Involvement of Glial Endothelin/Nitric Oxide in Delayed Neuronal Death of Rat Hippocampus After Transient Forebrain Ischemia

1. We examined time- and cell-type-dependent changes in endothelin (ET)-1-like immunoreactivity, ET receptors binding and nitric oxide (NO) synthase (NOS) activity in CA1 subfields of the hippocampus of stroke-prone spontaneously hypertensive rats subjected to a 10-min bilateral carotid occlusion and reperfusion.2. Microglia aggregated in accord with neuronal death and expressed a high density of ET_B receptors and an intense NOS activity in the damaged CA1 pyramidal cell layer, 7 days after the induced transient forebrain ischemia. The increased NOS activity and ET_B receptor in microglia disappeared 28 days after this transient ischemia.3. In contrast to microglia, astrocytes presented a moderate level of ET-1-like immunoreactivity, ET_B receptors, and NOS activity in all areas of the damaged CA1 subfields, 7 days after the ischemia. These events were further enhanced 28 days after the ischemia.4. In light of these findings, the possibility that the microglial and the astrocytic ET_B/NO system largely contributes to development of the neuronal death and to reconstitution of the damaged neuronal tissue, respectively, in the hippocampus subjected to a transient forebrain ischemia would have to be considered.     

Induction of cytosolic NADPH-diaphorase/nitric oxide synthase in reactive microglia/macrophages after quinolinic acid lesions in the rat striatum: an electron and light microscopical study

Induction of nitric oxide synthase and increased production of nitric oxide in microglia may play a crucial role in neuronal damage and neurodegenerative disorders. In the present study we have used light and electron microscopical NADPH-diaphorase histochemistry as the visualization procedure for nitric oxide synthase to investigate the time-course and subcellular patterns of NADPH-diaphorase expression in microglia/macrophages of quinolinic acid-lesioned rat striatum. For light microscopy, NADPH-diaphorase histochemistry sections were stained with nitroblue tetrazolium, while for ultrastructural analysis the tetrazolium salt 2-(2-benzothiazolyl)-5-styryl-3(4-phthalhydrazidyl) tetrazolium chloride (BSPT) was applied. Light microscopical inspection revealed a progressively increasing number of positive cells with increasing intensity of NADPH-diaphorase staining in microglia/macrophages from day 1 after quinolinic acid injection onward. Electron microscopical examination revealed a membrane bound NADPH-diaphorase in quiescent microglia as well as in activated microglia/macrophages through all stages of the lesion studied. Predominantly membranes of the nuclear envelope and the endoplasmic reticulum were labeled with BSPT-formazan, while in advanced stages selective membrane portions of mitochondria, Golgi apparatus and plasmalemma were also stained. From day 5 onward after lesion induction, a very distinctive type of NADPH-diaphorase was observed, forming accumulations of electron-dense grains that were distributed differentially throughout cytoplasmic areas and phagocytic vacuoles. Dynamics of expression, unique cytosolic localization and occurrence exclusively in activated microglia/macrophages suggest that this particular NADPH-diaphorase activity probably reflects the inducible isoform of nitric oxide synthase, whereas the membrane-bound precipitate may represent the neuronal and/or the endothelial isoform of the enzyme.     

The protective effect of aminoguanidine on cerebral ischemic damage in the rat brain

The NADPH-diaphorase (NADPH-d) histochemical technique is commonly used to localize the nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) in neural tissue. The expression of inducible nitric oxide synthase (iNOS) is induced in the late stage of cerebral ischemia, and NO produced by iNOS contributes to the delay in recovery from brain neuronal damage. The present study was performed to investigate whether the increase in nitric oxide production via inducible nitric oxide synthase was suppressed by the administration of aminoguanidine, a selective iNOS inhibitor, as it follows a decrease of NADPH-diaphorase activity (a marker for NOS) after four-vessel occlusion used as an ischemic model. The administration of aminoguanidine (100 mg/kg i.p., twice per day up to 3 days immediately after the ischemic insult) reduced the number of NADPH-diaphorase positive cells to control levels. Our results indicated that aminoguanidine suppressed NADPH-diaphorase activity, and also decreased the number of NADPH-diaphorase positive cells in the CA1 region of the hippocampus following ischemic brain injury.     

