Inhibition of nNOS and iNOS following hypoxia-ischaemia improves long-term outcome but does not influence the inflammatory response in the neonatal rat brain

In this study, we tested the hypothesis that combined inhibition of nNOS and iNOS will reduce neuronal damage and the inflammatory response induced by perinatal hypoxia-ischaemia (HI). In 12-day-old rats, HI was induced by right carotid artery occlusion followed by 90 min of 8% O2. Immediately upon reoxygenation, the rats were treated with NOS inhibitors (n = 24) or placebo (n = 24). Neuropathology was scored at 6 weeks after HI on a 4-point scale (n = 12 per group). The expression of heat shock protein 70 (HSP70) and mRNA expression for cytokines were measured 12 h after HI (n = 12 per group). Histopathological analysis showed that the ipsilateral hemisphere in the NOS inhibition group was less damaged than in the placebo group (p < 0.05). HI induced a significant increase in HSP70 levels (p < 0.05) in the ipsilateral hemispheres, which tended to be lower in the NOS inhibition group (p = 0.07). HI induced an increase in mRNA expression for IL-1beta, TNF-alpha and TNF-beta, but there was no difference between the ipsi- and contralateral hemispheres. Combined inhibition of nNOS and iNOS did not induce any change in cytokine expression. We conclude that the long-term neuroprotective effects of combined nNOS and iNOS inhibition were not achieved by an altered cytokine response.     

一氧化氮合酶在脑缺血再灌注中的双重作用

目的 探讨短暂脑缺血再灌注后大鼠脑内3型一氧化氮合酶(nitric oxide synthase，NOS)的表达及作用，为脑缺血治疗提供理论依据。方法 采用免疫组织化学方法，用3型NOS的多克隆抗体检测大鼠局灶性脑缺血2h再灌注15min及22h NOS在脑内的表达情况。结果 大鼠脑缺血2h再灌注15min，在脑缺血边缘区的血管壁及神经细胞出现内皮型一氧化氮合酶(endothelial nitric oxide synthase，eNOS)上调表达；脑缺血2h再灌注22h，在脑梗死区内表达神经元型一氧化氮合酶(neuronal mitric oxide synthase，nNOS)的神经细胞减少，并出现表达诱导型一氧化氮合酶(inducible nitric oxide synthase，iNOS)的胶质细胞，同时梗死边缘区血管及神经细胞出现eNOS及iNOS的上调表达。结论 在短暂脑缺血再灌注早期，缺血区周围可能有eNOS相关的保护机制；亚急性期eNOS及iNOS的保护及损伤机制并存；因此，在短暂脑缺血早期恢复灌注后予选择性iNOS抑制剂及促进eNOS活性有可能减少迟发性神经损伤。     

Time-related changes in constitutive and inducible nitric oxide synthases in the rat striatum in a model of Huntington's disease

Excitotoxicity and oxidative stress are mechanisms involved in the neuronal cell death induced by the intrastriatal injection of quinolinic acid (QUIN) as a model of Huntington's disease. Production of nitric oxide by nitric oxide synthase (NOS) has been proposed to participate in QUIN-induced neurotoxicity; however, the precise role of NOS in QUIN-induced toxicity still remains controversial. In order to provide further information on the role of NOS isoforms in QUIN toxicity, we performed real time RT-PCR and immunohistochemistry of inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) and determined Ca(2+)-dependent and Ca(2+)-independent NOS activity in a temporal course (3-48h), after an intrastriatal injection of QUIN to rats. NOS isoforms exhibited a transitory expression of mRNA and protein after QUIN infusion: eNOS increased between 3 and 24h, iNOS between 12 and 24h, while nNOS at 35 and 48h. Ca(2+)-independent activity (iNOS) did not show any change, while Ca(2+)-dependent activity (constitutive NOS: eNOS/nNOS) exhibited increased levels at 3h. Our results support the participation of Ca(2+)-dependent NOS isoforms during the toxic events produced at early times after QUIN injection.     

Differences in NOS protein expression and activity in the rat vestibular nucleus following unilateral labyrinthectomy

We used Western blotting to analyse the expression of different isoforms of nitric oxide synthase (NOS) in the rat vestibular nucleus complex (VNC) at various times following unilateral vestibular deafferentation (UVD), together with a radioenzymatic assay to compare NOS activity at the same time points. nNOS expression did not change significantly in the ipsilateral or contralateral VNC at any time following UVD. However, eNOS expression decreased significantly (P<0.05) in the contralateral VNC at 6 h post-UVD, recovering to normal levels by 50 h. iNOS was not expressed at any time following UVD. NOS activity demonstrated a significant increase in the contralateral VNC at 6 h post-UVD (P<0.05), recovering toward normal levels by 50 h.     

