Ramesh Raghupathi, Ph.D.
Associate Professor, Dept. Neurobiology and Anatomy, Drexel University College of Medicine
Email: ramesh.raghupathi@drexelmed.edu

Cellular Neurobiology of Brain Injury: Mechanisms of Cell Dysfunction/Death and Plasticity

Research and Interest

In the United States, traumatic brain injury (TBI) is a significant factor in the mortality and morbidity of individuals under the age of 45, comprising 1-2% of deaths from all causes. Survivors are faced with debilitating and long-term neurologic dysfunction that has a major impact on quality of life and carries a significant social and economic burden. The damage observed after TBI comprises both primary disruption of neural tissue related to the impact, and secondary mechanisms that develop over the weeks to months after the traumatic event. The spectrum of pathologies observed after TBI include focal contusions in the gray matter and diffuse injuries to axons in the white matter. It has been suggested that these pathologies are a consequence of the biomechanics of the impact, i.e., focal injuries occur due to contact forces to the head, while diffuse injuries are a result of non-contact, rotational forces to the brain. While aspects of focal pathology can be superimposed on diffuse brain injury (and vice versa), it is our belief that significant differences exist between the pathobiology of these two types of injuries that warrant the separate evaluation of mechanisms of damage in the cell body (soma) and the axon. Secondary mechanisms of neural damage are initiated immediately after impact and result in a number of cascades that affect both the neural tissue and the vasculature. In response to the impact, the brain becomes edematous leading to increases in intracranial pressure and subsequent neuronal death, which may be an underlying cause for the neurologic impairment. In turn, injured neurons are faced with imbalances in ionic homeostasis, over-activation of excitatory amino acid receptors, increases in intracellular calcium, increased free radical generation, and mitochondrial dysfunction that may underlie the eventual death of injured neurons. Concomitant with neuronal death and damage, axons are also subjected to mechanical forces that lead to traumatic axonal injury. Injury to axons is characterized by focal accumulations cytoskeletal proteins resulting in a swollen phenotype in the acute post-traumatic period. Over time these swollen axons undergo complete axotomy (Wallerian degeneration), a process that is associated with death of oligodendrocytes.

Biography

Ramesh Raghupathi graduated from Virginia Commonwealth University with a Ph.D. in Biochemistry and Molecular Biophysics. He did post-doctoral fellowships at the University of Connecticut Health Science Center and the University of Pennsylvania. He served on the faculty in the Department of Neurosurgery at the University of Pennsylvania School of Medicine. He was appointed to the faculty in the Department of Neurobiology and Anatomy at Drexel University College of Medicine in 2003.

Ongoing Projects

1. Caspase-mediated cell death in brain trauma (funded by NINDS, NS41561; 2002-2007). By identifying specific mechanisms the lead to cell death, the long-term goal of the studies outlined in this project is to develop appropriate, mechanism-based therapies for the head-injured patient. The hypothesis tested here is that post-traumatic neural cell death is a result of activation of the pro-apoptotic caspase family of cysteine proteases. Preliminary observations indicate that a certain number of cells that die following experimental TBI or closed head injury in humans exhibit activation of the apoptotic "executor" protease, caspase-3. In addition, in animals, the activation of caspase-3 is preceded by activation of the initiator caspases-8 and -9, albeit in a regionally distinct manner. Based on these observations, the lab seeks to utilize a combination of pharmacology and molecular genetics to elucidate whether (1) whether caspase-3 activation occurs directly as a result of activation of the "initiator" caspase-8, and/or indirectly as a result of mitochondrial pathway which requires Bax translocation, cytochrome c release and caspase-9 activation, (2) whether caspase-8 activation occurs as a result of activation of the tumor necrosis factor family of death receptors, (3) the role of Bax in mediating trauma-induced caspase-9 activation and subsequent caspase-3 activation and cell death, and, (4) whether post-traumatic inhibition of caspases-3, -8 and -9 will reduce the extent of injury-induced cell death.

2. Injury-severity dependent activation of the cell death-inducing proteases, caspases and calpains following brain injury (funded by NINDS, NS35712; 2001-2006, and NS08803; 2000-2005). The choice of an appropriate treatment paradigm for head-injured patients will depend on the severity of the injury. While it is simple to assume that cellular response is proportional to the severity of the injury, preliminary observations indicate that this is not so. The hypothesis being tested in these studies is that the severity of the mechanical injury controls the nature of the cellular response; in particular, increasing the severity of the injury shifts the nature of cell death from apoptosis to necrosis. In contrast, axonal injury may be more prevalent in mild injuries. In collaboration with Dr. David Meaney at the University of Pennsylvania (NS35712), we have demonstrated that subjecting organotypic hippocampal cultures to a stretch injury in vitro results predominantly in caspase-3 activation (apoptosis) at mild stretch levels; increasing the severity of stretch resulted in calpain activation (necrosis?). In contrast, preliminary observations in vivo indicate that calpain activation predominates over caspase-3 activation in the cortex at any injury severity. However, there are regional differences in calpain and caspase-3 activation as a function of injury severity. Current studies seek to elucidate the effect of glutamatergic receptor antagonism in alleviating the extent of protease activation at different injury severities - the hypothesis being that protease activation following mild injuries will be more amenable to glutamate receptor antagonism that that after moderate-severe injuries.

