Metabotropic glutamate receptor protein alterations after traumatic brain injury in rats.

Glutamate toxicity, mediated via ion channel-linked receptors, plays a key role in traumatic brain injury (TBI) pathophysiology. Excessive glutamate release after TBI also activates protein G-linked metabotropic glutamate receptors (mGluRs). We performed Western blot and immunohistochemical analysis with antibodies for group 1 and 2 mGluRs in hippocampal and cortex tissue at 7 and 15 days after lateral fluid-percussion TBI in rats. Protein homogenates of brain tissue were separated on 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to nitrocellulose, and incubated with either antibodies recognizing both mGluR2 and mGluR3 or antibodies against mGluR5. Equivalent protein loading of lanes was confirmed by using beta-actin antibody. Immunoreactive proteins were revealed with enhanced chemiluminescence and relative optical density of Western blots quantified by computerized image analysis. At 7 days after TBI, mGluR2/3 immunobinding ipsilateral to the fluid-percussion injury was reduced by 28% in hippocampus and 25% in cortex in comparison with the contralateral hemisphere (p < .05). mGluR5 immunobinding ipsilateral to the fluid-percussion injury was reduced by 20% in hippocampus and 27% in cortex (p < .05). At 15 days after TBI, the decreases in immunobinding were no longer significant. Immunohistochemical staining with the same antibodies revealed density changes congruent with the Western blot results. These data suggest that TBI produces an alteration in receptor protein expression that spontaneously recovers by 15 days after injury.

Differential consequences of lateral and central fluid percussion brain injury on receptor coupling in rat hippocampus.

We have identified alterations in the responses of muscarinic and metabotropic receptors in rat hippocampus that persist for at least 15 days after central fluid percussion injury. This study compares the effect of lateral fluid percussion and central fluid percussion on these responses. Moderate injury was obtained by displacement and deformation of the brain within the closed cranial cavity using a fluid percussion device positioned either centrally or laterally. Carbachol and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated polyphosphoinositide (PPI) hydrolysis was assayed in hippocampus from injured and sham-injured controls at 15 days following injury. At 15 days after central fluid percussion traumatic brain injury (TBI), the response to carbachol was enhanced by 30% and the response to trans-ACPD was enhanced by 75% compared to sham-injured animals. At 15 days after lateral fluid percussion TBI the response to trans-ACPD was enhanced by 40% both ipsilateral and contralateral to the side of injury. In contrast, the response to carbachol was enhanced by 29% contralateral to the side of injury but was diminished by 12% ipsilateral to the side of injury. Cresyl violet staining shows no hippocampal cell death after central fluid percussion injury or on the side contralateral to lateral fluid percussion injury but on the ipsilateral side cell death was identified in hippocampal area CA3. Thus, abnormal hippocampal cell signaling through the phosphoinositide pathway occurs in the absence of cell death and may contribute to cognitive impairment.

ICP monitoring in the rat: comparison of monitoring in the ventricle, brain parenchyma, and cisterna magna.

Various methods of continuous intracranial pressure (ICP) monitoring during experimental procedures in the rat have been described. However, no systematic comparison of ICP monitoring in the ventricle, brain parenchyma, and cisterna magna has been reported. Since accurate and reliable ICP measurements are important in experimental models of traumatic brain injury, the present study was conducted to compare simultaneous ICP measurements from ventricular, cisterna magna, and intraparenchymal monitors during ICP changes. Subdural hematoma was produced by infusion of 0.3 ml of autologous blood into the subdural space over 6 min. The ventricular and the intraparenchymal fiberoptic catheter produced reliable and comparable pressure recordings, that did not statistically differ (p = 0.4), throughout the one hour monitoring time. In contrast, the cisterna magna catheter was less reliable and produced significantly lower readings throughout the monitoring time (p<0.001). The intraparenchymal device produced greater cortical damage than the ventricular catheter. In conclusion, ventricular ICP monitoring is the preferred method under these circumstances, since it is accurate and induces least brain damage.

Comparison of behavioral deficits and acute neuronal degeneration in rat lateral fluid percussion and weight-drop brain injury models.

