Ramesh Raghupathi

The Dorothy Russell Memorial Lecture. The molecular and cellular sequelae of experimental traumatic brain injury : pathogenetic mechanisms

The mechanisms underlying secondary or delayed cell death following traumatic brain injury (TBI) are poorly understood. Recent evidence from experimental models of TBI suggest that diffuse and widespread neuronal damage and loss is progressive and prolonged for months to years after the initial insult in selectively vulnerable regions of the cortex, hippocampus, thalamus, striatum, and subcortical nuclei. The development of new neuropathological and molecular techniques has generated new insights into the cellular and molecular sequelae of brain trauma. This paper will review the literature suggesting that alterations in intracellular calcium with resulting changes in gene expression, activation of reactive oxygen species (ROS), activation of intracellular proteases (calpains), expression of neurotrophic factors, and activation of cell death genes (apoptosis) may play a role in mediating delayed cell death after trauma. Recent data suggesting that TBI should be considered as both an inflammatory and/or a neurodegenerative disease is also presented. Further research concerning the complex molecular and neuropathological cascades following brain trauma should be conducted, as novel therapeutic strategies continue to be developed.

Transplanted neural stem cells survive, differentiate, and improve neurological motor function after experimental traumatic brain injury.

OBJECTIVE Using the neural stem cell (NSC) clone C17.2, we evaluated the ability of transplanted murine NSCs to attenuate cognitive and neurological motor deficits after traumatic brain injury. METHODS Nonimmunosuppressed C57BL/6 mice (n = 65) were anesthetized and subjected to lateral controlled cortical impact brain injury (n = 52) or surgery without injury (sham operation group, n = 13). At 3 days postinjury, all brain-injured animals were reanesthetized and randomized to receive stereotactic injection of NSCs or control cells (human embryonic kidney cells) into the cortex-hippocampus interface in either the ipsilateral or the contralateral hemisphere. One group of animals (n = 7) was killed at either 1 or 3 weeks postinjury to assess NSC survival in the acute posttraumatic period. Motor function was evaluated at weekly intervals for 12 weeks in the remaining animals, and cognitive (i.e., learning) deficits were assessed at 3 and 12 weeks after transplantation. RESULTS Brain-injured animals that received either ipsilateral or contralateral NSC transplants showed significantly improved motor function in selected tests as compared with human embryonic kidney cell-transplanted animals during the 12-week observation period. Cognitive dysfunction was unaffected by transplantation at either 3 or 12 weeks postinjury. Histological analyses showed that NSCs survive for as long as 13 weeks after transplantation and were detected in the hippocampus and/or cortical areas adjacent to the injury cavity. At 13 weeks, the NSCs transplanted ipsilateral to the impact site expressed neuronal (NeuN) or astrocytic (glial fibrillary acidic protein) markers but not markers of oligodendrocytes (2′3′cyclic nucleotide 3′-phosphodiesterase), whereas the contralaterally transplanted NSCs expressed neuronal but not glial markers (double-labeled immunofluorescence and confocal microscopy). CONCLUSION These data suggest that transplanted NSCs can survive in the traumatically injured brain, differentiate into neurons and/or glia, and attenuate motor dysfunction after traumatic brain injury.

Insulin-like Growth Factor-1 (IGF-1) Improves both Neurological Motor and Cognitive Outcome Following Experimental Brain Injury

We evaluated the efficacy of insulin-like growth factor-1 (IGF-1) in attenuating neurobehavioral deficits following lateral fluid percussion (FP) brain injury. Male Sprague-Dawley rats (345-425 g, n = 88) were anesthetized and subjected to FP brain injury of moderate severity (2.4-2.9 atm). In Study 1, IGF-1 (1.0 mg/kg, n = 9) or vehicle (n = 14) was administered by subcutaneous injection at 15 min postinjury and similarly at 12-h intervals for 14 days. In animals evaluated daily for 14 days, IGF-1 treatment attenuated motor dysfunction over the 2-week period (P < 0.02). In Study 2, IGF-1 (4 mg/kg/day, n = 8 uninjured, n = 13 injured) or vehicle (n = 8 uninjured, n = 13 injured) was administered for 2 weeks via a subcutaneous pump implanted 15 min postinjury. IGF-1 administration was associated with increased body weight and mild, transient hypoglycemia which was more pronounced in brain-injured animals. At 2 weeks postinjury (P < 0.05), but not at 48 h or 1 week, brain-injured animals receiving IGF-1 showed improved neuromotor function compared with those receiving vehicle. IGF-1 administration also enhanced learning ability (P < 0.03) and memory retention (P < 0.01) in brain-injured animals at 2 weeks postinjury. Taken together, these data suggest that chronic, posttraumatic administration of the trophic factor IGF-1 may be efficacious in ameliorating neurobehavioral dysfunction associated with traumatic brain injury.

