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Biomedical subjects

R P Simon

Publications and source records attributed to R P Simon.

At least 37 records · Page 2Linked to original sources
PubMed2000

Alterations in bcl-2 and caspase gene family protein expression in human temporal lobe epilepsy.

OBJECTIVE: To address the role of cell death regulatory genes of the bcl-2 and caspase families in the neuropathology of human epilepsy using tissue extracted from patients undergoing temporal lobectomy for intractable seizures. METHODS: Using Western blotting and immunohistochemistry, the authors investigated the expression of bcl-2, bcl-xL, bax, caspase-1,and caspase-3 in temporal cortex samples from patients who had undergone temporal lobectomy surgery for intractable epilepsy (n = 19). Nonepileptic postmortem tissue from a brain bank served as control (n = 6). RESULTS: Western blot analysis demonstrated significant increases in levels of bcl-2 and bcl-xL protein in seizure brain compared to control. Cleavage of caspase-1 was evidenced by a reduction in levels of the 45 kDa proenzyme form and an increase in levels of the p10 fragment. Levels of the 32 kDa proenzyme form of caspase-3 were elevated in seizure patients, as were levels of the 12 kDa cleaved fragment. Bcl-2, bax, and caspase-3 immunoreactivity was increased predominantly in cells with the morphologic appearance of neurons, whereas bcl-xL immunoreactivity was increased in cells with the appearance of glia. DNA fragmentation was detected in some but not all sections from epileptic brain samples. CONCLUSIONS: Cell death regulatory genes of the bcl-2 and caspase families may play a role in ongoing neuropathologic processes in human epilepsy, and offer novel targets as an adjunct to anticonvulsant therapy.

Adolescent↗
PubMed2000

Expression of the RNA-binding protein TIAR is increased in neurons after ischemic cerebral injury.

T-cell restricted intracellular antigen-related protein (TIAR) is an RNA recognition motif-type RNA-binding protein that has been implicated in the apoptotic death of T-lymphocytes and retinal pigment epithelial cells. Western blots prepared with a monoclonal antibody against TIAR showed expression in normal rat hippocampus, and induction by 15 min of global cerebral ischemia. This increased expression was evident at 8 hr after ischemia and maximal at 24 hr, whereas expression at 72 hr was reduced below basal levels. Expression of TIAR protein was also increased in parietal cortex 6 and 24 hr after 90 min of focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, as well as in cultured cortical neurons and astroglia after exposure to hypoxia in vitro. Immunocytochemistry showed that increased expression of TIAR occurred mainly in the CA1 sector of hippocampus 24 hr after global ischemia, and in cortical and striatal neurons 24 hr after 20 or 90 min of focal ischemia. Double-labeling studies showed that TIAR protein expression was co-localized with DNA damage in neuronal cells. The findings suggest that TIAR may be involved in neuronal cell death after cerebral ischemic injury.

Animals↗
PubMed2000

Spatio-temporal profile of DNA fragmentation and its relationship to patterns of epileptiform activity following focally evoked limbic seizures.

The specific electrographic activity responsible for seizure-induced DNA damage remains little explored. We therefore examined the regional and temporal appearance of DNA fragmentation and cell death and its relationship to specific electrographic seizure patterns in a rat model of focally evoked limbic epilepsy. Animals received intra-amygdaloid injection of kainic acid (KA) to induce seizures for 45 min during continuous electroencephalographic (EEG) monitoring, after which diazepam (30 mg/kg) was administered. DNA polymerase I-mediated biotin-dATP nick translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) were used to detect single- and double-stranded DNA breaks, respectively. Injection of 0.01 microg KA induced seizures characterized by ictal fast activity but without consequent brain injury. By contrast, 0.1 microg KA induced an additional pattern of seizure activity characterized by bursts of high frequency polyspike paroxysmal discharges. In these animals, there was a significant reduction in numbers of pyramidal neurons within the ipsilateral and contralateral CA3 subfield of the hippocampus, detectable as little as 4 h following seizures. PANT- and TUNEL-positive cells appeared in similar numbers 16 h following seizure cessation within the CA3, declining after 72-96 h. Varying the duration of polyspike paroxysmal discharges determined that as little as 30 s elicited maximal injury. These data suggest single- and double-stranded DNA breaks are generated during the cell death process and are consequent on a specific component of seizure activity electrographically determined.

