Renuka M. Prasad

Immunohistochemical detection of the lipid peroxidation product 4-hydroxynonenal after experimental brain injury in the rat

This study examined the accumulation of the cytotoxic lipid peroxidation product 4-hydroxynonenal (HNE) after lateral fluid percussion (FP) brain injury in rats. A diffuse distribution of HNE-immunoreactivity (HNE-ir) was observed in the cortex and hippocampus of the ipsilateral, but not of the contralateral, hemisphere at 30 min, 6 h, 24 h, and 48 h after brain injury. The HNE-ir was well-localized in cell bodies of the ipsilateral cortex and the CA3 pyramidal layer in the ipsilateral hippocampus. Because HNEs interaction with certain proteins causes protein dysfunction and HNE, in vitro, causes neuronal cell damage, the present results suggest that HNEs interaction with neuronal proteins may contribute to neuronal damage in the ipsilateral cortex and hippocampus after brain injury.

Regional expression of Bcl-2 mRNA and mitochondrial cytochrome c release after experimental brain injury in the rat.

Regional levels of anti-apoptotic Bcl-2 mRNA and the cytosolic cytochrome c protein were measured after lateral fluid percussion (FP) brain injury in rats. Levels of Bcl-2 mRNA were significantly decreased in the injured left cortex (IC) and ipsilateral hippocampus (IH), but not in the contralateral right cortex (CC) and hippocampus (CH) after brain injury. Levels of Bcl-2 mRNA were significantly decreased as early as 2 h and stayed decreased as long as 48 h in the IC and IH after injury. Levels of the cytosolic cytochrome c protein were significantly increased in the IC and IH, but not in the CC and CH after brain injury. Levels of cytosolic cytochrome c were significantly increased in the IC at 30 min, 48 and 72 h, and in the IH at 2 h and as long as 72 h after injury. The increase of cytosolic cytochrome c suggests that the mitochondrial release of cytochrome is increased in the IC and IH after lateral FP brain injury. These data show that the reduction of anti-apoptotic Bcl-2 and increases of mitochondrial release of cytochrome c protein occur only in the IC and IH, regions which have been observed to undergo apoptosis and neuronal cell loss after lateral FP brain injury. Therefore, it is likely that the reduction of Bcl-2 and the increased cytochrome c protein in the cytosol contribute to the observed apoptosis and neuronal cell death in the IC and IH after lateral FP brain injury in rats.

Effects of chronic ethanol administration on expression of BDNF and trkB mRNAs in rat hippocampus after experimental brain injury

Previous evidence indicates that both chronic alcohol treatment and traumatic brain injury modulate expression of certain neurotrophins and neurotrophin receptors in cortical tissue. However, the combined effects of chronic alcohol and brain trauma on expression of neurotrophins and their receptors have not been investigated. In the present study, we examined the effects of 6 weeks of chronic ethanol administration on lateral fluid percussion (FP) brain injury-induced alterations in expression of mRNAs for the neurotrophin brain-derived neurotrophic factor (BDNF) and its high affinity receptor, trkB, in rat hippocampus. In both the control- (pair-fed isocaloric sucrose) diet and the chronic ethanol-diet groups, unilateral FP brain injury induced a bilateral increase in levels of both BDNF and trkB mRNAs in the dentate gyrus granule cell layer, and of BDNF mRNA in hippocampal region CA3. However, no significant differences in expression were found between the control-diet and ethanol-diet groups, in either the sham-injured or FP-injured animals. These findings suggest that 6 weeks of chronic ethanol administration does not alter the plasticity of hippocampal BDNF/trkB expression in response to experimental brain injury.

Regional Activities of Phospholipase C after Experimental Brain Injury in the Rat

Regional activities of phosphoinositide-specific phospholipase C (PLC) were measured after lateral fluid percussion (FP) brain injury in rats. The activity of PLC on phosphatidylinositol 4,5-bisphosphate (PIP2) in the rat cortex required calcium, and at 45 μM concentration it increased PLC activity by about ten-fold. The activity of PLC was significantly increased in the cytosol fraction in the injured (left) cortex (IC) at 5 min, 30 min and 120 min after brain injury. However, in the same site, increases were observed in the membrane fraction only at 5 min after brain injury. In both the contralateral (right) cortex (CC) and ipsilateral hippocampus (IH), the activity of PLC was increased in the cytosol only at 5 min after brain injury. These results suggest that increased activity of PLC may contribute to increases in levels of cellular diacylglycerol and inositol trisphosphate in the IC (the greatest site of injury), and to a smaller extent in the IH and CC, after lateral FP brain injury. It is likely that this increased PLC activity is caused by alteration in either the levels or activities of one or more of its isozymes (PLCβ, PLCγ, and PLCδ) after FP brain injury.

