Caspase-3 and calpain activities after acute and repeated ethanol administration during the rat brain growth spurt

Ethanol administration during the rat brain growth spurt triggers apoptotic neurodegeneration that appears to be mediated by caspase-3 activation. In order to gain more insight on the role of this caspase in ethanol-induced developmental neurotoxicity, we studied its expression and activity under different conditions of ethanol exposure during development. Furthermore, because of the cross-talk between caspase-3 and calpain we extended our study also at this protease. Ethanol was administered by gavage to rat pups as a single-day exposure on postnatal day (PN) 7 or from PN4 to PN10. Cleaved caspase-3 expression peaked in the cerebral cortex 12 h after ethanol treatment and returned to control values at 24 h. An identical pattern was found for caspase-3-like activity, that was increased only with the highest dose of ethanol tested (5 g/kg) and mostly in PN4. Repeated ethanol exposure, at a dose that was previously found to induce microencephaly, did not increase caspase-3 expression and activity although it decreased procaspase-3 expression and released mitochondrial cytochrome c. Repeated ethanol administration also increased calpain activity. These data show that acute and repeated ethanol administration differentially affect caspase-3 and calpain activity, suggesting that calpain activation may play a role in developmental neurotoxicity of ethanol.     

Neuroprotection by taurine in ethanol-induced apoptosis in the developing cerebellum

Background

Acute ethanol administration leads to massive apoptotic neurodegeneration in the developing central nervous system. We studied whether taurine is neuroprotective in ethanol-induced apoptosis in the mouse cerebellum during the postnatal period.

Methods

The mice were divided into three groups: ethanol-treated, ethanol+taurine-treated and controls. Ethanol (20% solution) was administered subcutaneously at a total dose of 5 g/kg (2.5 g/kg at time 1 h and 2.5 g/kg at 3 h) to the ethanol and ethanol+taurine groups. The ethanol+taurine group also received two injections of taurine (1 g/kg each, at time zero and at 4 h). To estimate apoptosis, immunostaining for activated caspase-3 and TUNEL staining were made in the mid-sagittal sections containing lobules I-X of the cerebellar vermis at 12 or 8 hours after the first taurine injection. Changes in the blood taurine level were monitored at each hour by reverse-phase high-performance liquid chromatography (HPLC).

Results

Ethanol administration induced apoptosis of Purkinje cells on P4 in all cerebellar lobules, most extensively in lobules IX and X, and on P7 increased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum. Administration of taurine significantly decreased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum on P7, but had no effect on Purkinje cells in P4 mice. The high initial taurine concentration in blood of the ethanol+taurine group diminished dramatically during the experiment, not being different at 13 h from that in the controls.

Conclusions

We conclude that the neuroprotective action of taurine is not straightforward and seems to be different in different types of neurons and/or requires prolonged maintenance of the high taurine concentration in blood plasma.

     

Ethanol preconditioning protects against ischemia/reperfusion-induced brain damage: role of NADPH oxidase-derived ROS

Ethanol preconditioning (EtOH-PC) refers to a phenomenon in which tissues are protected from the deleterious effects of ischemia/reperfusion (I/R) by prior ingestion of ethanol at low to moderate levels. In this study, we tested whether prior (24 h) administration of ethanol as a single bolus that produced a peak plasma concentration of 42-46 mg/dl in gerbils would offer protective effects against neuronal damage due to cerebral I/R. In addition, we also tested whether reactive oxygen species (ROS) derived from NADPH oxidase played a role as initiators of these putative protective effects. Groups of gerbils were administered either ethanol or the same volume of water by gavage 24 h before transient global cerebral ischemia induced by occlusion of both common carotid arteries for 5 min. In some experiments, apocynin, a specific inhibitor of NADPH oxidase, was administered (5 mg/kg body wt, i.p.) 10 min before ethanol administration. EtOH-PC ameliorated behavioral deficit induced by cerebral I/R and protected the brain against I/R-induced delayed neuronal death, neuronal and dendritic degeneration, oxidative DNA damage, and glial cell activation. These beneficial effects were attenuated by apocynin treatment coincident with ethanol administration. Ethanol ingestion was associated with translocation of the NADPH oxidase subunit p67(phox) from hippocampal cytosol fraction to membrane, increased NADPH oxidase activity in hippocampus within the first hour after gavage, and increased lipid peroxidation (4-hydroxy-2-nonenal) in plasma and hippocampus within the first 2 h after gavage. These effects were also inhibited by concomitant apocynin treatment. Our data are consistent with the hypothesis that antecedent ethanol ingestion at socially relevant levels induces neuroprotective effects in I/R by a mechanism that is triggered by ROS produced through NADPH oxidase. Our results further suggest the possibility that preconditioning with other pharmacological agents that induce a mild oxidative stress may have similar therapeutic value for suppressing stroke-mediated damage in brain.     