Nimodipine inhibits calcium-independent nitric oxide synthase activity in transient focal cerebral ischemia rats and cultured mouse astroglial cells

The effect of nimodipine on nitric oxide synthase (NOS) activities in brains in transient focal cerebral ischemia rats, in cultured mouse neurons and astroglial cells and bovine brain capillary endothelial cells (BCECs) was investigated. The administration of nimodipine (3 mg.kg(-1), p.o., twice a day, for 3 days) before middle cerebral artery (MCA) occlusion significantly reduced infarct size, decreased nitrite/nitrate (NOx) content and inhibited Ca2+-independent NOS activity in the infarct area. Nimodipine inhibited the Ca2+-independent NOS activity induced by lipopolysaccharide (LPS) + tumor necrosis factor alpha (TNF alpha) in mouse astroglial cells with an IC50 value of 0.036+/-0.003 mM and Ca2+-dependent NOS activity in mouse neurons with an IC50 value of 0.047+/-0.003 mM, but did not affect Ca2+-dependent NOS activity in BCECs. The inhibition of Ca2+-independent NOS activity by nimodipine in astroglial cells was competitive with respect to L-arginine. Nimodipine also inhibited the induction of Ca2+-independent NOS activity in vitro. These results suggest that nimodipine in addition to its cerebral vasodilating effect may protect brain from ischemic neuronal damage through modifying NOS activity.     

Changes in non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities after transient cerebral ischemia

Non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities, NADH-cytochrome c reductase rotenone insensitive (marker of the outer membrane) and cytochrome oxidase (marker of the inner membrane), were measured in rat brain hippocampus and striatum immediately after and 1, 4, and 7 days following the induction of complete transient ischemia (15 min) by the four vessel occlusion method. Furthermore citrate synthetase activity was measured with and without Triton X-100 in order to qualitatively evaluate the membrane permeability. Nonsynaptosomal mitochondrial membranes showed reduction of both activities only in the late reperfusion phase: NADH-CCRRi decreased in striatal mitochondria after 4–7 days and only after 7 days in the hippocampus. COX activity decreased only in striatal mitochondria 7 days after ischemia. Non-synaptosomal mitochondrial membrane permeability did not show changes. Synaptosomal mitochondria showed a decrease of NADH-CCRRi only at 7 days of reperfusion both in hippocampus and striatum, while COX activity decreased only during ischemia and returned to normal levels in the following days in the two areas considered. In summary, free mitochondria showed insensitiveness to ischemia but they risulted damaged in the late reperfusion phase, while mitochondria from the synaptic terminal showed ischemic damage, partially restored during reperfusion. The striatal mitochondria showed a major susceptibility to ischemia/repefusion damage, showing changes earlier than the hippocampal ones.     

Spatiotemporal Alterations of the NO/NOS Neuronal Pools Following Transient Abdominal Aorta Occlusion: Morphological and Biochemical Studies in the Rabbit

1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusion may lead to degeneration of specific spinal cord neurons and to irreversible loss of neurological function. The alteration of nitric oxide/nitric oxide synthase (NO/NOS) pool occurring after ischemic insult may play a protective or destructive role in neuronal survival of affected spinal cord segments.2. In the present study, the spatiotemporal changes of NOS following transient ischemia were evaluated by investigating neuronal NOS immunoreactivity (nNOS-IR), reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, and calcium-dependent NOS (cNOS) conversion of [³H] l-arginine to [³H] l-citrulline.3. The greatest levels of these enzymes and activities were detected in the dorsal horn, which appeared to be most resistant to ischemia. In that area, the first significant increase in NADPHd staining and cNOS catalytic activity was found immediately after a 15-min ischemic insult.4. Increases in the ventral horn were observed later (i.e., after a 24-h reperfusion period). While the most intense increase in nNOS-IR was detected in surviving motoneurons of animals with a shorter ischemic insult (13 min), the greatest increase of cNOS catalytic activity and NADPHd staining of the endothelial cells was found after stronger insult (15 min).5. Given that the highest levels of nNOS, NADPHd, and cNOS were found in the ischemia-resistant dorsal horn, and nNOS-IR in surviving motoneurons, it is possible that NO production may play a neuroprotective role in ischemic/reperfusion injury.     