Animals↗
PubMed2000

Bcl-w expression is increased in brain regions affected by focal cerebral ischemia in the rat.

Proteins of the bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult and may play a role in cell death following stroke. The recently isolated bcl-w gene encodes a pro-survival member of the bcl-2 family, which is widely expressed. However, it is not known whether bcl-w plays a role in determining cell survival after cerebral ischemia. Using Western blot analysis and immunocytochemistry, regional bcl-w protein expression was studied in rat brain 2, 6, 24 and 72 h following 20 min temporary middle cerebral artery occlusion (MCAO). Focal cerebral ischemia increased bcl-w protein expression within the caudate putamen and parietal cortex, as well as causing milder increases within frontal cortex. Immunocytochemically bcl-w was expressed within neurons (frontal and parietal cortex) and glia (caudate putamen) 24 h after MCAO. These data suggest that bcl-w could play a role in determining cell survival after cerebral ischemia.

Animals↗
PubMed2000

Increased expression of apoptosis-linked gene 2 (ALG2) in the rat brain after temporary focal cerebral ischemia.

Calcium is an important mediator of programmed cell death induced by transient cerebral ischemia, and calcium-binding proteins have been implicated in calcium-regulated signal transduction. Apoptosis-linked gene 2 is a calcium-binding protein required for cell death induced by different apoptotic stimuli. By Western blot analysis, we found that apoptosis-linked gene 2 protein was expressed in normal brains, and that expression increased in ischemic brains after 20 or 90 min of transient focal cerebral ischemia. Immunocytochemistry showed increased apoptosis-linked gene 2 protein expression in frontal cortex, a region where neurons underwent ischemic stress but still survived, after 20 or 90 min of focal cerebral ischemia. Apoptosis-linked gene 2 protein was also up-regulated in the ischemic border-zone of parietal cortex 24h after 20 min of focal ischemia, and was remarkably over-expressed in the caudate-putamen and parietal cortex, (where cells are destined to die) 24h after 90 min of ischemia. The expression pattern of apoptosis-linked gene 2 protein was similar to that of deoxyribonucleic acid damage detected by Klenow labeling assay. Our results suggest that apoptosis-linked gene 2 may be involved in the regulation of cell death after transient focal cerebral ischemia.

Animals↗
PubMed2000

Formation of the base modification 8-hydroxyl-2'-deoxyguanosine and DNA fragmentation following seizures induced by systemic kainic acid in the rat.

The formation of oxidative DNA damage as a consequence of seizures remains little explored. We therefore investigated the regional and temporal profile of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) formation, a hallmark of oxidative DNA damage and DNA fragmentation in rat brain following seizures induced by systemic kainic acid (KA). Formation of 8-OHdG was determined via HPLC with electrochemical detection, and single- and double-stranded DNA breaks were detected using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated nick end-labeling (TUNEL), respectively. Systemic KA (11 mg/kg) significantly increased levels of 8-OHdG within the thalamus after 2 h, within the amygdala/piriform cortex after 4 h, and within the hippocampus after 8 h. Levels remained elevated up to sevenfold within these areas for 72 h. Smaller increases in 8-OHdG levels were also detected within the parietal cortex and striatum. PANT-positive cells were detected within the thalamus, amygdala/piriform cortex, and hippocampus 24-72 h following KA injection. TUNEL-positive cells appeared within the same brain regions and over a similar time course (24-72 h) but were generally lower in number. The present data suggest oxidative damage to DNA may be an early consequence of epileptic seizures and a possible initiation event in the progression of seizure-induced injury to DNA fragmentation and cell death.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia.

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.

Animals↗
PubMed2000

Induction of oxidative DNA damage in the peri-infarct region after permanent focal cerebral ischemia.

