The modulatory role of nitric oxide in the regulation of proenkephalin and prodynorphin gene expressions induced by kainic acid in rat hippocampus

The effect of L-arginine (L-ARG), a nitric oxide donor, or Nomega-nitro-L-arginine (L-NAME), a nitric oxide synthase inhibitor, on the regulation of kainic acid (KA)-induced proenkephalin (proENK) and prodynorphin (proDYN) mRNA expressions in rat hippocampus was studied. The proENK and proDYN mRNA levels were markedly increased 6 h after KA (10 mg/kg, i.p.) administration. The elevations of both proENK and proDYN mRNA levels induced by KA was effectively inhibited by pre-administration of L-ARG (400 mg/kg, i.p.), but was not affected by pre-treatment with L-NAME (200 mg/kg, i.p.). The blockade of KA-induced proENK and proDYN mRNA levels by the pre-treatment with L-ARG was well correlated with proto-oncoprotein levels, such as c-Fos, Fra-2, FosB, JunD, JunB, and c-Jun, as well as AP-1 and ENKCRE-2 DNA binding activities. The pre-administration with L-NAME further increased KA-induced c-jun and c-fos mRNA levels in addition to their protein product levels, although the pre-treatment with L-NAME did not affect KA-induced FosB, Fra-2, JunB, and JunD protein levels at 6 h after treatment. In addition, the pre-administration with L-NAME further increased the KA-induced AP-1 and ENKCRE-2 DNA binding activities. Our results suggest that L-ARG plays an important role in inhibiting KA-induced proENK or proDYN mRNA expression, and its inhibitory action may be mediated through reducing the proto-oncoprotein levels, such as c-Fos, Fra-2, FosB, c-Jun, JunD, and JunB. In addition, L-NAME potentiated the c-Fos or c-Jun gene expression, as well as AP-1 or ENKCRE-2 DNA binding activity. However, these increases did not show the potentiative effect on KA-induced increases of proENK and proDYN mRNA level.     

Upregulation of bax protein levels in neurons following cerebral ischemia

The patterns of expression of the bcl-2, bax, and bci-X genes were examined immunohistochemically in neurons of the adult rat brain before and after 10 min of global ischemia induced by transient cardiac arrest. High levels of the cell death promoting protein Bax and concomitant low levels of the apoptosis-blocking protein Bcl-2 were found in some populations of neurons that are particularly sensitive to cell death induced by transient global ischemia, such as the CA1 sector of the hippocampus and the Purkinje cells of the cerebellum. Moreover, within 0.5 to 3 hr after an ischemic episode, immunostaining for Bax was markedly increased within neurons with morphological features of degeneration in many regions of the brain. Use of a two-color staining method for simultaneous analysis of Bax protein and in situ detection of DNA-strand breaks revealed high levels of Bax immunoreactivity in many neurons undergoing apoptosis. Postischemic elevations in Bax protein levels in the hippocampus, cortex, and cerebellum were also demonstrated by immunoblotting. At early times after transient ischemia, regulation of Bcl-2 and Bcl-x protein levels varied among neuronal subpopulations, but from 3 hr on, those neurons with morphological evidence of degeneration uniformly contained reduced levels of Bci-2 and particularly Bci-X immunoreactivity. The findings suggest that differential expression of some members of the bcl-2 gene family may play an important role in determining the relative sensitivity of neuronal subpopulations to ischemia and that postischemic alterations in the expression of bax, bcl-2, and bcl-x may contribute to the delayed neuronal cell death that occurs during the repurfusion phase after a transient ischemic episode.     

Effects of media composition on substrate removal by pure and mixed bacterial cultures

Morphological changes in the neurons of the gerbil hippocampus following 5 min of forebrain ischemia were examined using light and electron microscopy. Although non-pyramidal neurons in the CA1 region of the hippocampus survived through the full length of the observation period, up to six weeks after ischemia, they consistently demonstrated degenerative changes distinct from those of the well-known "delayed neuronal death" of CA1 pyramidal cells. When examined with the light microscope, CA1 non-pyramidal neurons were found to be shrunken and their nuclei and cytoplasm were hyperchromatic between seven days and six weeks after ischemia. When examined with the electron microscope, postischemic non-pyramidal neurons were found to have markedly electron-dense profiles; their cytoplasm contained numerous free ribosomes and heterogeneous smaller granular substances, the latter also filling the nuclei. However, there was no loss of ribosomes from the rough endoplasmic reticulum, and mitochondrial cristae were preserved, suggesting that these neurons were viable. CA1 non-pyramidal neurons were studied immunohistochemically using three types of monoclonal antibodies, one each against parvalbumin, a nonphosphorylated epitope on the 168,000 mol. wt and 200,000 mol. wt subunits of neurofilament proteins, and microtubule-associated protein 2. CA1 non-pyramidal neurons lost immunoreactivity to these neuron-specific substances six weeks after ischemia, suggesting that these degenerating cells lacked certain types of normal neuronal activity. We conclude that non-pyramidal neurons in the hippocampal CA1 region survive transient ischemia but undergo degenerative changes following complete loss of CA1 pyramidal cells. These changes may be due to depletion of presumptive target-derived trophic factors within the non-pyramidal neurons.     

