Postinjury magnesium treatment attenuates traumatic brain injury-induced cortical induction of p53 mRNA in rats.

Administration of magnesium has been shown to be neuroprotective in experimental models of traumatic brain injury (TBI). The present study examined the effect of magnesium on posttraumatic regional induction of p53, a gene associated with induction of cell death. Male Sprague-Dawley rats (350-400 g, n = 26) were anesthetized with sodium pentobarbital and subjected to either lateral fluid percussion brain injury of moderate severity (2.4-2.6 atm; n = 22) or sham surgery (n = 4). At 15 min postinjury, animals randomly received an intravenous bolus of either 125 micromol magnesium chloride (n = 12) or saline vehicle (n = 10). Expression of p53 mRNA was not observed in any uninjured animal. By 6 h postinjury in vehicle-treated, brain-injured animals, p53 mRNA was induced in the cortex, dentate hilus, and CA3 regions of the hippocampus and geniculate nuclei of the thalamus, ipsilateral to the impact site. Posttraumatic magnesium treatment significantly reduced the number of labeled cells in the injured cortex (P < 0.05), but not in the hippocampus or thalamus. p53 mRNA expression returned to near baseline in all animals by 24 h postinjury. These data suggest that the neuroprotective effects of magnesium treatment may be related, in part, to a downregulation in expression of a gene associated with induction of cell death and further support the utility of magnesium as a pharmacotherapy for TBI.

Survival and integration of transplanted postmitotic human neurons following experimental brain injury in immunocompetent rats.

OBJECT: Limitations regarding cell homogeneity and survivability do not affect neuronlike hNT cells, which are derived from a human teratocarcinoma cell line (Ntera2) that differentiates into postmitotic neurons with exposure to retinoic acid. Because NT2N neurons survive longer than 1 year after transplantation into nude mice brains, the authors grafted these cells into the brains of immunocompetent rats following lateral fluid-percussion brain injury to determine the long-term survivability of NT2N cell grafts in cortices damaged by traumatic brain injury (TBI) and the therapeutic effect of NT2N neurons on cognitive and motor deficits. METHODS: Seventy-two adult male Sprague-Dawley rats, each weighing between 340 and 370 g, were given an anesthetic agent and subjected to lateral fluid percussion brain injury of moderate severity (2.2-2.5 atm in 46 rats) or to surgery without TBI (shamoperation, 26 rats). Twenty-four hours postinjury, 10(5) NT2N cells (24 injured animals) or 3 microl of vehicle (22 injured and 14 control animals) was stereotactically implanted into the periinjured or control cerebral cortex. Motor function was assessed at weekly intervals and all animals were killed at 2 or 4 weeks after their posttraumatic learning ability was assessed using a Morris water maze paradigm. Viable NT2N grafts were routinely observed to extend human neural cell adhesion molecule-(MOC-1)immunoreactive processes into the periinjured cortex at 2 and 4 weeks posttransplantation, although no significant improvement in motor or cognitive function was noted. Inflammation identified around the transplant at both time points was assessed by immunohistochemical identification of macrophages (ED-1) and microglia (isolectin B4). CONCLUSIONS: Long-term survival and integration of NT2N cells in the periinjured cortex of immunocompetent rats provides the researcher with an important cellular system that can be used to study maturation, regulation, and neurite outgrowth of transplanted neurons following TBI.

Terminally differentiated human neurons survive and integrate following transplantation into the traumatically injured rat brain.

