Postinjury treatment with rolipram increases hemorrhage after traumatic brain injury.

The pathology caused by traumatic brain injury (TBI) is exacerbated by the inflammatory response of the injured brain. Two proinflammatory cytokines that contribute to inflammation after TBI are tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). From previous studies using the parasagittal fluid-percussion brain injury model, we reported that the anti-inflammatory drug rolipram, a phosphodiesterase 4 inhibitor, reduced TNF-α and IL-1ß levels and improved histopathological outcome when administered 30 min prior to injury. We now report that treatment with (±)-rolipram given 30 min after injury significantly reduced TNF-α levels in the cortex and hippocampus. However, postinjury administration of (±)-rolipram significantly increased cortical contusion volume and increased atrophy of the cortex compared with vehicle-treated animals at 10 days postinjury. Thus, despite the reduction in proinflammatory cytokine levels, histopathological outcome was worsened with post-TBI (±)-rolipram treatment. Further histological analysis of (±)-rolipram-treated TBI animals revealed significant hemorrhage in the contused brain. Given the well-known role of (±)-rolipram of increasing vasodilation, it is likely that (±)-rolipram worsened outcome after fluid-percussion brain injury by causing increased bleeding.

Talampanel, a novel noncompetitive AMPA antagonist, is neuroprotective after traumatic brain injury in rats.

Talampanel [(R)-7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo[4,5-h][2,3] benzodiazepine] is an orally active noncompetitive antagonist of the AMPA subtype of glutamate excitatory amino acid receptors. The purpose of this study was to determine whether treatment with talampanel would protect in a rat model of traumatic brain injury (TBI). Twenty-four hours prior to TBI, a fluid-percussion interface was positioned parasagittally over the right cerebral cortex. On the following day, fasted rats were anesthetized with 3% halothane, 70% nitrous oxide, and a balance of oxygen; mechanically ventilated and physiologically regulated; and subjected to right parieto-occipital parasagittal fluid-percussion injury (1.5-2.0 atm). The agent (talampanel, bolus infusion of 4 mg/kg followed by infusion of 4 mg/kg/h over 72 h) or vehicle was administered i.v. starting at either 30 min or 3 h after trauma. Seven days after TBI, brains were perfusion-fixed, coronal sections at various levels were digitized, and contusion areas were measured. Treatment with talampanel, when instituted 30 min after trauma, significantly reduced total contusion area compared to vehicle-treated rats (0.54 +/- 0.25 vs. 1.79 +/- 0.42 mm2, respectively). When talampanel treatment was begun at 3 h, the neuroprotective effect of the drug was lost. In addition, treatment with talampanel starting at 30 min significantly attenuated neuronal damage in all three subsectors of the hippocampal CA1 sector compared to vehicle-treated rats (normal-neuron counts, right (ipsilateral) medial CA1: 80.3 +/- 2.0 [talampanel] vs. 66.3 +/- 2.1 [vehicle] (mean +/- SEM); middle CA1: 71.5 +/- 2.0 vs. 60.3 +/- 2.2; lateral CA1: 74.5 +/- 3.0 vs. 63.0 +/- 3.2, respectively). By contrast, when talampanel treatment was begun at 3 h, normal pyramidal-neuron counts were almost identical in both groups. Our findings document that talampanel therapy instituted 30 min after trauma significantly reduces histological damage.

Apoptotic and antiapoptotic mechanisms after traumatic brain injury.

Caspase and inhibitor of apoptosis (IAP) expression was examined in rats subjected to moderate traumatic brain injury (TBI) using a parasagittal fluid-percussion brain insult (1.7 to 2.2 atm). Within 1 hour after injury, caspase-8 and -9, two initiators of apoptosis, were predominantly expressed in superficial cortical areas adjacent to the impact site and in the thalamus. Caspase-3, an effector caspase, was evident at 6 hours throughout the traumatized cerebral cortex and hippocampus. Moreover, the authors observed that XIAP, cIAP-1, and cIAP-2, members of the IAP family, were constitutively expressed in the brain. Colocalization of XIAP-immunolabled cells with cell-specific markers indicated that XIAP is expressed within neurons and a subpopulation of oligodendrocytes. Immunoblots of brain extracts revealed that the processed forms of caspase-8, -9, and -3 are present as early as 1 hour after trauma. The appearance of activated caspases corresponded with the detection of cleavage of XIAP into fragments after injury and a concomitant increase in the levels of cIAP-1 and cIAP-2 in the traumatized hemispheres. The current data are consistent with the hypotheses that caspases in both the extrinsic and intrinsic apoptotic pathways are activated after moderate TBI and that IAPs may have a protective role within the brain with alterations in levels and cleavage of IAPs that contribute to cell death in this setting.

