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.     

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.     

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

Levels of PLCγ, 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 PLCγ 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 PLCγ 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 PLCγ may also contribute to alterations in PIP₂ signal transduction pathway, particularly in the greatest injury site (LC) after lateral FP brain injury.     

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.     

Alterations in brain protein kinase C after experimental brain injury

Regional activities and levels of protein kinase C were measured after lateral fluid percussion brain injury in rats. At 5 min and 20 min after injury, neither cofactor-dependent nor -independent PKC activities in the cytosol and membrane fractions changed in the injured and contralateral cortices or in the ipsilateral hippocampus. Western blot analysis revealed decreases in the levels of cytosolic PKC α and PKC β in the injured cortex after brain injury. In the same site, a significant increase in the levels of membrane PKC α and PKC β was observed after injury. Although the level of PKC α did not change and that of PKC β decreased in the cytosol of the ipsilateral hippocampus, these levels did not increase in the membrane fraction after injury. The levels of PKC γ were generally unchanged in the cytosol and the membrane, except for its decrease in the cytosol of the hippocampus. There were no changes in the levels of any PKC isoform in either the cytosol or the membrane of the contralateral cortex after injury. The present results suggest a translocation of PKC α and PKC β from the cytosol to the membrane in the injured cortex after brain injury. The observation that such a translocation occurs only in the brain regions that undergo substantial neuronal loss suggests that membrane PKC may play a role in neuronal damage after brain injury.     

Temporal response and effects of excitatory amino acid antagonism on microtubule-associated protein 2 immunoreactivity following experimental brain injury in rats

Alterations in microtubule-associated protein 2 (MAP2) immunoreactivity following lateral fluid-percussion (FP) brain injury were investigated in rats with survival times ranging between 10 min and 7 days. MAP2 immunoreactivity was profoundly diminished in the cortex and hippocampus ipsilateral to the site of injury by 10 min and remained diminished up to 7 days after injury. Nissl staining and silver impregnation histochemistry demonstrated a correlation between the loss of MAP2 and neuronal degeneration. The effect of excitatory amino acid receptor antagonism on MAP2 immunoreactivity was evaluated by administering kynurenate or buffer 15 min after FP injury. Administration of kynurenate significantly attenuated the loss of MAP2 observed in the cortex two weeks after injury when compared to buffer treated control animals (P < 0.02). We conclude that significant and prolonged cytoskeletal changes occur following lateral FP brain injury, and that these alterations can be attenuated by blocking excitatory amino acid receptors.     

Temporal and spatial characterization of neuronal injury following lateral fluid-percussion brain injury in the rat

The pattern of neuronal injury following lateral fluid-percussion (FP) brain injury in the rat was systematically characterized at sequential time points to identify selectively vulnerable regions and to determine the temporal contribution of primary and delayed neuropathological events. Male Sprague-Dawley rats (n = 28) were killed 10 min, 2 h, 12 h, 24 h, 4 days, and 7 days following a lateral FP brain injury of moderate severity (2.2 atm), or 24 h after a sham injury. Brain sections were stained and analyzed using Nissl, acid fuchsin, and silver staining methods to identify regions with injured neurons or with visible lesions. Extensive numbers of acid fuchsin or silver-stained neurons were observed as early as 10 min after the FP brain injury in regions extending from the caudate/putamen to the pons. The frequency of injured neurons was greatest in the ipsilateral cortex, hippocampus, and thalamus, and a visible loss of Nissl-stained neurons was observed in these regions beginning at 12 h after the FP brain injury. Acid fuchsin-stained neurons were restricted to the same brain regions for all of the survival periods and gradually decreased in numbers between 24 h and 7 days after injury. These findings suggest that lateral FP brain injury in the rat produces a combination of focal cortical contusion and diffuse subcortical neuronal injury, which is present within minutes of the impact, progresses to a loss of neurons by 12 h, and does not markedly expand into other brain regions with survival periods up to 7 days. Furthermore, the acute onset and rapid evolution of the neuronal injury process may have important implications when considering a window of opportunity for pharmacological intervention. Received: 23 May 1995 / Revised, accepted: 15 September 1995     

