Mayfield Neurotrauma Research Laboratory

Overview
We are engaged in brain injury research. We identify effector genes that influence functional recovery (e.g., deficits in neurological reflexes, motor coordination, and cognitive ability) after traumatic brain injury (TBI). Using behavioral pharmacology, biochemistry, histopathology and molecular techniques, we study their effects on TBI-induced inflammatory responses, neural cell death, vascular permeability and metabolism. Genes that regulate arachidonic acid metabolism following TBI are the current focus of the lab.

The physical insult of traumatic brain injury (TBI) sets into motion a cascade of biochemical events that can result in secondary brain damage which can exacerbate even relatively mild and moderate injuries. Secondary injuries may be preventable. TBI initiates an inflammatory response and alters cerebrovascular reactivity. These may, if not properly regulated, be a cause of secondary brain injury.

Findings: Arachidonic acid & COX2
One part of this response to TBI is increased production of prostaglandins (PG). PGs can mediate vasodilatation, vasoconstriction, altered vascular permeability, platelet aggregation, as well as several inflammatory and neurochemical processes. PGs are synthesized from arachidonic acid and molecular oxygen by cyclooxygenases (COX), producing potentially damaging reactive oxygen free radicals in the process.

We have demonstrated a prolonged elevation of the inducible cyclooxygenase-2 (COX2) enzyme, in vitro and in vivo in neurons and glia. Prolonged COX2 overexpression following moderate to severe traumatic brain injury (TBI) results in a pathological increases in PGs and free radicals that may adversely affect functional recovery. Recently, increased brain COX2 expression has also been linked to a number of clinically important neuropathological conditions, including Alzheimer's disease, spinal cord injury, and cerebral infarction. The discrete localization and distinct regulation of COX2 gene expression in the brain provide a rational basis to suspect that perturbation of eicosanoid metabolism may play a significant role in a variety of neuropathological conditions.

We have confirmed that COX2 inhibitors improve functional recovery and attenuate neuronal cell death and inflammation in a rat model of TBI. COX2 inhibitors, administered up to 6h postinjury appear to benefit the brain injured rat through a variety of mechanisms. To investigate, we have developed a method to measure over 20 eicosanoids (arachidonic acid metabolites) from minute specimens of brain tissue. Further studies will measure eicosanoids formed in the brain after injury, and identify which eicosanoids formed in the presence of a COX2 inhibitor may benefit the injured brain. The aim is to develop specific therapeutic strategies to reverse the pathological changes caused by COX2.

We have also initiated drug studies using novel pharmaceuticals that block inflammation to look for improvements in functional deficits after traumatic brain injury. One such drug, a steroid (DHEA) analog, has proven effective at significantly protecting injured animals from severe losses of coordination and memory that accompany TBI. DHEA and its analogs are relatively new players in the treatment of inflammatory symptoms, but are currently being tested in humans for rheumatoid arthritis, diabetes, and lupus.

We wish to further develop these anti-inflammatory agents for treatment of human traumatic brain injury. To do so, we must characterize the dose and time frame that the drugs must be administered to be effective. In addition, these drugs are currently in a form that is completely insoluble in water, but to be effective in a trauma setting, would have to be administered intravenously. We are testing new forms of these agents that are more water soluble to determine if they are as effective as the original formulations.

COX2 is expressed in both neurons and astrocytes of the brain after traumatic brain injury (TBI). Dual-labeling fluorescence immunohistochemistry demonstrates cell type-specific expression of COX2. (A) A neuron (MAP2, right) in the injured cerebral cortex at 24 hours postinjury expresses COX2 (left). (B) Astrocytes (GFAP, right) also express COX2 (left) at 24 hours postinjury, adjacent section to A. (C) Neurons (MAP2, right) in the contralateral hippocampus at 6 hours postinjury express COX2.
(D) Astrocytes (GFAP, right) in the contralateral hippocampus at 6 hours postinjury express COX2 (left). (E) Lower Panel: COX2 expression changes over time in the brain after TBI. Normal cerebral cortex contains many low intensity COX2 neurons (left photo). Between 2 (second photo) and 24 (third photo) hours after TBI, increased numbers and intensity of COX2 neurons appeared (COX2/MAP2 labeling). At 3 days postinjury (far right), mostly COX2 astrocytes (COX2/GFAP labeling) were observed near the site of injury.

MAP2 stains a neuron-specific intermediate filament protein, GFAP stains an astrocyte-specific intermediate filament protein. Adapted from Strauss et al. 2000. CXI = injured cerebral cortex. pcl = pyramidal cell layer, gcl = granule cell layer, slm = stratum lacunosum-moleculare. Thick arrows point to astrocytes, thin arrows to neurons. White bar = 50 micrometers.