Aberrant expression of NOS isoforms in Alzheimer's disease is structurally related to nitrotyrosine formation

Various isoforms of the nitric oxide (NO) producing enzyme nitric oxide synthase (NOS) are elevated in Alzheimer's disease (AD) indicating a critical role for NO in the pathomechanism. NO can react with superoxide to generate peroxynitrite, a process referred to as oxidative stress, which is likely to play a role in AD. Peroxynitrite in turn, nitrates tyrosine residues to form nitrotyrosine which can be identified immunohistochemically. To study the potential structural link between the increased synthesis of NO and the deposition of nitrotyrosine in AD, we analyzed the expression of neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) in AD and control brain, and compared the localization with the distribution of nitrotyrosine. Nitrotyrosine was detected in neurons, astrocytes and blood vessels in AD cases. Aberrant expression of nNOS in cortical pyramidal cells was highly co-localized with nitrotyrosine. Furthermore, iNOS and eNOS were highly expressed in astrocytes in AD. In addition, double immunolabeling studies revealed that in these glial cells iNOS and eNOS are co-localized with nitrotyrosine. Therefore, it is suggested that increased expression of all NOS isoforms in astrocytes and neurons contributes to the synthesis of peroxynitrite which leads to generation of nitrotyrosine. In view of the wide range of isoform-specific NOS inhibitors, the determination of the most responsible isoform of NOS for the formation of peroxynitrite in AD could be of therapeutic importance in the treatment of Alzheimer's disease.     

Expression of endothelial nitric oxide synthase in the ischemic penumbra: relationship to expression of neuronal nitric oxide synthase and vascular endothelial growth factor

Expressional patterns of the endothelial and neuronal forms of nitric oxide synthase (NOS) in cerebral ischemia were studied utilizing a permanent middle cerebral artery occlusion (PMCAO) model. Motor performance and infarct volumes were determined in the rats. Immunohistochemical staining for eNOS, nNOS and neurofilament were performed at 1, 2, 3, 5, 7 and 14 days after PMCAO. Vascular endothelial growth factor (VEGF) expression was determined by in-situ hybridization. PMCAO caused a reproducible cortical infarct with motor deficits in the rats. Double immunohistochemical stainings indicated that eNOS and nNOS were induced in ischemic neurons. Most stained neurons were positive for both NOS forms but some reacted with only one NOS antibody. nNOS expression peaked at 24-48 h after PMCAO, stained mainly the cytoplasm of core neurons, and disappeared after the 3rd day. eNOS expression increased until the 7th day, stained mainly the cytoplasm and membrane of penumbral cells and disappeared by the 14th day after PMCAO. VEGF expression was significantly induced in the penumbral zone in a similar distribution to eNOS. The anatomical and temporal pattern of VEGF and eNOS induction in the brain after permanent ischemia suggest that these mediators may play a role in protecting penumbral tissue from additional ischemic damage.     

依达拉奉预处理对小鼠脑缺血再灌注损伤后皮质一氧化氮合酶表达的影响

目的探讨依达拉奉预处理对小鼠脑缺血再灌注(IR)损伤后皮质一氧化氮合酶(NOS)表达的影响。方法 48只健康ICR小鼠被分为假手术组、对照组和依达拉奉组。依达拉奉组和对照组分别给予依达拉奉3 mg/(kg.d)和同等体积的生理盐水腹腔注射共7 d,然后建立小鼠IR模型;缺血1 h、再灌注24 h时应用2,3,5-氯化三苯基四氮唑(TTC)染色法测量各组脑梗死体积,应用免疫组化法检测各组小鼠皮质神经元型、、诱导型和内皮型NOS(nNOS、iNOS、eNOS)阳性细胞数。结果与假手术组比较,对照组小鼠皮质nNOS、iNOS和eNOS阳性细胞数明显增多(均P<0.05);与对照组比较,依达拉奉组脑梗死体积明显缩小,皮质nNOS和iNOS阳性细胞数明显减少,eNOS阳性细胞数明显增多(均P<0.05)。结论依达拉奉预处理可以影响IR小鼠皮质nNOS、iNOS和eNOS的表达,发挥神经保护作用。     