Taurine protects cerebellar neurons of the external granular layer against ethanol-induced apoptosis in 7-day-old mice

Acute alcohol administration is harmful especially for the developing nervous system, where it induces massive apoptotic neurodegeneration leading to alcohol-related disorders of newborn infants. Neuroprotection against ethanol-induced apoptosis may save neurons and reduce the consequences of maternal alcohol consumption. Previously we have shown that taurine protects immature cerebellar neurons in the internal granular layer of cerebellum from ethanol-induced apoptosis. Now we describe a similar protective action for taurine in the external layer of cerebellum of 7-day-old mice. The mice were divided into three groups: ethanol-treated, ethanol + taurine-treated and controls. Ethanol (20% solution) was administered subcutaneously at a total dose of 5 g/kg (2.5 g/kg at time 0 h and 2.5 g/kg at 2 h) to the ethanol and ethanol + taurine groups. The ethanol + taurine group also received subcutaneously two injections of taurine (1 g/kg each, 1 h before the first dose of ethanol and 1 h after the second dose of ethanol). To verify apoptosis, immunostaining for activated caspase-3 and TUNEL staining were made in the mid-sagittal sections containing lobules I–X of the cerebellar vermis at 8 h after the first ethanol injection. Ethanol induced apoptosis in the cerebellar external granular layer. Taurine treatment significantly reduced the number of activated caspase-3-immunoreactive and TUNEL-positive cells. Taurine has thus a neuroprotective antiapoptotic action in the external granular layer of the cerebellum, preserving a number of neurons from ethanol-induced apoptosis.     

Ethanol-induced neuronal apoptosis in vivo requires BAX in the developing mouse brain

A single episode of ethanol intoxication triggers widespread apoptotic neurodegeneration in the infant rat or mouse brain. The cell death process occurs over a 6-16 h period following ethanol administration, is accompanied by a robust display of caspase-3 enzyme activation, and meets ultrastructural criteria for apoptosis. Two apoptotic pathways (intrinsic and extrinsic) have been described, either of which may culminate in the activation of caspase-3. The intrinsic pathway is regulated by Bax and Bcl-XL and involves Bax-induced mitochondrial dysfunction and release of cytochrome c as antecedent events leading to caspase-3 activation. Activation of caspase-8 is a key event preceding caspase-3 activation in the extrinsic pathway. In the present study, following ethanol administration to infant mice, we found no change in activated caspase-8, which suggests that the extrinsic pathway is not involved in ethanol-induced apoptosis. We also found that ethanol triggers robust caspase-3 activation and apoptotic neurodegeneration in C57BL/6 wildtype mice, but induces neither phenomenon in homozygous Bax-deficient mice. Therefore, it appears that ethanol-induced neuroapoptosis is an intrinsic pathway-mediated phenomenon involving Bax-induced disruption of mitochondrial membranes and cytochrome c release as early events leading to caspase-3 activation.     