Quantitative analysis of microvascular alterations in traumatic brain injury by endothelial barrier antigen immunohistochemistry.

Endothelial barrier antigen (EBA) is a protein triplet located in the plasma membrane of microvascular endothelium and selectively expressed in the normal nervous system. In this study, microvascular alterations following traumatic brain injury were studied using EBA immunohistochemistry. Anesthetized, physiologically regulated, normothermic Sprague-Dawley rats received moderate (1.5-2.0 atm) parieto-occipital parasagittal fluid-percussion traumatic brain injury (TBI). Control rats were subjected to similar anesthesia and physiological monitoring. Seven days after operative procedures, brains were perfusion-fixed, and coronal sections were reacted for EBA immunohistochemistry using a monoclonal antibody to rat EBA. Selected sections were reacted for isolectin B4 histochemistry. Computerized image analysis was used to compute numbers of EBA-immunopositive vascular profiles and mean vascular profile areas. In control brains, virtually all brain microvessels were clearly and positively immunostained, and antibody binding was specific for blood vessels. In rats with TBI, EBA immunoreactivity was greatly reduced in the zone of cortical contusion. Within the core contusion, fractional areas occupied by vascular profiles were markedly reduced (on average, by 57%), vascular profile counts were diminished, and lectin histochemistry revealed a robust inflammatory response with abundant macrophages. Taken together, these findings were thought to indicate frank microvascular destruction. At adjacent peri-contusional sites, the intensity of EBA immunostaining was also diminished; and vascular profile counts were reduced at adjacent cortical sites and homologous contralateral sites. The latter findings were interpreted as sublethal microvascular alterations possibly related to cerebral edema. The present results confirm that EBA is a specific immunohistochemical marker of normal central nervous system microvessels; that it is suitable for use in formaldehyde-fixed material; and that it is useful in quantitatively assessing microvascular alterations observed at contusional, peri-contusional and more remote sites following traumatic brain injury.

Diminution of metabolism/blood flow uncoupling following traumatic brain injury in rats in response to high-dose human albumin treatment.

OBJECT: The authors have recently demonstrated that high-dose human albumin is markedly neuroprotective in experimental traumatic brain injury (TBI) and cerebral ischemia. The pathophysiology of TBI involves acute uncoupling of cerebral glucose utilization and blood flow. The intent of this study was to establish whether the use of human albumin therapy in a model of acute TBI would influence this phenomenon. METHODS: Anesthetized, physiologically regulated rats received moderate (1.5-2 atm) fluid-percussion injury to the parietal lobe. Fifteen minutes after trauma or sham injury, rats in one group received human albumin (2.5 g/kg) administered intravenously and those in another group received 0.9% saline vehicle. At 60 minutes and 24 hours posttrauma, autoradiographic studies of local cerebral blood flow (LCBF) and local cerebral glucose utilization (LCMRglu) were conducted, and the LCMRglu/LCBF ratio was determined. Sham-injured rats had normal levels of LCBF and LCMRglu, and no differences between vehicle- and albumin-treated rats were evident. Sixty minutes after TBI, LCBF was moderately reduced bilaterally in vehicle-treated rats, whereas in albumin-treated animals, the LCBF contralateral to the side of injury was generally normal. Despite acutely depressed LCBF, LCMRglu in vehicle-treated rats at 60 minutes was paradoxically normal bilaterally, and foci of elevated LCMRglu were noted in the ipsilateral hippocampus and thalamus. By contrast, in albumin-treated rats studied 60 minutes post-TBI, reduced LCMRglu values were measured in the ipsilateral caudoputamen and parietal cortex, whereas LCMRglu in other ipsilateral and contralateral sites did not differ from that measured in sham-injured animals. The metabolism/blood flow ratio was normal in sham-injured rats, but became markedly elevated in vehicle-treated rats 60 minutes post-TBI (on average, by threefold ipsilaterally and 2.1-fold contralaterally). By contrast, the mean metabolism/blood flow ratio in albumin-treated animals was elevated by only 1.6-fold ipsilaterally and was normal contralaterally. Twenty-four hours after TBI, LCBF contralateral to the side of injury had generally returned to normal levels in the albumin-treated group. CONCLUSIONS: These results demonstrate that human albumin therapy benefits the posttraumatic brain by diminishing the pronounced metabolism > blood flow dissociation that would otherwise occur within the 1st hour after injury. Viewed together with our previous evidence of histological neuroprotection, these findings indicate that human albumin therapy may represent a desirable treatment modality for acute TBI.

