the middle cerebral artery (MCA area) and caudate putamen, MPO activity was significantly increased at 12 (MCA area, P < .01; caudate putamen, P < .05), 24 (MCA area, P < .05; caudate putamen, P < .01), and 72 hours (MCA area, P < .01; caudate putamen, P < .05) and returned to near-normal level by 168 hours after reperfusion. Brain MPO activity after transient MCA occlusion correlated well with the appearance of neutrophils. Depletion of neutrophils by RP3 treatment completely inhibited the increase in MPO activity in the ischemic brain after 24 hours of reperfusion. In addition, treatment with RP3 significantly attenuated the postischemic increase in brain water content at 24 hours after reperfusion. RP3 also significantly reduced the size of infarct area. These results indicate that the increase in brain MPO activity after transient focal ischemia virtually reflects the neutrophil infiltration and that neutrophil infiltration into the ischemic brain is implicated in postischemic brain injury.

Preconditioning of the brain with sublethal ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). In this study, we used NADPH-diaphorase histochemistry to investigate the postischemic changes of nitric oxide synthase (NOS) in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by 4-vessel occlusion for 3 min as an ischemic preconditioning. Three days after the preconditioning or sham operation, second ischemia was induced for 6 min. A transient increase in NADPH-diaphorase activity, beginning after 2 h and maximal after 1 day, was observed in CA1 pyramidal neurons of rats subjected to 3 min of preconditioning ischemia as well as 6 min of subsequent ischemia both with and without preconditioning. In addition, expression of NADPH-diaphorase activity was seen in reactive glial cells in the damaged CA1 region of animals subjected to 6 min of ischemia without preconditioning. Thus, direct involvement of increased NADPH-diaphorase activity in ischemic tolerance was not suggested because the increased NADPH-diaphorase activity preceded the induction of ischemic tolerance which takes place 1–7 days after preconditioning. However, the present findings suggest that the induction of neuronal NADPH-diaphorase activity occurs in response to cerebral ischemia.

Chronological changes of adenosine A1 receptor binding of the rat brain were determined by in vitro [3H]cyclohexyladenosine (CHA) autoradiography after 90 min of middle cerebral artery (MCA) occlusion and after such occlusion followed by different periods of recirculation. One day after the ischaemia, [3H]CHA binding sites decreased significantly in the cerebral cortex (p < 0.05) and lateral segment of the caudate putamen (p < 0.01), both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischaemic foci. On the contrary, there was no alteration on day 1, but 3 days after ischaemic insult, a significant decrease of [3H]CHA binding sites was first detected in the ipsilateral thalamus and the substantia nigra, both areas which had not been directly affected by the original ischaemic insult. This post-ischaemic delayed phenomenon observed in the thalamus and the substantia nigra developed concurrently with 45Ca accumulation, which was studied in our previous study. Based on the present study, alteration of adenosine A1 receptor binding activities is involved not only in the ischaemic foci, but also in the remote areas associated neuroanatomically with the ischaemic foci. We suggest that multi-focal post-ischaemic alterations of adenosine A1 binding activities may exacerbate clinical symptoms of patients at a chronic stage of stroke.

Nerve growth factor (NGF) synthesis in cultured mouse L-M fibroblast and astroglial cells can be increased after the treatment with L-threo-3,4-dihydroxyphenylserine (DOPS). Since the increase of NGF is not blocked by the treatment with decarboxylase inhibitor, DOPS may have direct effect to increase the NGF content. NGF and its receptor (NGFR) are suggested to play an important role in the neuronal survival and regeneration under pathologic conditions. In this study, we studied a possible protective effect of DOPS against the hippocampal CA1 cell death after transient forebrain ischaemia in gerbils in relation to the change of NGFR immunoreactivity. We found that treatment with DOPS (300 mg kg-1) in combination with a decarboxylase inhibitor (benserazide, 10 mg kg-1) protected ischaemic hippocampal CA1 cell against delayed neuronal death (neuronal density = 125 +/- 24 mm-1) as compared to the treatment with vehicle (49 +/- 11 mm-1) (p < 0.01). The immunoreactivity for NGFR was scarcely present in the sham-control CA1 area but was induced from 1 h and markedly expressed at 7 days after recirculation in the vehicle group. However, it was slightly and transiently induced from 8 h to 2 days in the DOPS plus benserazide treated group. These data suggest that the protective role of DOPS on the ischaemic hippocampal CA1 cells may act through the NGF and its receptor system.

