A model of posttraumatic epilepsy induced by lateral fluid-percussion brain injury in rats.

Although traumatic brain injury is a major cause of symptomatic epilepsy, the mechanism by which it leads to recurrent seizures is unknown. An animal model of posttraumatic epilepsy that reliably reproduces the clinical sequelae of human traumatic brain injury is essential to identify the molecular and cellular substrates of posttraumatic epileptogenesis, and perform preclinical screening of new antiepileptogenic compounds. We studied the electrophysiologic, behavioral, and structural features of posttraumatic epilepsy induced by severe, non-penetrating lateral fluid-percussion brain injury in rats. Data from two independent experiments indicated that 43% to 50% of injured animals developed epilepsy, with a latency period between 7 weeks to 1 year. Mean seizure frequency was 0.3+/-0.2 seizures per day and mean seizure duration was 113+/-46 s. Behavioral seizure severity increased over time in the majority of animals. Secondarily-generalized seizures comprised an average of 66+/-37% of all seizures. Mossy fiber sprouting was increased in the ipsilateral hippocampus of animals with posttraumatic epilepsy compared with those subjected to traumatic brain injury without epilepsy. Stereologic cell counts indicated a loss of dentate hilar neurons ipsilaterally following traumatic brain injury. Our data suggest that posttraumatic epilepsy occurs with a frequency of 40% to 50% after severe non-penetrating fluid-percussion brain injury in rats, and that the lateral fluid percussion model can serve as a clinically-relevant tool for pathophysiologic and preclinical studies.

No evidence for the presence of apolipoprotein epsilon4, interleukin-lalpha allele 2 and interleukin-1beta allele 2 cause an increase in programmed cell death following traumatic brain injury in humans.

BACKGROUND: Brain injury after trauma is an important cause of mortality and morbidity in society. There is evidence in both man and laboratory animals that in addition to necrosis, cell loss may occur as a result of programmed cell death (PCD). The cellular and molecular responses after head injury are partly influenced by genetic polymorphisms of apolipoprotein E and the pro-inflammatory cytokine IL-I. AIM: The principal aim of this study was to determine whether the presence of the ApoE epsilon4, IL- 1 alpha2 or IL- 1beta2 allele types influenced the amounts of PCD after head injury compared with controls. METHODS: Paraffin sections from the hippocampus of 38 patients (32 M : 6 F, aged 15 - 75, mean 38 years, survival 7- 576 hours; mean 36 hours) who died after a head injury were stained by Tunel histochemistry and quantified, and genotyping was undertaken by PCR "blind" to clinical detail. RESULTS: There were more Tunel+ cells (neurons and glia) after head injury than in controls with statistically increased numbers in all sectors of the hippocampus including the dentate fascia. However, there was no correlation between ApoEepsilon4, IL- 1 alpha allele 2 and IL- 1beta allele 2 and the amount of Tunel positivity. CONCLUSION: Given that both the ApoE and IL-1 influence outcome after various forms of acute brain injury, further work will be required to determine the mechanism underlying this relationship.

Traumatic brain injury alters the molecular fingerprint of TUNEL-positive cortical neurons In vivo: A single-cell analysis.

The cerebral cortex is selectively vulnerable to cell death after traumatic brain injury (TBI). We hypothesized that the ratio of mRNAs encoding proteins important for cell survival and/or cell death is altered in individual damaged neurons after injury that may contribute to the cell's fate. To investigate this possibility, we used amplified antisense mRNA (aRNA) amplification to examine the relative abundance of 31 selected candidate mRNAs in individual cortical neurons with fragmented DNA at 12 or 24 hr after lateral fluid percussion brain injury in anesthetized rats. Only pyramidal neurons characterized by nuclear terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) reactivity with little cytoplasmic staining were analyzed. For controls, non-TUNEL-positive neurons from the cortex of sham-injured animals were obtained and subjected to aRNA amplification. At 12 hr after injury, injured neurons exhibited a decrease in the relative abundance of specific mRNAs including those encoding for endogenous neuroprotective proteins. By 24 hr after injury, many of the mRNAs altered at 12 hr after injury had returned to baseline (sham-injured) levels except for increases in caspase-2 and bax mRNAs. These data suggest that TBI induces a temporal and selective alteration in the gene expression profiles or "molecular fingerprints" of TUNEL-positive neurons in the cerebral cortex. These patterns of gene expression may provide information about the molecular basis of cell death in this region after TBI and may suggest multiple avenues for therapeutic intervention.

Mechanisms of mitochondria-neurofilament interactions.