Intraischemic but not postischemic hypothermia prevents non-selective hippocampal downregulation of AMPA and NMDA receptor gene expression after global ischemia.

Hypothermia may afford histological neuroprotection induced by ischemia by preventing aberrant Ca2+ influx through NMDA (N-methyl-D-aspartic acid) or Ca2+-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors. Expression of hippocampal GluR1A, GluR2B, GluR3C and NMDAR1 (NR1) subunits was investigated by in situ hybridization at 1 and 7 days after 10-min transient global ischemia in the presence and absence of intraischemic or postischemic brain hypothermia (30 degrees C). At 1 day, normothermic ischemia markedly suppressed the expression of GluR1A, GluR2B, and GluR3C receptor mRNAs to a similar degree in the vulnerable CA1. Less vulnerable CA3a-c subregions were also acutely downregulated. NR1 mRNA expression was reduced in CA1 but to a lesser extent than AMPA mRNAs. At 7 days after normothermic ischemia, a time of marked CA1 cell loss, all three AMPA transcripts were nearly absent in CA1 while a percentage (33.9+/-7.2%) of NR1 mRNA remained. Intraischemic hypothermia fully blocked the damage and non-selective mRNA downregulations at 1 and 7 days. By contrast, postischemic hypothermia postponed neurodegeneration but only partially rescued the expression of AMPA and NR1 mRNAs at 7 days and not at 1 day after the insult. Therefore, hippocampal AMPA receptor mRNAs decline at a relatively similar rate after normothermic global ischemia and cellular neuroprotection by intraischemic hypothermia occurred independently of altered subunit composition of AMPA receptors. Since decreases persist within resistant neurons under the postischemic condition, AMPA receptor-mediated Ca2+ currents probably do not contribute to selective vulnerability.

Importance of posttraumatic hypothermia and hyperthermia on the inflammatory response after fluid percussion brain injury: biochemical and immunocytochemical studies.

The purpose of this study was to investigate: 1) the temporal and regional profile of polymorphonuclear leukocyte (PMNL) infiltration after moderate traumatic brain injury using the parasagittal fluid percussion model and 2) the effects of posttraumatic hypothermia (30 degrees C) and hyperthermia (39 degrees C) on the acute and subacute inflammatory response. We hypothesized that posttraumatic hypothermia would reduce the degree of PMNL accumulation whereas hyperthermia would exacerbate this response to injury. In the first series of experiments we quantitated the temporal profile of altered myeloperoxidase activity under normothermic (37 degrees C) conditions (n = 20). The rats were allowed to survive for 3 hours, 24 hours, 3 days, or 7 days after trauma, and brains were dissected into cortical and subcortical regions ipsilateral and contralateral to injury. Additional animals were perfused and fixed for the immunocytochemical visualization of myeloperoxidase (n = 15). In the second series of experiments, rats (n = 25) were killed 3 hours or 3 days after the 3-hour monitoring period of normothermia (36.5 degrees C), hypothermia (30 degrees C), or hyperthermia (39 degrees C) (n = 4 to 5 per group), and myeloperoxidase activity was again quantitated. In normothermic rats, the enzymatic activity of myeloperoxidase was significantly increased (P < 0.05) at 3 hours within the anterior cortical segment (213.97 +/- 56.2 versus control 65.5 +/- 52.3 U/g of wet tissue; mean +/- SD) and posterior (injured) cortical and subcortical segments compared to sham-operated rats (305.76 +/- 27.8 and 258.67 +/- 101.4 U/g of wet tissue versus control 62.8 +/- 24.8 and 37.28 +/- 35.6 U/g of wet tissue; P < 0.0001, P < 0.05, respectively). At 24 hours and 7-days after trauma only the posterior cortical region (P < 0.005, P < 0.05, respectively) exhibited increased myeloperoxidase activity. However, 3 days after trauma, myeloperoxidase activity was also significantly increased within the anterior cortical segment (P < 0.05) and in posterior cortical and subcortical regions compared to sham-operated cortex (P < 0.0001, P < 0.05, respectively). Immunocytochemical analysis of myeloperoxidase reactivity at 3 hours, 24 hours, 3- and 7-days demonstrated large numbers of immunoreactive leukocytes within and associated with blood vessels, damaged tissues, and subarachnoid spaces. Posttraumatic hypothermia and hyperthermia had significant effects on myeloperoxidase activity at both 3 hours and 3 days after traumatic brain injury. Posttraumatic hypothermia reduced myeloperoxidase activity in the injured and noninjured cortical and subcortical segments compared to normothermic values (P < 0.05). In contrast, posttraumatic hyperthermia significantly elevated myeloperoxidase activity in the posterior cortical region compared to normothermic values at both 3 hours and 3 days (473.5 +/- 258.4 and 100.11 +/- 27.58 U/g of wet tissue, respectively, P < 0.05 versus controls). These results indicate that posttraumatic hypothermia decreases early and more prolonged myeloperoxidase activation whereas hyperthermia increases myeloperoxidase activity. Temperature-dependent alterations in PMNL accumulation appear to be a potential mechanism by which posttraumatic temperature manipulations may influence traumatic outcome.