The present study evaluated the survival and integration of human postmitotic neurons (hNT) following transplantation into the traumatically injured rodent brain. Anesthetized male Sprague-Dawley rats (n = 47) were subjected to lateral fluid percussion brain injury of moderate severity (2.4-2.6 atm). Sham animals (n = 28) were surgically prepared, but did not receive brain injury. At 24 h following injury or sham surgery, the rats were re-anesthetized and approximately 100,000 hNT cells (freshly cultured or previously frozen) or vehicle were stereotactically injected into the ipsilateral cortex. Animals were examined for neuromotor function at 48 h, 7 days, and 14 days posttransplantation using a standard battery of motor tests. Animals were sacrificed at 2 weeks postinjury and viability of hNT grafts was assessed by Nissl staining and MOC-1 immunohistochemistry, which recognizes human neural cell adhesion molecules (NCAM) expressed on hNT cells. Transplanted hNT grafts remained viable in 83% of brain-injured animals at 2 weeks following transplantation of either fresh or frozen hNT cells. Glial fibrillary acidic protein (GFAP) immunohistochemistry revealed a marked increase in the number of reactive astrocytes following brain injury in both vehicle and hNT implanted animals. These reactive astrocytes appeared not to impede grafted cells from sending projections into host tissue. Despite the survival of transplanted cells in the traumatically injured brain, hNT cells had no significant effect on posttraumatic neurologic motor function during the acute posttraumatic period. Since hNT cells are transfectable, prolonged survival of these transplanted cells in the posttraumatic milieu suggests that grafted hNT cells may be a suitable means for delivery of therapeutic, exogenous proteins into the CNS for treatment of traumatic brain injury.

GABAA receptor activation attenuates excitotoxicity but exacerbates oxygen-glucose deprivation-induced neuronal injury in vitro.

We examined the effects of GABA receptor stimulation on the neuronal death induced by exogenously added excitatory amino acids or combined oxygen-glucose deprivation in mouse cortical cell cultures. Death induced by exposure to NMDA, AMPA, or kainate was attenuated by addition of GABA or the GABAA receptor agonist, muscimol, but not by the GABAB receptor agonist, baclofen. The antiexcitotoxic effect of GABAA receptor agonists was blocked by bicuculline or picrotoxin. In contrast, GABA or muscimol, but not baclofen, markedly increased the neuronal death induced by oxygen-glucose deprivation. Muscimol potentiation of neuronal death was associated with increased glutamate efflux to the bathing medium, and increased cellular 45Ca2+ accumulation; it was blocked by MK-801, but not NBQX, suggesting mediation by NMDA receptors. Bicuculline only weakly attenuated muscimol potentiation of oxygen-glucose deprivation-induced neuronal death, probably because it itself increased this death. Present results raise a note of caution in the proposed use of GABAA receptor stimulation to limit ischemic brain damage in vivo.

Potentiation of oxygen-glucose deprivation-induced neuronal death after induction of iNOS.

BACKGROUND AND PURPOSE: Previous studies have shown that brain ischemia and other insults can induce a marked increase in inducible nitric oxide synthase (iNOS) expression in astrocytes and some immune cells, but the biological significance of this phenomenon has not been elucidated. The purpose of the present study was to determine whether this induction of astrocyte iNOS alters neuronal vulnerability to severe hypoxic insults. METHODS: Astrocytic iNOS was induced by exposure of murine cortical cultures to interferon gamma in combination with either interleukin-1 beta or lipopolysaccharide. Cultures were exposed to combined oxygen-glucose deprivation. The extracellular concentration of glutamate was measured by high-performance liquid chromatography. N-Methyl-D-aspartate (NMDA) receptor activity was assessed by measurement of 45Ca2+ influx: neuronal death was assessed by morphological examination and quantitated by measurement of lactate dehydrogenase efflux to the bathing medium. RESULTS: In murine neocortical cell cultures containing neurons and astrocytes, neuronal injury induced by combined oxygen-glucose deprivation was not reduced by the addition of the nitric oxide synthase inhibitors NG-nitro-L-arginine or LG-nitro-arginine methyl ester. However, after induction of astrocyte iNOS activity with interferon gamma plus lipopolysaccharide or interleukin-1 beta, oxygen-glucose deprivation-induced neuronal injury was markedly enhanced and nitric oxide synthase inhibitors became protective. This iNOS-mediated potentiation was associated with a large increase in both extracellular glutamate accumulation and 45Ca2+ influx into neurons. The potentiation could be blocked by MK-801 but not CNQX, suggesting critical involvement of NMDA receptor activation. CONCLUSIONS: These results support the idea that nitric oxide production mediated by induced astrocytic iNOS can potentiate NMDA receptor-mediated neuronal death consequent to hypoxic-ischemic insults.