Mitochondria are localized to regions of the cell where ATP consumption is high and are dispersed according to changes in local energy needs. In addition to motion directed by molecular motors, mitochondrial distribution in neuronal cells appears to depend on the docking of mitochondria to microtubules and neurofilaments. We examined interactions between mitochondria and neurofilaments using fluorescence microscopy, dynamic light scattering, atomic force microscopy, and sedimentation assays. Mitochondria-neurofilament interactions depend on mitochondrial membrane potential, as revealed by staining with a membrane potential sensitive dye (JC-1) in the presence of substrates/ADP or uncouplers (valinomycin/carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone) and are affected by the phosphorylation status of neurofilaments and neurofilament sidearms. Antibodies against the neurofilament heavy subunit disrupt binding between mitochondria and neurofilaments, and isolated neurofilament sidearms alone interact with mitochondria, suggesting that they mediate the interactions between the two structures. These data suggest that specific and regulated mitochondrial-neurofilament interactions occur in situ and may contribute to the dynamic distribution of these organelles within the cytoplasm of neurons.

Experimental models of traumatic brain injury: do we really need to build a better mousetrap?

Approximately 4000 human beings experience a traumatic brain injury each day in the United States ranging in severity from mild to fatal. Improvements in initial management, surgical treatment, and neurointensive care have resulted in a better prognosis for traumatic brain injury patients but, to date, there is no available pharmaceutical treatment with proven efficacy, and prevention is the major protective strategy. Many patients are left with disabling changes in cognition, motor function, and personality. Over the past two decades, a number of experimental laboratories have attempted to develop novel and innovative ways to replicate, in animal models, the different aspects of this heterogenous clinical paradigm to better understand and treat patients after traumatic brain injury. Although several clinically-relevant but different experimental models have been developed to reproduce specific characteristics of human traumatic brain injury, its heterogeneity does not allow one single model to reproduce the entire spectrum of events that may occur. The use of these models has resulted in an increased understanding of the pathophysiology of traumatic brain injury, including changes in molecular and cellular pathways and neurobehavioral outcomes. This review provides an up-to-date and critical analysis of the existing models of traumatic brain injury with a view toward guiding and improving future research endeavors.

Delayed inhibition of Nogo-A does not alter injury-induced axonal sprouting but enhances recovery of cognitive function following experimental traumatic brain injury in rats.

Traumatic brain injury causes long-term neurological motor and cognitive deficits, often with limited recovery. The inability of CNS axons to regenerate following traumatic brain injury may be due, in part, to inhibitory molecules associated with myelin. One of these myelin-associated proteins, Nogo-A, inhibits neurite outgrowth in vitro, and inhibition of Nogo-A in vivo enhances axonal outgrowth and sprouting and improves outcome following experimental CNS insults. However, the involvement of Nogo-A in the neurobehavioral deficits observed in experimental traumatic brain injury remains unknown and was evaluated in the present study using the 11C7 monoclonal antibody against Nogo-A. Anesthetized, male Sprague-Dawley rats were subjected to either lateral fluid percussion brain injury of moderate severity (2.5-2.6 atm) or sham injury. Beginning 24 h post-injury, monoclonal antibody 11C7 (n=17 injured, n=6 shams included) or control Ab (IgG) (n=16 injured, n=5 shams included) was infused at a rate of 5 microl/h over 14 days into the ipsilateral ventricle using osmotic minipumps connected to an implanted cannula. Rats were assessed up to 4 weeks post-injury using tests for neurological motor function (composite neuroscore, and sensorimotor test of adhesive paper removal) and, at 4 weeks, cognition was assessed using the Morris water maze. Hippocampal CA3 pyramidal neuron damage and corticospinal tract sprouting, using an anterograde tracer (biotinylated dextran amine), were also evaluated. Brain injury significantly increased sprouting from the uninjured corticospinal tract but treatment with monoclonal antibody 11C7 did not further increase the extent of sprouting nor did it alter the extent of CA3 cell damage. Animals treated with 11C7 showed no improvement in neurologic motor deficits but did show significantly improved cognitive function at 4 weeks post-injury when compared with brain-injured, IgG-treated animals. To our knowledge, the present findings are the first to suggest that (1) traumatic brain injury induces axonal sprouting in the corticospinal tract and this sprouting may be independent of myelin-associated inhibitory factors and (2) that post-traumatic inhibition of Nogo-A may promote cognitive recovery unrelated to sprouting in the corticospinal tract or neuroprotective effects on hippocampal cell loss following experimental traumatic brain injury.