Hemodynamic rounds series II: pulmonic balloon valvuloplasty

We studied changes in expression of F3/contactin (F3), a neuron-specific adhesion molecule, in the gerbil hippocampus after transient forebrain ischemia for 5 min. By immunohistochemical techniques using F3 antibody, we found a biphasic change in immunoreactivity for F3 in the CA1 area after ischemia. Western blotting of F3 protein showed a similar biphasic change. F3 immunoblots decreased to 67% of the control at 1 week, but then they increased and attained 159% at 3 weeks and 152% at 5 weeks after ischemia. Immunoreactivity of a neurofilament (NF145) showed a similar biphasic change to F3 but to a lesser extent. In contrast, microtubule-associated protein 2 (MAP2) immunoreactivity uniformly decreased after ischemia. In situ hybridization revealed that F3 messenger RNA (mRNA) hybridization signals in CA1 area were greatly reduced 1 week after ischemia, while the signals in the CA3 area were unchanged and even increased 3 weeks after ischemia. Damage to CA3 neurons by hyperthermic ischemia blocked the F3 increase in area CA1. Our results suggest that the initial decrease in F3 following ischemia reflects loss of CA1 neurons and the late increase in F3, which shows that a similar time course with neurofilaments may be caused by neurite sprouting.     

Endothelin-like immunoreactivity in hippocampus following transient global cerebral ischemia. I. Neuronal and glial cells

The effect of transient, global ischemia on endothelin (ET) localization in CA1 area of hippocampus of the rats was investigated using post-embedding immunogold method. This paper provides immunocytochemical evidence that cerebral ischemia is accompanied by the increase of production of endothelin in CA1 area of the rat. This study focuses on the analysis ET-like immunoreactive neurons and glial cells in hippocampal CA1 area after long-term survival after ischemia: 1 week-12 months. One and two weeks after ischemia appearance of shrunken neurons and significant increase in ET-like immunoreactivity in astroglial cells as well as their proliferation was observed. Six and twelve months--marked immunoreactivity of endothelin in shrunken neurons and in perineuronal microglial macrophages was observed. The data suggest the possibility that the increased intracellular content of endothelin has a role in the development of neuronal death even following a long-term period after ischemia.     

Expression of zinc transporter gene, ZnT-1, is induced after transient forebrain ischemia in the gerbil

To elucidate the molecular mechanisms underlying neuronal death after transient forebrain ischemia, we cloned genes expressed after transient forebrain ischemia in the Mongolian gerbil by a differential display method. A gerbil homolog of rat zinc transporter, ZnT-1, which transports intracellular Zn2+ out of cells, was isolated. Its expression became detectable exclusively in pyramidal neurons of the CA1 region 12 hr after ischemia and reached a maximum from day 1 to day 2 as shown by in situ hybridization. By day 7, expression had disappeared entirely from the cells in the CA1 region, because the neurons had died. No other brain regions exhibited such a significant level of ZnT-1 mRNA expression during this period. Zn2+ was shown to accumulate in CA1 pyramidal neurons expressing ZnT-1 mRNA after the ischemia by using zinquin, a zinc-specific fluorescent dye. When primary hippocampal neurons were exposed to a high dose of Zn2+, ZnT-1 mRNA accumulated. These results suggest that the induction of ZnT-1 mRNA observed in CA1 neurons was caused by an increase in the intracellular Zn2+ concentration. It was reported recently that Zn2+ chelator blocked neuronal death after ischemia and that the influx of Zn2+ might be a key mechanism underlying neuronal death. The induction of ZnT-1 mRNA in CA1 pyramidal neurons fated to die after transient ischemia is of interest to the study of postischemic events and the molecular mechanisms underlying delayed neuronal death.     

