Conventional and functional proteomics using large format two-dimensional gel electrophoresis 24 hours after controlled cortical impact in postnatal day 17 rats.

Conventional and functional proteomics have significant potential to expand our understanding of traumatic brain injury (TBI) but have not yet been used. The purpose of the present study was to examine global hippocampal protein changes in postnatal day (PND) 17 immature rats 24 h after moderate controlled cortical impact (CCI). Silver nitrate stains or protein kinase B (PKB) phosphoprotein substrate antibodies were used to evaluate high abundance or PKB pathway signal transduction proteins representing conventional and functional proteomic approaches, respectively. Isoelectric focusing was performed over a nonlinear pH range of 3-10 with immobilized pH gradients (IPG strips) using supernatant from the most soluble cellular protein fraction of hippocampal tissue protein lysates from six paired sham and injured PND 17 rats. Approximately 1,500 proteins were found in each silver stained gel with 40% matching of proteins. Of these 600 proteins, 52% showed a twofold, 20% a fivefold, and 10% a 10-fold decrease or increase. Spot matching with existing protein databases revealed changes in important cytoskeletal and cell signalling proteins. PKB substrate protein phosphorylation was best seen in large format two-dimensional blots and known substrates of PKB such as glucose transporter proteins 3 and 4 and forkhead transcription factors, identified based upon molecular mass and charge, showed altered phosphorylation 24 h after injury. These results suggest that combined conventional and functional proteomic approaches are powerful, complementary and synergistic tools revealing multiple protein changes and posttranslational protein modifications that allow for more specific and comprehensive functional assessments after pediatric TBI.

The simple model versus the super model: translating experimental traumatic brain injury research to the bedside.

Despite considerable investigation in rodent models of traumatic brain injury (TBI), no novel therapy has been successfully translated from bench to bedside. Although well-described limitations of clinical trails may account for these failures, several modeling factors may also contribute to the lack of therapeutic translation from the laboratory to the clinic. Specifically, models of TBI may omit one or more critical, clinically relevant pathophysiologic features. In this invited review article, the impact of the limited incorporation of several important clinical pathophysiologic factors in TBI, namely secondary insults (i.e., hypotension and/or hypoxemia), coma, and aspects of standard neurointensive care monitoring and management strategies (i.e., intracranial pressure [ICP] monitoring and ICP-directed therapies, sedation, mechanical ventilation, and cardiovascular support) are discussed. Comparative studies in rodent and large animal models of TBI (which may, in some cases, represent super models) are also presented. We conclude that therapeutic breakthroughs will likely require a multidisciplinary approach, involving investigation in a range of models, including clinically relevant modifications of established animal models, along with development and application of new innovations in clinical trial design.

Histopathologic response of the immature rat to diffuse traumatic brain injury.

The purpose of this study was to characterize the histopathologic response of rats at postnatal day (PND) 17 following an impact-acceleration diffuse traumatic brain injury (TBI) using a 150-g/2-meter injury as previously described. This injury produces acute neurologic and physiologic derangements as well as enduring motor and Morris water maze (MWM) functional deficits. Histopathologic studies of perfusion-fixed brains were performed by gross examination and light microscopy using hematoxylin and eosin, Bielschowsky silver stain, and glial fibrillary acidic protein (GFAP) immunohistochemistry at 1, 3, 7, 28, and 90 day after injury. Gross pathologic examination revealed diffuse subarachnoid hemorrhage (SAH) at 1-3 days but minimal supratentorial intraparenchymal hemorrhage. Petechial hemorrhages were noted in ventral brainstem segments and in the cerebellum. After 1-3-day survivals, light microscopy revealed diffuse SAH and intraventricular hemorrhage (IVH), mild edema, significant axonal injury, reactive astrogliosis, and localized midline cerebellar hemorrhage. Axonal injury most commonly occurred in the long ascending and descending fiber tracts of the brainstem and occasionally in the forebrain, and was maximal at 3 days, but present until 7 days after injury. Reactive astrocytes were similarly found both in location and timing, but were also significantly identified in the hippocampus, white matter tracts, and corpus callosum. Typically, TBI produced significant diffuse SAH accompanied by cerebral and brainstem astrogliosis and axonal injury without obvious neuronal loss. Since this injury produces some pathologic changes with sustained functional deficits similar to TBI in infants and children, it should be useful for the further study of the pathophysiology and therapy of diffuse TBI and brainstem injury in the immature brain.

