Guy L. Clifton

Lack of effect of induction of hypothermia after acute brain injury

BACKGROUND Induction of hypothermia in patients with brain injury was shown to improve outcomes in small clinical studies, but the results were not definitive. To study this issue, we conducted a multicenter trial comparing the effects of hypothermia with those of normothermia in patients with acute brain injury. METHODS The study subjects were 392 patients 16 to 65 years of age with coma after sustaining closed head injuries who were randomly assigned to be treated with hypothermia (body temperature, 33 degrees C), which was initiated within 6 hours after injury and maintained for 48 hours by means of surface cooling, or normothermia. All patients otherwise received standard treatment. The primary outcome measure was functional status six months after the injury. RESULTS The mean age of the patients and the type and severity of injury in the two treatment groups were similar. The mean (+/-SD) time from injury to randomization was 4.3+/-1.1 hours in the hypothermia group and 4.1+/-1.2 hours in the normothermia group, and the mean time from injury to the achievement of the target temperature of 33 degrees C in the hypothermia group was 8.4+/-3.0 hours. The outcome was poor (defined as severe disability, a vegetative state, or death) in 57 percent of the patients in both groups. Mortality was 28 percent in the hypothermia group and 27 percent in the normothermia group (P=0.79). The patients in the hypothermia group had more hospital days with complications than the patients in the normothermia group. Fewer patients in the hypothermia group had high intracranial pressure than in the normothermia group. CONCLUSIONS Treatment with hypothermia, with the body temperature reaching 33 degrees C within eight hours after injury, is not effective in improving outcomes in patients with severe brain injury.

Clinical Trials in Head Injury

Secondary brain damage, following severe head injury is considered to be a major cause for bad outcome. Impressive reductions of the extent of brain damage in experimental studies have raised high expectations for cerebral neuroprotective treatment, in the clinic. Therefore multiple compounds were and are being evaluated in trials. In this review we discuss the pathomechanisms of traumatic brain damage, based upon their clinical importance. The role of hypothermia, mannitol, barbiturates, steroids, free radical scavengers, arachidonic acid inhibitors, calcium channel blockers, N-methyl-D-aspartate (NMDA) antagonists, and potassium channel blockers, will be discussed. The importance of a uniform strategic approach for evaluation of potentially interesting new compounds in clinical trials, to ameliorate outcome in patients with severe head injury, is proposed. To achieve this goal, two nonprofit organizations were founded: the European Brain Injury Consortium (EBIC) and the American Brain Injury Consortium (ABIC). Their aim lies in conducting better clinical trials, which incorporate lessons learned from previous trials, such that the succession of negative, or incomplete studies, as performed in previous years, will cease.

Marked Protection by Moderate Hypothermia after Experimental Traumatic Brain Injury

These experiments examined the effects of moderate hypothermia on mortality and neurological deficits observed after experimental traumatic brain injury (TBI) in the rat. Brain temperature was measured continuously in all experiments by intraparenchymal probes. Brain cooling was induced by partial immersion (skin protected by a plastic barrier) in a water bath (0°C) under general anesthesia (1.5% halothane/70% nitrous oxide/30% oxygen). In experiment I, we examined the effects of moderate hypothermia induced prior to injury on mortality following fluid percussion TBI. Rats were cooled to 36°C (n = 16), 33°C (n = 17), or 30°C (n = 11) prior to injury and maintained at their target temperature for 1 h after injury. There was a significant (p < 0.04) reduction in mortality by a brain temperature of 30°C. The mortality rate at 36°C was 37.5%, at 33°C was 41%, and at 30°C was 9.1%. In experiment II, we examined the effects of mod erate hypothermia or hyperthermia initiated after TBI or long-term behavioral deficits. Rats were cooled to 36°C (n = 10), 33°C (n = 10), or 30°C (n = 10) or warmed to 38°C (n = 10) or 40°C (n = 12) starting at 5 min after injury and maintained at their target temperatures for 1 h. Hypothermia-treated rats had significantly less beam-walking beam-balance, and body weight loss deficits compared to normothermic (38°C) rats. The greatest protection was observed in the 30°C hypothermia group. Since a temperature of 30°C can be induced in humans by surface cooling without coagulopathy or ventricular fibrillation, hypothermia to 30°C may have potential clinical value for treatment of human brain injury.