Amphetamine affects the behavioral outcome of lateral fluid percussion brain injury in the rat

This study examined the effects of (D)-amphetamine, methoxamine (an al-adrenergic receptor agonist), and prazosin (an al-adrenergic receptor antagonist) on the behavioral outcome of lateral fluid percussion brain injury. Rats trained to perform a beam walking task were subjected to brain injury of moderate severity (2.1-2.2 atm). At 10 min after injury, rats were treated with amphetamine, methoxamine or prazosin at two different dose levels. Amphetamine-treated animals displayed significantly lower impairment in beam walking ability from days 1 to 5 after brain injury. Neither methoxamine nor prazosin significantly affected the impairment in beam walking ability from day 1 to day 7 after injury. However, prazosin treatment at both dose levels increased the post-injury mortality and the incidences of failure to recovery from hemiplegia. Amphetamine-treatment at 4 mg/kg, but not at 2 mg/kg, improved the spatial learning abilities of the injured animals. Neither methoxamine nor prazosin affected the spatial learning abilities. These results indicate that amphetamine facilitated beam walking recovery and improved cognitive function after concussive fluid percussion injury. Although the methoxamine experiments suggest that the norepinephrine-α1-adrenergic receptor system may not be involved in the pathophysiology of fluid percussion brain injury, our results with amphetamine (beneficial effects) and prazosin (deleterious effects) and the results observed in other models of brain injury point out that further investigations are necessary to understand the role of a1-adrenergic receptors in brain injury.

Amphetamine administration improves neurochemical outcome of lateral fluid percussion brain injury in the rat

This study examined the effects of the administration of D-amphetamine on the regional accumulation of lactate and free fatty acids (FFAs) after lateral fluid percussion (FP) brain injury in the rat. Rats were subjected to either FP brain injury of moderate severity (1.9 to 2.0 atm) or sham operation. At 5 min after injury, rats were treated with either d-amphetamine (4 mg/kg, i.p.) or saline. At 30 min and 60 min after brain injury, brains were frozen in situ, and cortices and hippocampi were excised at 0 degrees C. In the saline-treated brain injured rats, levels of lactate were increased in the ipsilateral left cortex and hippocampus at 30 min and 60 min after injury. These increases were attenuated by the administration of D-amphetamine at 5 min after lateral FP brain injury. At 30 and 60 min after FP brain injury, increases in the levels of all individual FFAs (palmitic, stearic, oleic and arachidonic acids) and of total FFAs were also observed in the ipsilateral cortex of the saline-treated injured rats. These increases in the ipsilateral cortex and hippocampus were also attenuated by the administration of d-amphetamine. Neither levels of lactate nor levels of FFAs were increased in the contralateral cortex in the saline-treated injured rats at 30 min or 60 min after FP brain injury. The levels of lactate and FFAs in the contralateral cortex were also unaffected by the administration of D-amphetamine. These results suggest that the attenuation of increases in the levels of lactate and FFAs in the ipsilateral cortex and hippocampus may be involved in the amphetamine-induced improvement in behavioral outcome after lateral FP brain injury.

Kynurenate attenuates the accumulation of diacylglycerol and free fatty acids after experimental brain injury in the rat.

This study examined the effects of the administration of kynurenate, a non-specific excitatory amino acid (EAA) receptor subtype antagonist, on the regional accumulation of diacylglycerol (DG) and free fatty acids (FFAs) after lateral fluid percussion (FP) brain injury in the rat. After brain injury of moderate severity (2.0 atm), rats were treated with either kynurenate (200 mg/kg, i.v.) or saline at 5 min after injury. In the saline-treated brain-injured rats, levels of all individual DG-fatty acids (palmitic, stearic, oleic and arachidonic acids) and total DG-fatty acids were increased in the ipsilateral left cortex and hippocampus at 30 min and 60 min after injury. Kynurenate administration attenuated increases of individual and total DG-fatty acids in the ipsilateral cortex at 30 and 60 min and in the ipsilateral hippocampus at 30 min after FP brain injury. At 30 and 60 min after FP brain injury, increases in the levels of individual FFAs (palmitic, stearic, oleic and arachidonic acids) and of total FFAs were also observed in the ipsilateral cortex and hippocampus of the saline-treated injured rats. Kynurenate administration attenuated increases of all individual and total FFAs in the ipsilateral cortex and hippocampus either at 30 min alone or at both 30 min and 60 min after FP brain injury. In the contralateral cortex, levels of both DG-fatty acids and FFAs were not increased in the saline-treated injured rats and were also not affected by the administration of kynurenate. These results support the role of EAA receptor subtypes in the phospholipases-catalyzed formation of DG and FFAs in the ipsilateral cortex and hippocampus after lateral FP brain injury.