Genetic approaches to neurotrauma research: opportunities and potential pitfalls of murine models.

Genetic strategies provide new ways to define the molecular cascades that regulate the responses of the mammalian nervous system to injury. Genetic interventions also provide opportunities to manipulate and control key molecular steps in these cascades, so as to modify the outcome of CNS injury. Most current genetic strategies involve the use of mice, an animal that has not heretofore been used extensively for neurotrauma research. Therefore, one purpose of the present review is to consider how mice respond to neural trauma, focusing especially on recent information that reveals important differences between mice and rats, and between different inbred strains of mice. The second aim of this review is to provide a brief introduction to the opportunities, caveats, and potential pitfalls of studies that use genetically modified animals for neurotrauma research.

Structural and functional damage sustained by mitochondria after traumatic brain injury in the rat: Evidence for differentially sensitive populations in the cortex and hippocampus

The cellular and molecular pathways initiated by traumatic brain injury (TBI) may compromise the function and structural integrity of mitochondria, thereby contributing to cerebral metabolic dysfunction and cell death. The extent to which TBI affects regional mitochondrial populations with respect to structure, function, and swelling was assessed 3 hours and 24 hours after lateral fluid—percussion brain injury in the rat. Significantly less mitochondrial protein was isolated from the injured compared with uninjured parietotemporal cortex, whereas comparable yields were obtained from the hippocampus. After injury, cortical and hippocampal tissue ATP concentrations declined significantly to 60% and 40% of control, respectively, in the absence of respiratory deficits in isolated mitochondria. Mitochondria with ultrastructural morphologic damage comprised a significantly greater percent of the population isolated from injured than uninjured brain. As determined by photon correlation spectroscopy, the mean mitochondrial radius decreased significantly in injured cortical populations (361 ± 40 nm at 24 hours) and increased significantly in injured hippocampal populations (442 ± 36 at 3 hours) compared with uninjured populations (Ctx: 418 ± 44; Hipp: 393 ± 24). Calcium-induced deenergized swelling rates of isolated mitochondrial populations were significantly slower in injured compared with uninjured samples, suggesting that injury alters the kinetics of mitochondrial permeability transition (MPT) pore activation. Cyclosporin A (CsA)-insensitive swelling was reduced in the cortex, and CsA-sensitive and CsA-insensitive swelling both were reduced in the hippocampus, demonstrating that regulated MPT pores remain in mitochondria isolated from injured brain. A proposed mitochondrial population model synthesizes these data and suggests that cortical mitochondria may be depleted after TBI, with a physically smaller, MPT-regulated population remaining. Hippocampal mitochondria may sustain damage associated with ballooned membranes and reduced MPT pore calcium sensitivity. The heterogeneous mitochondrial response to TBI may underlie posttraumatic metabolic dysfunction and contribute to the pathophysiology of TBI.

Brain Trauma Induces Massive Hippocampal Neuron Death Linked to a Surge in β-Amyloid Levels in Mice Overexpressing Mutant Amyloid Precursor Protein

Although brain trauma is a risk factor for Alzheimers disease, no experimental model has been generated to explore this relationship. We developed a model of brain trauma in transgenic mice that overexpress mutant human amyloid precursor protein (PDAPP) leading to the appearance of Alzheimers disease-like beta-amyloid (Abeta) plaques beginning at 6 months of age. We induced cortical impact brain injury in the PDAPP animals and their wild-type littermates at 4 months of age, ie, before Abeta plaque formation, and evaluated changes in posttraumatic memory function, histopathology, and regional tissue levels of the Abeta peptides Abeta1-40 and Abeta1-42. We found that noninjured PDAPP mice had impaired memory function compared to noninjured wild-type littermates (P < 0.01) and that brain-injured PDAPP mice had more profound memory dysfunction than brain-injured wild-type littermates (P < 0.001). Although no augmentation of Abeta plaque formation was observed in brain-injured PDAPP mice, a substantial exacerbation of neuron death was found in the hippocampus (P < 0.001) in association with an acute threefold increase in Abeta1-40 and sevenfold increase in Abeta1-42 levels selectively in the hippocampus (P < 0.01). These data suggest a mechanistic link between brain trauma and Abeta levels and the death of neurons.