Early treatment with a novel inhibitor of lipid peroxidation (LY341122) improves histopathological outcome after moderate fluid percussion brain injury in rats.

OBJECTIVE: Reactive oxygen species are thought to participate in the pathobiology of traumatic brain injury (TBI). This study determined whether treatment with LY341122, a potent inhibitor of lipid peroxidation and an antioxidant, would provide neuroprotection in a rat model of TBI. METHODS: To investigate the efficacy of LY341122 in this parasagittal fluid percussion model (1.8-2.1 atm), the rats received oral administration of LY341122 (100 mg/kg) or vehicle 2 hours before and 4 hours after TBI (each group, n = 7). To investigate the therapeutic window for treatment, rats were treated with LY341122 or vehicle for 20 hours by femoral vein infusion starting at 5 minutes, 30 minutes, or 3 hours after TBI (each group, n = 5). Three days after injury, analysis of contusion volumes and the frequency of damaged cortical neurons was conducted. RESULTS: Oral administration of LY341122 before and after TBI led to a significant reduction in overall contusion volume (3.28 mm3+/-0.75 mm3 [mean +/- standard error of the mean] versus 1.32 mm3 +/- 0.33 mm3; P < 0.05) and also reduced the frequency of damaged cortical neurons (1191.7 +/- 267.1 versus 474.6 +/- 80.2; P < 0.05). In the second experiment, rats treated with LY341122 at 5 minutes or 30 minutes after TBI also demonstrated a significant reduction (P < 0.05) in contusion volume (1.92 mm3 +/- 0.64 mm3 or 1.59 mm3 +/- 0.50 mm3, respectively) compared with vehicle-treated rats (4.32 mm3 +/- 1.15 mm3). A significant reduction in total cortical necrotic neuron counts was also demonstrated in the 5-minute group (2243.8 +/- 265.3 versus 1457.8 +/- 265.3; P < 0.05). In contrast, histopathological outcome was not significantly improved when treatment was delayed until 3 hours after TBI. CONCLUSION: These data reinforce the hypothesis that lipid peroxidation and reactive oxygen species participate in the acute pathogenesis of TBI. Treatment delayed until 3 hours after TBI did not provide significant histopathological protection.

Sequential changes in glial fibrillary acidic protein and gene expression following parasagittal fluid-percussion brain injury in rats.