Regional levels of phospholipase Cγ after fluid percussion brain injury in the rat

Levels of PLCgamma, a phospholipase C (PLC) isozyme, were significantly increased in the cytosol in the injured left cortex (LC) at 5, 30 and 120 min after brain injury. In the same site, although levels of membrane PLCgamma did not alter at 5 and 30 min, they were found to be decreased at 2 h after brain injury. In general, the levels of both cytosolic and membrane PLCgamma were unaltered in the contralateral right cortex (RC), ipsilateral left hippocampus (LH) and contralateral right hippocampus (RH) between 5 and 120 min after brain injury. These results suggest that, in addition to well-proposed excitatory neurotransmitter-receptor systems, increased levels of PLCgamma may also contribute to alterations in PIP2 signal transduction pathway, particularly in the greatest injury site (LC) after lateral FP brain injury.

Effects of Binge Ethanol Administration on the Behavioral Outcome of Rats after Lateral Fluid Percussion Brain Injury

This study examined the effects of 4 weeks of binge ethanol administration (BEAn) on the behavioral outcome in rats after lateral fluid percussion (FP) brain injury. Rats were intragastrically given 7.5 mL/kg of either 40% ethanol in 5% glucose solution (3 g ethanol/kg; binge ethanol group), or 5% glucose solution (vehicle group), twice on Thursday and Friday of 3 consecutive weeks. Then rats from both groups were subjected to either lateral FP brain injury of moderate severity (1.8 atm) or to sham operation. Postinjury behavioral measurements revealed that brain injury caused significant spatial learning disability in both groups. There were no significant differences in mean search latencies in the sham animals between the vehicle and binge ethanol groups. On the other hand, the mean search latency of the binge ethanol group was significantly higher than that of the vehicle group in trial blocks 2 and 4. There were no significant differences in the target visits (expressed as mean zone difference [MZD]) during the probe trial between the injured animals of binge ethanol and vehicle groups. However, there was only a minor trend towards worsened MZD score in the binge-injured animals. Histologic analysis of injured animals from both injured ethanol and vehicle groups revealed similar extents of ipsilateral cortical and observable hippocampal damage. These results suggest that 4 weeks of binge ethanol treatment followed by ethanol intoxication at the time of injury worsens some aspects of the spatial learning ability of rats. This worsening is probably caused by subtle, undetectable morphologic damage by binge ethanol administration.

Regional expression of Par-4 mRNA and protein after fluid percussion brain injury in the rat.

Regional levels of prostate apoptosis response-4 (Par-4) protein and mRNA were measured after lateral fluid percussion (FP) brain injury in rats. Immunochemical studies indicated that Par-4 immunoreactivity (ir) is present in cortical neurons and hippocampal CA1-CA3 pyramidal neurons in uninjured rats. Increases of Par-4-ir were observed in the CA3 neurons of the ipsilateral hippocampus (IH), but not in injured left cortex (IC) at 48 h after FP brain injury. Levels of the Par-4 mRNA measured by RT-PCR assay and protein measured by Western blot procedure were significantly increased in the injured IC and IH, but not in the contralateral right cortex and hippocampus after brain injury. Levels of both Par-4 protein and mRNA were significantly increased in the IC and IH as early as 2 h and stayed elevated at 24 and 48 h after injury. These data show that the induction of proapoptotic Par-4 mRNA and protein occurs only in the IC and IH that have been observed to undergo apoptosis and neuronal cell loss after lateral FP brain injury. Because increased expression of Par-4 has been observed to contribute to apoptosis and cell death in cultured neurons, the present temporal pattern of Par-4 expression is consistent with a role for Par-4 in apoptosis and neuronal cell death after traumatic brain injury.