Chronic prenatal ethanol exposure-induced decrease of guinea pig hippocampal CA1 pyramidal cell and cerebellar Purkinje cell density

The brain is a key target of ethanol teratogenicity, in which ethanol can produce neurodegeneration in selected areas, including the hippocampus and cerebellum. The research objective was to test the hypothesis that chronic prenatal ethanol exposure, via maternal ethanol administration, produces differential time course of decreased linear density of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells. Timed pregnant guinea pigs received chronic oral administration of ethanol, isocaloric-sucrose/pair-feeding, or water throughout gestation (term, about gestational day (GD) 68), and the offspring were studied at GD 62 (near-term fetus), postnatal day (PD) 1 (neonate), PD 5, and PD 12 (early postnatal life). Ethanol treatment, compared with isocaloric-sucrose/pair-feeding and water treatments, decreased brain, hippocampal, and cerebellar weights at GD 62, PD 1, PD 5, and PD 12. Hippocampal CA1 pyramidal cell linear density and cerebellar Purkinje cell linear density were unaffected at GD 62. Ethanol treatment produced 25, 30, and 30% decreases in linear density of hippocampal CA1 pyramidal cells at PD 1, PD 5, and PD 12, respectively, and a 30% decrease in linear density of cerebellar Purkinje cells at PD 12 only. At PD 5, Purkinje cell profile linear density remained unaffected; however, ethanol treatment appeared to increase linear density of apoptotic Purkinje cell nuclei, as determined by a modified TUNEL method. The data demonstrate that chronic prenatal ethanol exposure produces apparent differential time course of decreased linear density of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells in the developing guinea pig.     

Neuroprotective Effects of Crocin Against Ethanol Neurotoxicity in the Animal Model of Fetal Alcohol Spectrum Disorders

Several experimental and clinical findings suggest that ethanol consumption during pregnancy activates an oxidative-inflammatory cascade followed by wide apoptotic neurodegeneration within several brain areas, including the hippocampus. Crocin can protect neurons because of its antioxidant, anti-inflammatory, and antiapoptotic effects. This study evaluated the crocin protective impact on ethanol-related neuroinflammation and neuronal apoptosis in the hippocampus of rat pups exposed to alcohol over postnatal days. Ethanol (5.25 g/kg) was administrated in milk solution (27.8 ml/kg) by intragastric intubation 2–10 days after birth. The animals received crocin (15, 30, and 45 mg/kg) 2–10 days after birth. The hippocampus-dependent memory and spatial learning were evaluated 36 days after birth using the Morris water maze task. Further, the concentrations of TNF-α and antioxidant enzymes were determined using ELISA assay to examine the antioxidant and anti-inflammatory activities. Also, immunohistochemical staining was performed to evaluate the glial fibrillary acidic protein (GFAP), Ionized calcium binding adaptor molecule 1(Iba-1), and caspase-3 expression. The administration of crocin significantly attenuated spatial memory impairment (P?<?0.01) after ethanol neurotoxicity. Also, crocin led to a significant enhancement in SOD (P?<?0.05) and GSH-PX (P?<?0.01), whereas it caused a reduction in the TNF-α and MDA concentrations compared to the ethanol group (P?<?0.01). Moreover, the hippocampal level of caspase-3 (P?<?0.01) and the number of GFAP and Iba-1-positive cells decreased in the crocin group (P?<?0.001). Crocin suppresses apoptotic signaling mediated by the oxidative-inflammatory cascade in rat pups exposed to ethanol after birth.

     

Novel osmotin attenuates glutamate-induced synaptic dysfunction and neurodegeneration via the JNK/PI3K/Akt pathway in postnatal rat brain

The glutamate-induced excitotoxicity pathway has been reported in several neurodegenerative diseases. Molecules that inhibit the release of glutamate or cause the overactivation of glutamate receptors can minimize neuronal cell death in these diseases. Osmotin, a homolog of mammalian adiponectin, is a plant protein from Nicotiana tabacum that was examined for the first time in the present study to determine its protective effects against glutamate-induced synaptic dysfunction and neurodegeneration in the rat brain at postnatal day 7. The results indicated that glutamate treatment induced excitotoxicity by overactivating glutamate receptors, causing synaptic dysfunction and neuronal apoptosis after 4 h in the cortex and hippocampus of the postnatal brain. In contrast, post-treatment with osmotin significantly reversed glutamate receptor activation, synaptic deficit and neuronal apoptosis by stimulating the JNK/PI3K/Akt intracellular signaling pathway. Moreover, osmotin treatment abrogated glutamate-induced DNA damage and apoptotic cell death and restored the localization and distribution of p53, p-Akt and caspase-3 in the hippocampus of the postnatal brain. Finally, osmotin inhibited glutamate-induced PI3K-dependent ROS production in vitro and reversed the cell viability decrease, cytotoxicity and caspase-3/7 activation induced by glutamate. Taken together, these results suggest that osmotin might be a novel neuroprotective agent in excitotoxic diseases.     