Microglia with an Endothelin ETB Receptor Aggregate in Rat Hippocampus CA1 Subfields Following Transient Forebrain Ischemia

Abstract: We examined endothelin (ET) receptors in the hippocampus CA1 subfields of stroke-prone spontaneously hypertensive rats subjected to a 10-min bilateral carotid occlusion and reperfusion. When delayed neuronal death had occurred in the pyramidal cell layer at 7 days after transient forebrain ischemia, the quantitative receptor autoradiographic method we used revealed a dramatic increase in number of ¹²⁵I-ET-1 binding sites in the hippocampus CA1 subfields. The highest number of de novo binding sites appeared in the area corresponding anatomically to the pyramidal cell layer with neuronal death. These binding sites were characteristically the ET_B receptor. The de novo ¹²⁵I-ET-1 binding was mainly present on microglia aggregating with a high density in the damaged pyramidal cell layer. As ET-1- and ET-3-like immunoreactivities were highly expressed within astrocytes in damaged neural tissue, the possibility that microglia with the ET_B receptor are activated to participate in the pathophysiology of ischemia-related neural tissue damage by astrocytic ET-1 and ET-3 produced in response to transient forebrain ischemia would have to be considered.     

Ornithine Decarboxylase Activity in In Vivo and In Vitro Models of Cerebral Ischemia

Ornithine decarboxylase (ODC) is considered the rate-limiting enzyme in polyamine biosynthesis, and an increase in putrescine after central nervous system (CNS) injury appears to be involved in neuronal death. Cerebral ischemia and reperfusion trigger an active series of metabolic events, which eventually lead to neuronal death. In the present study, ODC activity was evaluated following transient focal cerebral ischemia and reperfusion in rat. The middle cerebral artery (MCA) was occluded for 2 h in male rats with an intraluminal suture technique. Animals were sacrificed between 3 and 48 h of reperfusion following MCA occlusion, and ODC activity was assayed in cortex and striatum. ODC activity was also estimated in an in vitro ischemia model using primary rat cortical neuron cultures, at 6–24 h reoxygenation following 1 h oxygen-glucose deprivation (OGD). In cortex, following ischemia, ODC activity was increased at 3 h (P < .05), reached peak levels by 6–9 h (P < .001) and returned to sham levels by 48 h reperfusion. In striatum the ODC activity followed a similar time course, but returned to basal levels by 24 h. This suggests that ODC activity is upregulated in rat CNS following transient focal ischemia and its time course of activation is region specific. In vitro, ODC activity showed a significant rise only at 24 h reoxygenation following ischemic insult. The release of lactate dehydrogenase (LDH), an indicator for cell damage, was also significantly elevated after OGD. 0.25 mM -difluoromethylornithine (DFMO) inhibited ischemia-induced ODC activity, whereas a 10-mM dose of DFMO appears to provide some neuroprotection by suppressing both ODC activity and LDH release in neuronal cultures, suggesting the involvement of polyamines in the development of neuronal cell death.     

