Brain-injured Animals Research Articles

Cognitive outcome following brain injury and treatment with an inhibitor of Nogo-A in association with an attenuated downregulation of hippocampal growth-associated protein-43 expression

Central nervous system axons regenerate poorly after traumatic brain injury (TBI), partly due to inhibitors such as the protein Nogo-A present in myelin. The authors evaluated the efficacy of anti-Nogo-A monoclonal antibody (mAb) 7B12 administration on the neurobehavioral and cognitive outcome of rats following lateral fluid-percussion brain injury, characterized the penetration of the 7B12 or control antibodies into target brain regions, and evaluated the effects of Nogo-A inhibition on hemispheric tissue loss and sprouting of uninjured motor tracts in the cervical cord. To elucidate a potential molecular response to Nogo-A inhibition, we evaluated the effects of 7B12 on hippocampal GAP-43 expression. Beginning 24 hours after lateral fluid-percussion brain injury or sham injury in rats, the mAb 7B12 or control antibody was infused intracerebroventricularly over 14 days, and behavior was assessed over 4 weeks. Immunoreactivity for 7B12 or immunoglobulin G was detected in widespread brain regions at 1 and 3 weeks postinjury. The brain-injured animals treated with 7B12 showed improvement in cognitive function (p < 0.05) at 4 weeks but no improvement in neurological motor function from 1 to 4 weeks postinjury compared with brain-injured, vehicle-treated controls. The enhanced cognitive function following inhibition of Nogo-A was correlated with an attenuated postinjury downregulation of hippocampal GAP-43 expression (p < 0.05). Increased GAP-43 expression may be a novel molecular mechanism of the enhanced cognitive recovery mediated by Nogo-A inhibition after TBI in rats.

Repetitive Mild Non-Contusive Brain Trauma in Immature Rats Exacerbates Traumatic Axonal Injury and Axonal Calpain Activation: A Preliminary Report

Infants who experience inflicted brain injury (shaken-impact syndrome) present with subdural hematoma, brain atrophy, and ventriculomegaly, pathologic features that are suggestive of multiple incidences of brain trauma. To develop a clinically relevant model of inflicted brain injury in infants, the skulls of anesthetized 11-day-old rat pups were subjected to one, two, or three successive mild impacts. While skull fractures were not observed, a single impact to the intact skull resulted in petechial hemorrhages in the subcortical white matter, and double or triple impacts led to hemorrhagic tissue tears at 1 day postinjury. Whereas the singly impacted brain did not exhibit overt damage at 7 days, two impacts resulted in an enlarged ventricle and white matter atrophy; three impacts to the brain led to similar pathology albeit at 3 days postinjury. By 7 days, cortical atrophy was observed following three impacts. Reactive astrocytes were visible in the deep cortical layers below the impact site after two impacts, and through all cortical layers after three impacts. Swellings were observed in intact axons in multiple white matter tracts at 1 day following single impact and progressed to axonal disconnections by 3 days. In contrast, double or triple impacts resulted in axonal disconnections by 1 day postinjury; in addition, three impacts led to extensive axonal injury in the dorsolateral thalamus by 3 days. Calpain activation was observed in axons in subcortical white matter tracts in all brain-injured animals at 1 day and increased with the number of impacts. Despite these pathologic alterations, neither one nor two impacts led to acquisition deficits on the Morris water maze. While indicative of the graded nature of the pathologic response, these data suggest that repetitive mild brain injury in the immature rat results in pathologic features similar to those following inflicted brain injuries in infants.