This study documents the regional and temporal patterns of glial fibrillary acidic protein (GFAP) RNA and protein expression after parasagittal fluid-percussion (F-P) brain injury (1.7 to 2.2 atm) in male Sprague-Dawley rats. In situ hybridization was conducted in 28 rats with a 35S-labeled antisense riboprobe to GFAP at 0.5, 2, and 6 hours and 1, 3, and 30 days after traumatic brain injury (TBI) or sham procedures. Immunocytochemical staining of GFAP was conducted in 20 rats at 1, 3, 7, and 30 days after TBI or sham procedures. At 0.5 and 2 hours after TBI, increased GFAP mRNA was restricted to superficial cortical areas underlying the impact site. At 24 hours, increased GFAP mRNA was observed throughout the traumatized hemisphere except within the histopathologically vulnerable lateral parietal cortex and external capsule. Contralateral expression within the hippocampus and cingulate and lateral cortices was also observed. Three days after TBI, GFAP mRNA expression was prominent overlying pial surfaces, in cortical regions surrounding the contusion, and within the hippocampus and lateral thalamus. Immunocytochemical visualization of GFAP at 1 and 3 days demonstrated reactive astrocytes overlying the pial surface, surrounding the cortical contusion, and within ipsilateral white matter tracts, hippocampus, and lateral thalamus. At 30 days, GFAP mRNA and protein expression were present within the deeper cortical layers of the lateral somatosensory cortex and lateral thalamus and throughout ipsilateral white matter tracts. These data demonstrate a complex pattern of GFAP mRNA and protein expression within gray and white matter tracts following F-P brain injury. Patterns of GFAP gene expression may be a sensitive molecular marker for evaluating the global response of the brain to focal injury in terms of progressive neurodegenerative as well as regenerative processes.

Posttreatment with high-dose albumin reduces histopathological damage and improves neurological deficit following fluid percussion brain injury in rats.

We have recently shown that high-dose human serum albumin (HSA) therapy confers marked histological protection in experimental middle cerebral artery occlusion. Thus, the purpose of this study was to determine whether treatment with high-dose HSA would protect in a rat model of traumatic brain injury (TBI). Twenty-four hours prior to TBI, the fluid percussion interface was positioned parasagittally over the right cerebral cortex. On the following day, fasted rats were anesthetized with 3% halothane, 70% nitrous oxide, and 30% oxygen and received right parieto-occipital parasagittal fluid-percussion injury (1.5-2.0 atm). Cranial and rectal temperatures were monitored throughout the experiment and held at normothermic levels (36.5-37.5 degrees C) by a warming lamp above the animal's head. The agent (25% human serum albumin, HSA) or vehicle (sodium chloride 0.9%) was administered i.v. (1% of body weight) 15 min after trauma. Behavioral function was evaluated in all rats before and after TBI (at 2 h, 24 h, 48 h, 72 h, and 7 days). Neurological function was graded on a scale of 0-12 (normal score = 0; maximal score = 12). Seven days after TBI, brains were perfusion-fixed, coronal sections at various levels were digitized, and contusion areas in the superficial, middle and deep layers of cortex and in the underlying fimbria were measured. HSA significantly improved the neurological score compared to saline at 24 h, 72 h, and 7 days after TBI (6.0 +/- 0.6 [albumin] versus 8.4 +/- 0.5 [saline]; 3.6 +/- 0.7 versus 6.8 +/- 1.0; and 2.6 +/- 0.6 versus 5.7 +/- 0.8, respectively; p < 0.05). HSA therapy also significantly reduced total contusion area (0.89 +/- 0.2 versus 1.82 +/- 0.3 mm2; p = 0.02). Our findings document that high-concentration albumin therapy instituted 15 min after trauma significantly improves the neurological score and reduces histological damage. We believe that this pharmacological agent may have promising potential for the clinical treatment of brain injury.

Expression of brain-derived neurotrophic factor, nerve growth factor, and heat shock protein HSP70 following fluid percussion brain injury in rats.

Traumatic brain injury can induce the expression of stress-related and neurotrophic genes both within the injury site and in distant regions. These genes may affect severity of damage and/or be neuroprotective. We used in situ hybridization to assess the alterations in expression of the heat shock protein HSP70, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) genes in rat brain following moderate fluid-percussion (F-P) injury at various survival times. HSP70 gene expression was induced at and surrounding the injury site as early as 30 min after trauma. This elevated signal spread ventrally and laterally through the ipsilateral cortex and into the underlying white matter over the next few hours. In addition, there was elevated expression in the temporal hippocampus. BDNF was strongly upregulated in the granular cells of the dentate gyrus and in the CA3 hippocampus 2-6 h after injury. Cortical regions at and near the injury site showed no response at the mRNA level. NGF mRNA increased over the granular cells of the dentate gyrus at early time points. There was also a weaker secondary induction of the NGF gene in the contralateral dentate gyrus of some animals. Cortical response was observed in the entorhinal cortex, bilaterally, but not at the injury site. All three of the studied genes responded quickly to injury, as early as 30 min. The induction of gene expression for neurotrophins in regions remote from areas with histopathology may reflect coupling of gene expression to neuronal excitation, which may be associated with neuroprotection and plasticity.