NAD(P)H oxidase, superoxide dismutase, catalase, glutathione peroxidase and nitric oxide synthase expression in subacute spinal cord injury

Primary trauma to the spinal cord triggers a cascade of cellular and molecular events that promote continued tissue damage and expansion of the lesion for extended periods following the initial injury. Oxidative and nitrosative stresses play an important role in progression of spinal cord injury (SCI). In an attempt to explore the biochemical origin of oxidative/nitrosative stress associated with secondary SCI, we studied expression of the superoxide (O2*-)-generating enzyme, NAD(P)H oxidase, antioxidant enzymes [superoxide dismutase (CuZn SOD, Mn SOD), catalase, glutathione peroxidase (GPX)], nitric oxide synthases (NOS) and a byproduct of NO-O2*- interaction (nitrotyrosine) in the spinal cord tissues of rats 16 h and 14 days after surgical resections of a 5-mm segment of the cord below T8 or sham-operation. Immunodetectable NAD(P)H oxidase subunits (gp91phox and P67phox), Mn SOD, inducible NOS (iNOS), endothelial NOS (eNOS), and nitrotyrosine were elevated in the transected cords on day 1 and day 14. Neuronal NOS (nNOS) was unchanged on day 1 and significantly depressed on day 14. GPX was unchanged on day 1 and significantly elevated on day 14. Catalase was unchanged in the cord tissue surrounding the transection site at both points. Thus, concurrent upregulations of NAD(P)H oxidase, eNOS and iNOS (but not nNOS), work in concert to maintain oxidative and nitrosative stress in the injured cord tissue.     

Focal protein synthesis inhibition in a model of neonatal hypoxic-ischemic brain injury

Cerebral hypoxia-ischemia was produced in 1-week-old rats by exposing them to 8% O2-92% N2 commencing 4 to 6 h after unilateral carotid artery ligation. Protein synthesis rates were measured in cerebral cortex, caudate-putamen, thalamus, and lateral septal nuclei during 2 h of hypoxia and at three times (15 min, 10 h, and 18 h) after a 3.5-h hypoxic exposure. Protein synthesis was inhibited in the ipsilateral but not contralateral forebrain during the brief hypoxic exposure, and in both hemispheres during early recovery after a 3.5 h exposure, which was sufficient to produce brain injury in the ipsilateral hemisphere. At 10 h after hypoxia, protein synthesis rates in the contralateral forebrain had recovered to control values, but in vulnerable structures of the ipsilateral forebrain, recovery of protein synthesis was transient and incomplete (cerebral cortex) or remained at the low values found immediately after hypoxia (caudate-putamen). The early development of abnormal patterns of protein synthesis in vulnerable brain regions during hypoxia and its persistence in some rats during recovery when irreversible cell injury becomes manifest suggests a possible role for abnormal protein metabolism in the evolution of irreversible brain cell damage.     

Distinct distribution and time-course changes in neuronal nitric oxide synthase and inducible NOS in the paraventricular nucleus following lipopolysaccharide injection

Nitric oxide (NO) is known to be involved in the modulation of neuroendocrine function. To clarify the role of different isoforms of NO synthase (NOS) in the neuroendocrine response to immune challenge, the expressions of neuronal NOS (nNOS) and inducible NOS (iNOS) genes in the hypothalamus following lipopolysaccharide (LPS) injection were examined using in situ hybridization. NOS activity was also determined by NADPH-diaphorase (NADPH-d) histochemistry. LPS (25 mg/kg) or sterile saline was injected intraperitoneally to male Wistar rats and the rats sacrificed 30 min, or 1, 2, 3, 5, 12 or 24 h after injection. nNOS mRNA expression in the paraventricular nucleus (PVN) was significantly increased 2 h after LPS injection. iNOS mRNA, which was not detected until 2 h after LPS injection, was significantly increased in the PVN 3 h after LPS injection. Both RNA expressions had returned to basal levels by 12 h after LPS injection. The number of NADPH-d positive cells was significantly increased 5 h after LPS injection. iNOS expression was more robust in parvocellular PVN, while nNOS was distributed mainly in the magnocellular PVN. Double in situ hybridization histochemistry revealed that some of the iNOS- (48.4%) or nNOS-positive cells (34. 3%) in the parvocellular PVN expressed CRF mRNA. The results demonstrate that LPS-induced sepsis causes significant increases in nNOS and iNOS gene expression with different time-courses and distributions, and that iNOS mRNA was more frequently co-localized with CRF-producing parvocellular neurons in the PVN. Thus, NO produced by iNOS and nNOS may play an important role in the neuroendocrine response to an immune challenge. Distinct differences in the distribution and time-course changes of iNOS and nNOS suggest different roles for the hypothalamic-pituitary-adrenal axis and/or neurohypophyseal system.     