Tissue sparing and functional recovery following experimental traumatic brain injury is provided by treatment with an anti‐myelin‐associated glycoprotein antibody

Axonal injury is a hallmark of traumatic brain injury (TBI) and is associated with a poor clinical outcome. Following central nervous system injury, axons regenerate poorly, in part due to the presence of molecules associated with myelin that inhibit axonal outgrowth, including myelin-associated glycoprotein (MAG). The involvement of MAG in neurobehavioral deficits and tissue loss following experimental TBI remains unexplored and was evaluated in the current study using an MAG-specific monoclonal antibody (mAb). Anesthetized rats (n=102) were subjected to either lateral fluid percussion brain injury (n=59) or sham injury (n=43). In surviving animals, beginning at 1 h post-injury, 8.64 microg anti-MAG mAb (n=33 injured, n=21 sham) or control IgG (n=26 injured, n=22 sham) was infused intracerebroventricularly for 72 h. One group of these rats (n=14 sham, n=11 injured) was killed at 72 h post-injury for verification of drug diffusion and MAG immunohistochemistry. All other animals were evaluated up to 8 weeks post-injury using tests for neurologic motor, sensory and cognitive function. Hemispheric tissue loss was also evaluated at 8 weeks post-injury. At 72 h post-injury, increased immunoreactivity for MAG was seen in the ipsilateral cortex, thalamus and hippocampus of brain-injured animals, and anti-MAG mAb was detectable in the hippocampus, fimbria and ventricles. Brain-injured animals receiving anti-MAG mAb showed significantly improved recovery of sensorimotor function at 6 and 8 weeks (P<0.01) post-injury when compared with brain-injured IgG-treated animals. Additionally, at 8 weeks post-injury, the anti-MAG mAb-treated brain-injured animals demonstrated significantly improved cognitive function and reduced hemispheric tissue loss (P<0.05) when compared with their brain-injured controls. These results indicate that MAG may contribute to the pathophysiology of experimental TBI and treatment strategies that target MAG may be suitable for further evaluation.

Energy Metabolic Changes in the Early Post-injury Period Following Traumatic Brain Injury in Rats

Impaired cerebral energy metabolism may be a major contributor to the secondary injury cascade that occurs following traumatic brain injury (TBI). To estimate the cortical energy metabolic state following mild and severe controlled cortical contusion (CCC) TBI in rats, ipsi-and contralateral cortical tissues were frozen in situ at 15 and 40 min post-injury and adenylate (ATP, ADP, AMP) levels were analyzed using high-performance liquid chromatography (HPLC) and the energy charge (EC) was calculated. At 15 min post-injury, mildly brain-injured animals showed a 43% decrease in cortical ATP levels and a 2.4-fold increase in AMP levels (P < 0.05), and there was a significant reduction of the ipsilateral cortical EC when compared to sham-injured animals (P < 0.05). At 40 min post-injury, the ipsilateral adenylate levels and EC had recovered to the values observed in the sham-injury group. In the severe CCC group, there was a 51% decrease in ipsilateral cortical ATP levels and a 5.3-fold increase in AMP levels with a significant reduction of cortical EC at 15 min post-injury (P < 0.05). At 40 min post-injury, a 2.6-fold ipsilateral increase in AMP levels and an 11% and 44% decrease in EC and ATP levels, respectively, remained (P < 0.05). A 37-38% reduction of the total adenylate pool was observed ipsilaterally in both CCC severity groups at the early time-point, and a 19% and 28% decrease remained in the mild and severe CCC groups, respectively, at 40 min post-injury. Significant contralateral ATP and EC changes were only observed in the severe CCC group at 40 min post-injury (P < 0.05). The energy-requiring secondary injury cascades that occur early post-injury do not challenge the brain tissue to the extent of ATP depletion and may provide a window of opportunity for therapeutic intervention.

Neural progenitor cells engineered to secrete GDNF show enhanced survival, neuronal differentiation and improve cognitive function following traumatic brain injury

We sought to evaluate the potential of C17.2 neural progenitor cells (NPCs) engineered to secrete glial cell line-derived neurotrophic factor (GDNF) to survive, differentiate and promote functional recovery following engraftment into the brains of adult male Sprague-Dawley rats subjected to lateral fluid percussion brain injury. First, we demonstrated continued cortical expression of GDNF receptor components (GFRalpha-1, c-Ret), suggesting that GDNF could have a physiological effect in the immediate post-traumatic period. Second, we demonstrated that GDNF over-expression reduced apoptotic NPC death in vitro. Finally, we demonstrated that GDNF over-expression improved survival, promoted neuronal differentiation of GDNF-NPCs at 6 weeks, as compared with untransduced (MT) C17.2 cells, following transplantation into the perilesional cortex of rats at 24 h post-injury, and that brain-injured animals receiving GDNF-C17.2 transplants showed improved learning compared with those receiving vehicle or MT-C17.2 cells. Our results suggest that transplantation of GDNF-expressing NPCs in the acute post-traumatic period promotes graft survival, migration, neuronal differentiation and improves cognitive outcome following traumatic brain injury.