To address the role of oxidative DNA damage in focal cerebral ischemia lacking reperfusion, we investigated DNA base and strand damage in a rat model of permanent middle cerebral artery occlusion (MCAO). Contents of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and apurinic/apyrimidinic abasic sites (AP sites), hallmarks of oxidative DNA damage, were quantitatively measured in nuclear DNA extracts from brains obtained 4-72 h after MCAO. DNA single- and double-strand breaks were detected on coronal brain sections using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), respectively. Levels of 8-OHdG and AP sites were markedly elevated 16-72 h following MCAO in the frontal cortex, representing the peri-infarct region, but levels did not significantly change within the ischemic core regions of the caudateputamen and parietal cortex. PANT- and TUNEL-positive cells began to be detectable 4-8 h following MCAO in the caudate-putamen and parietal cortex and reached maximal levels at 72 h. PANT- and TUNEL-positive cells were also detected 16-72 h after MCAO in the lateral frontal cortex within the infarct border, where many cells also showed colocalization of DNA single-strand breaks and DNA fragmentation. In contrast, levels of PANT-positive cells alone were transiently increased (16 h after MCAO) in the medial frontal cortex, an area distant from the infarct zone. These data suggest that within peri-infarct brain regions, oxidative injury to nuclear DNA in the form of base and strand damage may be a significant and contributory cause of secondary expansion of brain damage following permanent focal ischemia.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Involvement of caspase-3-like protease in the mechanism of cell death following focally evoked limbic seizures.

The cysteine protease caspase-3 may be involved in the mechanism of cell death following seizures. Using a rat model of focally evoked limbic epilepsy with continuous electroencephalography monitoring, we investigated seizure-induced changes in caspase-3 protein expression and processing, enzyme activity, and the in vivo effect of caspase-3 inhibition. Seizures were induced by intraamygdaloid injection of kainic acid (0.1 microg) and were terminated after 45 min by diazepam (30 mg/kg) administration. Animals were killed 0-72 h following diazepam administration. Levels of the 32-kDa proenzyme form of caspase-3 were unaffected by seizures. Levels of the 17-kDa cleaved (active) fragment of caspase-3 were almost undetectable in control brain, but were increased significantly at 4 and 24 h within ipsilateral hippocampus and cortex in seizure animals. Caspase-3-like protease activity was increased within the ipsilateral hippocampus at 8 and 24 h following seizures. Caspase-3 immunoreactivity was increased within the vulnerable ipsilateral CA3/CA4 subfield at 24 and 72 h following seizures and was associated predominantly, but not exclusively, with neurons exhibiting DNA fragmentation. The putatively selective caspase-3 inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone significantly improved neuronal survival bilaterally within the hippocampal CA3/CA4 subfields following seizures. Collectively, these data suggest that caspase-3 may play a significant role in the mechanism by which neurons die following seizures.

Animals↗
PubMed2000

Overexpression of the cell death suppressor Bcl-w in ischemic brain: implications for a neuroprotective role via the mitochondrial pathway.

Bcl-w is a newly described cell death suppressor member of the Bcl-2 gene family. As these genes may have a role in the outcome of ischemic brain injury, the regional expression of Bcl-w protein in rat brain was examined at 6 to 72 hours after 90 minutes of transient middle cerebral artery occlusion. Bcl-w protein, although constitutively expressed at low levels in nonischemic brain, was found to be overexpressed in ischemic brain at all time points studied. Up-regulation of Bcl-w protein was particularly abundant in the penumbral region of the cortex and mainly in cells lacking DNA fragmentation. In the cortical penumbra, Bcl-w protein was detected predominantly in neurons and showed mitochondrial localization, as determined using double-label immunohistochemistry. Bcl-w expression was also detectable, to a lesser extent, in reactive astrocytes in the infarct border zone and in microvessel walls in the infarct regions. At the mechanistic level, incubation of isolated brain mitochondria with the addition of recombinant Bax or high concentration of calcium resulted in release of cytochrome c from the mitochondria. In the presence of recombinant Bcl-w protein, however, the release of cytochrome c induced by Bax or calcium was largely inhibited. Further, recombinant Bcl-w protein inhibited calcium-induced loss of mitochondrial transmembrane potential, indicative of permeability transition, in a dose-dependent manner. These results suggest that Bcl-w may be an endogenous neuroprotectant against ischemic neuronal death and that, like its analogues such as Bcl-2 and Bcl-x-long, Bcl-w may achieve this protection via the mitochondrial death-regulatory pathway.

Animals↗
PubMed2000

Suppression of endogenous bcl-2 expression by antisense treatment exacerbates ischemic neuronal death.