We investigated the effects of age and nafidrofuryl 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 gamma-aminobutyric acid-A (GABAA) 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.

We measured cerebral metabolic rates for glucose (CMRGlc) in the remote brain areas of 7 patients, who were affected by unilateral cortical infarction, at a chronic stage using 2[18F]-fluoro-2-deoxy-D-glucose and positron emission tomography. There were significant decreases of CMRGlc compared with each control value (p < 0.01), not only in the cerebral cortex directly damaged by the ischaemic insult, but also in the ipsilateral thalamus and in the contralateral cerebellum, areas in which no lesions had been detected by MRI or CT scan. The present study indicates that different mechanisms may be responsible for multi-focal metabolic disturbances in the remote areas after stroke. We suggest that these multi-focal brain dysfunctions may exacerbate clinical symptoms at a chronic stage of stroke.

The influence of transient forebrain ischemia on the temporal alteration of NMDA receptor (NMDAR1) mRNAs in the hippocampal formation was analysed by in situ hybridization, RNA blot and in vitro receptor autoradiography ([3H]3-((+)2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid). In the CA1 subfield of the hippocampus, decrease of NMDAR1 mRNA levels was not observed until death of the CA1 pyramidal cells. Reactive astrocytes, which appear after CA1 pyramidal cell death, expressed few NMDAR1. The CA3 and dentate gyrus also showed a reduction of NMDAR1 mRNA levels 7 days after ischemia. Temporal profiles of NMDAR1 mRNA levels correlated well with those of NMDA receptor-binding in the CA1 and CA3/dentate gyrus. These results indicate that NMDA receptors in the CA1 are predominantly located on the CA1 pyramidal cells and that these receptors are preserved during early recirculation period. Histologically intact neurons in the CA3 and dentate gyrus also had marked modulation in glutamatergic neurotransmission after ischemia.

We investigated the effects of vinconate and pentobarbital against the alterations in spirodecanone binding in the gerbil striatum and hippocampus 5 h and 7 days after 10 min of cerebral ischemia, using receptor autoradiography. Vinconate and pentobarbital were given intraperitoneally 10 and 30 min prior to ischemic insult, respectively. The spirodecanone binding in vehicle-treated gerbils subjected to ischemia was unchanged in the brain 5 h after recirculation, compared with that in sham-operated animals. Seven days after ischemia, a significant elevation in the spirodecanone binding was observed in the striatum and the stratum radiatum of the hippocampal CA1 sector and the hippocampal CA3 sector of the vehicle-treated animals. Other regions showed no significant change in the binding. Vinconate and pentobarbital showed no significant change in the striatum and hippocampus 5 h after ischemia. However, the administration of vinconate inhibited a significant elevation in the spirodecanone binding in the lateral striatum and the stratum radiatum of hippocampal CA1 sector 7 days after ischemia. Pentobarbital also prevented a significant elevation only in the lateral striatum. A histological study revealed that cerebral ischemia caused severe neuronal damage in the lateral striatum and hippocampal CA1 and CA3 sectors. However, ischemic neuronal damage was not observed in the dentate gyrus. An immunohistochemical study also showed that numerous reactive astrocytes were evident in the hippocampus, particularly in the hippocampal CA1 sector, 7 days after ischemia. The present study demonstrates that cerebral ischemia can cause a conspicuous elevation in spirodecanone binding in the striatum and hippocampus. They also suggest that the postischemic elevation in the spirodecanone binding is partly prevented by treatment with vinconate and pentobarbital. These results suggest that the postischemic elevation in spirodecanone binding sites may reflect expression of reactive astrocytes.