Delayed Transplantation of Human Neurons following Brain Injury in Rats: A Long-Term Graft Survival and Behavior Study

The NTera2 (NT2) cell line is a homogeneous population of cells, which, when treated in vitro with retinoic acid, terminally differentiate into postmitotic neuronal NT2N cells. Although NT2N neurons transplanted in the acute (24 h postinjury) period survive for up to 1 month following experimental traumatic brain injury (TBI), nothing is known of their ability to survive for longer periods or of their effects when engrafted during the chronic postinjury period. Adult male Sprague-Dawley rats (n = 348; 360-400 g) were initially anesthetized and subjected to severe lateral fluid-percussion (FP) brain injury or sham injury. At 1 month postinjury, only brain-injured animals showing severe neurobehavioral deficits received cryopreserved NT2N neurons stereotaxically transplanted into three sites in the peri-injured cortex (n = 18). Separate groups of similarly brain-injured rats received human fibroblast cells (n = 13) or cell suspension vehicle (n = 14). Sham-injured animals (no brain injury) served as controls and received NT2N transplants (n = 24). All animals received daily immunosuppression for three months. Behavioral testing was performed at 1, 4, 8, and 12 weeks post-transplantation, after which animals were sacrificed for histological analysis. Nissl staining and anti-human neuronal specific enolase (NSE) immunostaining revealed that NT2N neurons transplanted in the chronic post-injury period survived up to 12 weeks post-transplantation, extended processes into the host cortex and immunolabeled positively for synaptophysin. There were no statistical differences in cognitive or motor function among the transplanted brain-injured groups. Long-term graft survival suggests that NT2N neurons may be a viable source of neural cells for transplantation after TBI and also that these grafts can survive for a prolonged time and extend processes into the host cortex when transplanted in the chronic post-injury period following TBI.

Caspase-mediated cell death predominates following engraftment of neural progenitor cells into traumatically injured rat brain

Neural progenitor cells (NPCs) have been shown to be a promising therapy for cell replacement and gene transfer in neurological diseases including traumatic brain injury (TBI). However, NPCs often survive poorly after transplantation despite immunosuppression, and the mechanisms of graft cell death are unknown. In this study, we evaluated caspase- and calpain-mediated mechanisms of cell death of neonatal mouse C17.2 progenitor cells, transplanted at 24 h following lateral fluid percussion brain injury (FP) in rats. Adult Male Sprague–Dawley rats ( n = 30) were subjected to lateral FP injury ( n = 18) or sham surgery ( n = 12). C17.2 cells labeled with green fluorescent dye (CMFDA) were engrafted in the perilesional deep cortex, and animals were sacrificed at 24 h, 72 h and 1 week post-transplantation. Pro-apoptotic caspase-mediated cleavage products (Ab246) and calpain-mediated cleavage products (Ab38) were detected in the engrafted cells using immunohistochemistry. Only 2 to 4.5% of grafted NPCs were found to survive at 24 h post-transplantation, regardless of injury status of the host brain, although brain-injured animals had significantly fewer graft cells than sham-injured animals. Limited caspase and calpain-mediated graft cell death was observed in both sham- and brain-injured animals, and caspase-mediated graft cell death was significantly greater than calpain-mediated graft cell death in all animals. Brain-injured animals had significantly increased caspase-mediated graft cell death compared to sham-injured animals. These results suggest that both the caspase and calpain family of proteases are involved in graft cell death, and that caspase-mediated apoptotic graft cell death predominates in the acute post-traumatic period following TBI.