Serum extravasation and cytoskeletal alterations following traumatic brain injury in rats

Disruption of the blood-brain barrier (BBB) and neuronal cytoskeletal damage were evaluated in two commonly used rat models of traumatic brain injury. Adult rats received a lateral cortical impact (CI) or lateral fluid percussion (FP) injury of mild or moderate severity or a sham procedure. Six hours after trauma, the brains were removed and analyzed with immunocytochemical techniques for alterations in the serum protein, IgG, and the cytoskeletal protein, microtubule-associated protein 2 (MAP2). Both models induced profound alterations in these proteins in the ipsilateral cortex and hippocampus compared to sham-injured controls. Following an injury of moderate severity, the CI injury resulted in greater IgG extravasation in the cortex and hippocampus than the FP injury. Conversely, after a mild injury, IgG extravasation in the hippocampus was greater for FP than CI. All of the animals in the CI group and most of the FP group showed a loss of MAP2 in the hippocampus. The specific subregions within the cortex and hippocampus that were affected by the injury varied between models, despite having identical impact sites. These data demonstrate that there are both similarities and differences between a CI and FP injury on vascular and neuronal cystoskeletal integrity, which should be considered when utilizing these animal models to study selected features of human head injury.     

Changes in neuropeptide Y after experimental traumatic brain injury in the rat.

We utilized a model of fluid percussion (FP) brain injury in the rat to examine the hypothesis that alterations in brain neuropeptide Y (NPY) concentrations occur following brain injury. Male rats (n = 44) were subjected to FP traumatic brain injury. One group of animals (n = 38) was killed at 1 min, 15 min, 1 h, or 24 h after brain injury, and regional brain homogenates were analyzed for NPY concentrations using radioimmunoassay. A second group of animals (n = 6) was killed for NPY immunocytochemistry. Concentrations of NPY in the injured left parietal cortex were significantly elevated at 15 min post injury (p less than 0.05). No changes were observed in other brain regions. NPY-immunoreactive fibers were seen at 15 min post injury predominantly in the injured cortex and adjacent hippocampus. These temporal changes in NPY immunoreactivity, together with previous observations concerning posttraumatic changes in regional CBF in these same areas, suggest that an increase in region NPY concentrations after brain injury may be involved in part in the pathogenesis of posttraumatic hypoperfusion.     

The maxi-K channel opener BMS-204352 attenuates regional cerebral edema and neurologic motor impairment after experimental brain injury.

Large-conductance, calcium-activated potassium (maxi-K) channels regulate neurotransmitter release and neuronal excitability, and openers of these channels have been shown to be neuroprotective in models of cerebral ischemia. The authors evaluated the effects of postinjury systemic administration of the maxi-K channel opener, BMS-204352, on behavioral and histologic outcome after lateral fluid percussion (FP) traumatic brain injury (TBI) in the rat. Anesthetized Sprague-Dawley rats (n = 142) were subjected to moderate FP brain injury (n = 88) or surgery without injury (n = 54) and were randomized to receive a bolus of 0.1 mg/kg BMS-204352 (n = 26, injured; n = 18, sham), 0.03 mg/kg BMS-204352 (n = 25, injured; n = 18, sham), or 2% dimethyl sulfoxide (DMSO) in polyethylene glycol (vehicle, n = 27, injured; n = 18, sham) at 10 minutes postinjury. One group of rats was tested for memory retention (Morris water maze) at 42 hours postinjury, then killed for evaluation of regional cerebral edema. A second group of injured/sham rats was assessed for neurologic motor function from 48 hours to 2 weeks postinjury and cortical lesion area. Administration of 0.1 mg/kg BMS-204352 improved neurologic motor function at 1 and 2 weeks postinjury (P < 0.05) and reduced the extent of cerebral edema in the ipsilateral hippocampus, thalamus, and adjacent cortex (P < 0.05). Administration of 0.03 mg/kg BMS-204352 significantly reduced cerebral edema in the ipsilateral thalamus (P < 0.05). No effects on cognitive function or cortical tissue loss were observed with either dose. These results suggest that the novel maxi-K channel opener BMS-204352 may be selectively beneficial in the treatment of experimental TBI.     