The behavioral and histological effects of the lateral fluid percussion (LFP) brain injury model were compared with the weight drop impact-acceleration model with 10 min of secondary hypoxia (WDIA + H). LFP injury resulted in significant motor deficits on the beam walk and inclined plane, and memory deficits on the radial arm maze and Morris water maze. Motor deficits following LFP remained throughout 6 weeks of behavioral testing. WDIA + H injury produced significant motor deficits on the beam walk and inclined plane immediately following injury, but these effects were transient and recovered by 14 days post-injury. In contrast to the LFP injury, the WDIA + H injured animals showed no memory deficits on the radial arm maze and Morris water maze. In order to determine if the differences in behavioral outcome between models were due to differences in injury mechanism or injury severity, 10 LFP-injured animals were matched with 10 WDIA-injured animals based on injury severity (i.e., time to regain righting reflex after brain injury). The LFP-matched injury group showed greater impairment than the WDIA + H matched injury group on the radial arm maze and Morris water maze. Histological examination of LFP-injured brains with Fluoro-Jade staining 24 h, 48 h, and 7 days post-injury revealed degenerating neurons in the cortex, thalamus, hippocampus, caudate-putamen, brainstem, and cerebellum, with degenerating fibers tracts in the corpus callosum and other major tracts throughout the brain. Fluoro-Jade staining following WDIA+H injury revealed damage to fibers in the optic tract, lateral olfactory tract, corpus callosum, anterior commissure, caudate-putamen, brain stem, and cerebellum. While both models produce reliable and characteristic behavioral and neuronal pathologies, their differences are important to consider when choosing a brain injury model.

Effects of mu opioid agonist and antagonist on neurological outcome following traumatic brain injury in the rat.

We examined the effects of an exogenous mu opioid agonist and antagonist on systemic physiology and neurological outcome following TBI in the rat. Experiment I: [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) (0.1 nMol or 0.3 nMol in 5 microliters) (n = 10) or artificial CSF (n = 10) was administered 5 min prior to fluid-percussion brain injury (2.1 atmospheres). Motor performance was assessed on days 1-5 after TBI. The mu receptor agonist, DAMGO significantly reduced both beam-walking latency and body weight loss after injury (p < 0.05). DAMGO-treated rats (n = 5) did not differ from CSF-treated rats (n = 5) on either systemic arterial blood pressure or heart rate responses to injury. Experiment II: Beta-funaltrexamine (beta-FNA) (20.0 nMol in 5.0 microliters) (n = 10) or artificial CSF (n = 10) was administered (icv) to rats 5 min prior to fluid-percussion brain injury (1.8 atmospheres). Motor performance was assessed on days 1-5 after TBI. The mu receptor antagonist, beta-FNA, significantly increased beam-walking latency after injury (p < 0.05). beta-FNA-treated rats (n = 5) did not differ from CSF-treated rats (n = 5) on either systemic arterial blood pressure or heart rate responses to injury. Experiment III: Neither beta-FNA nor DAMGO affected motor performance in uninjured rats. These results suggest that activation of mu opioid receptors by exogenous agonists may provide protection against deficits in motor performance produced by fluid percussion brain injury.

Metabotropic glutamate antagonist, MCPG, treatment of traumatic brain injury in rats.

The metabotropic glutamate receptor (mGluR) antagonist, alpha-methyl-4-carboxyphenylglycine (MCPG) was administered into the left lateral ventricle 5 min prior to fluid percussion traumatic brain injury (TBI) in the rat. A single 5.0 microliters ventricular infusion of the active isomer. (+)-MCPG (0.2 mumol), significantly reduced beam walking motor deficits on days 1-5 after injury and learning/memory deficits measured on days 11-15 after injury. Neither a lower dose of (+)-MCPG (0.2 mumol) affected behavioral outcome. (+)-MCPG (0.2 mumol) did not affect systemic hemodynamic responses to injury. These results suggest that TBI induced activation of mGluRs contributes to behavioral morbidity and that blockade of certain mGluR subtypes (mGluR1, mGluR5 and/or mGluR2) may reduce these pathophysiological responses.

Dicyclomine, an M1 muscarinic antagonist, reduces infarct volume in a rat subdural hematoma model.

The rat subdural hematoma (SDH) model produces a zone of ischemic brain damage within the hemisphere beneath the SDH. Previous studies have measured large increases in extracellular acetylcholine during cerebral ischemia in the rat. We examined infarct volume after selectively blocking muscarinic M1 receptors with dicyclomine during SDH. Rats were anesthetized with isoflurane (2%), intubated, and femoral artery and vein cannulated. Autologous blood (0.375 ml) was injected (0.05 ml/min) under the dura of the right parietal cortex. Dicyclomine (5 mg/kg, i.v.) was injected at 5 min after and again at 2 h after completion of the subdural blood infusion. Blood pressure and intracranial pressure (ICP) were continuously measured. At 4 h after SDH rats were euthanized, brains sectioned, and immunoreacted with glia fibrillary acidic protein. Cortical infarct volume was quantified in coronal brain sections at 0.7-mm intervals from +1.0 mm to -3.9 mm relative to bregma. Infarct volume in drug-treated rats (n = 10) 22.1 +/- 6.99 mm3 was significantly smaller (p < 0.02) than vehicle treated rats (n = 10) 56.7 +/- 9.59 mm3. ICP, blood pressure and cerebral perfusion pressure were not significantly different between groups. These data suggest that activation of M1 muscarinic receptors during an ischemic event may contribute to the development of subsequent pathology.