Pharmacologic Inhibition of Poly(ADP-Ribose) Polymerase Is Neuroprotective Following Traumatic Brain Injury in Rats

The nuclear enzyme poly(ADP-ribose) polymerase (PARP), which has been shown to be activated following experimental traumatic brain injury (TBI), binds to DNA strand breaks and utilizes nicotinamide adenine dinucleotide (NAD) as a substrate. Since consumption of NAD may be deleterious to recovery in the setting of CNS injury, we examined the effect of a potent PARP inhibitor, GPI 6150, on histological outcome following TBI in the rat. Rats (n = 16) were anesthetized, received a preinjury dose of GPI 6150 (30 min; 15 mg/kg, i.p.), subjected to lateral fluid percussion (FP) brain injury of moderate severity (2.5-2.8 atm), and then received a second dose 3 h postinjury (15 mg/kg, i.p.). Lesion area was examined using Nissl staining, while DNA fragmentation and apoptosis-associated cell death was assessed with terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labeling (TUNEL) with stringent morphological evaluation. Twenty-four hours after brain injury, a significant cortical lesion and number of TUNEL-positive/nonapoptotic cells and TUNEL-positive/apoptotic cells in the injured cortex of vehicle-treated animals were observed as compared to uninjured rats. The size of the trauma-induced lesion area was significantly attenuated in the GPI 6150-treated animals versus vehicle-treated animals (p < 0.05). Treatment of GPI 6150 did not significantly affect the number of TUNEL-positive apoptotic cells in the injured cortex. The observed neuroprotective effects on lesion size, however, offer a promising option for further evaluation of PARP inhibition as a means to reduce cellular damage associated with TBI.

Hyperthermia following traumatic brain injury: a critical evaluation.

Hyperthermia, frequently seen in patients following traumatic brain injury (TBI), may be due to posttraumatic cerebral inflammation, direct hypothalamic damage, or secondary infection resulting in fever. Regardless of the underlying cause, hyperthermia increases metabolic expenditure, glutamate release, and neutrophil activity to levels higher than those occurring in the normothermic brain-injured patient. This synergism may further compromise the injured brain, enhancing the vulnerability to secondary pathogenic events, thereby exacerbating neuronal damage. Although rigorous control of normal body temperature is the current standard of care for the brain-injured patient, patient management strategies currently available are often suboptimal and may be contraindicated. This article represents a compendium of published work regarding the state of knowledge of the relationship between hyperthermia and TBI, as well as a critical examination of current management strategies.

Tissue tears in the white matter after lateral fluid percussion brain injury in the rat: relevance to human brain injury.

Abstract A characteristic feature of severe diffuse axonal injury in man is radiological evidence of the “shearing injury triad” represented by lesions, sometimes haemorrhagic, in the corpus callosum, deep white matter and the rostral brain stem. With the exception of studies carried out on the non-human primate, such lesions have not been replicated to date in the multiple and diverse rodent laboratory models of traumatic brain injury. The present report describes tissue tears in the white matter, particularly in the fimbria of Sprague-Dawley rats killed 12, 24, and 48 h and 7 days after lateral fluid percussion brain injury of moderate severity (2.1–2.4 atm). The lesions were most easily seen at 24 h when they appeared as foci of tissue rarefaction in which there were a few polymorphonuclear leucocytes. At the margins of these lesions, large amounts of accumulated amyloid precursor protein (APP) were found in axonal swellings and bulbs. By 1 week post-injury, there was macrophage infiltration with marked astrocytosis and early scar formation. This lesion is considered to be due to severe deformation of white matter and this is the first time that it has been identified reproducibly in a rodent model of head injury under controlled conditions.

The tumor-suppressor gene, p53, is induced in injured brain regions following experimental traumatic brain injury

A growing body of evidence suggests that neurons undergo apoptotic cell death following traumatic brain injury (TBI). Since the expression of several tumor suppressor and cell cycle genes have been implicated in neuronal apoptosis, the present study used in situ hybridization (ISH) histochemistry to evaluate the regional and temporal patterns of expression of the mRNAs for the tumor suppressor gene, p53, and the cell cycle gene, cyclin D1, following lateral fluid-percussion (FP) brain injury in the rat. Anesthetized adult male Sprague-Dawley rats (n=16) were subjected to lateral FP brain injury of moderate severity (2.4-2.7 atm), while sham controls (n=6) were surgically prepared but did not receive brain injury. Animals were killed by decapitation at 6 h (n=6 injured and 2 sham), 24 h (n=6 injured and 2 sham), or 3 days (n=4 injured and 2 sham), and their brains processed for ISH. Little to no expression of p53 mRNA was observed in sham brains. At 6 h post-injury, p53 mRNA was induced predominantly in cells that are vulnerable to TBI, such as those in the contused cortex, lateral and medial geniculate nuclei of the thalamus, and the CA(3) and hilar neurons of the hippocampus. Increased p53 mRNA was also detected in hippocampal CA(1) neurons, cells that are relatively resistant to FP brain injury. Levels of p53 mRNA returned to sham levels in all regions of the injured brain by 24 h. In contrast to p53, cyclin D1 mRNA was detectable in the brains of uninjured animals and was not altered by brain injury. These results suggest that the tumor suppressor gene p53, but not cyclin D1, is upregulated and may participate in molecular response to TBI.