Mechanism of ethanol enhancement of apoptosis and caspase activation in serum-deprived PC12 cells

Neuronal death is one of the most prominent consequences of alcohol exposure during development. Ethanol-induced neuronal death appears to involve apoptosis. The objective of the present study was to characterize the effect of ethanol on neuronal cell viability and to determine the mechanism by which ethanol enhances apoptosis in neural cells. For these studies the rat pheochromocytoma (PC12) cells were used. PC12 cells were incubated for 24 h in the presence or absence of 100 mM ethanol. Apoptosis was induced by serum withdrawal. Ethanol in the presence of serum-containing media did not alter cell viability, while incubation of PC12 cells in serum-free media resulted in a significant increase in cell death that was further significantly increased by 35% in cells exposed to ethanol. The temporal response of the PC12 cells to serum withdrawal was studied over a period of 22 h. At least 18 h of ethanol exposure was necessary to observe a significant increase in death for cells incubated in serum-free media. An increase in the caspase-3 activity in PC12 cells deprived of serum was observed that was further increased by ethanol exposure. This increase of caspase-3 activity was correlated with an enhancement of caspase-9 activity. Ethanol exposure increased the amount of cytosolic cytochrome c in PC12 cells incubated in serum-free media but did not alter the level of cytochrome c in cells incubated in serum. Finally, a 26% increase was observed in the number of cells with depolarized mitochondria due to ethanol treatment. The present study implicates an increase in the mitochondrial outer membrane permeability as a potential mechanism of enhancement of apoptosis in serum-deprived PC12 cells by ethanol.     

Tau Phosphorylation and Cleavage in Ethanol-Induced Neurodegeneration in the Developing Mouse Brain

Previous studies indicated that ethanol-induced neurodegeneration in postnatal day 7 (P7) mice, widely used as a model for the fetal alcohol spectrum disorders, was accompanied by glycogen synthase kinase-3β (GSK-3β) and caspase-3 activation. Presently, we examined whether tau, a microtubule associated protein, is modified by GSK-3β and caspase-3 in ethanol-treated P7 mouse forebrains. We found that ethanol increased phosphorylated tau recognized by the paired helical filament (PHF)-1 antibody and by the antibody against tau phosphorylated at Ser199. Ethanol also generated tau fragments recognized by an antibody against caspase-cleaved tau (C-tau). C-tau was localized in neurons bearing activated caspase-3 and fragmented nuclei. Over time, cell debris and degenerated projections containing C-tau appeared to be engulfed by activated microglia. A caspase-3 inhibitor partially blocked C-tau formation. Lithium, a GSK-3β inhibitor, blocked ethanol-induced caspase-3 activation, phosphorylated tau elevation, C-tau formation, and microglial activation. These results indicate that tau is phosphorylated by GSK-3β and cleaved by caspase-3 during ethanol-induced neurodegeneration in the developing brain.     

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.     

Blockade of ionotropic glutamate receptors produces neuronal apoptosis through the Bax-cytochrome C-caspase pathway: the causative role of Ca2+ deficiency

