Ronald L. Hayes

Effects of scopolamine treatment on long-term behavioral deficits following concussive brain injury to the rat

Scopolamine (0.1, 1.0, or 10.0 mg/kg) or saline was systemically (i.p.) administered to rats 15 min prior to concussive fluid percussion brain injury. Animals pretreated with the 1.0 mg/kg dose exhibited significantly (P less than 0.05) less motor deficits and less body weight loss and recovered to baseline performance sooner than saline-treated rats. Mortality and associated convulsions were significantly lower in rats pretreated with the 1.0 mg/kg dose of scopolamine. A 1.0 mg/kg dose of scopolamine administered (i.p.) 30 s after injury also significantly reduced behavioral deficits. No differences were observed between saline- and scopolamine-treated animals in either the incidence or duration of transient apnea following injury. A 1.0 mg/kg dose of scopolamine administered (i.p.) 15 min prior to epidural clip compression of the spinal cord had no effect on the severity of motor function deficits assessed by an inclined plane test. The data from these experiments suggest muscarinic cholinergic involvement in at least some of the long-term behavioral deficits following mild and moderate levels of brain injury. These results suggest that muscarinic cholinergic antagonists may prove beneficial in the treatment of human head injury.

Outcome measures for clinical trials involving traumatically brain-injured patients: report of a conference.

A conference was held in Houston, Texas, on October 8-9, 1991, to develop recommendations for outcome measures for clinical trials in traumatic brain injury. Participants, all experts in this area, discussed and agreed on treatments for patients with severe brain injury (Glasgow Coma Score [GCS] < or = 8) and moderate brain injury (GCS, 9-12). A parallel trial design was recommended rather than a factorial, sequential, or crossover design. It was agreed that stratifying randomization based on motor score alone or on a combination of motor score and age would result in increased power. Acute stage measurements, such as cerebral blood flow, cerebrospinal fluid biochemistry, and evoked potentials, were recommended only when they satisfied a specific hypothesis. Functional outcome measures were recommended as the primary outcome measure for severe brain injury (GCS, 3-8). Either the Glasgow Outcome Scale or Disability Rating Scale, measured at 6 months after injury, were recommended as the primary outcome measure for severe brain injury (GCS, < or = 8). For patients with moderately severe brain injury (GCS, 9-12), the Disability Rating Scale at 3 months after injury was recommended as the primary outcome measure. The Neurobehavioral Rating Scale appears to be a satisfactory instrument for measuring behavioral changes. Specific neuropsychological measures were recommended as supplementary outcome measures for both severe and moderate brain injury, consistent with a 1.5-hour period available for testing.

Increased expression of brain‐derived neurotrophic factor but not neurotrophin‐3 mRNA in rat brain after cortical impact injury

Levels of brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT3) mRNA expression were measured in a rodent model of traumatic brain injury (TBI) following unilateral injury to the cerebral cortex. To obtain reliable data on the co‐expression of neurotrophin genes, adjacent coronal sections from the same rat brains were hybridized in situ with BDNF and NT3 cRNA probes. BDNF mRNA increased at 1, 3, and 5 hr after unilateral cortical injury in the cortex ipsilateral to the injury site and bilaterally in the dorsal hippocampus. NT3 mRNA did not change significantly following injury. Our results suggest that TBI produces rapid increases in BDNF mRNA expression in rat brain without changes in NT3 mRNA expression, a finding which differs from studies of ischemia and seizures. It is possible that increased levels of BDNF mRNA rather than NT3 are important components of pathophysiological responses to TBI.