Histopathologic consequences of hyperglycemic cerebral ischemia during hypothermic cardiopulmonary bypass in pigs.

BACKGROUND: This study examined whether 34 degrees C or 31 degrees C hypothermia during global cerebral ischemia with hyperglycemic cardiopulmonary bypass (CPB) in surviving pigs improves electroencephalographic (EEG) recovery and histopathologic scores when compared with normothermic animals. METHODS: Anesthetized pigs were placed on CPB and randomly assigned to 37 degrees C (n = 9), 34 degrees C (n = 10), or 31 degrees C (n = 8) management. After increasing serum glucose to 300 mg/dL, animals underwent 15 minutes of global cerebral ischemia by temporarily occluding the innominate and left subclavian arteries. Following reperfusion, rewarming, and termination of CPB, animals were recovered for 24 (37 degrees C animals) or 72 hours (34 degrees C and 31 degrees C animals). Daily EEG signals were recorded, and brain histopathology from cortical, hippocampal, and cerebellar regions was graded by an independent observer. RESULTS: Before ischemia, serum glucose concentrations were similar in the 37 degrees C (307+/-9 mg/dL), 34 degrees C (311+/-14 mg/dL), and 31 degrees C (310+/-15) groups. By the first postoperative day, EEG scores in 31 degrees C animals (4.2+/-0.6) had returned to baseline and were greater than those in the 34 degrees C (3.4+/-0.5) and 37 degrees C (2.5+/-0.4) groups (p < 0.05, respectively, between groups). Cooling to 34 degrees C showed selective improvement over 37 degrees C in hippocampal, temporal cortical, and cerebellar regions, but the greatest improvement in all regions occurred with 31 degrees C. Cumulative neuropathology scores in 31 degrees C animals (13.5+/-2.2) exceeded 34 degrees C (6.8+/-2.2) and 37 degrees C (1.9+/-2.1) animals (p < 0.05, respectively, between groups). CONCLUSIONS: Hypothermia during CPB significantly reduced the morphologic consequences of severe, temporary cerebral ischemia under hyperglycemic conditions, with the greatest protection at 31 degrees C.

Adenovirus-mediated transfer and expression of beta-gal in injured hippocampus after traumatic brain injury in mice.

In models of focal cerebral ischemia, adenoviral gene transfer is often attenuated or delayed versus naive. After controlled cortical impact (CCI)-induced traumatic brain injury in mice, CA1 and CA3 hippocampus exhibit delayed neuronal death by 3 days, with subsequent near complete loss of hippocampus by 21 days. We hypothesized that adenoviral-mediated expression of the reporter gene beta-Galactosidase (beta-Gal) in hippocampus would be attenuated after CCI in mice. C57BL6 mice (n = 16) were subjected to either CCI to left parietal cortex or sham (burr hole). Adenovirus carrying the beta-Gal gene (AdlacZ; 1 x 10(9) plaque-forming units [pfu]/mL) was then injected into left dorsal hippocampus. At 24 or 72 h, beta-Gal expression was quantified (mU/mg protein). Separate mice (n = 10) were used to study beta-Gal spatial distribution in brain sections. Beta-Gal expression in left hippocampus was similar in shams at 24 h (48.4 +/- 4.1) versus 72 h (68.8 +/- 8.8, not significant). CCI did not reduce beta-Gal expression in left hippocampus (68.8 +/- 8.8 versus 88.1 +/- 7.0 at 72 h, sham versus CCI, not significant). In contrast, CCI reduced beta-Gal expression in right (contralateral) hippocampus versus sham (p < 0.05 at both 24 and 72 h). Beta-Gal was seen in many cell types in ipsilateral hippocampus, including CA3 neurons. Despite eventual loss of ipsilateral hippocampus, adenovirus-mediated gene transfer was surprisingly robust early after CCI providing an opportunity to test novel genes targeting delayed hippocampal neuronal death.