Local neutrophil influx following lateral fluid-percussion brain injury in rats is associated with accumulation of complement activation fragments of the third component (C3) of the complement system

Traumatic brain injury can lead to locally destructive secondary events mediated by several inflammatory components. Following lateral fluid-percussion (FP) brain injury in rats, we examined cortical and hippocampal sections for neutrophil infiltration and accumulation of complement component C3. Neutrophil influx into the brain after injury was detected by an improved myeloperoxidase (MPO) microassay and manual cell counting, while C3 accumulation was detected using immunocytochemistry. MPO levels were elevated in the injured cortical tissue, whereas C3 immunoreactivity was increased in both injured cortical and ipsilateral hippocampal sections. These results show that the FP model of head injury leads to an intense local inflammatory reaction and subsequent tissue destruction.     

N-acetylcysteine attenuates early induction of heme oxygenase-1 following traumatic brain injury

Traumatic brain injury (TBI) results in a cascade of events that includes the production of reactive oxygen species. Heme oxygenase-1 (HO-1) is induced in glial cells following head trauma, suggestive of oxidative stress. We have studied the temporal and spatial effects of the antioxidant N-acetylcysteine (NAC) on HO-1 levels following lateral fluid-percussion injury by immunoblotting and immunohistochemistry. In the injured cerebral cortex, maximal HO-1 induction was seen 6 h post-TBI and was maintained for up to 24 h following the insult, while the ipsilateral hippocampus and thalamus showed marked induction at 24 h postinjury. In all three brain regions, little or no HO-1 immunoreactivity was observed on the contralateral side. Astrocytes exhibited positive immunoreactivity for HO-1 in the injured cerebral cortex, hippocampus, and thalamus, while some neurons and microglia were also immunoreactive in the injured cortex. The administration of NAC 5 min following TBI resulted in a marked reduction in this widespread induction of HO-1, concomitant with a decrease in the volume of injury in all three brain regions. Together, these findings indicate that HO-1 induction is related to both oxidative and injury characteristics of the affected tissue, suggesting that protein expression of this gene is a credible marker of oxidative damage in this model of TBI.     

Selective temporal and regional alterations of Nogo-A and small proline-rich repeat protein 1A (SPRR1A) but not Nogo-66 receptor (NgR) occur following traumatic brain injury in the rat

Axons show a poor regenerative capacity following traumatic central nervous system (CNS) injury, partly due to the expression of inhibitors of axonal outgrowth, of which Nogo-A is considered the most important. We evaluated the acute expression of Nogo-A, the Nogo-66 receptor (NgR) and the novel small proline-rich repeat protein 1A (SPRR1A, previously undetected in brain), following experimental lateral fluid percussion (FP) brain injury in rats. Immunofluorescence with antibodies against Nogo-A, NgR and SPRR1A was combined with antibodies against the neuronal markers NeuN and microtubule-associated protein (MAP)-2 and the oligodendrocyte marker RIP, while Western blot analysis was performed for Nogo-A and NgR. Brain injury produced a significant increase in Nogo-A expression in injured cortex, ipsilateral external capsule and reticular thalamus from days 1-7 post-injury (P < 0.05) compared to controls. Increased expression of Nogo-A was observed in both RIP- and NeuN positive (+) cells in the ipsilateral cortex, in NeuN (+) cells in the CA3 region of the hippocampus and reticular thalamus and in RIP (+) cells in white matter tracts. Alterations in NgR expression were not observed following traumatic brain injury (TBI). Brain injury increased the extent of SPRR1A expression in the ipsilateral cortex and the CA3 at all post-injury time-points in NeuN (+) cells. The marked increases in Nogo-A and SPRR1A in several important brain regions suggest that although inhibitors of axonal growth may be upregulated, the injured brain is also capable of expressing proteins promoting axonal outgrowth following TBI.     

Diffuse prolonged depression of cerebral oxidative metabolism following concussive brain injury in the rat: a cytochrome oxidase histochemistry study.