Preconditioning the brain with sublethal cerebral ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). The purpose of this study is to investigate the role of low-molecular weight stress proteins, 27-kDa heat shock protein (HSP27) and alpha B crystallin, in ischemic tolerance. We measured the content of these proteins with enzyme immunoassay in the rat hippocampus and cerebral cortex following 6 min of ischemia with and without preconditioning with 3 min of ischemia and 3 days of reperfusion. We also visualized the localization of HSP27 immunohistochemically in comparison with that of HSP70. A 3-min period of ischemia caused a 2.4-fold increase in HSP27 content in the hippocampus after 3 days. Immunohistochemical localization of HSP27 was found in glial cells in all subregions of the hippocampus, whereas HSP70 immunostaining was seen only in CA1 pyramidal neurons. HSP27 content in the hippocampus decreased 2 h after 6 min of ischemia. HSP27 content progressively increased in the unpreconditioned hippocampus after 1 and 3 days, but returned to preischemic levels in the preconditioned hippocampus. HSP27 and HSP70 immunostaining was seen in CA1 pyramidal neurons after 1 day both with and without preconditioning. After 3 and 7 days, an intense HSP27 staining was observed in reactive glial cells in the CA1 without preconditioning, whereas the staining decreased in the preconditioned hippocampus. HSP70 staining was seen only in neurons at these time points. We observed no significant changes in HSP27 content in the cerebral cortex although neurons in the third and fifth layers were immunostained after 1 and 3 days. We observed no alterations in alpha B crystallin content after ischemia both in the hippocampus and the cortex. The present study demonstrated that cerebral ischemia induces HSP27 expression but not alpha B crystallin. Both HSP27 and HSP70 induction had a good temporal correlation with the induction of ischemic tolerance. However, different sites of action were suggested because the localization and cell types of HSP27 induction were quite different from those of HSP70 induction. The result suggests that it is unlikely that HSP27 is directly involved in the protection afforded by ischemic preconditioning.

We studied the postischemic alteration of muscarinic acetylcholine binding sites in the rat brain using in vitro [3H]quinuclidinyl benzilate (QNB) autoradiography. Transient ischemia was induced by the occlusion of the middle cerebral artery (MCA) for 90 min and such occlusion followed by various recirculation periods of up to 4 weeks. After 90 min of ischemia followed by 3 days of recirculation, [3H]QNB binding sites were found to be significantly decreased in the cerebral cortex (P < 0.01) and lateral segment of the caudate putamen (P < 0.05), both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischemic foci. Moreover, 3 days after the ischemia, significant decreases of [3H]QNB binding sites were observed in the ipsilateral thalamus and the amygdala, and also in the substantia nigra 1 week after the ischemia, areas which had not been directly affected by the original ischemic insult. This postischemic phenomenon observed in the thalamus and the substantia nigra developed concurrently with 45Ca accumulation, which was detected there in our previous study. These results indicate that alteration of muscarinic acetylcholine binding sites may be involved not only in the ischemic foci, but also in the exo-focal remote areas, in which delayed neuronal degeneration due to neuronal network disturbances after the ischemia was observed. We suggest that multifocal postischemic alterations of muscarinic acetylcholine binding sites may exacerbate the clinical symptoms of patients during the chronic stage of stroke.

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.

We studied the chronological changes of protein kinase C (PKC) and muscarinic acetylcholine receptor binding activities of the rat brain which were determined by using [3H]phorbol 12,13-dibutyrate (PDBu) and [3H]quinuclidinyl benzilate (QNB) autoradiographic methods, respectively, after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion, followed by different periods of recirculation. After the ischemic insult followed by 3 h of recirculation, [3H]PDBu binding sites were found to be significantly decreased in the cerebral cortex and lateral segment of the caudate putamen, both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischemic foci. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, a significant decrease of [3H]QNB binding sites was first detected in those ischemic foci. Moreover, 3 days after ischemic insult, both [3H]PDBu and [3H]QNB binding sites were concurrently reduced in the ipsilateral thalamus and 1 week after the ischemia, in the substantia nigra, in which both areas had not been directly affected by the original ischemic insult. These alterations of PKC in the postischemic brain areas developed concurrently with 45Ca accumulation, which was detected in our previous study. These results suggest that postischemic alterations of second-messenger (PKC) and neurotransmitter receptor systems were involved not only in the ischemic foci due to ischemia-induced energy failure, but also in the exo-focal remote areas prior to the histologic changes where neuronal damage might be caused by transsynaptic delayed degeneration.