Intraischemic mild hypothermia increases hippocampal CA1 blood flow during forebrain ischemia.

The hippocampal CA1 sector is selectively vulnerable to forebrain ischemia but protected by mild hypothermia. However, the consequence of intraischemic hypothermia on CA1 blood flow during the insult has not been adequately characterized. The effects of mild intraischemic hypothermia on relative changes in regional hippocampal CA1 blood flow were recorded continuously using laser Doppler flowmetry (LDF) during and 30 min after 6 min of forebrain ischemia. Six experimental groups (n=6/group) of fasted male Wistar rats were compared. Groups 1, 3 and 5 consisted of normothermic rats that underwent either 6 (for CBF measurements) and 6 or 10 (for 7 day survival-CA1 neuronal death measurements) min of transient forebrain ischemia using bilateral carotid clamping and hemorrhagic hypotension. Groups 2, 4 and 6 rats were subjected to mild hypothermia (34 degrees C) before, during, and 30 min after 6 (for CBF measurements) and 6 or 10 (for 7 day survival-CA1 neuronal death measurements) min of transient forebrain ischemia. CA1 blood flow and electroencephalogram (EEG) were continuously recorded. During the ischemic insult there were intergroup differences in the magnitude of CBF decreases in the CA1 region. In both groups 1 and 2, CBF returned to preischemic values within 1 min of reperfusion but hypothermic rats had more sustained hyperemia. Hypothermic rats had a quicker recovery of EEG activity and less delayed CA1 neuronal death (group 2 versus 4). These data suggest ischemic blood flow to the CA1 sector was altered by intraischemic mild hypothermia which may contribute to the greater benefit of intraischemic hypothermic neuroprotection.

Isoflurane improves long-term neurologic outcome versus fentanyl after traumatic brain injury in rats.

Despite routine use of fentanyl in patients after traumatic brain injury (TBI), it is unclear if it is the optimal sedative/analgesic agent. Isoflurane is commonly used in experimental TBI. We hypothesized that isoflurane would be neuroprotective versus fentanyl after TBI. Rats underwent controlled cortical impact (CCI) and received 4 h of N2O/O2 (2:1) and either fentanyl (10 microg/kg i.v. bolus, 50 microg/kg/h infusion) or isoflurane (1% by inhalation) with controlled ventilation. Shams underwent identical preparation, without CCI. Functional outcome (beam balance, beam walking, Morris water maze [MWM] tasks) was assessed over 20 days. Lesion volume and hippocampal neuron survival were quantified on day 21. Additional rats underwent identical CCI and anesthesia with intracranial pressure (ICP) monitoring, and brain water content was assessed. Motor and MWM performances were better in injured rats treated with isoflurane versus fentanyl (p < 0.05). CA1 hippocampal damage was attenuated in isoflurane-treated rats (p < 0.05). Fentanyl-treated rats had higher mean arterial blood pressure after injury (p < 0.05); however, ICP and brain water were similar between groups. Isoflurane improved functional outcome and attenuated damage to CA1 versus fentanyl in rats subjected to CCI. Isoflurane may be neuroprotective by augmenting cerebral blood flow and/or reducing excitotoxicity, not by reducing ICP or brain water content. Alternatively, fentanyl may be detrimental. Isoflurane may mask beneficial effects of novel agents tested in TBI models. Additionally, fentanyl may not be optimal early after TBI in humans.

Evaluation of combined fibroblast growth factor-2 and moderate hypothermia therapy in traumatically brain injured rats.