Synthesis and in vitro T-cell immunogenicity of conjugates with dual specificity: attachment of epitope peptides of 16 and 38 kDa proteins from Mycobacterium tuberculosis to branched polypeptide

A brief period of bilateral carotid occlusion (BCO)-induced forebrain ischemia in gerbils triggers neuronal degeneration and the subsequent expression of amyloid precursor protein (APP), b-amyloid protein (b-AP), and apolipoprotein E (APO-E) in the selectively vulnerable CA1 region of the hippocampus. The increase in immunoreactivity is secondary to the postischemic degeneration of the CA1 neurons and is largely astrocyte-derived as evidenced by a simultaneous increase in glial fibrillary acidic protein (GFAP) staining. Oxygen radical-induced lipid peroxidation has been strongly suggested to play a role in postischemic neuronal damage and Alzheimer's disease. Recent literature suggests a possible link between early oxidative stress and APP overexpression. Therefore, the present investigation examined the effect of two novel brain-penetrating pyrrolopyrimidine lipid peroxidation inhibitors (PNU-101033E and PNU-104067F) on CA1 neurodegeneration and the subsequent increase in APP, b-AP, APO-E, and GFAP immunostaining at 4 days after a 5-minute episode of forebrain ischemia. Using an antibody for lipid peroxidation-derived malondialdehyde (MDA)-modified proteins, the authors also examined the effects of PNU-104067F on MDA immunostaining 2 days after ischemia, before completion of the neuronal loss. At 2 days, the authors also evaluated microglial activation using an antibody to surface major histocompatibility complex class II antigen expressed by activated microglia. Gerbils were treated at 30 mg/kg orally 30 minutes before the BCO and 2 hours after ischemia, followed by daily dosing for the next day (microglia and MDA) and the successive 3 days for APP, b-AP, APO-E, and GFAP immunostaining. APP and APO-E staining was significantly suppressed by 50% and 66%, respectively, with either compound. b-AP immunoreactivity was decreased 56% with both compounds, and GFAP expression was significantly decreased 53% (PNU-101033E) and 60.5% (PNU-104067F). There was a concomitant partial sparing of the CA1 hippocampal neurons by both PNU-101033E and PNU-104067F (P < .01) as determined by cresyl violet histochemistry. PNU-104067F significantly inhibited lipid peroxidation-derived MDA immunostaining and microglia activation (P < .05) at 48 hours after ischemia. Brain-penetrable lipid peroxidation inhibitors may provide attenuation of various glial response proteins after ischemic injury, probably secondary to neuronal protection.     

Effect of naftidrofuryl (LS129) on axonal growth

Wistar rats, eight days old, were subjected to permanent bilateral forebrain ischemia, followed by hypoxia for 15 minutes. A cerebral infarct, mainly involving the cerebral neocortex, hippocampus, amygdala, striatum and subcortical white matter was produced. Neurons and glia showing punctate chromatin condensation and karyorrhectic cells were observed 12 hours after hypoxia-ischemia. Their number increased during the first two days and recruitment of cells with degenerating nuclei occurred until day five. In situ labeling of nuclear DNA fragmentation stained many normal-appearing nuclei, as well as punctate chromatin condensations and nuclear fragments in karyorrhectic cells. Delayed neuronal death in the CA1 area of the hippocampus was observed after 20 minutes of transient forebrain ischemia in the adult gerbil. In situ labeling of nuclear DNA fragmentation demonstrated stained punctate chromatin condensation in a few degenerating cells at 48 hours post-ischemia. Substantial labeling of CA1 neurons occurred in the fourth day. Agarose gel electrophoresis of extracted brain DNA from ischemic infant rats and adult gerbils showed a ladder-type pattern which is typical of nuclear DNA fragmentation into oligonucleosomal fragments (internucleosomal cleavage). These findings suggest that endonuclease(s) activation may play a role in cell death induced by different forms of hypoxia-ischemia.     

Degradation of cytokeratin intermediate filaments by calcium-activated proteases (calpains) in vitro: implications for formation of Mallory bodies

A replication defective adenoviral vector containing the E. coli lacZ gene (AdCMVnLacZ) was directly injected into right hippocampus and lateral ventricle immediately after 5 min of transient global ischemia in gerbils. The relations between the lacZ gene expression and DNA fragmentation or heat shock protein 72 (HSP72) immunoreactivity were examined up to 21 days post ischemia. The lacZ gene was transiently expressed at 1 day in the hippocampus except around the CA1 region, while a large number of the periventricular cells strongly expressed the lacZ gene from 8 h to 7 days. In CA1 layer, terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) positive cells, which were present only adjacent to the needle track at 8 h to 1 day, became more extensive in the whole CA1 layer at 3 to 7 days. TUNEL-positive cells were also detected around the DG at 1 day, around the needle track at 8 h to 3 days, and in the choroid plexus cells at 7 days. HSP72 staining was detected in the subiculum at 1 to 3 days, the dentate granule cells at 8 h to 1 day, and in the CA3 or CA4 pyramidal cells at 1 to 3 days. Some lacZ expressing cells were double-positive with HSP72 in DG, while the majority of those were distinguished from the TUNEL-positive cells. Pyramidal neurons were almost completely lost in the CA1 sector at 7 days after the ischemia. The present study demonstrates the successful LacZ gene transfer into the hippocampus and ventricle of postischemic gerbil brain except in the vulnerable CA1 layer by adenoviral vector injection. However, adenovirus-mediated gene transfer may induce indirect apoptotic cell death in the DG and ventricle, in addition to direct traumatic injury around the needle track.     