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

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

Dietary Sutherlandia and Elderberry Mitigate Cerebral Ischemia-Induced Neuronal Damage and Attenuate p47phox and Phospho-ERK1/2 Expression in Microglial Cells

Sutherlandia (Sutherlandia frutescens) and elderberry (Sambucus spp.) are used to promote health and for treatment of a number of ailments. Although studies with cultured cells have demonstrated antioxidative and anti-inflammatory properties of these botanicals, little is known about their ability to mitigate brain injury. In this study, C57BL/6 J male mice were fed AIN93G diets without or with Sutherlandia or American elderberry for 2 months prior to a 30-min global cerebral ischemia induced by occlusion of the bilateral common carotid arteries (BCCAs), followed by reperfusion for 3 days. Accelerating rotarod assessment at 24 h after BCCA occlusion showed amelioration of sensorimotor impairment in the mice fed the supplemented diets as compared with the ischemic mice fed the control diet. Quantitative digital pathology assessment of brain slides stained with cresyl violet at 3 days after ischemia/reperfusion (I/R) revealed significant reduction in neuronal cell death in both dietary groups. Immunohistochemical staining for ionized calcium-binding adapter molecule-1 demonstrated pronounced activation of microglia in the hippocampus and striatum in the ischemic brains 3 days after I/R, and microglial activation was significantly reduced in animals fed supplemented diets. Mitigation of microglial activation by the supplements was further supported by the decrease in expression of p47phox, a cytosolic subunit of NADPH oxidase, and phospho-ERK1/2, a mitogen-activated protein kinase known to mediate a number of cytoplasmic processes including oxidative stress and neuroinflammatory responses. These results demonstrate neuroprotective effect of Sutherlandia and American elderberry botanicals against oxidative and inflammatory responses to cerebral I/R.     

Differential NOS expression in freshwater and aestivating Protopterus dolloi (lungfish): heart vs kidney readjustments.

African lungfish Protopterus dolloi is an obligatory air-breather, which aestivates in a cocoon during the dry season. Aestivation associates with functional modifications in many tissues and organs, including heart and kidney. Due to its pleiotropic modulatory effects, nitric oxide (NO), generated by nitric oxide synthases (NOSs), may coordinate organ rearrangement, allowing adaptive adjustments under stressful environmental conditions. By immunofluorescence, Western blotting and NADPH-diaphorase, we examined cardiac and renal localization and activity of NOSs isoforms in both freshwater (FW) and aestivating [6 days (6DA) and 40 days (40DA) of estivation] P. dolloi. In heart and kidney endothelial NOS (eNOS) is the major isoform with respect to inducible and neuronal NOS (iNOS and nNOS, respectively). Cardiac eNOS locates in the epicardium, the trabecular endothelial endocardium, and myocardiocytes of both FW and aestivating fish. Western blotting revealed that cardiac eNOS expression increases in 6DA, but decreases in 40DA fish. In FW fish kidney eNOS is present in vascular endothelial cells and in podocytes of renal corpuscles. In tubular epithelial cells it is restricted to the apical pole. With aestivation, both renal localization and expression of eNOS increase. NADPH-diaphorase revealed an enhancement of cardiac and renal NOS activities during aestivation. Results suggest that in P. dolloi NO contributes, in an autocrine-paracrine fashion, to cardiac and renal readjustments during aestivation. Our findings are of evolutionary interest, since they document for the first time the presence of a NOS system in a ancestral fish, indicative of deep phylogenetic roots of NO bio-synthesis.     

Direct Angiotensin AT2 Receptor Stimulation Using a Novel AT2 Receptor Agonist,Compound 21, Evokes Neuroprotection in Conscious Hypertensive Rats

Background

In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents.

Methods and Results

Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury.

Conclusion

These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development.     