Long-term neuroprotection with 2-iminobiotin, an inhibitor of neuronal and inducible nitric oxide synthase, after cerebral hypoxia-ischemia in neonatal rats.

The short- and long-term neuroprotective effects of 2-iminobiotin, a selective inhibitor of neuronal and inducible nitric oxide synthase, were studied in 12-day-old rats following hypoxia-ischemia. Hypoxia-ischemia was induced by occlusion of the right carotid artery followed by 90 minutes of hypoxia (FiO2 0.08). Immediately on reoxygenation, 12 and 24 hours later the rats were treated with vehicle or 2-iminobiotin at a dose of 5.5, 10, 30, or 60 mg/kg per day. Histologic analysis of brain damage was performed at 6 weeks after hypoxia-ischemia. To assess early changes of cerebral tissue, levels of HSP70, nitrotyrosine, and cytochrome c were determined 24 hours after reoxygenation. Significant neuroprotection was obtained using a dose of 30 mg/kg per day of 2-iminobiotin. Levels of HSP70 were increased in the ipsilateral hemisphere in both groups (P<0.05), but the increase was significantly (P<0.05) less in the rats receiving the optimal dose of 2-iminobiotin (30 mg/kg per day). Hypoxia-ischemia did not lead to increased levels of nitrotyrosine, nor did 2-iminobiotin influence levels of nitrotyrosine. In contrast, hypoxia-ischemia induced an increase in cytochrome c level that was prevented by 2-iminobiotin. In conclusion, 2-iminobiotin administered after hypoxia-ischemia provides long-term neuroprotection. This neuroprotection is obtained by mechanisms other than a reduction of nitrotyrosine formation in proteins.     

Nitric oxide synthase and arginase expression in the vestibular nucleus and hippocampus following unilateral vestibular deafferentation in the rat

The aim of this study was to investigate the possible relationship between changes in neuronal and endothelial nitric oxide synthase (nNOS and eNOS) and arginase expression in the vestibular nucleus complex and the hippocampus (CA1, CA2/3 and the dentate gyrus (DG) at 10 h or 2 weeks following a unilateral vestibular deafferentation (UVD) in rats. There were no significant differences in nNOS or arginase II expression in the ipsilateral or contralateral VNC at either 10 h or 2 weeks post-UVD. For eNOS, there was only a significant decrease in expression in the ipsilateral VNC at 2 weeks post-UVD (P<0.01). In the hippocampus, the only significant difference in nNOS expression was a decrease in the ipsilateral DG at 2 weeks post-UVD (P<0.05). There was a significant decrease in eNOS expression in the contralateral CA2/3 region at 10 h post-UVD (P<0.01). The only other significant change in eNOS was an increase in the contralateral DG at 10 h post-UVD (P<0.01). Although arginase II was expressed in all regions of the hippocampus, there were no significant differences in arginase II expression at any time point following UVD. These results suggest that the changes in NOS expression that occur in the VNC and hippocampus following UVD are not correlated with one another or with changes in arginase II.     

Inflammatory Cytokines are in Action: Brain Plasticity and Recovery after Brain Ischemia Due to Delayed Melatonin Administration

ObjectivesPost-ischemic inflammation leads to apoptosis as an indirect cause of functional disabilities after the stroke. Melatonin may be a good candidate for the stroke recovery because of its anti-inflammatory effects. Therefore, we investigated the effect of melatonin on inflammation in the functional recovery of brain by evaluating ipsilesional and contralesional alterations.Materials and MethodsMelatonin (4 mg/kg/day) was intraperitoneally administered into the mice from the 3^rd to the 55^th day of the post-ischemia after 30 min of middle cerebral artery occlusion.ResultsMelatonin produced a functional recovery by reducing the emigration of the circulatory leukocytes and the local microglial activation within the ischemic brain. Overall, the expression of the inflammation-related genes reduced upon melatonin treatment in the ischemic hemisphere. On the other hand, the expression level of the inflammatory cytokine genes raised in the contralateral hemisphere at the 55^th day of the post-ischemia. Furthermore, melatonin triggers an increase in the iNOS expression and a decrease in the nNOS expression in the ipsilateral hemisphere at the earlier times in the post-ischemic recovery. At the 55^th day of the post-ischemic recovery, melatonin administration enhanced the eNOS and nNOS protein expressions.ConclusionsThe present molecular, biological, and histological data have revealed broad anti-inflammatory effects of melatonin in both hemispheres with distinct temporal and spatial patterns at different phases of post-stroke recovery. These outcomes also established that melatonin act recruitment of contralesional rather than of ipsilesional.     