Utilizing a lateral fluid percussion injury as a model of cerebral concussion, rats were studied histochemically measuring the degree of cytochrome oxidase activity present within different structures at different times following injury. After concussion, the cerebral cortex ipsilateral to the site of injury exhibited a diffuse decrease in its level of chromotome oxidase (CO) activity beginning at as soon as one day and lasting for up to 10 days after the insult. The ipsilateral dorsal hippocampus also exhibited an injury-induced decrease in CO activity, however, it was not as severe as in the cortex. These results indicate that oxidative metabolism is depressed primarily within the cerebral cortex and hippocampus for several days following a cerebral concussion. We propose that this period of metabolic depression may delineate a period of time during which the injured brain is unable to function normally and thus would be vulnerable to a second insult.     

Hypoxia-ischemia induces heat shock protein-like (HSP72) immunoreactivity in neonatal rat brain

The expression of heat shock protein immunoreactivity in rat brain was evaluated in a model of neonatal hypoxia-ischemia. One-week-old rats were subjected to left carotid artery coagulation and exposure to 8% O2/92% N2 for 2 h (moderate injury) or 3.5 h (severe injury). Animals were sacrificed 1, 12 and 24 h after the hypoxic insult. Cells immunoreactive for the 72 kDa heat shock protein (HSP72) were observed in ipsilateral cortex as early as 1 h after the termination of the hypoxia. After 12 h, neurons in the ipsilateral hippocampus and cortex stained intensely for HSP72 immunoreactivity in the moderately injured group. In the severely injured brains, bilateral staining was observed in neurons and vessels of the hippocampus and cortex. Therefore, cells containing HSP72 immunoreactivity may serve as an early marker for neuronal injury from hypoxia-ischemia in the neonatal rat brain and more importantly may illustrate previously unrecognized areas of central nervous system vulnerability.     

大鼠脑损伤后谷氨酸引起乳酸含量变化的作用机制

目的探讨大鼠脑损伤后谷氨酸引起乳酸含量变化的作用机制。方法28只大鼠随机分为对照组、犬尿烯酸（KYN）灌注组、Ouabain灌注组及Ba^2＋灌注组,每组7只。在建立大鼠局部脑损伤模型前后,应用微透析技术将格林液、KYN、Ouabain和Ba2＋分别灌注到各组大鼠致伤脑区,在致伤前45min开始和致伤后90min内,观察3种药物对脑损伤后乳酸含量的影响。结果对照组伤前乳酸含量为（0.28±0.07）mmol/L,脑损伤后引起乳酸含量迅速升高,伤后15min达到峰值（0.75±0.18）mmol/L,乳酸含量持续升高60min后逐渐下降至接近伤前水平。Ouabain灌注组及KYN灌注组伤后乳酸含量升高幅度减弱,持续时间缩短。Ba^2＋灌注组乳酸含量升高幅度增加,持续时间延长。结论脑损伤后异常增加的谷氨酸作用于兴奋性氨基酸受体偶联离子通道,引起细胞外K^＋增加,使Na^＋-K^＋泵活性增强,继而导致糖酵解代谢水平增高,乳酸含量增加。     

The tumor-suppressor gene, p53, is induced in injured brain regions following experimental traumatic brain injury.

A growing body of evidence suggests that neurons undergo apoptotic cell death following traumatic brain injury (TBI). Since the expression of several tumor suppressor and cell cycle genes have been implicated in neuronal apoptosis, the present study used in situ hybridization (ISH) histochemistry to evaluate the regional and temporal patterns of expression of the mRNAs for the tumor suppressor gene, p53, and the cell cycle gene, cyclin D1, following lateral fluid-percussion (FP) brain injury in the rat. Anesthetized adult male Sprague-Dawley rats (n=16) were subjected to lateral FP brain injury of moderate severity (2.4-2.7 atm), while sham controls (n=6) were surgically prepared but did not receive brain injury. Animals were killed by decapitation at 6 h (n=6 injured and 2 sham), 24 h (n=6 injured and 2 sham), or 3 days (n=4 injured and 2 sham), and their brains processed for ISH. Little to no expression of p53 mRNA was observed in sham brains. At 6 h post-injury, p53 mRNA was induced predominantly in cells that are vulnerable to TBI, such as those in the contused cortex, lateral and medial geniculate nuclei of the thalamus, and the CA(3) and hilar neurons of the hippocampus. Increased p53 mRNA was also detected in hippocampal CA(1) neurons, cells that are relatively resistant to FP brain injury. Levels of p53 mRNA returned to sham levels in all regions of the injured brain by 24 h. In contrast to p53, cyclin D1 mRNA was detectable in the brains of uninjured animals and was not altered by brain injury. These results suggest that the tumor suppressor gene p53, but not cyclin D1, is upregulated and may participate in molecular response to TBI.     