Both the exogenous administration of fibroblast growth factor-2 (FGF-2) or the induction of moderate hypothermia have been shown to attenuate histopathology and improve functional outcome after traumatic brain injury (TBI). Since combined therapeutic strategies may be more beneficial than single therapies, we examined the potential synergistic effect of FGF-2 combined with moderate hypothermia treatment induced 10 min after TBI on functional and histological outcome following controlled cortical impact (CCI) injury. Fifty male Sprague-Dawley rats were randomized to one sham and four CCI treatment groups: Sham+vehicle (VEH); FGF-2 (45 microg/kg/h for 3 h i.v.)+Normothermia (37+/-0.5 degrees C); FGF-2+Hypothermia (32+/-0.5 degrees C for 3 h); VEH+Norm; VEH+Hypo. Vestibulomotor performance on the beam balance and beam-walk (BW) tasks on post-operative days 1-5 and spatial memory acquisition in the Morris water maze (MWM) on days 14-18 were assessed. After 4 weeks survival, histological evaluations (CA(1) and CA(3) cell counts and lesion volume) were performed. MWM performance improved in all treatment groups, but combined treatment was not more efficacious than either alone. The FGF-2+Hypo group performed significantly better than the other injured treatment groups in the BW task. Lastly, no significant group differences in beam balance or histological outcome were observed. These data suggest a suboptimal and incomplete synergy of combined FGF-2 and hypothermia treatment. These data may indicate that either our dose of FGF-2 or combination therapy was not optimized in our model.

Transient electroencephalographic suppression with initiation of cardiopulmonary bypass in pigs: blood versus nonblood priming.

Electroencephalographic (EEG) changes have been reported with cardiopulmonary bypass (CPB). We tested whether the type of priming solution (blood versus nonblood) affected the EEG. Twenty-six anesthetized pigs (29.5+/-1.6 kg) were cannulated for CPB primed with 1 liter plasmalyte and 500 ml 6% hetastarch (nonblood prime). EEG signals were recorded during the initiation of normothermic CPB. Three minutes later, animals were weaned from CPB and allowed to stabilize. CPB was reinstituted using the animals' hemodiluted blood as prime. We found that with nonblood prime, abrupt and marked EEG suppression lasting 12.6+/-0.7 s was found in all animals, followed by gradual resumption of baseline EEG activity. In contrast, CPB with blood prime caused no detectable EEG changes. We conclude that severe reductions in EEG activity occur after initiating CPB with nonblood prime; these reductions are not seen when using blood prime. The cause of EEG suppression is unknown, but may represent transient impairment of oxygen delivery to the brain caused by nonblood perfusion.

Lamotrigine attenuates cortical glutamate release during global cerebral ischemia in pigs on cardiopulmonary bypass.

BACKGROUND: The dose-response effects of pretreatment with lamotrigine (a phenyltriazine derivative that inhibits neuronal glutamate release) in a porcine cerebral ischemia model during cardiopulmonary bypass were studied. METHODS: Sagittal sinus catheters and cortical microdialysis catheters were inserted into anesthetized pigs. Animals undergoing normothermic cardiopulmonary bypass were pretreated with lamotrigine 0, 10, 25, or 50 mg/kg (n = 10 per group). Fifteen minutes of global cerebral ischemia was produced, followed by 40 min of reperfusion and discontinuation of cardiopulmonary bypass. Cerebral oxygen metabolism was calculated using cerebral blood flow (radioactive microspheres) and arterial-venous oxygen content gradients. Concentrations of microdialysate glutamate and aspartate were quantified; electroencephalographic signals were recorded. After cardiopulmonary bypass, blood and cerebrospinal fluid were sampled for S-100B protein, and a biopsy was performed on the cerebral cortex for metabolic profile. RESULTS: Lamotrigine caused dose-dependent reductions in systemic vascular resistance so that additional fluid was required to maintain venous return. Concentrations of glutamate and aspartate did not change during reperfusion after 50 mg/kg lamotrigine in contrast to fivefold and twofold increases, respectively, with lower doses. There were no intergroup differences in cerebral metabolism, electroencephalographic scores, cortical metabolites, brain lactate, or S-100B protein concentrations in the cerebrospinal fluid and blood. CONCLUSIONS: Lamotrigine 50 mg/kg significantly attenuated excitatory neurotransmitter release during normothermic cerebral ischemia during cardiopulmonary bypass without improving other neurologic parameters. Lamotrigine caused arterial and venous dilation, which limits its clinical usefulness.