Previous studies have shown that overexpression of bcl-2 in transgenic mice or by viral vectors protects the brain against cerebral ischemia. However, it is not known whether bcl-2, which is endogenously expressed in response to ischemia, exerts a protective effect. To address this question, the authors blocked the endogenous expression of bcl-2 after ischemia using antisense oligodeoxynucleotides (ODN). Antisense, sense, scrambled ODN, or vehicles were infused in the lateral ventricle of the rat for 24 hours after 30 minutes of temporary middle cerebral artery occlusion. Twenty-four hours later the brains were removed and bcl-2 protein expression was assayed by Western blot. Antisense ODN, but not sense or scrambled ODN treatment, significantly inhibited bcl-2 protein expression after ischemia. Bcl-2 protein expression was also studied 24 hours after 60 minutes of temporary middle cerebral artery occlusion in vehicle and antisense ODN-treated rats. After 60 minutes of ischemia and vehicle treatment, bcl-2 was expressed in many neurons in the ventral cortical mantle and the medial striatum. After antisense ODN treatment there were few neurons in this region expressing bcl-2, instead most neurons TUNEL labeled. Treatment with the antisense ODN, but not sense ODN, increased infarction volume as determined by cresyl violet staining 72 hours after ischemia compared with vehicle controls. These results suggested that endogenously expressed bcl-2 promoted survival in ischemic neurons and was not simply an epiphenomenon in neurons already destined to live or die.

Animals↗
PubMed1999

Cannabinoids and neuroprotection in global and focal cerebral ischemia and in neuronal cultures.

Marijuana and related drugs (cannabinoids) have been proposed as treatments for a widening spectrum of medical disorders. R(+)-[2, 3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate (R(+)-WIN 55212-2), a synthetic cannabinoid agonist, decreased hippocampal neuronal loss after transient global cerebral ischemia and reduced infarct volume after permanent focal cerebral ischemia induced by middle cerebral artery occlusion in rats. The less active enantiomer S(-)-WIN 55212-3 was ineffective, and the protective effect of R(+)-WIN 55212-2 was blocked by the specific central cannabinoid (CB1) cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide-hydrochloride. R(+)-WIN 55212-2 also protected cultured cerebral cortical neurons from in vitro hypoxia and glucose deprivation, but in contrast to the receptor-mediated neuroprotection observed in vivo, this in vitro effect was not stereoselective and was insensitive to CB1 and CB2 receptor antagonists. Cannabinoids may have therapeutic potential in disorders resulting from cerebral ischemia, including stroke, and may protect neurons from injury through a variety of mechanisms.

Animals↗
PubMed1999

Parallel dose-response studies of the voltage-dependent Na+ channel antagonist BW619C89, and the voltage-dependent Ca2+ channel antagonist nimodipine, in rat transient focal cerebral ischaemia.

We have compared two classes of putative neuroprotectants, the voltage-dependent Na+ channel antagonist BW619C87 [4-amino-2-(4-methyl-1-piperazinyl)-5-(2,3,5-trichlorophenyl) pyrimidine], and the voltage-dependent Ca2+ channel antagonist nimodipine, in a rat model of transient focal cerebral ischaemia. BW619C87 (10-50 mg/kg) or nimodipine (10-100 microg/kg) were injected intravenously 5 min before induction of 2 h transient focal cerebral ischaemia via intraluminal thread occlusion of the middle cerebral artery. BW619C87 was a potent neuroprotectant over the range tested, maximally reducing the volume of hemispheric ischaemic damage by 51% at the 50 mg/kg dose. Nimodipine maximally reduced ischaemic damage by 33% at the 50 microg/kg dose, although the maximal level of neuroprotection afforded by BW619C89 and nimodipine was not significantly different. This is the first study to compare these two classes of drug directly in a model of middle cerebral artery occlusion with reperfusion, and it supports the effectiveness of both as neuroprotectants.

Animals↗
PubMed1999

In situ detection of neuronal DNA strand breaks using the Klenow fragment of DNA polymerase I reveals different mechanisms of neuron death after global cerebral ischemia.

Ischemic cell injury in the brain may involve a cascade of programmed cell death. DNA damage may be either a catalyst or a consequence of this cascade. Therefore, the induction of DNA strand breaks in the rat brain following transient global ischemia was examined using (a) the Klenow labeling assay, identifying DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) with protruding 5' termini, and (b) terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), detecting DNA DSBs with protruding 3' termini or blunt ends. Klenow-positive staining occurred within 2 h of reperfusion and increased with increasing durations of reperfusion. DNA damage detected with the Klenow labeling assay preceded that of TUNEL expression in the caudate putamen, reticular thalamus, thalamus, and cortex. However, in CA1, DNA SSBs were not detected until 72 h of reperfusion and occurred simultaneously with DSBs. Thus, the time course and fragmentation characteristics of DNA damage differ between the hippocampal CA1 and other selectively vulnerable brain regions. This distinct pattern suggests that the delayed neuronal death in CA1 following transient global ischemia may occur via an apoptotic mechanism different from that of other brain regions.