Influence of early posttraumatic hypothermia therapy on local cerebral blood flow and glucose metabolism after fluid-percussion brain injury.

OBJECT: Using autoradiographic image averaging, the authors recently described prominent foci of marked glucose metabolism-greater-than-blood-flow uncoupling in the acutely traumatized rat brain. Because hypothermia is known to ameliorate injury in this and other injury models, the authors designed the present study to assess the effects of posttraumatic therapeutic hypothermia on the local cerebral metabolic rate of glucose (LCMRglu) and local cerebral blood flow (LCBF) following moderate parasagittal fluid-percussion head injury (FPI) in rats. METHODS: Either cranial hypothermia (30 degrees C) or normothermia (37 degrees C) was induced for 3 hours in matched groups of rats immediately after FPI; LCMRglu and LCBF were assessed 3 hours after concluding these temperature manipulations. In rats subjected to FPI, regardless of whether normothermia or hypothermia ensued, LCBF was reduced relative to the sham-injury groups. In addition, when FPI was followed by hypothermia (FPI-30 degrees C group), the subsequent LCBF was significantly lower (35-38% on average) than in FPI-37 degrees C rats. Statistical mapping of LCBF difference imaging data revealed confluent cortical and subcortical zones of significantly reduced LCBF (largely ipsilateral to the prior injury) in FPI-30 degrees C rats relative to the FPI-37 degrees C group. Local glucose utilization was reduced in both hemispheres of FPI-37 degrees C rats relative to the sham-injury group and was lower in the right (traumatized) hemisphere than in the left. However, LCMRglu values were largely unaffected by temperature manipulation in either the FPI or sham-injury groups. The LCMRglu/LCBF ratio was nearly doubled in FPI-30 degrees C rats relative to the FPI-37 degrees C group, in a diffuse and bihemispheric fashion. Linear regression analysis comparing LCMRglu and LCBF revealed that the FPI-37 degrees C and FPI-30 degrees C data sets were completely nonoverlapping, whereas the two sham-injury data sets were intermixed. CONCLUSIONS: Despite its proven neuroprotective efficacy, early posttraumatic hypothermia (30 degrees C for 3 hours) nonetheless induces a moderate decline in cerebral perfusion without the (anticipated) improvement in cerebral glucose utilization, so that a state of mild metabolism-greater-than-blood-flow dissociation is perpetuated.

Uncoupling of local cerebral glucose metabolism and blood flow after acute fluid-percussion injury in rats.

We assessed local cerebral glucose metabolism (lCMRGlc) and blood flow (lCBF) interrelationships in the first hour after parasagittal fluid-percussion head injury (FPI) in rats. Matched series were studied autoradiographically for lCMRGlc and lCBF with 2-[14C]deoxyglucose and 14C-labeled iodoantipyrine, respectively. Three-dimensional autoradiographic-image mapping was to generate average data sets from which a mean ICMRGlc-to-lCBF ratio data set was derived. lCBF in neocortical regions ipsilateral to the trauma were depressed, on average, by 44% compared with sham-FPI rats, whereas contralateral lCBF values were not altered. By contrast, ICMRGlc was elevated in many cortical and subcortical sites of both hemispheres; this amounted to 1.3- to 1.4-fold increases in neocortical regions in the thalamus and 1.6- to 1.7-fold increases in the hippocampus. The lCMRGlc-to-lCBF ratio data revealed striking elevations both ipsilateral (P = 7 x 10(-7) and contralateral to the FPI (P = 0.003). The extent of metabolism-flow uncoupling, on average, amounted to 2.5-fold in the ipsilateral hippocampus and neocortex and 1.7-fold contralaterally. The loci of pronounced metabolism-flow dissociation corresponded closely to the previously documented histological distribution of neuronal necrosis. Our findings resemble events occurring in the acute focal ischemic penumbra and suggest that similar injury mechanisms may be operative.