Using the techniques of molecular biology, recent experimental studies have shown that cerebral ischemia induces a variety of changes in gene expression in the brain. During the early postischemic stages, protein synthesis in the brain is generally suppressed, but specific genes are expressed and their corresponding proteins may be synthesized, such as immediate-early gene products (c-fos, c-jun, and zinc finger gene), heat-shock proteins, and amyloid precursor protein. The ability of neurons to induce such stress responses, which depends on both the severity of ischemia and the intrinsic nature of the neuronal populations, may be directly associated with neuronal death and survival after ischemia. Nerve growth factor and fibroblast growth factor are also induced after ischemia and may be related to repair processes, in which a role of glial cells is suggested. Postischemic events that may be associated with the altered gene expression include (1) induction of tolerance to ischemia after pretreatment with sublethal ischemia, (2) slow, progressive neuronal changes and the development of neuronal plasticity after ischemia, and (3) delayed neuronal changes in remote areas outside the cerebral ischemic focus. Because a variety of harmful stresses, including ischemia, elicit the same stress response and because this response is induced when total protein synthesis in the brain is nearly completely suppressed, this response may be vital to cell survival and repair. A successful induction of this response may induce resistance and survival of neurons after ischemia. However, failure or abortion of the response and persistent stresses may lead to neuronal death and possibly long-term changes and degeneration.

We studied the alterations in binding of cyclic AMP as an indicator of particulate cyclic AMP-dependent protein kinase binding activity following transient cerebral ischemia in Mongolian gerbils and examined the effects of vinconate and pentobarbital against alterations in the binding. Animals were allowed to survive for 5 h and 7 days after 10 min of cerebral ischemia induced by bilateral occlusion of common carotid arteries. [3H]Cyclic AMP binding was significantly reduced in the hippocampus 5 h after ischemia, whereas the striatum showed no significant change in the binding. Seven days after ischemia, a severe reduction of [3H]cyclic AMP binding was noted in the dorsolateral striatum, hippocampal CA1 and CA3 sectors, and dentate gyrus. Intraperitoneal administration of vinconate (100 or 300 mg/kg) showed a significant elevation of [3H]cyclic AMP binding in the striatum, stratum pyramidale of hippocampal CA1 and CA3 sectors, and dentate gyrus 5 h after ischemia. By contrast, the intraperitoneal administration of pentobarbital (40 mg/kg) showed no significant alteration of [3H]cyclic AMP binding in most of these regions. However, vinconate and pentobarbital prevented a significant reduction of [3H]cyclic AMP binding in the dorsolateral striatum and stratum pyramidale of hippocampal CA3 sector 7 days after ischemia, although both drugs failed to prevent damage to the hippocampal CA1 sector. These results suggest that alteration in cyclic AMP binding may not be a major factor in causing ischemic neuronal damage.

Age-related alterations in major neurotransmitter receptors and voltage dependent calcium channels were analyzed by receptor autoradiography in the gerbil brain. [3H]Quinuclidinyl benzilate (QNB), [3H]cyclohexyladenosine (CHA), [3H]muscimol, [3H]MK-801, [3H]SCH 23390, [3H]naloxone, and [3H]PN200-110 were used to label muscarinic acetylcholine receptors, adenosine A1 receptors, gamma-aminobutyric acidA (GABAA) receptors, N-methyl-D-aspartate (NMDA) receptors, dopamine D1 receptors, opioid receptors, and voltage dependent calcium channels, respectively. In middle-aged gerbils (16 months old), the hippocampus exhibited a significant elevation in [3H]QNB, [3H]MK-801, [3H]SCH 23390, [3H]naloxone, and [3H]PN200-110 binding, whereas [3H]CHA and [3H]muscimol binding showed a significant reduction in this area, compared with that of young animals (1 month). On the other hand, the cerebellum showed a significant alteration in [3H]QNB, [3H]CHA, and [3H]naloxone binding and the striatum also exhibited a significant alteration in [3H]SCH 23390 and [3H]CHA binding in middle-aged gerbils. The neocortex showed a significant elevation only in [3H]CHA binding in middle-aged animals. The nucleus accumbens and thalamus also showed a significant alteration only in [3H]muscimol binding. However, the hypothalamus and substantia nigra exhibited no significant alteration in these bindings in middle-aged gerbils. These results demonstrate the age-related alterations of various neurotransmitter receptors and voltage dependent calcium channels in most brain regions. Furthermore, they suggest that the hippocampus is most susceptible to aging processes and is altered at an early stage of senescence.