Animals↗
PubMed1999

Relationship between seizure-induced transcription of the DNA damage-inducible gene GADD45, DNA fragmentation, and neuronal death in focally evoked limbic epilepsy.

We investigated the temporal and spatial profile of mRNA transcription for the growth arrest and DNA damage-inducible gene GADD45, DNA fragmentation, and neuronal death in rat brain following focally evoked limbic seizures. GADD45 mRNA was detected by in situ hybridization, whereas fragmented DNA was detected using in situ nick end-labeling by the large (Klenow) fragment of DNA polymerase I. Kainic acid (0.1 microg) was injected into the right amygdala of rats to induce seizures for 45 min, after which diazepam (30 mg/kg) was administered. GADD45 mRNA, DNA fragmentation, and cell death were quantified bilaterally within six limbic brain regions 0-96 h following seizure cessation. All animals underwent seizures of equivalent severity and duration as determined electrographically. In situ hybridization detected bilateral up-regulation of GADD45 mRNA throughout the CA1, CA3, and dentate gyrus of the hippocampus, the piriform and retrosplenial cortices, and the thalamus within 1 h of seizure termination. GADD45 mRNA levels remained elevated for up to 6 h, declining to baseline within all structures by 16 h. Klenow-positive cells were only found within the CA3 pyramidal layer of the ipsilateral hippocampus and appeared 16-72 h following seizure cessation. Morphologic cell death was also restricted to the CA3 subfield. These data demonstrate that focally evoked limbic seizures trigger early bihemispheric GADD45 mRNA transcription within connected limbic structures, whereas subsequent DNA fragmentation and cell death are restricted to selectively vulnerable brain regions.

Amygdala↗
PubMed1999

Molecular cloning of a cell cycle regulation gene cyclin H from ischemic rat brain: expression in neurons after global cerebral ischemia.

Gene expression plays an important role in determining the fate of neurons after ischemia. To identify additional genes that promote survival or execute programmed cell death in ischemic neurons, a subtractive cDNA library was constructed from hippocampus of rats subjected to global ischemia. With use of a differential screening technique, a cDNA was identified that was up-regulated after ischemia. The cDNA was found to have high homology with human cyclin H at both the nucleotide level (89%) and the amino acid level (93%). Northern blotting detected cyclin H mRNA in nonischemic and ischemic brains. In situ hybridization studies revealed that cyclin H message was found in hippocampal neurons in nonischemic brain. After ischemia, expression was increased primarily in the dentate gyrus and CA3 regions of hippocampus. Expression of cyclin H protein, detected by western blotting of hippocampal tissue, was increased after global ischemia, but expression of cyclins B1 and D1 and other related cell cycle genes (Cdk7 and Cdc2) was not increased. Cyclin H immunoreactivity was found exclusively within neurons. After ischemia, there was increased immunoreactivity within neurons in dentate gyrus, CA3, and cortex. Thus, cyclin H is expressed in normal postmitotic neurons and expression is increased in neurons that are ischemic yet survive. These results suggest that cyclin H may have functions in neurons other than cell cycle regulation, including other known functions such as DNA repair.

Amino Acid Sequence↗
PubMed1998

Bilateral intranigral NMDA infusion suppresses neuronal injury without affecting the duration of kainic acid-induced seizures in rats.

The substantia nigra pars reticulata (SNpr) is recognized as an important modulator of seizures within the limbic system. We have investigated the effects of N-methyl-D-aspartate (NMDA) infusion into SNpr upon seizure-related neuronal injury (assessed by expression of the 72-kDa heat shock protein - HSP 72) induced by systemic kainic acid (KA) in rats. Three to four days following implantation of guide cannulae for drug administration into SNpr, KA (7 mg/kg) was injected intravenously to induce seizures. Bilateral intranigral infusion of NMDA (20 nmol) 15 min prior to KA injection, suppressed the expression of HSP 72 in the hippocampal CA1 region without affecting seizure duration. These results support the involvement of NMDA receptors within SNpr in modulating neuronal injury following KA-induced limbic seizures.

Animals↗