Combined therapy affects outcomes differentially after mild traumatic brain injury and secondary forebrain ischemia in rats.

Muscarinic and NMDA receptors contribute to post-traumatic hypersensitivity to secondary ischemia. However, the effect of these receptor antagonists on behavior and CA1 neuronal death after traumatic brain injury (TBI) with acute (1 h after TBI) forebrain ischemia has not been systematically assessed. We examined cognitive and motor dysfunction and the relationship of behavior deficits to neuronal death in this model using muscarinic and NMDA antagonists. Three behavioral groups (n=10/group) of Wistar rats were subjected to mild TBI and 6 min of forebrain ischemia imposed 1 h after TBI with 45 days survival. Motor and spatial memory performance were assessed using the rotarod task and Morris water maze. Seven additional groups (n=6/group) were evaluated only for CA1 death after 7 days survival following sham, individual or combined injury with and without drug treatments. Rats were given 0.3 mg/kg MK-801 (M) and 1.0 mg/kg scopolamine (S) alone or combined (M-S) before or 45 min after TBI. Rotarod performance was tested at days 1-5 and maze performance on days 11-15 and 40-44 after M-S treatment. The 7-day studies showed M-S treatment (p<0.01) reduced CA1 neuronal death better than either S or M alone. Behavioral groups had inadvertent post-ischemic hypothermia that decreased CA1 death and likely influenced behavioral morbidity. M-S given before TBI (p<0.01) decreased memory deficits on day 15, while M-S treatment given after TBI was ineffective. Unexpectedly, M-S treatment before or after TBI produced transient motor deficits (p<0. 01). Memory improvement occurred independent of CA1 death.

Traumatic brain injury in rats results in increased expression of Gap-43 that correlates with behavioral recovery.

Traumatic brain injury is associated with behavioral deficits, often in the absence of histopathological or ultrastructural changes. To determine whether membrane remodeling occurs, immunocytochemical techniques were used and the density and distribution of GAP-43 were measured. GAP-43 is a membrane-bound protein, which, when phosphorylated, is thought to regulate metabolic pathways involved in membrane remodeling and neurite growth. Moderate central fluid percussion injury (FPI, 1.9-2.2 atm.) was performed on anesthetized, spontaneously hypertensive Wistar rats (SHR). Behavioral reflex recovery was consistent with moderate levels of brain injury. One, 3, 5, 7 and 9 days after injury, both sham control (n = 4) and FPI (n = 4) animals were sacrificed, the brains were removed, cryosectioned and processed. Density measurements were taken from histological sections taken at interaural 6.20 mm and bregma -2.80 mm and were found to be statistically greater (P < 0.05) than background grey matter readings in the agranular cortices, the frontal, hindlimb, parietal 1 and 2 cortices, and the hippocampus and dentate gyrus, excluding the pyramidal and granular cell layers. Density measurements taken in forelimb and hindlimb cortical regions correlate with forelimb and hindlimb recovery in foot-fault and beam balance tests (P < 0.05). We interpret these data to indicate neuronal membrane remodeling as a result of the disruption of neuronal membranes due to the impact and shearing forces associated with the FPI. The disruption and remodeling of neuronal membranes are in areas that are consistent with the loss and recovery of locomotor and spatial behavior as a result of FPI.

Mild traumatic brain injury does not modify the cerebral blood flow profile of secondary forebrain ischemia in Wistar rats.