Densitometric analysis of cytochrome oxidase in ischemic rat brain

Elevated brain lactate during incomplete ischemia is thought to contribute to the irreversibility of cell damage by interference with mitochondrial respiratory function, that should be evident in reduced cytochrome oxidase (CO) activity. In this study changes in the density of CO staining in a stroke model in the rat were assessed. Brains were analyzed subsequent to 30 min of ischemia followed by 30 min of reperfusion. The effects of postischemic treatment with sodium dichloroacetate (DCA)--a compound used to decrease lactate, were also evaluated. Examination of lateral cortex, hippocampus, and corpus striatum showed different intensities of CO in a distribution consistent with known regional variations in metabolic activity of the forebrain. Known laminar staining patterns in lateral cortex and areal patterns in the hippocampus were also confirmed. Comparable regions in ischemic forebrain were stained less densely for CO than controls. Image analysis demonstrated that the density of CO: (a) was greater in lateral cortex than hippocampus in control; (b) in ischemics was reduced by an equal degree in cortex and hippocampus; (c) lacked regional uniformity in ischemic rats; and (d) was not changed by DCA treatment in the majority of cases of ischemia. Our results suggest that lactate may not be the major determinant of 'selective vulnerability'. Despite elevated lactate levels in lateral cortex when compared to hippocampus in a previous study, the proportionate decrease in CO activity in lateral cortex and hippocampus was equal. However, there was a considerable decrease in CO activity subsequent to high brain lactate and some ischemic hemispheres appeared to respond to DCA treatment. Therefore, the role of excessive lactate in the exacerbation of 'selective vulnerability' warrants further evaluation. CO histochemistry can be used successfully to determine the distribution of pathology and the quality of fixation of ischemic forebrain. Densitometric measurements allowed comparative assessment of degrees of injury and the effects of treatment in discrete anatomical regions. This kind of analysis may allow localization of pathology within specific cellular circuits.     

Early neuronal expression of tumor necrosis factor-alpha after experimental brain injury contributes to neurological impairment

Tumor necrosis factor-alpha (TNF alpha) is a pleiotropic cytokine involved in inflammatory cascades associated with CNS injury. To examine the role of TNF alpha in the acute pathophysiology of traumatic brain injury (TBI), we studied its expression, localization and modulation in a clinically relevant rat model of non-penetrating head trauma. TNF alpha levels increased significantly in the injured cortex at 1 and 4, but not at 12, 24 or 72 h after severe lateral fluid-percussion trauma (2.6-2.7 atm). TNF alpha was not elevated after mild injury. At 1 and 4 h after severe TBI, marked increases of TNF alpha were localized immunocytochemically to neurons of the injured cerebral cortex. A small population of astrocytes, ventricular cells and microvessels, also showed positive TNF alpha staining, but this expression was not injury-dependent. Macrophages that were present in a hemorrhagic zone along the external capsule, corpus callosum and alveus hippocampus at 4 h after TBI did not express TNF alpha. Intracerebroventricular administration of a selective TNF alpha antagonist--soluble TNF alpha receptor fusion protein (sTNFR:Fc) (37.5 microg)--at 15 min before and 1 h after TBI, improved performance in a series of standardized motor tasks after injury. In contrast, intravenous administration of sTNFR:Fc (0.2, 1 or 5 mg/kg) at 15 min after trauma did not improve motor outcome. Collectively, this evidence suggests that enhanced early neuronal expression of TNF alpha after TBI contributes to subsequent neurological dysfunction.