Global ischemia induces downregulation of Glur2 mRNA and increases AMPA receptor-mediated Ca2+ influx in hippocampal CA1 neurons of gerbil

Transient, severe forebrain or global ischemia leads to delayed cell death of pyramidal neurons in the hippocampal CA1. The precise molecular mechanisms underlying neuronal cell death after global ischemia are as yet unknown. Glutamate receptor-mediated Ca2+ influx is thought to play a critical role in this cell death. In situ hybridization revealed that the expression of mRNA encoding GluR2 (the subunit that limits Ca2+ permeability of AMPA-type glutamate receptors) was markedly and specifically reduced in gerbil CA1 pyramidal neurons after global ischemia but before the onset of neurodegeneration. To determine whether the change in GluR2 expression is functionally significant, we examined the AMPA receptor-mediated rise in cytoplasmic free Ca2+ level ([Ca2+]i) in individual CA1 pyramidal neurons by optical imaging with the Ca2+ indicator dye fura-2 and by intracellular recording. Seventy-two hours after ischemia, CA1 neurons that retained the ability to fire action potentials exhibited a greatly enhanced AMPA-elicited rise in [Ca2+]i. Basal [Ca2+]i in these neurons was unchanged. These findings provide evidence for Ca2+ entry directly through AMPA receptors in pyramidal neurons destined to die. Downregulation of GluR2 gene expression and an increase in Ca2+ influx through AMPA receptors in response to endogenous glutamate are likely to contribute to the delayed neuronal death after global ischemia.     

Pathophysiology and treatment of cerebral ischemia

This article describes the pathophysiology of, and treatment strategy for, cerebral ischemia. It is useful to think of an ischemic lesion as a densely ischemic core surrounded by better perfused "penumbra" tissue that is silent electrically but remains viable. Reperfusion plays an important role in the pathophysiology of cerebral ischemia. Magnetic resonance imaging (MRI) and histological studies in rat focal ischemia models using transient middle cerebral artery (MCA) occlusion indicate that reperfusion after an ischemic episode of 2- to 3-hour duration does not result in reduction of the size of the infarct. Brief occlusion of the MCA produces a characteristic, cell-type specific injury in the striatum where medium-sized spinous projection neurons are selectively lost; this injury is accompanied by gliosis. Transient forebrain ischemia leads to delayed death of the CA1 neurons in the hippocampus. Immunohistochemical and biochemical investigations of Ca2+/calmodulin-dependent protein kinase II(CaM kinase II) and protein phosphatase (calcineurin) after transient forebrain ischemia demonstrated that the activity of CaM kinase II was decreased in the CA1 region of the hippocampus early (6-12 hours) after ischemia. However, calcineurin was preserved in the CA1 region until 1.5 days after the ischemic insult and then lost; a subsequent increase in the morphological degeneration of neurons was observed. We hypothesized that an imbalance of Ca2+/calmodulin dependent protein phosphorylation-dephosphorylation may be involved in delayed neuronal death after ischemia. In the treatment of acute ischemic stroke, immediate recanalization of the occluded artery, using systemic or local thrombolysis, is optimal for restoring the blood flow and rescuing the ischemic brain from complete infarction. However, the window of therapeutic effectiveness is very narrow. The development of effective neuroprotection methods and the establishment of reliable imaging modalities for an early and accurate diagnosis of the extent and degree of the ischemia are imperative.     