Neuronal and inducible nitric oxide synthase expression and protein nitration in rat cerebellum after oxygen and glucose deprivation

A perfusion model of global cerebral ischemia was used for the immunohistochemical study of changes in the glutamate-nitric oxide (NO) system in the rat cerebellum and cerebellar nuclei during a 0-14 h reperfusion period after 30 min of oxygen and glucose deprivation, with and without administration of 1.5 mM N(omega)-nitro-L-arginine methyl ester (L-NAME). While immunostaining for N-methyl-D-aspartate receptor subunit 1 (NMDAR1) showed no marked changes during the reperfusion period, neuronal NO synthase (nNOS) immunostaining increased in stellate and basket cells, granule cells and neurons of the cerebellar nuclei. However, global cerebellar nNOS concentrations determined by Western blotting remained largely unchanged in comparison with actin expression. Inducible NOS (iNOS) immunostaining appeared in Purkinje cells and neurons of the cerebellar nuclei after 2-4 h of reperfusion and intensified during the 6-14 h period. This was reflected by an increase in global cerebellar iNOS expression determined by Western blotting. Immunostaining for protein nitrotyrosine was seen in Purkinje cells, stellate and basket cells, neurons of the cerebellar nuclei and glial cells in controls, and showed a progressive translocation in Purkinje cells and neurons of the cerebellar nuclei from an initial perinuclear or nuclear location towards the periphery. At the end of the reperfusion period the Purkinje cell apical dendrites were notably retracted and tortuous. Prior and concurrent L-NAME administration eliminated nitrotyrosine immunostaining in controls and blocked or reduced most of the postischemic changes observed. The results suggest that while nNOS expression may be modified in certain cells, iNOS is induced after a 2-4 h period, and that changes in protein nitration may be associated with changes in cell morphology.     

Intracellular calcium accumulation during the evolution of hypoxic-ischemic brain damage in the immature rat

An excessive intracellular accumulation of calcium (Ca2+) in neurons and glia has been proposed to represent a major 'final common pathway' for cell death arising from hypoxia-ischemia. To clarify the role of altered calcium flux into the perinatal brain undergoing hypoxic-ischemic damage, 7-day postnatal rats underwent unilateral common carotid artery ligation followed by systemic hypoxia with 8% oxygen. This insult is known to produce brain damage in the form of selective neuronal death or infarction largely limited to the cerebral hemisphere ipsilateral to the arterial occlusion. Either prior to or following hypoxia-ischemia, the rat pups received a s.c. injection of 45CaCl2, and specimens of blood, cerebrospinal fluid (CSF), and brain were obtained for isotopic measurements and the calculation of the extent of brain intracellular radioactivity. During hypoxia-ischemia, there was a modest increase in intracellular Ca2+ radioactivity (+28-47%) in both cerebral hemispheres only after 2 h of hypoxia-ischemia. During recovery from 2 h of hypoxia-ischemia, intracellular Ca2+ accumulated progressively only in the ipsilateral cerebral hemisphere for up to 24 h, during which interval intracellular Ca2+ decreased in the contralateral hemisphere. No such progressive accumulation was noted during recovery in animals previously exposed to only 1 h of hypoxia-ischemia. The results suggest that a disruption of intracellular Ca2+ homeostasis is a major contributing factor in the evolution of perinatal hypoxic-ischemic brain damage. Ca2+ accumulation is a relatively modest and late event during the hypoxic-ischemic phase, and a progressive overload occurs during the recovery phase only if infarction occurs. The question remains as to whether or not the intracellular Ca2+ overload occurring during recovery is a contributor to or a consequence of the ultimate brain damage.     