The rat hippocampus is hypersensitive to secondary cerebral ischemia after mild traumatic brain injury (TBI). An unconfirmed assumption in previous studies of mild TBI followed by forebrain ischemia has been that antecedent TBI did not alter cerebral blood flow (CBF) dynamics in response to secondary ischemia. Using laser Doppler flowmetry (LDF), relative changes in regional hippocampal CA1 blood flow (hCBF) were recorded continuously to quantitatively characterize hCBF before, during, and after 6 min of forebrain ischemia in either normal or mildly traumatized rats. Two experimental groups of fasted male Wistar rats were compared. Group 1 (n = 6) rats were given 6 minutes of transient forebrain ischemia using bilateral carotid clamping and hemorrhagic hypotension. Group 2 (n = 6) rats were subjected to mild (0.8 atm) fluid percussion TBI followed 1 h after trauma by 6 min of transient forebrain ischemia. The laser Doppler flow probe was inserted stereotactically to measure CA1 blood flow. The electroencephalogram (EEG) was continuously recorded. During the forebrain ischemic insult there were no intergroup differences in the magnitude or duration of the decrease in CBF in CA1. In both groups, CBF returned to preischemic values within one minute of reperfusion but traumatized rats had no initial hyperemia. There were no intergroup differences in the CBF threshold when the EEG became isoelectric. These data suggest that the ischemic insult was comparable either with or without antecedent TBI in this model. This confirms that this model of TBI followed by forebrain ischemia is well suited for evaluating changes in the sensitivity of CA1 neurons to cerebral ischemia rather than assessing differences in relative ischemia.

Hypothermic modulation of cerebral ischemic injury during cardiopulmonary bypass in pigs.

BACKGROUND: The aim of this study was to determine whether progressive levels of hypothermia (37, 34, 31, or 28 degrees C) during cardiopulmonary bypass (CPB) in pigs reduce the physiologic and metabolic consequences of global cerebral ischemia. METHODS: Sagittal sinus and cortical microdialysis catheters were inserted into anesthetized pigs. Animals were placed on CPB and randomly assigned to 37 degrees C (n = 10), 34 degrees C (n = 10), 31 degrees C (n = 11), or 28 degrees C (n = 10) management. Next 20 min of global cerebral ischemia was produced by temporarily ligating the innominate and left subclavian arteries, followed by reperfusion, rewarming, and termination of CPB. Cerebral oxygen metabolism (CMRO2) was calculated by cerebral blood flow (radioactive microspheres) and arteriovenous oxygen content gradient. Cortical excitatory amino acids (EAA) by microdialysis were measured using high-performance liquid chromatography. Electroencephalographic (EEG) signals were graded by observers blinded to the protocol. After CPB, cerebrospinal fluid was sampled to test for S-100 protein and the cerebral cortex was biopsied. RESULTS: Cerebral oxygen metabolism increased after rewarming from 28 degrees C, 31 degrees C, and 34 degrees C CPB but not in the 37 degrees animals; CMRO2 remained lower with 37 degrees C (1.8 +/- 0.2 ml x min[-1] x 100 g[-1]) than with 28 degrees C (3.1 +/- 0.1 ml x min[-1] x 100 g[-1]; P < 0.05). The EEG scores after CPB were depressed in all groups and remained significantly lower in the 37 degrees C animals. With 28 degrees C and 31 degrees C CPB, EAA concentrations did not change. In contrast, glutamate increased by sixfold during ischemia at 37 degrees C and remained significantly greater during reperfusion in the 34 degrees C and 37 degrees C groups. Cortical biopsy specimens showed no intergroup differences in energy metabolites except two to three times greater brain lactate in the 37 degrees C animals. S-100 protein in cerebrospinal fluid was greater in the 37 degrees C (6 +/- 0.9 microg/l) and 34 degrees C (3.5 +/- 0.5 microg/l) groups than the 31 degrees C (1.9 +/- 0.1 microg/l) and 28 degrees C (1.7 +/- 0.2 microg/l) animals. CONCLUSIONS: Hypothermia to 28 degrees C and 31 degrees C provides significant cerebral recovery from 20 min of global ischemia during CPB in terms of EAA release, EEG and cerebral metabolic recovery, and S-100 protein release without greater advantage from cooling to 28 degrees C compared with 31 degrees C. In contrast, ischemia during 34 degrees C and particularly 37 degrees C CPB showed greater EAA release and evidence of neurologic morbidity. Cooling to 31 degrees C was necessary to improve acute recovery during global cerebral ischemia on CPB.

Cerebral metabolic consequences of hypotensive challenges in hemodiluted pigs with and without cardiopulmonary bypass.