Effects of muscarinic receptor antagonism on the phosphatidylinositol bisphosphate signal transduction pathway after experimental brain injury.

Hippocampal levels of fatty acids extracted from phosphatidylinositol 4,5-bisphosphate (PIP2), free fatty acids (FFA), and lactate were measured after central fluid percussion traumatic brain injury (TBI) in rats. At 5 min after injury, there was a decrease in fatty acids extracted from PIP2 suggesting a decrease in PIP2. At the same time point, total FFA increased in saline-treated TBI rats. Levels of arachidonic acid were significantly decreased in PIP2, while at the same time arachidonic and stearic acids increased in FFA in saline-treated TBI rats. No significant alterations in PIP2-derived fatty acids or FFA were observed at 20 min after TBI. Hippocampal concentrations of lactate were significantly elevated at 5 and 20 min after injury in saline-treated rats. In general, these alterations were blunted by preinjury administration of the muscarinic antagonist, scopolamine. These results suggest that the PIP2 signal transduction pathway is activated in the hippocampus at the onset of central fluid percussion TBI and that the enhanced phospholipase C-catalyzed phosphodiestric breakdown of PIP2 is a major mechanism of liberation of FFA in these sites immediately after such injury. The blunting of PIP2 and FFA alterations in animals treated with scopolamine suggests that activation of muscarinic receptors significantly contributes to the phospholipase C (PLC) signal transduction pathophysiology in TBI. The attenuation of lactate accumulation in scopolamine-treated rats suggests that TBI-induced muscarinic receptor activation also contributes to increased glycolytic metabolism and/or ionic imbalances.

The effect of groups II and III metabotropic glutamate receptor activation on neuronal injury in a rodent model of traumatic brain injury.

OBJECTIVE: The role of metabotropic glutamate receptor activation after traumatic brain injury (TBI) is not well understood. In vitro studies suggest that activation of Groups II and III metabotropic glutamate receptors may provide some degree of neuroprotection and may be potential targets for the development of therapeutic strategies. Thus, we examined the effects of Group II and Group III selective agonists on neuronal degeneration after in vivo TBI. METHODS: Fifty male Sprague-Dawley rats were subjected to lateral fluid percussion brain injury immediately followed by an intracranial injection of 2-(2',3')-dicarboxycyclopropylglycine (DCG-IV) (Group II) or (R,S)-4-phosphonophenylglycine (Group III) in the CA2 and CA3 areas of the hippocampus. DCG-IV was injected at doses of 20 fmol, 100 fmol, and 500 fmol, and (R,S)-4-phosphonophenylglycine was injected at 8 nmol, 40 nmol, and 200 nmol. Vehicle injection control groups were used for comparison with each drug group. All animals were killed 24 hours after TBI was induced. Four 50-microm brain sections were obtained from each animal and stained for degenerating neurons with the fluorochrome Fluoro-Jade. Two independent, blinded investigators counted the number of degenerating (Fluoro-Jade-positive) neurons in the CA2 and CA3 areas of the hippocampus of each brain section. RESULTS: Compared with vehicle, the 500-fmol dose of DCG-IV significantly reduced the number of Fluoro-Jade-positive degenerating neurons (P < 0.001). Lower doses of DCG-IV were associated with a decreased but not statistically significant number of Fluoro-Jade-positive neurons. In contrast, (R,S)-4-phosphonophenylglycine had no significant effect on the number of degenerating neurons. CONCLUSION: Administration of selective Group II metabotropic glutamate receptor agonists protects neurons against in vivo TBI. These receptors may thus be a promising target for future neuroprotective drugs.

Brain injury-induced enhanced limbic epileptogenesis: anatomical and physiological parallels to an animal model of temporal lobe epilepsy.