The CA3 pyramidal cells receive mossy fibers from the dentate granule cells, and the damage to CA3 pyramidal cells induces aberrant mossy fiber sprouting near the site of normal targets. Fetal CA1 and CA3 subregions were separately transplanted into the host CA3 subfield, where CA3 pyramidal neurons were previously damaged with kainic acid. In vitro [3H]inositol 1,4,5-trisphosphate (IP3) receptor autoradiography was used to analyze the purity of CA1 neurons. CA1 transplants had high IP3 receptor density compatible with adult CA1 subfield, while CA3 transplants had trace IP3 receptors. Animals with successful graft-host connections exhibited no aberrant mossy fiber sprouting, and mossy fibers invaded into the fetal CA3 grafts. However, animals without direct host mossy fibers-grafts connection showed aberrant mossy fiber sprouting in the CA3 subfield. These observations demonstrate that aberrant mossy fiber sprouting was suppressed by molecular cues presented on the grafted neurons and that [3H]IP3 receptor autoradiography provides an excellent marker for the identification of the CA1 pyramidal cells in grafts.

The alterations in morphology and Ca 2+ currents (I Ca ) were assessed in PC12 cells, which were (1) control (untreated), (2) treated with nerve growth factor (NGF), and (3) deprived of NGF. NGF treatment induced neuronal differentiation morphologically and increased the current density of I Ca , and especially that of the ω-conotoxin (ω-CgTX)-sensitive component. NGF-deprived cells manifested morphological deterioration and did not exhibit remarkable changes in the current density of the total I Ca and dihydropyridine components, whereas the ω-CgTX-sensitive I Ca component was decreased significantly compared with that in NGF-treated cells. The results suggest that the total function of Ca 2+ channels, including the dihydropyridine component, does not seem to be lost at the initial stage of neuronal deterioration, whereas ω-CgTX-sensitive Ca 2+ channels lost their function early.

Using quantitative receptor autoradiography, spirodecanone binding was evaluated in the gerbil hippocampus 1 h-1 month after cerebral ischaemia of 10 min. The spirodecanone binding was unaffected in the hippocampus up to 48 h after ischaemia. Thereafter, increased binding was found in the stratum radiatum of hippocampal CA1 sector 7 days and 1 month after ischaemia. Other hippocampal regions showed no significant alterations in the spirodecanone binding. A histological study revealed that the hippocampal CA1 sector was severely damaged 7 days and 1 months after ischaemia. These results demonstrate that spirodecanone binding sites are located on interneurones or glial cells in the hippocampal CA1 sector.

To evaluate the mechanism of tolerance to ischemia, inductions of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs in gerbil hippocampus were compared with in situ hybridization between cases of a single 3.5-min period of forebrain ischemia and a 3.5-min period of ischemia 2 days after 2-min pretreatment with ischemia. Immunohistochemistry for HSP70 protein and morphological studies were also performed in the same brains up to 7 days after the reperfusion. Following a single 3.5-min period of ischemia, HSP70 and HSC70 mRNAs were induced in all hippocampal cells. However, the hippocampal CA1 cells produced only a minimum of HSP70 protein, and the cells were almost lost by 7 days. Following 3.5 min of ischemia after 2-min pretreatment, large populations of the CA1 cells survived at 7 days. The peak time of the HSP70 and HSC70 mRNA induction shifted to an earlier period of reperfusion in all hippocampal cells as compared with the case of a single episode of ischemia. The peak of HSP70 and HSC70 mRNA induction shifted from 1 day to 3 h in the CA1 cells. The CA1 cells produced strongly immunoreactive HSP70 from 3 hr to 2 days. These results suggest that pretreatment with an initial period of ischemia (for 2 min) accelerated HSP70 and HSC70 gene expression at the transcriptional level, ameliorated the translational disturbance of HSP70 mRNA to protein, and saved the CA1 cells from subsequent lethal ischemia (for 3.5 min). These changes of heat shock gene expression might play important roles in the acquisition of ischemic tolerance of hippocampal CA1 neurons.

Distribution of heat shock protein (HSP) 90 alpha mRNA induction after 10 min of transient global ischemia was investigated in gerbil hippocampus by in situ hybridization. A small amount of HSP90 alpha mRNA was normally present in hippocampal cells and the mRNA was further induced with a peak at 8 h after ischemia. In hippocampal CA1 cells that are vulnerable to ischemia, HSP90 alpha mRNA was continuously induced by 1 day and finally diminished at 2 days. The temporal profile of HSP90 alpha mRNA induction in hippocampal CA1 cells was similar to that of HSP70 mRNA reported previously, suggesting a cooperative role of HSP90 alpha with other HSPs after ischemia.