亚低温减少沙土鼠脑缺血后延迟性神经元死亡机制的研究

目的：研究亚低温对脑缺血后延迟性神经元死亡的影响及其与海马羟自由基产生以及纹状体多巴胺和ＡＴＰ含量变化的关系。方法：沙土鼠前脑缺血再灌注模型,缺血１０ｍｉｎ,应用病理检查方法判断海马ＣＡｌ锥体细胞死亡的数目。动物随机分为假手术组、缺血组、缺血再灌注组和亚低温缺血再灌注组。高效液相加电化学检测器方法测定海马羟自由基和纹状体多巴胺的含量,高效液相紫外检测器法测定纹状体ＡＴＰ含量。结果：亚低温条件下沙土     

Chronic inhibition of nitric oxide synthase activity by N(G)-nitro-L-arginine induces nitric oxide synthase expression in the developing rat cerebellum

Studies on chronic inhibition of nitric oxide synthase (NOS) in the CNS suggest a plastic change in nitric oxide (NO) synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor N(G)-nitro-l-arginine (l-NNA) was examined on NO production, NO-sensitive cyclic guanosine monophosphate (cGMP) levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5-200mg/kg) to 21-day-old rats reduced NOS activity and NO concentration dose dependently by 70-90% and the tissue cGMP level by 60-80%. By contrast, chronic application of l-NNA between postnatal days 4-21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10-50%. Chronic treatment of 10mg/kg l-NNA induced neuronal (n)NOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50mg/kg) of l-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced inducible (i)NOS expression & activity in glial cells. Treatments did not affect endothelial (e)NOS expression. The administration of the biologically inactive isomer D-NNA (50mg/kg) or saline was ineffective. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced cronich inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.     

Aminoguanidine inhibits caspase-3 and calpain activation without affecting microglial activation following neonatal transient cerebral ischemia

Microglial cells, the resident macrophages of the CNS, can be both beneficial and detrimental to the brain. These cells play a central role as mediators of neuroinflammation associated with many neurodegenerative states, including cerebral ischemia. Because microglial cells are both a major source of inducible nitric oxide synthase (iNOS)/nitric oxide (NO) production locally in the injured brain and are activated by NO-mediated injury, we tested whether iNOS inhibition reduces microglial activation and ischemic injury in a neonatal focal ischemia-reperfusion model. Post-natal day 7 rats were subjected to a 2 h transient middle cerebral artery (MCA) occlusion. Pups with confirmed injury on diffusion-weighted magnetic resonance imaging (MRI) during occlusion were administered 300 mg/kg/dose aminoguanidine (AG) or vehicle at 0, 4 and 18 h after reperfusion, and animals were killed at 24 or 72 h post-reperfusion. The effect of AG on microglial activation as judged by the acquisition of ED1 immunoreactivity and proliferation of ED1-positive cells, on activation of cell death pathways and on injury volume, was determined. The study shows that while AG attenuates caspase 3 and calpain activation in the injured tissue, treatment does not affect the rapidly occurring activation and proliferation of microglia following transient MCA occlusion in the immature rat, or reduce injury size.     

Expression of TNF and TNF receptors (p55 and p75) in the rat brain after focal cerebral ischemia.

Cerebral ischemia induces a rapid and dramatic up-regulation of tumor necrosis factor (TNF) protein and mRNA, but the cellular sources of TNF in the ischemic brain have not been defined. The diverse activities of TNF are mediated via ligand interaction with two distinct receptors, p55 and p75, which activate separate intracellular signal transduction pathways, leading to distinct biological effects. Since the effects of cerebral ischemia on TNF receptor (TNFR) expression are unknown, we examined the cellular localization and protein expression of TNF and its two receptors in the rat cerebral cortex in response to permanent middle cerebral artery (MCA) occlusion. The results indicate that focal. cerebral ischemia up-regulates expression of TNF and both TNFRs within the ischemic cortex. The most abundant type of TNF immunoreactivity (IR) was a punctate and filamentous pattern of transected cellular processes; however, cell bodies of neurons, astrocytes, and microglia, as well as infiltrating polymorphonuclear (PMN) leukocytes also showed TNF IR. Brain vasculature displayed TNF IR not only within endothelial cells but also in the perivascular space. MCA occlusion induced significant up-regulation of TNF receptors, with p55 IR appearing within 6 hr, significantly before the appearance of p75 IR at 24 hr after the onset of ischemia. Since p55 has been implicated in transducing cytotoxic signalling of TNF, these results support the proposed injurious role of excessive TNF produced during the acute response to cerebral ischemia.     