Pharmacology of traumatic brain injury.

The intensity of experimental and clinical research to identify a neuroprotective drug for the treatment of traumatic brain injury is motivated by the devastating morbidity and mortality of this condition. Encouraging experimental work has led so far to disappointing clinical trials and the identification of new potential therapeutic targets is critically dependent on a better understanding of the chronic pathophysiology triggered by the initial insult. Future advances in the pharmacological treatment of traumatic brain injury are likely to include the evaluation of sequentially timed therapies combining multiple and targeted agents, and manipulation of the newly discovered neurogenic potential of the adult brain together with the refinement of traditional interventions to block specific cytotoxic cascades.

Regional heterogeneity in the response of astrocytes following traumatic brain injury in the adult rat.

The regional distribution and temporal appearance of astrocytes expressing glial fibrillary acidic protein (GFAP), S100 protein, and vimentin were determined in a nonpenetrating lateral fluid percussion (LFP) brain injury model. Following injury, reactive astrocytes were observed in the subcortical white matter tracts as early as 1 day, in the hippocampus and injured cortex by 3 days, and in the thalamus by 1 week. Reactive astrocytes in the injured cortex, subcortical white matter tracts, and CA3 region of the hippocampus were all vimentin positive at 1 month post-injury. These astrocytes had a distinct morphology characterized by an enlarged cell body and long intertwined processes. In contrast, reactive astrocytes in the thalamic nuclei never expressed vimentin, and displayed an enlarged cell body with thick shortened processes. An increase in S100 protein was detected in all reactive astrocytes following LFP brain injury. Quantitative assessment of GFAP, S100, and vimentin polypeptides confirmed the immunohistochemical evaluation. Our data indicate that although astrogliosis mirrors the spatial pattern of post-traumatic neuronal cell loss, the expression of vimentin and the cellular morphology of the cells were regionally distinct, suggesting that astrogliosis may be modulated by factors present in the post-traumatic brain.

Acute cytoskeletal alterations and cell death induced by experimental brain injury are attenuated by magnesium treatment and exacerbated by magnesium deficiency.

Traumatic brain injury results in a profound decline in intracellular magnesium ion levels that may jeopardize critical cellular functions. We examined the consequences of preinjury magnesium deficiency and post-traumatic magnesium treatment on injury-induced cytoskeletal damage and cell death at 24 h after injury. Adult male rats were fed either a normal (n = 24) or magnesium-deficient diet (n = 16) for 2 wk prior to anesthesia and lateral fluid percussion brain injury (n = 31) or sham injury (n = 9). Normally fed animals were then randomized to receive magnesium chloride (125 micromol, i.v., n = 10) or vehicle solution (n = 11) at 10 min postinjury. Magnesium treatment reduced cortical cell loss (p < 0.05), cortical alterations in microtubule-associated protein-2 (MAP-2) (p < 0.05), and both cortical and hippocampal calpain-mediated spectrin breakdown (p < 0.05 for each region) when compared to vehicle treatment. Conversely, magnesium deficiency prior to brain injury led to a greater area of cortical cell loss (p < 0.05 compared to vehicle treatment). Moreover, brain injury to magnesium-deficient rats resulted in cytoskeletal alterations within the cortex and hippocampus that were not observed in vehicle- or magnesium-treated animals. These data suggest that cortical cell death and cytoskeletal disruptions in cortical and hippocampal neurons may be sensitive to magnesium status after experimental brain injury, and may be mediated in part through modulation of calpains.

Recent advances in neurotrauma.

The frequency of and outcome from acute traumatic brain injury (TBI) in humans are detailed together with a classification of the principal focal and diffuse pathologies, and their mechanisms in extract laboratory models are outlined. Particular emphasis is given to diffuse axonal injury, which is a major determinant of outcome. Cellular and molecular cascades triggered by injury are described with reference to the induction of axolemmal and cytoskeletal abnormalities, necrotic and apoptotic cell death, the role of Ca2+, cytokines and free radicals, and damage to DNA. It is concluded that TBI in humans is heterogeneous, reflecting various pathologies in differing proportions in patients whose genetic background (APOE gene polymorphisms) contributes to the outcome at 6 months. Although considerable progress has been made in the understanding of TBI, much remains to be determined. However, a deeper understanding of the pathophysiological events may lead to the possibility of improving outcome from rational targeted therapy.

Prolonged calpain-mediated spectrin breakdown occurs regionally following experimental brain injury in the rat.