Superoxide dismutase improves posttraumatic cortical blood flow in rats.

Oxygen free radicals, such as the superoxide anion, are known to mediate damage to the cerebral microcirculation following traumatic brain injury. The purpose of this study was to determine if superoxide dismutase (SOD), a scavenger of superoxide anion, could alter posttraumatic cortical blood flow. Following barbiturate anesthesia, rats were surgically prepared for moderate fluid percussion brain injury. Cortical blood flow contralateral to the site of injury was measured using laser-Doppler flowmetry. Laser-Doppler flowmetry assesses flow by measuring cell volume and velocity, which are multiplied electronically to give flow. Starting 10 min before injury, animals received either superoxide dismutase (24,000 U/kg bolus, followed by continuous infusion of 1600 U/kg/min) or an equal volume of saline. Blood pressure, heart rate, and cortical blood flow were measured up to 1 h posttrauma. Rats receiving superoxide dismutase had significantly higher cortical blood flow posttrauma (F = 6.91, p < 0.02). One hour posttrauma, the blood flow in SOD-treated rats was 89 +/- 8% of preinjury baseline, whereas this value was only 66 +/- 6% of control in saline-treated rats. SOD caused not only greater blood velocity but also less reduction in cortical blood volume after injury. There were no significant differences between the groups with respect to blood pressure or heart rate. This study further supports the role of oxygen radical-mediated cerebrovascular dysfunction following traumatic brain injury and is the first to show the beneficial effect of SOD on cortical blood flow following fluid percussion brain injury.

The effect of acute cocaine or lidocaine on behavioral function following fluid percussion brain injury in rats.

One of the goals of our laboratory is to examine how the presence of drugs of abuse will influence traumatic brain injury. Previous studies in our laboratory have shown that cocaine or lidocaine treatment before experimental fluid percussion brain injury in rats reduces the cortical hypoperfusion normally found in the early posttraumatic period. The purpose of the current study was to determine if pretreatment with cocaine or lidocaine is also associated with changes in trauma-induced suppression of reflexes and motor and cognitive dysfunction that occurs following traumatic brain injury (TBI). Twenty-four hours after surgical preparation, rats were randomly assigned to a saline or drug pretreatment group, cocaine (0.5, 2, or 5 mg/kg) or lidocaine (2 mg/kg), which was injected via the tail vein. None of the drug pretreatments worsened injury. Lidocaine and cocaine decreased the duration of suppression of some neurological reflexes and reduced posttraumatic body weight losses. Lidocaine and cocaine both decreased postinjury motor deficits. Lidocaine and cocaine did not affect cognitive function on days 11-15 postinjury. The mechanism by which lidocaine improves acute neurological and motor function following brain injury is unknown, but may involve improved posttraumatic cortical blood flow, as seen in our previous study. Our results, along with other studies showing lidocaine to be neuroprotective in animal models of ischemia, suggest that studies of the effect of posttraumatic administration of lidocaine are warranted.

Acute cocaine administration alters posttraumatic blood pressure and cerebral blood flow in rats.