Neuronal Damage Using Fluoro-Jade B Histofluorescence and Gliosis in the Gerbil Septum Submitted to Various Durations of Cerebral Ischemia

The extent of neuronal damage/death in some brain regions is highly correlated to duration time of transient ischemia. In the present study, we carried out neuronal degeneration/death and glial changes in the septum 4 days after 5, 10, 15, and 20 min of transient cerebral ischemia using gerbils. To examine neuronal damage, Fluoro-Jade B (F-J B, a marker for neuronal degeneration) histofluorescence staining was used. F-J B positive (⁺) cells were detected in the septo-hippocampal nucleus (SHN) of the septum only in the 20 min ischemia-group; the mean number of F-J B⁺ neurons was 14.9 ± 2.5/400 μm² in a section. Gliosis of astrocytes and microglia was examined using anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1), respectively. In all the ischemia-groups, GFAP- and Iba-1-immunoreactive astrocytes and microglia, respectively, were increased in number, and apparently tended to be increased in their immunoreactivity. Especially, in the 20 min ischemia-group, the number and immunoreactivity of Iba-immunoreactive microglia was highest and strongest in the ischemic SHN 4 days after ischemia–reperfusion. In brief, our findings showed that neuronal damage/death in the SHN occurred and gliosis was apparently increased in the 20 min ischemia-group at 4 days after ischemia–reperfusion.     

Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats

Recent evidence indicates that cell-cycle regulating proteins are involved in apoptotic process in post-mitotic neurons. In this study, we examined cell-cycle regulators for G1/S cell-cycle progression after a transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion. In the cerebral frontoparietal cortex, we observed a marked induction of Cyclin D1 (a coactivator of Cdks), and proliferating cell nuclear antigen (PCNA), together with upregulated Cdk kinase activities. This process is accompanied with multiple phosphorylation of retinoblastoma (Rb) protein at Cdk phosphorylation sites in neurons from the ischemic cortex. We further examined DNA synthesis by the incorporation of BrdU, a nucleotide analog that incorporates into newly synthesized DNA. Within 24-h of reperfusion after 60-min occlusion, substantial BrdU-positive neurons were observed in the ischemic cortex. Inhibition of Cdk4 activity during this ischemia/reperfusion is highly neuroprotective. These results suggest that ischemia/reperfusion cerebral damage induces signalings at the G1/S cell-cycle transition, and may constitute a critical step in the neuronal apoptotic pathway in ischemia/reperfusion induced neuronal damage.     

Distribution of intracardiac neurones and nerve terminals that contain a marker for nitric oxide,NADPH-diaphorase,in the guinea-pig heart

There is strong evidence that NADPH-diaphorase can be used as a marker for neurones that employ nitric oxide as a messenger molecule. In the present study, the NADPH-diaphorase activity of intracardiac neurones and nerve terminals in whole-mount stretch preparations and sections of the newborn and adult guinea-pig atria and interatrial septum has been examined histochemically. Together with epicardial, endothelial and endocardial cells, which displayed some NADPH-diaphorase staining, a subpopulation of intracardiac neurones exhibited moderate-heavy labelling for NADPH-diaphorase, while the majority of neurones were only lightly stained or negative. Intracardiac ganglia containing positive neuronal cell bodies were located between the epicardial cells and atrial myocytes in four main regions: in association with the superior and inferior vena cavae, the points of entry of the pulmonary veins, and within the interatrial septum. Nerve terminals exhibiting NADPH-diaphorase activity were seen throughout the atrial tissue, forming basket-like endings around intracardiac neuronal cell bodies; varicose terminals were also observed on atrial myocytes and other non-neuronal structures. A proportion of the nerve fibres was clearly of intrinsic origin, other terminals may well have originated from neuronal cell bodies present outside the heart.