We tested the hypothesis that progressive aortic hypotension with bicarotid occlusion produces greater reductions in cerebral blood flow (CBF) and more flow-metabolism mismatching with hemodilution during cardiopulmonary bypass (CPB) than with hemodilution alone. In Yorkshire pigs randomized to hemodilution with CPB (n = 10) or hemodilution without CPB (control; n = 9), the effects of bicarotid ligation and graded hypotension on CBF (microspheres), the electroencephalogram (EEG), and cortical energy metabolites were examined. After bicarotid ligation, systemic flow was reduced for 15-min intervals of 80, 60, and 40 mm Hg aortic pressure, followed by a cortical brain biopsy. At baseline, CBF was lower in CPB (58 +/- 3 mL.100g-1.min-1) than control (90 +/- 3 mL.100 g-1.min-1., P < 0.05) animals, as was cerebral oxygen metabolism (3.1 +/- 0.1 vs 4.2 +/- 0.2 mL.min-1.100g-1; P < 0.05). Although CBF remained 40% lower at each level of hypotension in CPB than control animals (P < 0.05), EEG scores showed no intergroup differences, indicating similar flow-metabolism matching. Brain metabolites were similar between CPB and control groups (adenosine triphosphate, 9.6 +/- 2.4 vs 12.4 +/- 1.9 mumol/g; adenosine diphosphate, 6.0 +/- 0.7 vs 6.3 +/- 0.4 mumol/g; adenosine monophosphate, 4.8 +/- 0.9 vs 3.8 +/- 0.8 mumol/g; creatine phosphate, 8.3 +/- 1.8 vs 7.9 +/- 1.0 mumol/g; and lactate, 178.4 +/- 20.2 vs 150.8 +/- 13.9 mumol/g). Thus, despite significantly lower CBF during hypotension with bicarotid occlusion in hemodiluted animals during normothermic CPB, cortical electrical activity and the balance between flow and metabolism did not differ from those in control animals without CPB.

Time course of increased vulnerability of cholinergic neurotransmission following traumatic brain injury in the rat.

We have previously shown that spatial memory changes following experimental traumatic brain injury (TBI) include long-term changes that are (1) 'overt': detected by routine behavioral assessments, or (2) 'covert': undetected in the absence of a secondary pharmacological challenge, such as by the cholinergic antagonist, scopolamine. Our objective in this study was to extend this finding by characterizing the time course of recovery of overt and covert spatial memory performance following two magnitudes of experimental TBI. The Morris water maze was used to assess cognitive performance. Rats received either moderate magnitude (6 m/s, 1.77 mm deformation) or low magnitude (6 m/s, 1 mm deformation) impacts through a lateral craniectomy under isoflurane anesthesia. Sham rats underwent identical surgical procedures but were not injured. To avoid motor deficits, water maze testing started two weeks post-injury. Rats were given four trials per day for seven consecutive days. For each trial, latency to find a hidden platform was timed. On the sixth, rats were injected (i.p.) with scopolamine (1 mg/kg) 15 min prior to maze testing. The next day, rats were retested. This testing regimen was repeated, beginning 4, 6, and 10 weeks post-TBI. Results showed that, while the low-magnitude injury produced no overt spatial memory deficits, the moderate-magnitude group exhibited overt deficits during the first test regimen. Also, while both injury magnitudes produced an enhanced sensitivity to spatial memory impairment by scopolamine at two weeks post-TBI, this covert deficit persisted only in the severe group at 4, 6, and 10 weeks post-TBI. Qualitative light microscopy showed that both injury groups had graded cortical necrosis. However, underlying subcortical structures such as the hippocampus appeared intact, with no overt cellular or parenchymal damage to the neuropil. These data suggest three distinct stages of functional recovery: (1) the initial period when overt deficits are present, (2) a period following recovery from overt deficits within which covert deficits can be reinstated by a pharmacological challenge, and (3) a period following recovery from both overt and covert deficits. Covert deficits can persist long after the recovery of overt deficits and, like other neurological deficits, the rate of recovery is dependent on the magnitude of TBI. Finally, spatial memory deficits can occur in the absence of light microscopic evidence of cell death in the hippocampus.

Are the pathobiological changes evoked by traumatic brain injury immediate and irreversible?