Transient neurophysiological changes in CA3 neurons and dentate granule cells after severe forebrain ischemia in vivo

Transient neurophysiological changes in CA3 neurons and dentate granule cells after severe forebrain ischemia in vivo. J. Neurophysiol. 80: 2860-2869, 1998. The spontaneous activities, evoked synaptic responses, and membrane properties of CA3 pyramidal neurons and dentate granule cells in rat hippocampus were compared before ischemia and 相似文献   

Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia

Delayed neuronal death after transient cerebral ischemia may be mediated, in part, by the induction of apoptosis-regulatory gene products. Caspase-3 is a newly characterized mammalian cysteine protease that promotes cell death during brain development, in neuronal cultures, and in other cell types under many different conditions. To determine whether caspase-3 serves to regulate neuronal death after cerebral ischemia, we have (1) cloned a cDNA encoding the rat brain caspase-3; (2) examined caspase-3 mRNA and protein expression in the brain using in situ hybridization, Northern and Western blot analyses, and double-labeled immunohistochemistry; (3) determined caspase-3-like activity in brain cell extracts; and (4) studied the effect of caspase-3 inhibition on cell survival and DNA fragmentation in the hippocampus in a rat model of transient global ischemia. At 8-72 hr after ischemia, caspase-3 mRNA and protein were induced in the hippocampus and caudate-putamen (CPu), accompanied by increased caspase-3-like protease activity. In the hippocampus, caspase-3 mRNA and protein were predominantly increased in degenerating CA1 pyramidal neurons. Proteolytic activation of the caspase-3 precursor was detected in hippocampus and CPu but not in cortex at 4-72 hr after ischemia. Double-label experiments detected DNA fragmentation in the majority of CA1 neurons and selective CPu neurons that overexpressed caspase-3. Furthermore, ventricular infusion of Z-DEVD-FMK, a caspase-3 inhibitor, decreased caspase-3 activity in the hippocampus and significantly reduced cell death and DNA fragmentation in the CA1 sector up to 7 d after ischemia. These data strongly suggest that caspase-3 activity contributes to delayed neuronal death after transient ischemia.     

Fumonisin B1 contamination of maize and experimental acute fumonisin toxicosis in pigs

As part of the stress response, the 72 kDa heat shock protein (hsp72) is induced in neurons after ischemic and traumatic brain injury (TBI). To examine the stress response after TBI with secondary insult, we examined the regional and cellular expression of hsp72 mRNA and protein after controlled cortical impact (CCI) injury with secondary hypoxemia and mild hypotension in rats. Rats were killed at 6, 8, 24, 72, or 168 h after trauma. Naive and sham-operated rats were used as controls. Brains were removed, and in situ hybridization (n = 2/group), immunocytochemistry (n = 4/group), and Western blot analysis (n = 3 to 5/group) for hsp72 was performed. Hsp72 mRNA was expressed in neurons in the ipsilateral cortex, CA3 region of the hippocampus, hilus, and dentate gyrus at 6 h. Hsp72 mRNA was expressed primarily in the ipsilateral cortex, at 24 h, and by 72 h hsp72 mRNA expression returned to near basal levels. Hsp72 protein was seen in ipsilateral cortical neurons, hilar neurons, and neurons in the medial aspect of the CA3 region of the hippocampus (CA3-c) at 24 h. At 72 h, hsp72 immunoreactivity was reduced versus 24 h in these same regions, but it was increased versus baseline. Western blot analysis confirmed an increase in hsp72 protein in the ipsilateral cortex. The regional pattern of hsp72 mRNA induction in neurons was similar to the pattern of protein expression after CCI, with the exceptions that hsp72 mRNA, but not protein, was expressed in the dentate gyrus and the lateral aspect of the CA3 region of the hippocampus (CA3-a). The stress response, as detected by hsp72 expression, is induced in some neurons in some regions that are selectively vulnerable to delayed neuronal death in this model of TBI. The failure to translate some proteins including hsp72 may be associated with delayed neuronal death in certain hippocampal regions after TBI.     

Dexamethasone inhibits ischemia-induced transient reduction of neurotrophin-3 mRNA in rat hippocampal neurons

Dexamethasone (DEX) increases the expression of neurotrophin-3 (NT-3) in normal rat hippocampal neurons, whereas transient forebrain ischemia reduces the NT-3 mRNA level. The effect of DEX on the expression of NT-3 mRNA in injured brain cells after ischemia has not been investigated, however. Using in situ hybridization and ribonuclease protection assay methods, we studied NT-3 mRNA expression in rats with and without DEX administration after transient forebrain ischemia. Without DEX treatment, NT-3 mRNA was down-regulated in the hippocampal neurons at 2, 4, 12 h and returned to basal levels 24 h following ischemia. With DEX treatment, however, NT-3 mRNA showed no change at 2, 4 and 12 h and increased 24 h after ischemia. The results indicate that DEX inhibits ischemia-induced NT-3 mRNA down-regulation during the first 12 h and up-regulates NT-3 mRNA 24 h after ischemia. DEX administration might be effective in influencing some of the pathophysiological effects of ischemia in the hippocampus.