Blockade of ionotropic glutamate receptors induces neuronal cell apoptosis. We investigated if mitochondria-mediated death signals would contribute to neuronal apoptosis following administration of glutamate antagonists. The administration of MK-801 and CNQX (MK-801/CNQX), the selective antagonists of N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors, produced widespread neuronal death in neonatal rat brain and cortical cell cultures. MK-801/CNQX-induced neuronal apoptosis was prevented by zVAD-fmk, a broad inhibitor of caspases, but insensitive to inhibitors of calpain or cathepsin D. Activation of caspase-3 was observed within 6-12 h and sustained over 36 h after exposure to MK-801/CNQX, which cleaved PHF-1 tau, the substrate for caspase-3. Activation of caspase-3 was blocked by high K+ and mimicked by BAPTA-AM, a selective Ca2+ chelator. Reducing extracellular Ca2+, but not Na+, activated caspase-3, suggesting an essential role of Ca2+ deficiency in MK-801/CNQX-induced activation of caspases. Cortical neurons treated with MK-801/CNQX triggered activation of caspase-9, release of cytochrome c from mitochondria, and translocation of Bax into mitochondria. The present study suggests that blockade of ionotropic glutamate receptors causes caspase-3-mediated neuronal apoptosis due to Ca2+ deficiency that is coupled to the sequential mitochondrial death pathway.     

The anti-inflammatory and anti-apoptotic effects of nesfatin-1 in the traumatic rat brain

Nesfatin-1 has been demonstrated to possess anti-inflammatory and anti-apoptotic effects in the rat brain with subarachnoid hemorrhage. The study was designed to investigate the influence of nesfatin-1 on inflammatory responses and neuronal cell apoptosis after traumatic brain injury. Wistar rats were subjected to 5, 10 or 20 μg/kg of nesfatin-1 at designed time points (0.5, 2, 4 or 8h after head trauma) intraperitoneally. Rats were sacrificed at hours 2, 6 and 12, as well as day 1, 2, 3 and 5 after head trauma. The administration of 10 or 20 μg/kg of nesfatin-1 at hour 0.5 after head trauma could significantly suppress gene expressions of nuclear factor kappa-B, lessen concentrations of tumor necrosis factor-alpha, interleukin-1beta and interleukin-6, diminish caspase-3 activity as well as reduce number of apoptotic neuronal cells in traumatic rat brain tissues (P<0.05), but the administration of 5 μg/kg of nesfatin-1 not (P>0.05). Moreover, 20 μg/kg nesfatin-1 also significantly suppressed the inflammation and neuronal cell apoptosis when applied 2, 4 or 8h after head trauma. However, a clear concentration-response or time-response relationship was not found. These findings suggest that nesfatin-1 may inhibit nuclear factor kappa-B-dependent inflammatory responses, and lessen caspase-3-mediated neuronal cell apoptosis after traumatic brain injury in rats.     

Chlorpromazine protects against apoptosis induced by exogenous stimuli in the developing rat brain

Background

Chlorpromazine (CPZ), a commonly used antipsychotic drug, was found to play a neuroprotective role in various models of toxicity. However, whether CPZ has the potential to affect brain apoptosis in vivo is still unknown. The purpose of this study was to investigate the potential effect of CPZ on the apoptosis induced by exogenous stimuli.

Methodology

The ethanol treated infant rat was utilized as a valid apoptotic model, which is commonly used and could trigger robust apoptosis in brain tissue. Prior to the induction of apoptosis by subcutaneous injection of ethanol, 7-day-old rats were treated with CPZ at several doses (5 mg/kg, 10 mg/kg and 20 mg/kg) by intraperitoneal injection. Apoptotic cells in the brain were measured using TUNEL analysis, and the levels of cleaved caspase-3, cytochrome c, the pro-apoptotic factor Bax and the anti-apoptotic factor Bcl-2 were assessed by immunostaining or western blot.

Findings

Compared to the group injected with ethanol only, the brains of the CPZ-pretreated rats had fewer apoptotic cells, lower expression of cleaved caspase-3, cytochrome c and Bax, and higher expression of Bcl-2. These results demonstrate that CPZ could prevent apoptosis in the brain by regulating the mitochondrial pathway.

Conclusions

CPZ exerts an inhibitory effect on apoptosis induced by ethanol in the rat brain, intimating that it may offer a means of protecting nerve cells from apoptosis induced by exogenous stimuli.     