Very early hypothermia induction in patients with severe brain injury (the National Acute Brain Injury Study: Hypothermia II): a randomised trial

BACKGROUND The inconsistent effect of hypothermia treatment on severe brain injury in previous trials might be because hypothermia was induced too late after injury. We aimed to assess whether very early induction of hypothermia improves outcome in patients with severe brain injury. METHODS The National Acute Brain Injury Study: Hypothermia II (NABIS: H II) was a randomised, multicentre clinical trial of patients with severe brain injury who were enrolled within 2·5 h of injury at six sites in the USA and Canada. Patients with non-penetrating brain injury who were 16-45 years old and were not responsive to instructions were randomly assigned (1:1) by a random number generator to hypothermia or normothermia. Patients randomly assigned to hypothermia were cooled to 35°C until their trauma assessment was completed. Patients who had none of a second set of exclusion criteria were either cooled to 33°C for 48 h and then gradually rewarmed or treated at normothermia, depending upon their initial treatment assignment. Investigators who assessed the outcome measures were masked to treatment allocation. The primary outcome was the Glasgow outcome scale score at 6 months. Analysis was by modified intention to treat. This trial is registered with ClinicalTrials.gov, NCT00178711. FINDINGS Enrolment occurred from December, 2005, to June, 2009, when the trial was terminated for futility. Follow-up was from June, 2006, to December, 2009. 232 patients were initially randomised a mean of 1·6 h (SD 0·5) after injury: 119 to hypothermia and 113 to normothermia. 97 patients (52 in the hypothermia group and 45 in the normothermia group) did not meet any of the second set of exclusion criteria. The mean time to 35°C for the 52 patients in the hypothermia group was 2·6 h (SD 1·2) and to 33°C was 4·4 h (1·5). Outcome was poor (severe disability, vegetative state, or death) in 31 of 52 patients in the hypothermia group and 25 of 56 in the normothermia group (relative risk [RR] 1·08, 95% CI 0·76-1·53; p=0·67). 12 patients in the hypothermia group died compared with eight in the normothermia group (RR 1·30, 95% CI 0·58-2·52; p=0·52). INTERPRETATION This trial did not confirm the utility of hypothermia as a primary neuroprotective strategy in patients with severe traumatic brain injury.

Fluid thresholds and outcome from severe brain injury.

Objective To determine, by retrospective analysis, critical thresholds for intracranial pressure, mean arterial pressure, cerebral perfusion pressure, and fluid balance associated with poor outcome in patients with severe brain injury. Design Retrospective review of patient data from the prospective, randomized, multicenter National Acute Brain Injury Study: Hypothermia, comparing outcome results at 6 months after injury with intracranial pressure, mean arterial pressure, cerebral perfusion pressure, and fluid balance measurements recorded during the 96-hr period after randomization. Setting Emergency departments and intensive care units in 11 metropolitan tertiary care university hospitals. Patients A total of 392 patients, aged 16–65 yrs, with severe, nonpenetrating brain injuries and a Glasgow Coma Scale score of 3–8 after resuscitation, who were enrolled in a study designed to determine the treatment effect of moderate hypothermia in patients with severe brain injury. Intervention Standard brain injury treatment for 193 randomly assigned patients and standard treatment plus hypothermia for 48 hrs for 199 patients. Measurements and Main Results Intracranial pressure levels of 20, 25, and 30 mm Hg, mean arterial pressure levels of 70 and 80 mm Hg, cerebral perfusion pressure levels of 50, 60, and 70 mm Hg, and fluid balance levels in quartiles were examined for their effect on outcome as measured by the Glasgow Outcome Scale at 6 months after injury. When considered separately, any of the following—intracranial pressure >25 mm Hg, mean arterial pressure <70 mm Hg, or cerebral perfusion pressure <60 mm Hg and fluid balance lower than −594 mL—was associated with an increased percentage of patients with poor outcome. When the variables were combined into a stepwise logistic regression model, Glasgow Coma Scale score at admission, age, mean arterial pressure <70 mm Hg, fluid balance lower than −594 mL, and intracranial pressure > 25 mm Hg, in that order, were the most powerful variables in determining outcome. Conclusions Exceeding thresholds of intracranial pressure, mean arterial pressure, cerebral perfusion pressure, and fluid volume may be detrimental to severe brain injury outcome. Fluid balance lower than −594 mL was associated with an adverse effect on outcome, independent of its relationship to intracranial pressure, mean arterial pressure, or cerebral perfusion pressure.

A Comparison of Magnetoencephalography, MRI, and V-EEG in Patients Evaluated for Epilepsy Surgery

Summary: Purpose: To determine the efficacy and relative contribution of several diagnostic methods [ictal and interictal scalp and intracranial EEG, magnetic resonance imaging (MRI), and magnetoencephalography (MEG)] in identifying the epileptogenic zone for resection.