Development of Posttraumatic Hyperthermia after Traumatic Brain Injury in Rats is Associated with Increased Periventricular Inflammation

Posttraumatic hyperthermia (PTH) is a noninfectious elevation in body temperature that negatively influences outcome after traumatic brain injury (TBI). We sought to (1) characterize a clinically relevant model and (2) investigate potential cellular mechanisms of PTH. In study I, body temperature patterns were analyzed for 1 week in male rats after severe lateral fluid percussion (FP) brain injury (n=75) or sham injury (n=17). After injury, 27% of surviving animals experienced PTH, while 69% experienced acute hypothermia with a slow return to baseline. A profound blunting or loss of circadian rhythmicity (CR) that persisted up to 5 days after injury was experienced by 75% of brain-injured animals. At 2 and 7 days after injury, patterns of cell loss and inflammation were assessed in selected brain thermoregulatory and circadian centers. Significant cell loss was not observed, but PTH was associated with inflammatory changes in the hypothalamic paraventricular nucleus (PVN) by one week after injury. In brain-injured animals with altered CR, reactive astrocytes were bilaterally localized in the suprachiasmatic nucleus (SCN) and the PVN. Occasional IL-1beta+/ED-1+ macrophages/microglia were observed in the PVN and SCN exclusively in brain-injured animals developing PTH. In animals with PTH there was a significant positive correlation (r=0.788, P<0.01) between the degree of postinjury hyperthermia and the total number of cells positive for inflammatory markers within selected thermoregulatory and circadian nuclei. In study II, a separate group of animals underwent the same injury and temperature monitoring paradigm as in study I, but had additional physiologic data obtained, including vital signs, arterial blood gases, white blood cell counts, and C-reactive protein levels. All parameters remained within normal ranges after injury. These data suggest that PTH and the alteration in CR of temperature may be due, in part, to acute reactive astrocytosis and inflammation in hypothalamic centers responsible for both thermoregulation and CR.

Experimental Traumatic Brain Injury Modulates the Survival, Migration, and Terminal Phenotype of Transplanted Epidermal Growth Factor Receptor-activated Neural Stem Cells

We have previously shown that constitutively active epidermal growth factor receptor signaling enhances the survival and motility of engrafted neural stem cells (NSCs) when transplanted into normal adult brain. In the present study, using the C17.2 NSC line stably transfected with the constitutively active epidermal growth factor receptor vIII, we sought to evaluate the phenotype of NSCs after engraftment into the milieu of traumatic head injury. We performed intracerebral NSC transplantation with C17.2 NSCs overexpressing the active epidermal growth factor receptor vIII receptor into the ipsilateral (n=17) or contralateral (n=19) corpus callosum at 48 hours after severe experimental traumatic brain injury (TBI) or after sham injury (n=12) in rats. All sham-injured animals (100%) showed NSC graft survival, compared with 65% of brain-injured animals receiving ipsilateral NSC transplants, and only 10% of brain-injured animals had surviving transplants after engraftment into the contralateral uninjured corpus callosum. A marked elevation of nerve growth factor (pg/mg protein) was observed at 72 hours after injury in the injured hemisphere (x=80 +/- 10 pg/mg) compared with the contralateral uninjured hemisphere (35 +/- 0 pg/mg) (P <0.05), and this elevation of nerve growth factor may have contributed to enhanced survival of engrafted NSCs. In uninjured control animals, NSC transplants proliferated actively, as evidenced by incorporation of bromodeoxyuridine. After TBI, however, transplanted NSCs failed to proliferate, regardless of the site of implantation. Morphologically, NSCs transplanted into the injured brain showed extensive process formation suggestive of a more differentiated phenotype, in contrast to NSCs engrafted into uninjured brain that appear undifferentiated, with round soma and no processes. NSCs transplanted into the corpus callosum of brain-injured animals also expressed NG2, a pro-oligodendrocyte marker that was not seen in cells transplanted into uninjured brain. Although migration of NSCs was much more pronounced in the uninjured brain, 2 weeks after TBI, NSCs transplanted into the ipsilateral corpus callosum were found to have migrated to the injury cavity. Moreover, NSCs transplanted into the corpus callosum contralateral to the site of injury were observed crossing the corpus callosum by 2 weeks after transplantation. Our results suggest that the environment associated with acute experimental TBI can significantly modulate the phenotype and migratory patterns of the engrafted NSC. These findings have particularly important implications for transplantation of NSCs into the traumatically injured nervous system.