3. Role of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in neuronal and glial cell death after brain trauma (funded by NINDS, NS08803; 2000-2005). In addition to proteases, cell dysfunction and death following brain injury may be mediated by protein kinases. In vitro, neuron death following an insult appears to be dependent on the activation of JNK and a concomitant inactivation of ERK. In contrast, our observations following experimental brain trauma in the rat suggest that both ERK and JNK are activated in neurons and glia, in regions that exhibit cell damage and/or death. In addition, JNK activation was observed in axons, suggestive of a role in traumatic axonal injury. The objectives of this proposal, using a combination of pharmacology and molecular genetics, are to determine the contribution of JNK and ERK activation in the activation of the caspase-3, and, to examine the possibility that neuronal and glial death following TBI in animals and humans is associated with coordinate alterations in expression of multiple death-associated genes. In addition to using JNK- and ERK-specific inhibitors, the anti-apoptotic effect of JNK following TBI will be tested by subjecting mice that are deficient in JNK to either gray matter or white matter injury. Finally, individual neurons and oligodendrocytes, from either human head-injured tissue or injured rat brain, exhibiting activated caspase-3 (indicative of the commitment to apoptosis), will be evaluated for the expression of cell death associated using laser capture microdissection and RNA amplification. Some of these studies are done in collaboration with Dr. Kathryn Saatman at the University of Kentucky.  

Lab Personnel

Saori Shimizu, M.D., Ph.D. (Post-doctoral fellow. Project: Role of cell cycle proteins in apoptotic neuronal death)
Veronica Holod, B.S. (Research Technician. Projects: Roles of Bax and JNK in traumatic apoptosis; NDMA receptor-induced activation of caspase-3 and calpain following brain trauma)
Kelly LeBrun, B.S. (Research Technician. Projects: Role of JNK and ERK in traumatic apoptosis)
Michael Franklin, B.S. (Research Technician. Project: Traumatic Brain Injury in the immature rat)
Penda Powell, B.S. (Graduate Student. Project: Role of cdk5 in traumatic apoptosis).
Rachael Paz. (Undergraduate student. Project: Role of Akt in traumatic apoptosis).

Collaborators & Colleagues

David F. Meaney, Ph.D., Department of Bioengineering, University of Pennsylvania
Kathryn E. Saatman, Ph.D., Department of Physiology, University of Kentucky
Jimmy Huh, M.D., Department of Anesthesia and Critical Care, Children's Hospital of Philadelphia.

Selected Publications

1. Conti, A.C., Raghupathi, R., Trojanowski, J.Q., McIntosh, T.K. Experimental Brain Injury Induces Regionally Distinct Apoptosis During the Acute and Delayed Post-Traumatic Period. J. Neurosci. 18:5663-5672, 1998.
2. Raghupathi, R., Fernandez, S.C., Murai, H., Trusko, S.P., Scott, R.W., Nishioka, W.K., McIntosh, T.K. Bcl-2 Overexpression Attenuates Cortical Cell Loss after Traumatic Brain Injury in Transgenic Mice. J. Cereb. Blood Flow Metab. 18:1259-1269, 1998.
3. Bain, A.C., Raghupathi, R., Meaney, D.F. Dynamic stretch correlates to both morphological abnormalities and electrophysiological impairment in a model of traumatic axonal injury. J. Neurotrauma 18:499-511, 2001.
4. Raghupathi, R., Conti, A.C., Graham, D.I., Krajewski, S., Reed, J.C., Grady, M.S., McIntosh, T.K. Mild traumatic brain injury induces apoptotic cell death in the cortex that is preceded by decreases in Bcl-2 immunoreactivity. Neuroscience 110:605-616, 2002.
5. Raghupathi, R., Margulies, S.S. Traumatic axonal injury following closed head injury in the neonatal pig. J.Neurotrauma 19:843-853, 2002.
6. Raghupathi, R., Strauss, K.I., Zhang,C., Krajewski, S., Reed, J.C., McIntosh, T.K. Temporal alterations in cellular Bax:Bcl-2 ratio following traumatic brain injury in the rat. J. Neurotrauma 20:421-435, 2003.
7. Raghupathi, R., Muir, J.K., Fulp, C.T., Pittman, R.N., McIntosh, T.K. Acute alterations in mitogen-activated protein kinases following traumatic brain in the rat: implications for post-traumatic cell death. Exp. Neurol. 183:438-448, 2003.
8. Raghupathi, R. Cell death mechanisms following traumatic brain injury. Brain Pathology 14:215-222, 2004.
9. DeRidder, M.N., Raghupathi, R., Meaney, D.F. Evidence of coordinate activation of apoptotic and necrotic pathways following stretch injury. J. Neurotrauma 20:1110, 2003.
10. Clouse, A.K., Wanderer, J.W., Pape, R.L., Siman, R., Saatman, K.E., Raghupathi, R. Coordinate and differential patterns of calpain and caspase-3 activation following brain trauma in the mouse. J. Neurotrauma 20:1109, 2003.
11. Marciano, P.G., Brettschneider, J., Manduchi, E., Davis, J.E., Eastman, S., Raghupathi, R., Saatman, K.E., Speed, T.P., Stoeckert, C.J., Eberwine, J.H., McIntosh, T.K. Neuron-specific mNRA complexity responses during hippocampal apoptosis after traumatic brain injury. J. Neurosci. 24:2866-2876, 2004.

Patents

Patent No. US 6,326,146: O'Dell, D.M., Raghupathi, R., McIntosh, T.K., Crino, P., Eberwine, J. Method of determining multiple mRNAs in dying cells. 2001.