A calpain inhibitor attenuates cortical cytoskeletal protein loss after experimental traumatic brain injury in the rat

The capacity of a calpain inhibitor to reduce losses of neurofilament 200-, neurofilament 68- and calpain 1-mediated spectrin breakdown products was examined following traumatic brain injury in the rat. Twenty-four hours after unilateral cortical impact injury, western blot analyses detected neurofilament 200 losses of 65% (ipsilateral) and 36% (contralateral) of levels observed in naive, uninjured rat cortices. Neurofilament 68 protein levels decreased only in the ipsilateral cortex by 35% relative to naive protein levels. Calpain inhibitor 2, administered 10 min after injury via continuous arterial infusion into the right external carotid artery for 24 h, significantly reduced neurofilament 200 losses to 17% and 3% relative to naive neurofilament 200 protein levels in the ipsilateral and contralateral cortices, respectively. Calpain inhibitor administration abolished neurofilament 68 loss in the ipsilateral cortex and was accompanied by a reduction of putative calpain-mediated neurofilament 68 breakdown products. Spectrin breakdown products mediated by calpain 1 activation were detectable in both hemispheres 24 h after traumatic brain injury and were substantially reduced in animals treated with calpain inhibitor 2 both ipsilaterally and contralaterally to the site of injury. Qualitative immunofluorescence studies of neurofilament 200 and neurofilament 68 confirmed western blot data, demonstrating morphological protection of neuronal structure throughout cortical regions of the traumatically injured brain. Morphological protection included preservation of dendritic structure and reduction of axonal retraction balls. In addition, histopathological studies employing hematoxylin and eosin staining indicated reduced extent of contusion at the injury site. These data indicate that calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons after experimental traumatic brain injury in vivo.

μ-Calpain activation and calpain-mediated cytoskeletal proteolysis following traumatic brain injury

Abstract: Increasing evidence suggests that excessive activation of the calcium‐activated neutral protease μ‐calpain could play a major role in calcium‐mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of μ‐calpain after unilateral cortical impact injury in vivo, the ratio of the 76‐kDa activated isoform of μ‐calpain to its 80‐kDa precursor was measured by western blotting. This μ‐calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24–48 h after injury. We also investigated the effect of μ‐calpain activation on proteolysis of the neuronal cytoskeletal protein α‐spectrin. Immunoreactivity for α‐spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of α‐spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of α‐spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of μ‐calpain autolysis, calpain‐mediated α‐spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although μ‐calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain‐mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent μ‐calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.

Sulforaphane enhances aquaporin-4 expression and decreases cerebral edema following traumatic brain injury.

Brain edema, the infiltration and accumulation of excess fluid causing an increase in brain tissue volume, often leads to a rise in intracranial pressure and is a key contributor to the morbidity and mortality associated with traumatic brain injury (TBI). The cellular and molecular mechanisms contributing to the development/resolution of TBI‐associated brain edema are poorly understood. Aquaporin‐4 (AQP4) water channel is expressed at high levels in brain astrocytes, and the bidirectional transport of water through these channels is critical for the maintenance of brain water homeostasis. By using a rodent injury model, we show that TBI decreased AQP4 level in the injury core and modestly increased it in the penumbra region surrounding the core. Postinjury administration of sulforaphane (SUL), an isothiocyanate present in abundance in cruciferous vegetables such as broccoli, attenuated AQP4 loss in the injury core and further increased AQP4 levels in the penumbra region compared with injured animals receiving vehicle. These increases in AQP4 levels were accompanied by a significant reduction in brain edema (assessed by percentage water content) at 3 days postinjury. These findings suggest that the reduction of brain edema in response to SUL administration could be due, in part, to water clearance by AQP4 from the injured brain.

Altered expression of novel genes in the cerebral cortex following experimental brain injury

Damage to the cerebral cortex results in neurological impairments such as motor, attention, memory and executive dysfunctions. To examine the molecular mechanisms contributing to these deficits, mRNA expression was profiled using high-density cDNA microarray hybridization after experimental cortical impact injury in mice. The mRNA levels at 2 h, 6 h, 24 h, 3 days and 14 days after injury were compared with those of control animals. This revealed 86 annotated genes and 24 expression sequence tags (ESTs) as being differentially expressed with a 1.5-fold or greater change. Quantitative real-time PCR analysis was used to independently verify these results for selected genes. Seven functional classes of genes were found to be altered following injury, including transcription factors, signal transduction genes and inflammatory proteins. While a few of these genes have been previously reported to be differentially regulated following injury, the most of the genes have not been previously implicated in traumatic brain injury (TBI) pathophysiology. For example, consistent with previous reports, the transcription factor c-jun and the neurotrophic factor bdnf mRNA levels were altered as a result of TBI. Among the novel genes, the mRNA levels for the high mobility group protein 1 (hmg-1), the regulator of G-protein signaling 2 (rgs-2), the transforming growth factor beta inducible early growth response (tieg), the inhibitor of DNA binding 3 (id3), and the heterogeneous nuclear ribonucleoprotein H (hnrnp h) were changed following injury. The functional significance of these genes in neurite outgrowth, neuronal regeneration, and plasticity following injury are discussed.