Differential Effects of the Anticonvulsant Topiramate on Neurobehavioral and Histological Outcomes following Traumatic Brain Injury in Rats

The efficacy of topiramate, a novel therapeutic agent approved for the treatment of seizure disorders, was evaluated in a model of traumatic brain injury (TBI). Adult male rats were anesthetized (sodium pentobarbital, 60 mg/kg, i.p.), subjected to lateral fluid percussion brain injury (n = 60) or sham injury (n = 47) and randomized to receive either topiramate or vehicle at 30 min (30 mg/kg, i.p.), and 8, 20 and 32 h postinjury (30 mg/kg, p.o.). In Study A, memory was evaluated using a Morris water maze at 48 h postinjury, after which brain tissue was evaluated for regional cerebral edema. In Study B, animals were evaluated for motor function at 48 h and 1, 2, 3, and 4 weeks postinjury using a composite neuroscore and the rotating pole test and for learning ability at 4 weeks. Brains were analyzed for hemispheric tissue loss and hippocampal CA3 cell loss. Topiramate had no effect on posttraumatic cerebral edema or histologic damage when compared to vehicle. At 48 h, topiramate treatment improved memory function in sham but not brain-injured animals, while at one month postinjury it impaired learning performance in brain-injured but not sham animals. Topiramate significantly improved composite neuroscores at 4 weeks postinjury and rotating pole performance at 1 and 4 weeks postinjury, suggesting a potentially beneficial effect on motor function following TBI.

Induction of prolonged electrographic seizures in vitro has a defined threshold and is all or none: implications for diagnosis of status epilepticus.

To study whether induction of prolonged (>30-min duration) in vitro electrographic seizure discharges resembling status epilepticus (SE) is graded or all-or-none, and to determine the critical factors mediating SE induction. Prolonged electrographic seizure discharges were induced in combined hippocampal-entorhinal cortical (HEC) brain slices by electrical stimulation of the Schaeffer collaterals. Discharges were recorded by using field-potential electrodes in the dentate gyrus, CA3, CA1, and entorhinal cortex. Slices were prepared from rats that were (a). 21- to 30-day-old naive, (b). 60- to 120-day old naive, (c). epileptic, and (d). status post a prior traumatic brain injury. Induction of SE discharges was dependent on the duration, but not amplitude of the preceding stimulus train-induced afterdischarge in HEC slices from 21- to 30-day-old control, brain-injured, and epileptic animals, but not from 60- to 120-day-old animals. In slices from 21- to 30-day-old control animals, once afterdischarges exceeded 4 min in duration, SE was induced in 50% of slices, and after >or=6 min 37 s seizure activity; SE was induced in 95% of slices. A defined SE threshold also was evident in brain-damaged rats, including rats in which an epileptic condition was induced by pilocarpine injection 4-16 weeks before recording, and rats subjected to a fluid percussive head trauma 1-8 weeks before recording. However, in these brain-damaged animals, mean SE threshold was considerably lower (24 and 44 s, respectively). HEC slices from 60- to 120-day-old controls for the brain-injured and epileptic animals did not develop SE even after 20 stimulations, demonstrating the pronounced effect of brain injury and epilepsy on the development of SE in the HEC slice preparation compared with that in age-matched controls. In vitro, SE discharges have a defined temporal threshold for initiation. Once a seizure exceeds 6-7 min in duration in control animals, and 30-55 s in brain-damaged animals, the probability of SE induction is greatly increased. This demonstrates that brain injury lowers the afterdischarge duration required to produce SE and suggests that brains injured from trauma or SE are more susceptible to develop status epilepticus.

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

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. 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. 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). 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.