Calpain, a calcium-activated neutral protease family, has been implicated in the neuropathologic sequelae accompanying various neurological disorders. We have characterized the distribution and time course of calpain activation following brain injury in the rat, using a monoclonal antibody that recognizes calpain-generated breakdown products (BDPs) of spectrin. Adult male Sprague-Dawley rats received lateral fluid percussion brain injury of moderate severity (2.2-2.4 atm, n = 35) or served as controls (uninjured, n = 12). One group of animals (n = 21) were sacrificed at either 30 minutes (min), 1 day, or 3 days post-injury, and selected brain regions were prepared for Western blot analysis. The remaining animals (n = 26) were sacrificed at 90 min, 4 hours (h), 1 day, or 7 days post-injury, and immunohistochemistry was performed. Spectrin BDPs were found predominantly in the hemisphere ipsilateral to the injury site, located primarily in cortical and hippocampal regions which exhibit neuronal death. Calpain-mediated spectrin breakdown was detected at 90 min in dendrites and axons, and by 4 h in neuronal perikarya. By 1 day post-injury, cortical and hippocampal regions of calpain activation had increased in size. Delayed spectrin breakdown was observed in the thalamus, both at 3 days and 7 days after injury. These results suggest that calpain may play an important role in the neurodegenerative process following brain injury.

Characterization of diffuse axonal pathology and selective hippocampal damage following inertial brain trauma in the pig.

Dynamic deformation applied to white matter tracts is a common feature of human brain trauma, and may result in diffuse axonal injury (DAI). To produce DAI in an experimental model, we have utilized nonimpact inertial loading to induce brain trauma in miniature swine. This species was chosen due to its large gyrencephalic brain with substantial white matter domains. Twenty anesthetized (2% isoflurane) miniature swine were subjected to pure impulsive centroidal rotation 110 degrees in the coronal plane in 4 to 6 ms; peak accelerations ranged from 0.6 to 1.7 x 10(5) rad/s2. Seven days following injury, the brains were fixed (4% paraformaldehyde). Histopathologic examination was performed on 40 microns sections stained with cresyl violet (Nissl), antibodies targeting neurofilament (axonal damage), GFAP (astrocytes), and pig IgG (protein extravasation). Widespread multifocal axonal injury was observed in combination with gliosis throughout the brain, most commonly in the root of gyri and at the interface of the gray and white matter. Very little vascular disruption was noted in regions of axonal injury. Neuronal damage was primarily found in the CA1 and CA3 subfields of the hippocampus. These results suggest that this model is clinically relevant and useful for evaluating mechanisms of inertial brain trauma.

Experimental investigation of cerebral contusion: histopathological and immunohistochemical evaluation of dynamic cortical deformation.

We used a new approach, termed dynamic cortical deformation (DCD), to study the neuronal, vascular, and glial responses that occur in focal cerebral contusions. DCD produces experimental contusion by rapidly deforming the cerebral cortex with a transient, nonablative vacuum pulse of short duration (25 milliseconds) to mimic the circumstances of traumatic injury. A neuropathological evaluation was performed on brain tissue from adult rats sacrificed 3 days following induction of either moderate (4 psi, n = 6) or high (8 psi, n = 6) severity DCD. In all animals, DCD produced focal hemorrhagic lesions at the vacuum site without overt damage to other regions. Examination of histological sections showed localized gross tissue and neuronal loss in the cortex at the injury site, with the volume of cell loss dependent upon the mechanical loading (p < 0.001). Axonal pathology shown with neurofilament immunostaining (SMI-31 and SMI-32) was observed in the subcortical white matter inferior to the injury site and in the ipsilateral internal capsule. No axonal injury was observed in the contralateral hemisphere or in any remote regions. Glial fibrillary acidic protein (GFAP) immunostaining revealed widespread reactive astrocytosis surrounding the necrotic region in the ipsilateral cortex. This analysis confirms that rapid mechanical deformation of the cortex induces focal contusions in the absence of primary damage to remote areas 3 days following injury. Although it is suggested that massive release of neurotoxic substances from a contusion may cause damage throughout the brain, these data emphasize the importance of combined injury mechanisms, e.g. mechanical distortion and excitatory amino acid mediated damage, that underlie the complex pathology patterns observed in traumatic brain injury.

Evolution of neurofilament subtype accumulation in axons following diffuse brain injury in the pig.