Nicotinamide inhibits alkylating agent-induced apoptotic neurodegeneration in the developing rat brain

Background

Exposure to the chemotherapeutic alkylating agent thiotepa during brain development leads to neurological complications arising from neurodegeneration and irreversible damage to the developing central nerve system (CNS). Administration of single dose of thiotepa in 7-d postnatal (P7) rat triggers activation of apoptotic cascade and widespread neuronal death. The present study was aimed to elucidate whether nicotinamide may prevent thiotepa-induced neurodegeneration in the developing rat brain.

Methodology/Principal Findings

Neuronal cell death induced by thiotepa was associated with the induction of Bax, release of cytochrome-c from mitochondria into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1). Post-treatment of developing rats with nicotinamide suppressed thiotepa-induced upregulation of Bax, reduced cytochrome-c release into the cytosol and reduced expression of activated caspase-3 and cleavage of PARP-1. Cresyl violet staining showed numerous dead cells in the cortex hippocampus and thalamus; post-treatment with nicotinamide reduced the number of dead cells in these brain regions. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and immunohistochemical analysis of caspase-3 show that thiotepa-induced cell death is apoptotic and that it is inhibited by nicotinamide treatment.

Conclusion

Nicotinamide (Nic) treatment with thiotepa significantly improved neuronal survival and alleviated neuronal cell death in the developing rat. These data demonstrate that nicotinamide shows promise as a therapeutic and neuroprotective agent for the treatment of neurodegenerative disorders in newborns and infants.     

Ethanol induced NF-{kappa}B activation protects against cell injury in cultured rat gastric mucosal epithelium

Ethanol is a well-established irritant inducing inflammation in gastric mucosa, but the effects at the cellular level remain unclear. This study investigates NF-kappaB activation in gastric mucosal cells by ethanol and assesses the effects of heat shock pretreatment in this ulcerogenic situation. Rat gastric mucosal epithelia were exposed to ethanol for different time periods. Heat shock was induced by incubating the cells at 42 degrees C for 1 h prior to the experiments. For evaluation of NF-kappaB activation, the nuclear fraction of the cell lysates was analyzed with an EMSA or an ELISA-based assay. Caspase-3 (a promoter of apoptosis) activity was measured with a time-resolved fluorescence based assay, cell viability with a tetrazolium assay, and cell membrane integrity with a LDH assay. Ethanol (1-5%) induced NF-kappaB activation, reaching a maximum after 3 h, and also led to moderately increased COX-2 expression. Heat shock pretreatment and the intracellular calcium chelator BAPTA were able to inhibit ethanol-induced NF-kappaB activation. Heat shock pretreatment decreased ethanol-induced caspase-3 activation, decreased cell membrane damage, and retained cellular viability. Inhibition of NF-kappaB activation by NEMO-binding peptide, by decreasing RelA expression, or by inhibiting COX-2 activity by CAY-14040 promoted the effects of ethanol, such as increased caspase-3 activity and decreased cell viability. In conclusion, ethanol induces NF-kappaB activation via a calcium-dependent pathway and induces COX-2 expression. Inhibition of the NF-kappaB activation or COX-2 activity potentiates apoptosis and cell damage induced by ethanol, suggesting a protective role for NF-kappaB activation and COX-2 expression.     

Selective,reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic-ischemic brain injury

Hypoxia-ischemia (H-I) in the developing brain results in brain injury with prominent features of both apoptosis and necrosis. A peptide-based pan-caspase inhibitor is neuroprotective against neonatal H-I brain injury, suggesting a central role of caspases in brain injury. Because previously studied peptide-based caspase inhibitors are not potent and are only partially selective, the exact contribution of specific caspases and other proteases to injury after H-I is not clear. In this study, we explored the neuroprotective effects of a small, reversible caspase-3 inhibitor M826. M826 selectively and potently inhibited both caspase-3 enzymatic activity and apoptosis in cultured cells in vitro. In a rat model of neonatal H-I, M826 blocked caspase-3 activation and cleavage of its substrates, which begins 6 h and peaks 24 h after H-I. Although M826 significantly reduced DNA fragmentation and brain tissue loss, it did not prevent calpain activation in the cortex. This activation, which is associated with excitotoxic/necrotic cell injury, occurred within 30 min to 2 h after H-I even in the presence of M826. Similar to calpain activation, we found evidence of caspase-2 processing within 30 min to 2 h after H-I that was not affected by M826. Caspase-2 processing appeared to be secondary to calpain-mediated cleavage and was not associated with caspase-2 activation. These data suggest that caspase-3 specifically contributes to delayed cell death and brain injury after neonatal H-I and that calpain activation is associated with and likely a marker for the early component of excitotoxic/necrotic brain injury previously demonstrated in this model.     