Age-related changes of the nitric oxide system in the rat brain

This work examines the age-related changes of the NO pathway in the central nervous system (CNS), analyzing nitric oxide synthase (NOS) isoform expression, the level of nitrotyrosine-modified proteins, and the NOS activity in the cerebral cortex, decorticated brain (basal ganglia, thalamus, hypothalamus, tegtum and tegmentum) and cerebellum of young, adult and aged rats. Our data demonstrate that the different NOS isoforms are not uniformly expressed across the CNS. In this sense, the nNOS and eNOS isoenzymes are expressed mainly in the cerebellum and decorticated brain, respectively, while the iNOS isoenzyme shows the highest level in cerebellum. Concerning age, in the cerebral cortex nNOS significantly increased its expression only in adult animals; meanwhile, in the cerebellum the eNOS expression decreased whereas iNOS increased in adult and aged rats. No age-related changes in any isoform were found in decorticated brain. NOS activity, determined by nitrate plus nitrite quantification, registered the highest levels in the cerebellum, where the significant increase detected with aging was probably related to iNOS activity. The number of nitrotyrosine-modified immunoreactive bands differed among regions; thus, the highest number was detected in the decorticated brain while the cerebellum showed the least number of bands. Finally, bulk protein nitration increased in cerebral cortex only in adult animal. No changes were found in the decorticated brain, and the decrease detected in the cerebellum of aged animals was not significant. According to these results, the NO pathway is differently modified with age in the three CNS regions analyzed.     

Biopterin in the acute phase of hypoxia-ischemia in a neonatal pig model

To clarify the participation of inducible NOS (iNOS) in the hypoxia-ischemia, we examined iNOS and its tetrahydrobiopterin co-factor in the cerebral cortex and plasma in a newborn-piglet model. We also investigated the role of hypothermia in iNOS expression and biopterin production. Male newborn piglets were ventilated 6% oxygen for 45 min. Their common carotid arteries were clamped during hypoxia. Then they were resuscitated with 30% oxygen (HI group). Piglets of the hypothermia group were treated as the HI group and their body was cooled to 35.5 degrees C after hypoxic-ischemic insults. Sham-treated piglets were also reserved. In the HI group, iNOS was present in neurons and macrophages of the cerebral cortex 12h after the insult. The concentrations of nitrite and nitrate were elevated in the cerebral cortex 12h after hypoxic-ischemic insults but the biopterin level was unchanged. The plasma biopterin concentration after the insult (377.9+/-78.7 nM) was five times higher than before the insult (80.1+/-4.3 nM); this level peaked 4h after the insult (604.8+/-200.9 nM) and only slightly decreased after 12h (445.9+/-57.8 nM). In the hypothermia group, no iNOS expression was observed 12h after the insult. The plasma biopterin concentration after the insult (464.2+/-92.3 nM) was similar to that in the HI group, but was suppressed by 4h of hypothermia (229.3+/-106.8 nM). In this study, neuronal iNOS expression and increase of NO production were found in the acute phase of hypoxia-ischemia. Brain biopterin did not increase in hypoxia-ischemia although plasma biopterin was five-fold elevated. The discrepancy may also affect hypoxic-ischemic organ damage.     

Calcium accumulation during the evolution of hypoxic-ischemic brain damage in the immature rat

An excessive accumulation of calcium in neuronal and other tissues has been postulated to represent a "final common pathway" for cell death arising from hypoxia-ischemia. To clarify the role of altered calcium flux into and distribution within the perinatal brain undergoing hypoxic-ischemic injury, 7-day postnatal rats underwent unilateral common carotid artery ligation followed by 3 h of hypoxia with 8% oxygen. This insult is known to produce brain damage confined to the cerebral hemisphere ipsilateral to the arterial occlusion in greater than 90% of the animals. Either before or after hypoxia-ischemia, the animals received a subcutaneous injection of [45Ca]Cl2, and their brains were subjected to 45Ca autoradiography at 0-1, 5, 24, and 72 h, 7 or 15 days thereafter. During hypoxia-ischemia, calcium flux into the ipsilateral cerebral hemisphere was prominent in 13 of 14 rat pups, especially in neocortex, hippocampus, striatum, and thalamus. Calcium accumulation also occurred to a variable degree (6 of 14 animals) in the contralateral cerebral hemisphere. During recovery, radioactivity in the contralateral cerebral hemisphere was no longer apparent, whereas in the ipsilateral hemisphere, the extent of calcium accumulation was mild in four of six at 1 h, moderate in three of six at 5 h, moderate to intense in six of seven and six of seven at 24 and 72 h, respectively, and intense in three of three and two of two animals at 7 and 15 days, respectively. As during hypoxia-ischemia, the distribution of the radioactivity was most prominent in those structures that are known to be vulnerable to hypoxic-ischemic injury.(ABSTRACT TRUNCATED AT 250 WORDS)