Although accumulation of neurofilament (NF) proteins in axons has been recognized as a prominent feature of brain trauma, the temporal course of the accumulation of specific NF subtypes has not been well established. In the present study, 17 miniature swine were subjected to nonimpact inertial brain injury. At 3 hours (h), 6 h, 24 h, 3 days, 7 days, and 10 days post-trauma, immunohistochemical analysis was performed to determine axonal accumulation of NF-light (NF-L), the rod and sidearm domains and sidearm phosphorylation states of NF-medium (NF-M), and heavy (NF-H). We found that NF-L accumulation was easily identified in damaged axons by 6 h post-trauma, but NF-M and H accumulation was not clearly visualized until 3 days following injury. In addition, the axonal accumulation of NF-M and H appeared to be primarily comprised of the sidearm domains. While the accumulating NF was found to be predominantly dephosphorylated, we also detected accumulation of phosphorylated NF. Finally, we found that developing axonal pathology may proceed either towards axotomy with discrete terminal bulb formation or towards the development of varicose swellings encompassing long portions of axons. These findings suggest that there is a differential temporal course in NF subtype disassembly, dephosphorylation, and accumulation in axons following initial brain trauma and that these processes occur in morphologically distinct phenotypes of maturing axonal pathology.

Prolonged disruption of plasma beta-endorphin dynamics after trauma in the nonhuman primate.

Recent evidence has suggested that a circadian rhythm exists for plasma beta-endorphin-like immunoreactivity. The purpose of the present study was to examine the long term effects of surgical trauma on plasma beta-endorphin dynamics. Blood samples for RIA were obtained from female baboons every 4 h for three 48-h periods: one beginning 1 week before surgical trauma, the second 30 min after surgical trauma, and the third 1 week after surgical trauma. Animals were subjected to laparotomy and 30-min anesthesia (n = 8), 5-min surgical trauma under 30-min anesthesia (low trauma; n = 8), or 20-min surgical trauma under 30-min anesthesia (high trauma; n = 8). Computer analysis of beta-endorphin levels as a function of clock time demonstrated a true preoperative circadian rhythm for all animals, with a mean of 87.9 pg/ml. In the immediate 48-h postoperative period, a postoperative alteration in circadian beta-endorphin dynamics occurred that was correlated with the severity of trauma. A disruption of circadian rhythms of plasma beta-endorphin occurred in the high trauma group only, in which it persisted for longer than 1 week after trauma. These studies establish a relationship between the alteration of circadian rhythmicity of plasma beta-endorphin-like immunoreactivity and the magnitude of trauma and injury.

Cellular responses to experimental brain injury.

Little is known regarding the molecular (genomic) events associated with the pathophysiology of traumatic brain injury (TBI). This review focusses on the experimental efforts to date elucidating the acute alterations in expression of immediate early genes (IEGs), heat shock proteins (HSPs) and cytokines following experimental brain injury. The immediate early genes, c-fos, c-jun and junB were observed to be bilaterally induced in the cortex and hippocampus as early as 5 min following lateral fluid-percussion (FP) brain injury in the rat. While levels of c-fos and junB mRNA returned to control levels by 2h, c-jun mRNA remained elevated up to 6h post-injury. Increased levels of mRNA for the inducible heat-shock protein (hsp72) were observed up to 12h following injury and were restricted to the cortex ipsilateral to the impact site. Mild induction of the glucose-regulated proteins (grp78 and grp94), which share sequence homology with hsp72, was apparent in the ipsilateral cortex. The cytokines IL-1 beta and TNF alpha were induced at 1h following FP brain injury and remained elevated up to 6h post-injury. These data, while indicative of the complex genomic response to TBI, are also suggestive of the trauma-induced activation of multiple signal transduction pathways.

Effects of traumatic brain injury on regional cerebral blood flow in rats as measured with radiolabeled microspheres.

To clarify the effect of experimental brain injury on regional CBF (rCBF), repeated rCBF measurements were performed using radiolabeled microspheres in rats subjected to fluid-percussion traumatic brain injury. Three consecutive microsphere injections in six uninjured control rats substantiated that the procedure induces no significant changes in hemodynamic variables or rCBF. Animals were subjected to left parietal fluid-percussion brain injury of moderate severity (2.1-2.4 atm) and rCBF values were determined (a) prior to injury and 15 min and 1 h following injury (n = 7); and (b) prior to injury and 30 min and 2 h following injury (n = 7). At 15 min post injury, there was a profound reduction of rCBF in all brain regions studied (p less than 0.01). Although rCBF in the hindbrain had recovered to near-normal by 30 min post injury, rCBF in both injured and contralateral (uninjured) forebrain areas remained significantly suppressed up to 1 h post injury. At 2 h post injury, recovery of rCBF to near-normal values was observed in all brain regions except the focal area of injury (left parietal cortex) where rCBF remained significantly depressed (p less than 0.01). This prolonged focal oligemia at the injury site was associated with the development of reproducible cystic necrosis in the left parietotemporal cortex at 4 weeks post injury. Our results demonstrate that acute changes in rCBF occur following experimental traumatic brain injury in rats and that rCBF remains significantly depressed up to 2 h post injury in the area circumscribing the trauma site.