Ethanol but not acetaldehyde induced changes in brain taurine: a microdialysis study

Summary. Research has suggested that catalase plays a role in mediating ethanols psychopharmacological effects. Catalase is an enzyme that oxidizes ethanol to acetaldehyde. It has been reported that when catalase activity is reduced by 3-amino-1,2,4-triazole (AT), rats reduce their intake and preference for ethanol. The present study assessed the effects of AT on the brain amino acids levels following ethanol administration in Wistar rats. The study consisted of three parts. In the first part, we found no effects of acute and chronic intraperitoneally administered acetaldehyde on amino acids dialysate levels in nucleus accumbens. In the second part, AT was administered five hours prior to ethanol or its vehicle. Ethanol significantly affected the levels of taurine in rat pre-treated with AT. In the final part, ethanol was administered following the pre-treatment with AT but the dependent variable was the concentration of ethanol in the brain.     

Induction of ethanol dependence increases signal peptidase mRNA levels in rat brain

Differential Northern blot hybridization was used as a screening tool to identify mRNAs that respond quantitatively to the induction of ethanol dependence. Adult male rats were treated with repeated, high doses of ethanol for 4 consecutive days. This regimen resulted in the development of tolerance and dependence upon ethanol. RNA isolated from the ethanol-dependent rat brains was used to construct a cDNA library. One cDNA was identified that hybridized to a mRNA which increased in rat brain during the ethanol treatment. Sequence analysis of the cDNA indicated that it recognized a mRNA in rat brain which was very similar to that which encodes the 18 kDa subunit of canine signal peptidase. The rat signal peptidase mRNA was observed to increase in brain nearly 2-fold within 48 h after the initiation of ethanol treatment. Ethanol did not significantly alter -actin mRNA levels during the treatment period. These results support the existence of an ethanol-responsive signal peptidase mRNA in rat brain.     

Ethanol-Induced Oxidative Stress: The Role of Binaphthyl Diselenide as a Potent Antioxidant

It is widely accepted that oxidative stress plays a central role in alcohol-induced pathogenesis. The protective effect of binaphthyl diselenide (NapSe)2 was investigated in ethanol (Etoh)-induced brain injury. Thirty male adult Wistar rats were divided randomly into five groups of six animals each and treated as follows: (1) The control group received the vehicle (soy bean oil, 1 mL/kg, p.o.). (2) Ethanol group of animals was administered with ethanol (70% v/v, 2 mL/kg, p.o.). (3) (NapSe)2 1 mg/kg, 1 mL/kg plus ethanol 70% (v/v, 2 mL/kg, p.o. (5) (NapSe)2 10 mg/kg, 1 mL/kg) plus ethanol 70% (v/v, 2 mL/kg, p.o). After acute treatment, all rats were sacrificed by decapitation. Evidence for oxidative stress in rat brain was obtained from the observed levels of thiobarbituric acid reactive species, of non-protein thiol (NPSH) groups, and of ascorbic acid, as well as from the activities of catalase (CAT) and of superoxide dismutase (SOD). (NapSe)2 compensated the deficits in the antioxidant defense mechanisms (CAT, SOD, NPSH, and ascorbic acid), and suppressed lipid peroxidation in rat brain resulting from Etoh administration. It was concluded that ethanol exposure causes alterations in the antioxidant defense system and induces oxidative stress in rat brain. (NaPSe)2 at 5 mg/kg restored the antioxidant defenses in rat brain and mitigated the toxic effects of alcohol, suggesting that could be used as a potential therapeutic agent for alcohol-induced oxidative damage in rat brain.