Increased expression of c-fos mRNA and AP-1 transcription factors after cortical impact injury in rats

Levels of c-fos mRNA and AP-1 transcription factors co-expression were measured in a controlled lateral cortical impact model of traumatic brain injury (TBI) in rats. Ipsilateral cerebral cortex and bilateral hippocampal c-fos mRNA increases were revealed by in situ hybridization after lateral cortical impact injury. Based on regional in situ hybridization data, we employed semi-quantitative RT-PCR methods to study the temporal profile of changes in the ipsilateral cortex at the site of injury. We found that TBI produces transient increases of c-fos mRNA expression in the ipsilateral cerebral cortex at 5 min postinjury, which peaks at 1 h postinjury and subsides by 1 day postinjury. Gel shift nuclear protein binding assays showed that AP-1 transcription factor binding was robustly increased in injured cerebral cortex at 1 h, 3 h, 5 h and 1 day after injury. These data indicate that TBI can produce significant increases in c-fos expression and subsequent upregulation of the AP-1 transcription factors. Thus, AP-1 transcription factors modulation of downstream gene expression may be an important component of pathophysiological responses to TBI.

Outcome Measures for Clinical Trials Involving Traumatically Brain-Injured Patients

A conference was held in Houston, Texas, on October 8-9, 1991, to develop recommendations for outcome measures for clinical trials in traumatic brain injury. Participants, all experts in this area, discussed and agreed on treatments for patients with severe brain injury (Glasgow Coma Score [GCS] < or = 8) and moderate brain injury (GCS, 9-12). A parallel trial design was recommended rather than a factorial, sequential, or crossover design. It was agreed that stratifying randomization based on motor score alone or on a combination of motor score and age would result in increased power. Acute stage measurements, such as cerebral blood flow, cerebrospinal fluid biochemistry, and evoked potentials, were recommended only when they satisfied a specific hypothesis. Functional outcome measures were recommended as the primary outcome measure for severe brain injury (GCS, 3-8). Either the Glasgow Outcome Scale or Disability Rating Scale, measured at 6 months after injury, were recommended as the primary outcome measure for severe brain injury (GCS, < or = 8). For patients with moderately severe brain injury (GCS, 9-12), the Disability Rating Scale at 3 months after injury was recommended as the primary outcome measure. The Neurobehavioral Rating Scale appears to be a satisfactory instrument for measuring behavioral changes. Specific neuropsychological measures were recommended as supplementary outcome measures for both severe and moderate brain injury, consistent with a 1.5-hour period available for testing.

Sphingosine-1-phosphate induces apoptosis of cultured hippocampal neurons that requires protein phosphatases and activator protein-1 complexes

In this study, we report that mobilization of internal Ca2+ by sphingosine-1-phosphate, a metabolite of ceramide, induces apoptosis in cultured hippocampal neurons. This sphingosine-1-phosphate-induced apoptosis is dependent upon the activation of protein phosphatases, possibly calcineurin and phosphatase 2A (or a related phosphatase). In addition, pretreatment of neurons with double-stranded oligonucleotides containing the metallothionein phorbol-12-myristate-13-acetate response element sequence as transcription factor decoys suppressed apoptosis. In contrast, double-stranded oligonucleotides containing either the c-jun or SV40 phorbol-12-myristate-13-acetate response element sequences were ineffective. Electrophoretic mobility shift assays and supershift assays revealed that c-Fos-containing activator protein- complexes preferentially bound the metallothionein phorbol-12-myristate-13-acetate response element sequence-containing oligonucleotides. Furthermore, antisense oligonucleotides to c-fos and c-jun were also protective. The apoptotic death of hippocampal neurons has been hypothesized to contribute to the cognitive impairments observed following insults to the brain. While increases in intracellular calcium are thought to be key mediators of neuronal apoptosis, the biochemical cascade(s) activated as a result of increased Ca2+ which mediates apoptosis of hippocampal neurons is (are) not well understood. The findings presented in this study suggest that mobilization of internal calcium via prolonged exposure of sphingosine-1-phosphate induces apoptosis of hippocampal neurons in culture. Sustained increases in intracellular calcium activate a phosphatase cascade that includes calcineurin and a phosphatase 2A-like phosphatase, and leads to the expression of genes containing metallothionein phorbol-12-myristate-13-acetate response element (TGAGTCA)-type enhancer sequences. The expression of genes containing TGAGTCA-type enhancer sequences appears to be essential for sphingosine-1-phosphate-induced apoptosis of hippocampal neurons.