Cocaine abuse is widespread, and it is possible that its two main pharmacological actions, sympathomimetic and local anesthetic, could influence the blood pressure and cerebral blood flow response to brain injury, which occurs with increased frequency in drug abusers. We tested this hypothesis in ventilated barbiturate-anesthetized rats. Brain injury was induced using the fluid-percussion method, and cortical blood flow was measured using laser-Doppler flowmetry. Saline, cocaine, methamphetamine, or lidocaine was administered 10 min before injury. Upon injury, both cocaine- and saline-pretreated rats showed a similar acute hypertensive phase, which was followed by a period of more pronounced hypotension in the cocaine group (68 +/- 4 vs. 100 +/- 6 mmHg). Cortical blood flow increased dramatically 3-15 s following injury-induced hypertension in both the cocaine and saline groups (approximately 230-260%), but then fell below preinjury values within minutes. At 1 h postinjury, the blood flow in the saline group was 53 +/- 6% of the preinjury value, while in the cocaine group, flow was 74 +/- 7% of preinjury baseline. Similar to the cocaine-treated animals, methamphetamine also caused a more pronounced hypotensive event, but blood flow was not significantly different from saline controls. Lidocaine did not alter posttraumatic blood pressure but did significantly elevate blood flow throughout the 1-h postinjury period. At 60 min posttrauma, blood flow in the lidocaine group was 80 +/- 10% of the preinjury value. The mechanism by which cocaine alters blood pressure and blood flow after injury is not entirely certain.(ABSTRACT TRUNCATED AT 250 WORDS)

Cocaine potentiates the blood pressure and cerebral blood flow response to norepinephrine in rats.

Acute drug-induced hypertension is known to have adverse consequences on the cerebral vasculature. Cocaine abuse has been reported to be associated with an increased frequency of hemorrhagic or ischemic stroke. The purpose of this study was to determine whether cocaine alters the blood pressure or cerebral blood flow response to exogenous norepinephrine. A craniectomy was made over the parietal cortex in rats and cortical blood flow changes were measured using laser-Doppler flowmetry. Ten minutes after cocaine (1 mg/kg, i.v.) or saline, increasing doses of norepinephrine (0.01-10 micrograms/kg, i.v.) were given by bolus injection and changes in blood pressure and flow were monitored. Cocaine produced a transient 27 +/- 5% increase in blood pressure and a 38 +/- 9% increase in blood flow. Cocaine significantly potentiated the blood pressure and cerebral blood flow responses produced by submaximal pressor doses of norepinephrine (0.01-0.6 microgram/kg, i.v.). In summary, cocaine causes a rapid, transient increase in blood pressure and cortical blood flow and potentiates the magnitude and duration of the pressure and flow response to norepinephrine. Repetitive blood pressure elevations in cocaine abusers is one of the proposed mechanisms leading to damage of cerebral vessels. These results may be relevant to an increased frequency of cerebrovascular accidents in cocaine-abusing individuals.

Continuous monitoring of posttraumatic cerebral blood flow using laser-Doppler flowmetry.

Traumatic brain injury causes alterations in cerebral blood flow that are thought to influence secondary pathophysiology and neurologic outcome in humans. Since it is difficult to study early changes in blood flow in head-injured patients, animal models of brain injury must be employed. However, techniques to monitor brain blood flow in animals are labor intensive and generally provide discontinuous flow measurements. The present study examines the application of laser-Doppler flowmetry for measurement of cerebral blood flow following experimental brain injury. This method allows continuous monitoring of local cerebral blood flow before, during, and after injury. Rats (n = 9) were prepared for lateral fluid percussion injury under barbiturate anesthesia. Injury (2.10 +/- 0.02 atm) was induced over the right parietal cortex, and blood flow was monitored in the contralateral cortex. Seconds after the peak hypertension after injury, blood flow in the left parietal cortex increased 226% +/- 18% (means +/- SEM). This increase was transient, with blood flow falling below control values within minutes. Five minutes after injury, blood flow was 83% +/- 8% of control, and at 1 h, this value had fallen to 56% +/- 6%. Blood flow at 60 min was 93% +/- 5% of control in the sham-injured group (n = 10). The reduction in cerebral blood flow in our laser-Doppler study was of similar magnitude as previously reported in rats injured at a similar intensity when blood flow was examined with radiolabeled microspheres. Given these results, we believe laser-Doppler flowmetry can be used to continuously monitor posttraumatic blood flow following experimental brain injury.