Bilateral ischemic tolerance of rat hippocampus induced by prior unilateral transient focal ischemia: relationship to c-fos mRNA expression.

The purpose of this study was to determine whether transient unilateral (2 h) middle cerebral artery occlusion (MCAo) is capable of inducing bilateral ischemic tolerance in hippocampal CA1 neurons when temporary bilateral forebrain ischemia by two-vessel occlusion (2VO) is carried out 3 days later, and to explore the relationship of this tolerance to the regional expression of c-fos and hsp-70 mRNA. Rats were sacrificed 4 days after 2VO, and normal-appearing neurons in CA1 subregions were counted. Rats subjected to MCAo and 2VO showed significant protection of CA1 neurons in both hippocampi, whereas rats which underwent sham MCAo and 2VO typically had severe bilateral destruction of CA1 neurons (normal neuron counts, ipsilateral medial CA1: 59.8 +/- 7.2 vs 16.6 +/- 7.8 (mean +/- s.e.m.); middle CA1: 50.0 +/- 4.7 vs 16.0 +/- 8.8; lateral CA1: 43.5 +/- 5.7 vs 13.8 +/- 6.3; contralateral, medial CA1: 52.3 +/- 6.3 vs 17.0 +/- 6.4; middle CA1: 43.3 +/- 4.7 vs 19.8 +/- 8.1; lateral CA1: 45.5 +/- 4.6 vs 26.0 +/- 10.3, respectively). This neuronal tolerance was preceded by the early bilateral induction of c-fos mRNA, which may in turn lead to expression of critical target genes that promote cell recovery.

Computer-assisted image-averaging strategies for the topographic analysis of in situ hybridization autoradiographs.

We report the application of a computer-based image-averaging strategy to the quantitative topographic analysis of in situ hybridization autoradiographs, based upon a disparity-analysis algorithm. We illustrate this approach for a representative antisense riboprobe-the astrocytic marker, glial fibrillary acid protein (GFAP)-in the setting of fluid-percussion brain injury in rats. Sequential coronal autoradiographs in individual animals are first digitized and aligned by disparity analysis. Next, coronal sections of all brains of a given experimental group are placed in register with one another, using a common anatomic reference level. One brain of the series serves as a template, and corresponding sections of other brains are mapped into its contour at each level. In this manner, average and standard deviation image data sets may be generated. With thresholding techniques, individual data sets can be dichotomized with respect to a chosen threshold, and frequency maps can be generated at each coronal level, displaying numbers of brains showing supra-threshold levels of mRNA at each pixel location. Pixel-by-pixel statistical comparison of data sets obtained under two different conditions (e.g., 30 min vs. 24 h following brain trauma) is then feasible. A digitized functional-anatomic brain atlas may be fitted to the images to assist analysis. Computer-based image analysis of in situ hybridization autoradiographs greatly extends the utility and applicability of this technique.

Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model.

BACKGROUND AND PURPOSE: The purpose of the present study was to evaluate a modified method of intraluminal suture occlusion of the middle cerebral artery (MCA) on the volume of brain infarction and on neurobehavioral function in rats subjected to a temporary focal ischemic insult. METHODS: Male Sprague-Dawley rats were anesthetized with halothane and subjected to 60 minutes or 2 hours of temporary MCA occlusion (MCAo) by an intraluminal thread. In one group of rats, the suture was coated with poly-L-lysine, while in a second group, a conventional uncoated suture was used. Behavioral function was evaluated at 50 to 60 minutes after occlusion and during a 3-day period after MCAo. Three days after MCAo brains were perfusion-fixed and infarct volumes were measured. RESULTS: In rats with 60-minute MCAo, only 3 of 7 animals with uncoated sutures had infarcts, whereas in the group with poly-L-lysine-coated sutures, all rats (n = 7) exhibited infarction (P = .009, Fisher's exact test). With 2 hours of MCAo, total infarct volume (corrected for brain edema) was significantly larger in rats with poly-L-lysine-coated sutures than in the group with uncoated sutures (mean +/- SEM, 122.1 +/- 4.8 versus 67.0 +/- 18.2 mm3, respectively; P = .03; n = 4 in each group). In the 2-hour MCAo study, infarct volumes in the uncoated-suture group tended to be variable and inconsistent (coefficient of variation, 54%) compared with the group in which sutures were coated with poly-L-lysine, in which a highly consistent infarct was produced (coefficient of variation of infarct volume, 8%). CONCLUSIONS: Reversible MCAo in which a poly-L-lysine-coated intraluminal suture was used proved to be a reliable and effective modification of this technique, yielding consistently larger infarcts and greatly reduced interanimal variability.