Neuroprotective and behavioral efficacy of nerve growth factor—transfected hippocampal progenitor cell transplants after experimental traumatic brain injury

Immortalized neural progenitor cells derived from embryonic rat hippocampus (HiB5), were transduced ex vivo with the gene for mouse nerve growth factor (NGF) to secrete NGF (NGF-HiB5) at 2 ng/hr/10(5) cells in culture. Fifty-nine male Wistar rats weighing 300 to 370 g each were anesthetized with 60 mg/kg sodium pentobarbital and subjected to lateral fluid-percussion brain injury of moderate severity (2.3-2.4 atm, 34 rats) or sham injury (25 rats). At 24 hours postinjury, 2 microl (150,000 cells/microl) of [3H]thymidine-labeled NGF-HiB5 cells were transplanted stereotactically into three individual sites in the cerebral cortex adjacent to the injury site (14 rats). Separate groups of brain-injured rats received nontransfected (naive [n])-HiB5 cells (12 animals) or cell suspension vehicle (eight animals). One week postinjury, animals underwent neurological evaluation for motor function and cognition (Morris water maze) and were killed for histological, autoradiographic, and immunocytochemical analysis. Viable HiB5 cell grafts were identified in all animals, together with reactive microglia and macrophages located throughout the periinjured parenchyma and grafts (OX-42 immunohistochemistry). Brain-injured animals transplanted with either NGF-HiB5 or n-HiB5 cells displayed significantly improved neuromotor function (p < 0.05) and spatial learning behavior (p < 0.005) compared with brain-injured animals receiving microinjections of vehicle alone. A significant reduction in hippocampal CA3 cell death was observed in brain-injured animals receiving transplants of NGF-HiB5 cells compared with those receiving n-HiB5 cells or vehicle (p < 0.025). This study demonstrates that immortalized neural stem cells that have been retrovirally transduced to produce NGF can markedly improve cognitive and neuromotor function and rescue hippocampal CA3 neurons when transplanted into the injured brain during the acute posttraumatic period.

Surgical Treatment of Mental Illness: Impact of Imaging

Surgical Treatment of Mental Illness: Impact of Imaging

Neurobehavioral protection by the neuronal calcium channel blocker ziconotide in a model of traumatic diffuse brain injury in rats.

Abnormal accumulation of intracellular calcium following traumatic brain injury (TBI) is thought to contribute to a cascade of cellular events that lead to neuropathological conditions. Therefore, the possibility that specific calcium channel antagonists might exert neuroprotective effects in TBI has been of interest. The focus of this study was to examine whether Ziconotide produces such neuroprotective effects. The authors report that the acceleration-deceleration model of TBI developed by Marmarou, et al., induces a long-lasting deficit of neuromotor and behavioral function. The voltage-sensitive calcium channel blocker Ziconotide (also known as SNX-111 and CI-1009) exerts neuroprotective effects in this model of diffuse brain injury (DBI) in rats. The dose and time of injection of Ziconotide chosen for the present study was based on the authors' previous biochemical studies of mitochondria. Rats were trained in a series of motor and memory tasks, following which they were subjected to DBI using the Marmarou, et al., model. At 3, 5, and 24 hours, all rats were injected with 2 mg/kg Ziconotide for a total cumulative dose of 6 mg/kg Ziconotide. Control brain-injured animals were injected with an equal volume of saline vehicle at each of these time points. The rats were tested for motor and cognitive performance at 1, 3, 7,14, 21, 28, 35, and 42 days postinjury. Saline-treated rats displayed severe motor and cognitive deficits after DBI. Compared with saline-treated control animals, rats treated with Ziconotide displayed better motor performance during inclined plane, beam balance, and beam walk tests; improved memory while in the radial arm maze; and improved learning while in the Morris water maze. These results demonstrated that the acceleration-deceleration model, which had been developed by Marmarou, et al., induces severe motor and cognitive deficits. We also demonstrated that Ziconotide exhibits substantial neuroprotective activity in this model of TBI. Improvement was observed in both motor and cognitive tasks, even though treatment was not initiated until 3 hours after injury. These findings support the development of neuronal N-type calcium channel antagonists as useful therapeutic agents in the treatment of TBI.