Traumatic brain injury has long been thought to evoke immediate and irreversible damage to the brain parenchyma and its intrinsic vasculature. In this review, we call into question the correctness of this assumption by citing two traumatically related brain parenchymal abnormalities that are the result of a progressive, traumatically induced perturbation. In this context, we first consider the pathogenesis of traumatically induced axonal damage to show that it is not the immediate consequence of traumatic tissue tearing. Rather, we illustrate that it is a delayed consequence of complex axolemmal and/or cytoskeletal changes evoked by the traumatic episode which then lead to cytoskeletal collapse and impairment of axoplasmic transport, ultimately progressing to axonal swelling and disconnection. Second, we consider the traumatized brain's increased neuronal sensitivity to secondary ischemic insult by showing that even after mild traumatic brain injury, CA1 neuronal cell loss can be precipitated by the induction of sublethal ischemic insult within 24 hrs of injury. In demonstrating this increased sensitivity to secondary insult, evidence is provided that it is triggered by the neurotransmitter storm evoked by traumatic brain injury, allowing for sublethal neuro-excitation. In relation to this phenomenon, the protective effect of receptor antagonists are discussed, as well as the concept that this relatively prolonged posttraumatic brain hypersensitivity offers a potential window for therapeutic intervention. Collectively, it is felt that both examples of the brain parenchyma's response to traumatic brain injury show that the resulting pathobiology is much more complex and progressive than previously envisioned, and as such, rejects many of the previous beliefs regarding the pathobiology of traumatic brain injury.

Enhanced vulnerability to secondary ischemic insults after experimental traumatic brain injury.

Both experimental traumatic brain injury and clinical traumatic brain injury appear to render the brain more vulnerable to a second ischemic insult. The mechanisms of enhanced vulnerability to subsequent ischemia appear to include a reduced ability to increase cerebral blood flow in response to hypotension, hypoxemia, or acute anemia and increased tissue sensitivity to ischemia. Although numerous mediators may be involved in increased tissue sensitivity, those that particularly merit investigation include oxygen free radicals, glutamate, arachidonate metabolites, calcium ions, and protein kinase C.

Hyperglycemia during hypothermic canine cardiopulmonary bypass increases cerebral lactate.

BACKGROUND: Hyperglycemia frequently occurs during cardiopulmonary bypass (CPB), although its direct effects on cerebral perfusion and metabolism are not known. Using a canine model of hypothermic CPB, we tested whether hyperglycemia alters cerebral blood flow and metabolism and cerebral energy charge. METHODS: Twenty anesthetized dogs were randomized into hyperglycemic (n = 10) and normoglycemic (n = 10) groups. The hyperglycemic group received an infusion of D50W, and the normoglycemic animals received an equal volume of 0.9% NaCl. Both groups underwent 120 min of hypothermic (28 degrees C) CPB using membrane oxygenators, followed by rewarming and termination of CPB. Cerebral blood flow (radioactive microspheres) and the cerebral metabolic rate for oxygen were measured intermittently during the experiment and brain tissue metabolites were obtained after bypass. RESULTS: Before CPB, the glucose-treated animals had higher serum glucose levels (534 +/- 12 mg/dL; mean +/- SE) than controls (103 +/- 4 mg/dL; P < 0.05), and this difference was maintained throughout the study. Cerebral blood flow and metabolism did not differ between groups at any time during the experiment. Sagittal sinus pressure was comparable between groups throughout CPB. Tissue high-energy phosphates and water contents were similar after CPB, although cerebral lactate levels were greater in hyperglycemic (37.2 +/- 5.7 mumol/g) than normoglycemic animals (19.7 +/- 3.7 mumol/g; P < 0.05). After CPB, pH values of cerebrospinal fluid for normoglycemic (7.33 +/- 0.01) and hyperglycemic (7.34 +/- 0.01) groups were similar. CONCLUSIONS: Hyperglycemia during CPB significantly increases cerebral lactate levels without adversely affecting cerebral blood flow and metabolism, cerebrospinal fluid pH, or cerebral energy charge.