Local cerebral glucose utilization and cytoskeletal proteolysis as indices of evolving focal ischemic injury in core and penumbra.

To ascertain the tempo of progression to irreversible injury in focal ischemia, we subjected halothane-anesthetized Sprague-Dawley rats to photochemically induced distal middle cerebral artery occlusion (dMCAO) combined with permanent ipsilateral and 1 h contralateral common carotid artery occlusions. Head temperature was maintained at 36 degrees C. At times centered at either 1.5 or 3 h post-dMCAO, the rate of local glucose metabolism (lCMRgl) was measured by 2-deoxyglucose autoradiography, and cytoskeletal proteolysis was assessed regionally by an immunoblotting procedure to detect spectrin breakdown products. At 1.5 h (n = 5), the cortical ischemic core was already severely hypometabolic (lCMRgl 15.5 +/- 10.8 mumol 100 g-1 min-1, mean +/- SD), whereas the cortical penumbral zone was hypermetabolic (69.0 +/- 9.7). (The lumped constant was verified to be unchanged by methylglucose studies). Neutral red pH studies at this time point showed that both the core and penumbral zones were equally acidotic. By 3 h post-dMCAO (n = 6), lCMRgl in the penumbral zone had fallen to low levels (15.4 +/- 2.2 mumol 100 g-1 min-1) equal to those of the ischemic core (16.7 +/- 4.5). Correspondingly, spectrin breakdown in the ischemic core was advanced at both 2 and 3.5 h post-dMCAO (36 +/- 18% and 33 +/- 18% of total spectrin, respectively), whereas in the penumbral zone spectrin breakdown was less extensive and more highly variable at both times (22 +/- 23% and 29 +/- 16%). We conclude that irreversible deterioration of the ischemic core, as evidenced by the onset of local cytoskeletal proteolysis, begins within 2 h of middle cerebral artery occlusion. In the ischemic penumbra, the transition from glucose hyper- to hypometabolism occurs by 3.5 h and is associated with a milder and more variable degree of spectrin breakdown.

Cerebral energy metabolism in rats studied by phosphorus nuclear magnetic resonance using surface coils.

Phosphorus nuclear magnetic resonance with surface coils was used to investigate the regional metabolism of the rat brain in vivo under conditions of normoxia, severe hypoxemia, partial necrosis, and partial ischemia. The results show an increase in sugar phosphate and/or inorganic phosphate with injury in accordance with in vivo assays. The technique provides a powerful means of monitoring the metabolism of stroke and its response to therapy in vivo.

A topographic measurement of brain pH.

The ability of neutral red to serve as an internal pH indicator in biological systems was tested in the rat brain after parenteral adminstration of the dye. The injected dye is avidly taken up by the brain cells and is distributed mainly in intracellular organelles. The ratio of optical signals recorded from the frozen brain at 445 and 530 nm and compared with calibration curves provides pH values. Color transition of neutral red ranges from 6.0 to 8.0 pH units. Topographic application of this method is particularly useful in studying local pH changes in brains with developing ischemic foci.

A pictorial representation of endogenous brain ATP by a bioluminescent method.

The layering of a luciferin-luciferase solution on brain slices makes endogenous ATP visible. Rat brains, frozen in situ and sliced at 16 micrometer thickness at a temperature of--15 degrees C, were fixed by a ternary mixture of ethanol, formalin and dioxane at--20 degrees C for 15 min, and dried at 40 degrees C for 12 h. Luciferin, luciferase and a small quantity of magnesium sulfate were dissolved into an arsenate buffer solution containing 1% polyvinylpyrrolidone, 2% gelatin and 1% glycerol. The solution was then frozen into a column, sliced at 40 micrometer thickness at--15 degrees C and placed on the precooled brain slice. A luminiferous luciferin-ATP reaction begins when the brain slice reaches room temperature and persists for more than 10 min. This technique therefore makes possible the optical and/or photographic determination of the endogenous concentration of brain ATP. Capability of the technique is also demonstrated.