Brain-derived neurotrophic factor administration after traumatic brain injury in the rat does not protect against behavioral or histological deficits

Brain-derived neurotrophic factor has been shown to be neuroprotective in models of excitotoxicity, axotomy and cerebral ischemia. The present study evaluated the therapeutic potential of brain-derived neurotrophic factor following traumatic brain injury in the rat. Male Sprague–Dawley rats ( N=99) were anesthetized and subjected to lateral fluid percussion brain injury of moderate severity (2.4–2.8 atm) or sham injury. Four hours after injury, the animals were reanesthetized, an indwelling, intraparenchymal cannula was implanted, and infusion of brain-derived neurotrophic factor or phosphate-buffered saline vehicle was initiated from a mini-osmotic pump and continued for two weeks. In Study 1 ( N=48), vehicle or 12 μg/day of brain-derived neurotrophic factor was infused into the dorsal hippocampus. In Study 2 ( N=51), vehicle or brain-derived neurotrophic factor at a high (12 μg/day) or low dose (1.2 μg/day) was infused into the injured parietal cortex. All animals were evaluated for neurological motor function at two days, one week and two weeks post-injury. Cognitive function (learning and memory) was assessed at two weeks post-injury using a Morris Water Maze. At two weeks post-injury, neuronal loss in the hippocampal CA3 and dentate hilus and in the injured cortex was evaluated. In Study 2, neuronal loss was also quantified in the thalamic medial geniculate nucleus. All of the above outcome measures demonstrated significant deleterious effects of brain injury ( P<0.05 compared to sham). However, post-traumatic brain-derived neurotrophic factor infusion did not significantly affect neuromotor function, learning, memory or neuronal loss in the hippocampus, cortex or thalamus when compared to vehicle infusion in brain-injured animals, regardless of the infusion site or infusion dose ( P>0.05 for each). In contrast to previous studies of axotomy, ischemia and excitotoxicity, our data indicate that brain-derived neurotrophic factor is not protective against behavioral or histological deficits caused by experimental traumatic brain injury using the delayed, post-traumatic infusion protocol examined in these studies.

Conditioning effects of repetitive mild neurotrauma on motor function in an animal model of focal brain injury

A weight drop model of brain injury was used to determine the effects of repetitive mild brain injury on motor function, heat shock protein and glial fibrillary acidic protein expression in the anesthetized, adult male, Sprague–Dawley rat. Repetitive mild brain injury was produced when animals received a series of three mild injuries spaced three days apart. A separate group of repetitive mild injured animals also received a subsequent severe brain injury between three and five days after the last mild injury. All animals were trained on a beam-walking test prior to surgery. The mild, repetitive mild and repetitive mild plus severe brain injury groups showed no motor deficits in the beam-walking test, whereas the animals with only severe brain injury showed significant motor deficits (increase in number of footslips) in the beam-walking test that recovered within eight days after injury. Both repetitive mild plus severe injury and severe injury only animals had cortical necrotic cavities of similar size in the region of the hindlimb motor cortex. Both the repetitive mild and severe brain-injured animals had marked heat shock protein 27 kDa and glial fibrillary acidic protein staining in the cerebral cortex. Fluoro-Jade, heat shock protein 27 kDa and 72 kDa labeling indicated that there were widespread effects on cortical, subcortical and spinal neurons and glial cells after repetitive mild brain injury. These results suggest that repetitive mild brain injury conditions the brain so that subsequent brain injury at the same site has no effect on motor function. Furthermore, repetitive mild injury-induced activation of processes distant to the primary injury site may have a role in activation of secondary sites involved in recovery of motor function.

Three months of chronic ethanol administration and the behavioral outcome of rats after lateral fluid percussion brain injury.

