No abstract

Secondary hypoxic/ischemic injuries, stemming from reductions in cerebral blood flow are important contributing factors in progressive neuronal dysfunction after brain trauma. A greater preclinical understanding of how brain trauma leads to secondary hypoxia/ischemia is necessary in the development of posttraumatic brain injury (TBI) therapeutics. To this end, we examined the density of microvascular coverage in the injured and contralateral cortical hemispheres using two intensities of fluid percussion trauma in rats. A silicone microangiography technique showed a significant loss in microvascular density in 2 atmosphere (atm) (16.9 ± 3.8%) and 3 atm (15.7 ± 1.3%) injured animals relative to sham animals (29.9±2.5%; P < 0.01). RECA-1 immunohistochemistry indicated that capillary changes involved a reduction in capillary number and diameter. Reduction in microvascular density was shown to be a diffuse phenomenon occurring up to 4 mm rostral and caudal to the injury epicenter. Recovery of microvasculature occurred by 2 weeks after injury only in the 2 atm injury group. Expression of HIF1a and increased vascular endothelial growth factor expression were observed in the ipsilateral hippocampus suggesting sufficiently impaired microcirculation resulting in the expression of hypoxic-response proteins. Collectively, the results indicate diffuse and heterogeneous microvascular alterations as well as endogenous expression of neuroprotective and neovascularization pathways after TBI.

Constitutive and activity-dependent regulation of the AMPA receptor GluR2 content is recognized as an important mediator of both neuronal plasticity and vulnerability to excitotoxic neuron death. In the latter case, inclusion of GluR2 protects against glutamate excitotoxicity in CNS disease by lowering receptor single-channel conductance and preventing deleterious calcium influx. We investigated the hypothesis that aberrations in GluR2 trafficking after in vitro and in vivo cerebral trauma contribute to excitotoxicity and associated calcium-dependent cell death processes. First, in an in vitro model of traumatic brain injury (TBI), we observed PICK1 and N-methyl-D-aspartic acid (NMDA) receptor-dependent phosphorylation and internalization of GluR2. The contributing cell signaling mechanisms involved enhanced binding between PKCa (the kinase that phosphorylates GluR2) and PICK1 (its PDZ-binding partner), and a novel protein interaction between PKCa and the NMDA receptor scaffolding protein PSD-95. Functionally, these phenomena enhanced single cell AMPAR mEPSCs and protracted calcium extrusion. In vivo TBI similarly promoted GluR2 phosphorylation and internalization, with enhanced expression of calcium-permeable AMPARs in the injured hippocampus. Peptide-mediated perturbation of the PKCa/PICK1 protein interaction after trauma preserved surface GluR2 expression, attenuated AMPAR-mediated toxicity, and occluded the sensitivity of neuronal physiology to calcium-permeable AMPAR antagonists. These findings suggest that experimental TBI promotes the expression of injurious GluR2-lacking AMPARs, thereby enhancing cellular vulnerability to secondary excitotoxicity. Traumatic brain injury (TBI) continues to be a leading cause of morbidity and disability in North America. 1 At the cellular level, excitotoxic stimulation of N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamatergic receptors has a major function in the deregulation of calcium homeostasis and acute cell swelling after TBI, ultimately leading to secondary neuronal cell death hours to days after the primary injury. 1 Unfortunately, global antagonism of these ionotropic channels (which has generally targeted the highly calciumpermeable NMDARs) is a clinically impractical approach because of interference with physiological receptor function. 2 As such, it remains of critical importance to identify novel potential intracellular targets of anti-excitotoxic therapy to mitigate delayed secondary cell loss, and improve functional outcome after TBI.Physiologically, AMPA receptors mediate the majority of excitatory ionotropic neurotransmission in the CNS. 3 Pathologically, however, AMPA receptors can also mediate excitotoxic neuronal damage when GluR2 subunits are absent from the tetrameric receptor complex 4 (composed of four subunits, GluR1-4). This is because receptors devoid of the GluR2 subunit (e.g. homomers of GluR1) exhibit two-to three-fold increases in single-channel conductance, increased calcium and zinc permeab...