Traumatic brain injury (TBI) is a leading cause of symptomatic epilepsy in young adults. This study examined physiological and anatomical epileptogenic consequences of a prior incident of TBI in rats. Rats were subjected to a fluid percussion brain injury one week prior to experimentation, and in vitro electrophysiological recording studies were conducted using combined hippocampal-entorhinal cortical slices (HEC slices). Results were compared to sham operated controls and rats in which a condition of chronic temporal lobe epilepsy was induced by a 2 h bout of pilocarpine-induced status epilepticus 2 months prior to recording (PILO). In field potential recording, PILO HEC slices evidenced a greater degree of disinhibition in Ca1 than did TBI or control slices. TBI slices showed greater disinhibition in the dentate gyrus than did PILO or control rats. In in vitro kindling experiments, 86% of TBI HEC slices generated self-sustaining epileptic activity within 9 stimulus trains. This type of activity was never triggered in control slices. HEC slices prepared from PILO animals generated self-sustaining epileptic activity with fewer stimulus trains than did TBI slices. In anatomical studies, both TBI and PILO hippocampi evidenced significant loss of neurons within the hilar region. TBI induces a series of changes within the limbic system of rats, which are qualitatively similar in many aspects but quantitatively less severe than changes seen in rats with chronic temporal lobe epilepsy. These physiological and anatomical TBI-associated alterations in the limbic system may contribute to the development of epilepsy following head trauma.

Group I metabotropic glutamate antagonist reduces acute neuronal degeneration and behavioral deficits after traumatic brain injury in rats.

Recent studies indicate that acute activation of Group I mGluRs following traumatic brain injury (TBI) contributes to the ensuing pathophysiology. The present study examined the effects of post-TBI administration of the selective mGluR1 antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) on acute neuronal degeneration in the hippocampus and long-term sensorimotor and learning/memory outcome. In Experiment 1, 26 rats received 0.4, 2.0, or 10.0 nmol AIDA or artificial CSF vehicle infusion into the hippocampus starting 5 min postinjury. At 24 h after TBI characteristic pyramidal cell degeneration was observed in Fluoro-Jade-stained coronal sections of the CA2/3 sectors of the dorsal hippocampus. The mean (+/-SEM) number of Fluoro-Jade-positive neurons in the 10 nmol AIDA group (184 +/- 32) was significantly less (P < 0.05) than the vehicle group (310 +/- 47). In Experiment 2, 20 rats were trained on sensorimotor and memory tasks prior to parasagittal fluid percussion TBI. Rats were administered 10 nmol AIDA or vehicle as in Experiment 1. Rats were assessed on beam walking and radial arm maze (RAM) performance weekly for 6 weeks after TBI. Acquisition of a Morris water maze (MWM) task was assessed on days 11-15 after TBI. The AIDA-treated group had significantly reduced deficits in beam walk, MWM, and RAM performance compared to the vehicle-treated group. These data indicate that injury-induced acute activation of mGluR1 receptors contributes to both the cellular pathology and the behavioral morbidity associated with TBI.

Inhibition of nitric oxide synthase potentiates hypertension and increases mortality in traumatically brain-injured rats.

We examined the effects of N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), on mortality, morbidity, and cardiovascular parameters following traumatic brain injury (TBI) in the rat. Rats were anesthetized with 2% isoflurane prior to moderate (2.0 atmosphere), central fluid percussion TBI. Temporalis muscle temperature was maintained at 37 +/- 0.5 degrees C. L-NAME (10 mg/kg iv) was administered once at either 5 min before, 5 min after, or 15 min after TBI. Sensorimotor deficits and spatial learning/ memory deficits were assessed after injury. Separate groups of rats were monitored for cardiovascular parameters. Preinjury administration of L-NAME significantly increased mortality from 13 (vehicle) to 70% (associated with pulmonary edema), whereas postinjury, L-NAME had no effect on mortality (14 and 25%). L-NAME administered at 5 or 15 min after injury had no significant effect on motor performance or cognitive performance deficits associated with TBI. L-NAME in uninjured rats increased arterial blood pressure by 25 mmHg within 2 min. L-NAME injected 5 min before TBI greatly prolonged the hypertensive episode associated with TBI (1 min in vehicle vs 60 min in L-NAME). L-NAME injected 5 min after TBI caused a sustained 35 mmHg increase in blood pressure. These findings suggest that acute inhibition of NOS has detrimental consequences on mortality that may be owing to its cardiovascular effects.

[Low-affinity binding sites of glucocorticoid].

The low-affinity glucocorticoid binding sites (LAGS) with steroid specificity were demonstrated in hepatic cytosol, cerebral cytosol, and thymocytes of rat. The Kd of LAGS was 1-10 mumol.L-1 as estimated by Scatchard, pseudo-scatchard, and competitive analysis. The specific binding of dexamethasone 1-100 nmol.L-1 was roughly parallel with its inhibition of [3H]UdR incorporation in thymocytes of rat, and both were blocked by mifepristone (RU-486), the competitive antagonist of glucocorticoids. These results suggest that the pharmacological activity of glucocorticoid might be mediated at least partially by LAGS.