This study examined the effects of 3 months of chronic ethanol administration (CEAn) on the behavioral outcome in rats after lateral fluid percussion (FP) brain injury. Rats were given either an ethanol liquid diet (ethanol diet groups) or a pair-fed isocaloric sucrose control diet (control diet groups) for 3 months. Then, rats from both diet groups were subjected to either lateral FP brain injury of moderate severity (1.8 atm) or to sham operation. Postinjury behavioral measurements revealed that brain injury caused significant spatial learning disability in both diet groups. There were no significant differences in spatial learning ability in the sham or brain-injured animals between the control and ethanol diets. However, a trend towards cognitive impairment in the sham animals and a trend towards reduced deficits in the brain-injured animals were observed in the ethanol diet group. Histologic analysis of injured animals from both diet groups revealed similar extents of ipsilateral cortical and hippocampal CA3 damage. These results, in general, suggest that 3 months of CEAn does not significantly alter the behavioral and morphologic outcome of experimental brain injury.

Positive and negative modulation of the GABA A receptor and outcome after traumatic brain injury in rats

Glutamate-mediated excitotoxicity has been shown to contribute to cellular dysfunction following traumatic brain injury (TBI). Increasing inhibitory function through stimulation of γ-aminobutyric acid (GABA A) receptors may attenuate excitotoxic effects and improve outcome. The present experiment examined the effects of diazepam, a positive modulator at the GABA A receptor, on survival and cognitive performance in traumatically brain-injured animals. In experiment 1, 15 min prior to central fluid percussion brain injury, rats ( n=8 per group) were injected (i.p.) with saline or diazepam (5 mg/kg or 10 mg/kg). Additional rats ( n=8) were surgically prepared but not injured (sham-injury). Rats pre-treated with the 5 mg/kg dose of diazepam had significantly lower mortality (0%) than injured, saline-treated rats (53%). Also, diazepam-treated (5 mg/kg) rats had significantly shorter latencies to reach the goal platform in the Morris water maze test performed 11–15 days post-injury. In experiment 2, at 15 min post-injury, rats were given either saline ( n=5) or 5 mg/kg diazepam ( n=6). Rats treated with diazepam did not differ in mortality from injured rats treated with vehicle. However, rats treated with diazepam at 15 min post-injury had significantly shorter latencies to reach the goal platform in the Morris water maze than injured, vehicle-treated rats. In experiment 3, the post-injury administration of bicuculline (1.5 mg/kg, n=8), a GABA A antagonist, increased Morris water maze goal latencies compared to injured animals treated with saline ( n=8). These results suggest that enhancing inhibitory function during the acute post-injury period produces beneficial effects on both survival and outcome following experimental TBI.

The Novel Compound LOE 908 Attenuates Acute Neuromotor Dysfunction but Not Cognitive Impairment or Cortical Tissue Loss Following Traumatic Brain Injury in Rats

Experimental traumatic brain injury (TBI) initiates massive disturbances in Ca2+ concentrations in the brain that may contribute to neuronal damage. Intracellular Ca2+ may be elevated via influx through voltage-operated cation channels, ligand-gated ionotropic channels, and store-operated cation channels (SOCs). In the present study, we evaluated the neurobehavioral and histological effects of acute posttraumatic administration of (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di[2-(2 ,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908), a broad spectrum inhibitor of voltage-operated cation channels and SOCs. Male Sprague-Dawley rats (n = 53) were trained in the Morris water maze, anesthetized (60 mg/kg pentobarbital, i.p.), and subjected to lateral fluid percussion brain injury (2.5-2.7 atm; n = 38) or surgery without injury (n = 15). At 15 min postinjury, animals were randomized to receive intravenous administration of either a high dose of LOE 908 (4 mg/kg bolus followed by 160 mg/kg over 24 h; n = 13), a low dose of LOE 908 (2 mg/kg bolus followed by 80 mg/kg over 24 h; n = 12), or vehicle (n = 13). Uninjured controls received the high dose of LOE 908 (n = 8) or vehicle (n = 7). Treatment with either dose of LOE 908 significantly improved neuromotor function at 48 h postinjury when compared to vehicle treatment. Although a significant deficit in visuospatial memory was observed in brain-injured animals at this timepoint when compared to uninjured animals, neither dose of LOE 908 attenuated injury-induced cognitive dysfunction. Histological evaluation revealed that neither dose of LOE 908 affected cortical lesion size at 48 h postinjury. These data suggest that broad spectrum cation channel blockers may be beneficial in the treatment of neurological motor dysfunction when administered in the acute posttraumatic period.