Alterations in regional cerebral blood flow following brain injury in the rat.

To elucidate the temporal changes in regional cerebral blood flow (rCBF) after experimental traumatic brain injury, serial rCBF measurements were made during a 24-h period following fluid-percussion (F-P) traumatic brain injury in the rat. Brain injury of 2.2 atm was induced over the left parietal cortex and serial measurements of rCBF were performed using the radiolabeled microsphere method. rCBF values were obtained prior to injury and at 15 and 30 min and 1, 2, 4, and 24 h postinjury. At 15 min postinjury, there was a profound, wide-spread reduction in rCBF in all brain regions studied (p less than 0.05). At 30 min and 1 h postinjury, all brain regions except pons-medulla and cerebellum showed significantly reduced rCBF compared to the preinjury values (p less than 0.05). By 2 h postinjury, however, a significant focal reduction of rCBF was observed only in the cerebral tissue surrounding the trauma site (p less than 0.05); rCBF in the remaining brain regions had recovered to the preinjury levels. By 4 h postinjury, rCBF had returned to normal in all brain regions studied. This recovery of rCBF was still evident at 24 h postinjury. The present study demonstrates that, following the experimental traumatic brain injury in the rat, (a) an initial global suppression of rCBF occurs up to 1 h postinjury; (b) at the trauma site, a more persistent focal reduction of rCBF occurs; and (c) these alterations in rCBF after trauma dissolve by 4 h postinjury.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in neuropeptide Y after experimental traumatic brain injury in the rat.

We utilized a model of fluid percussion (FP) brain injury in the rat to examine the hypothesis that alterations in brain neuropeptide Y (NPY) concentrations occur following brain injury. Male rats (n = 44) were subjected to FP traumatic brain injury. One group of animals (n = 38) was killed at 1 min, 15 min, 1 h, or 24 h after brain injury, and regional brain homogenates were analyzed for NPY concentrations using radioimmunoassay. A second group of animals (n = 6) was killed for NPY immunocytochemistry. Concentrations of NPY in the injured left parietal cortex were significantly elevated at 15 min post injury (p less than 0.05). No changes were observed in other brain regions. NPY-immunoreactive fibers were seen at 15 min post injury predominantly in the injured cortex and adjacent hippocampus. These temporal changes in NPY immunoreactivity, together with previous observations concerning posttraumatic changes in regional CBF in these same areas, suggest that an increase in region NPY concentrations after brain injury may be involved in part in the pathogenesis of posttraumatic hypoperfusion.

Behavioral efficacy of posttraumatic calpain inhibition is not accompanied by reduced spectrin proteolysis, cortical lesion, or apoptosis.

Administration of the selective calpain inhibitor AK295 has been shown to attenuate motor and cognitive dysfunction after brain trauma in rats. To explore mechanisms underlying the behavioral efficacy of posttraumatic calpain inhibition, we investigated histologic consequences of AK295 administration. Anesthetized Sprague-Dawley rats received lateral fluid percussion brain injury of moderate severity (2.2 to 2.4 atm) or served as uninjured controls. At 15 minutes after injury, animals were randomly assigned to receive a 48-hour infusion of either 2 mmol/L AK295 (120 to 140 mg/kg) or saline via the carotid artery. At 48 hours and 1 week after injury, spectrin fragments generated specifically by calpain were detected by Western blotting and immunohistochemistry, respectively, in saline-treated, brain-injured animals. Interestingly, equivalent spectrin breakdown was observed in AK295-treated animals when cortical and hippocampal regions were evaluated. Similarly, administration of the calpain inhibitor did not attenuate cortical lesion size or the numbers of apoptotic cells in the cortex, subcortical white matter, or hippocampus, as verified by terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick-end labeling and morphology, at 48 hours after injury. These data suggest that an overt reduction in spectrin proteolysis, cortical lesion, or apoptotic cell death at 48 hours or 1 week is not required for behavioral improvements associated with calpain inhibition by AK295 after experimental brain injury in rats.