Postinjury scopolamine administration in experimental traumatic brain injury

A single bolus dose of scopolamine (1.0 mg/kg) or saline (equal volume) was injected (i.p.) at 15, 30 or 60 min after fluid percussion traumatic brain injury in the rat. Scopolamine administered at 15 min postinjury significantly reduced beam walking deficits and body weight loss assessed for 5 days after injury. Scopolamine treatment at 30 or 60 min postinjury had no effect on behavioral outcome assessed for 5 days after injury. Plasma concentrations of scopolamine were measured with a radioreceptor assay. The plasma half-life for scopolamine was 21.6 min in injured rats and 17.3 min in normal rats (P less than 0.05). These results, along with evidence from previous studies, suggest that a brief period of excessive neuronal excitation can produce relatively long-lasting behavioral deficits. The temporal effectiveness of receptor antagonist intervention in this process appears to be brief.

Blood-Brain Barrier Permeability Changes after Experimental Subarachnoid Hemorrhage

Basic mechanisms underlying cerebrovascular permeability responses to subarachnoid hemorrhage (SAH) are still to be defined in detail. Previous investigations examining the occurrence of blood-brain barrier (BBB) breakdown after SAH in the experimental setting have yielded conflicting results. In a rat model of SAH, we assessed BBB changes by means of the quantitative [14C]-alpha-aminoisobutyric acid technique. Experiments were carried out on the second day post-SAH. In blood-injected rats [14C]-alpha-aminoisobutyric acid transport across the BBB increased significantly in cerebral cortices and cerebellar gray matter, averaging 1.3 to 1.5 times control values. The present data indicate that SAH induces well-defined changes in BBB function, possibly involved in the pathogenesis of post-SAH cerebral dysfunction in humans. Results reported here have also potential clinical implications for the management of aneurysm patients.

Traumatic brain injury causes a decrease in M2 muscarinic cholinergic receptor binding in the rat brain

Numerous studies indicate that an acute, excessive activation of muscarinic acetylcholine receptors (mAChR) contributes to the pathophysiological sequela of TBI. The present study examined the effect of moderate fluid percussion traumatic brain injury (TBI) on binding to M1 and M2 mAChR subtypes in the hippocampal formation and adjacent cortex using quantitative autoradiography. Injured animals along with concurrent controls were sacrificed by in situ freezing at 3 h or 24 h following TBI. Slide-mounted tissue sections were incubated in either [3H]pirenzepine (23 nM) for M1 or [3H]AFDX384 (9 nM) for M2 mAChR subtype labeling. Binding of [3H]pirenzepine to the M1 mAChR subtype was not significantly altered by TBI when compared to sham-injured animals. [3H]AFDX384 binding to the M2 mAChR subtype was significantly decreased at 24 h in hippocampal CA2-3 region and dorsal blade of the dentate gyrus (P < 0.05). The differences observed between M1 and M2 subtypes suggests that these muscarinic subtypes may differentially contribute to the pathophysiology of TBI.

Neurochemical aspects of head injury: Role of excitatory neurotransmission

Historically, studies of traumatic brain Injury CTBI) have focused on metabolic, cerebral vascular, and structural pathology. However, significant recent progress has been made in understanding the biochemical features of TBI. Studies have determined that TBI can produce excessive excitation of neurons in the period immediately following impact. This excessive excitation of neurons can produce enduring pathologic changes in cell function that could result In many of the neurologic deficits characteristic of TBI. Clinically, these observations suggest that the administration of various pharmacologlc agents that can blunt excessive excitation could improve the outcome of head-injured patients, if given soon after injury.