Effect of prior receptor antagonism on behavioral morbidity produced by combined fluid percussion injury and entorhinal cortical lesion

We have used an animal model of traumatic brain injury (TBI) that incorporates both the neurotransmitter toxicity of fluid percussion TBI and deafferentation of bilateral entorhinal cortical (BEC) lesion to explore whether administration of muscarinic cholinergic or N-methyl-D-aspartate glutamatergic antagonists prior to injury ameliorates cognitive morbidity. Fifteen minutes prior to moderate central fluid percussion TBI, rats were given intraperitoneal injections of either scopolamine (1.0 mg/kg) or MK-801 (0.3 mg/kg) and 24 hr later underwent BEC lesion. Body weight was followed for 5 days postinjury, as was beam balance and beam walk performance to assure motor recovery prior to spatial memory testing. Each group was assessed for spatial memory deficits with the Morris water maze at short term (days 11–15) and long-term (60–64 days) postinjury intervals and then compared with untreated combined insult and sham-injured controls. Results showed that each drug significantly elevated body weight relative to untreated injured cases. Both scopolamine and MK-801 reduced beam balance deficits, whereas neither drug had a significant effect on beam walk deficits. Interestingly, short-term cognitive deficits assessed on days 11–15 were differentially affected by the two drugs: MK-801 pretreatment enhanced the recovery of spatial memory performance, whereas scopolamine pretreatment did not. Long-term (days 60–64) deficits in spatial memory were not altered by pretreatment with either drug. Our results suggest that, unlike fluid percussion TBI alone, behavioral impairment may require more select intervention when deafferentation is part of the head trauma pathology. J. Neurosci. Res. 49:197–206, 1997. © 1997 Wiley-Liss, Inc.     

Glutamate antagonism during secondary deafferentation enhances cognition and axo-dendritic integrity after traumatic brain injury

The combination of central fluid percussion traumatic brain injury (TBI) followed 24 h later by a bilateral entorhinal cortical deafferentation (BEC) produces profound cognitive morbidity. We recently showed that MK-801 given prior to TBI in this insult improved spatial memory for up to 15 days. In the present study we examine whether MK-801 treatment of the BEC component in the combined insult model affects cognitive recovery. Two strategies for drug treatment were tested. Fifteen minutes prior to the BEC lesion in the combined insult, rats were given i.p. doses of either 3 mg/kg (acute group) or 1 mg/kg (chronic group) MK-801. The acute group received no further injections, whereas the chronic group received 1 mg/kg MK-801 i.p. twice a day for 2 days post-BEC lesion. Two additional groups of animals received BEC lesion alone and either acute or chronic MK-801 treatment identical with the combined insult cases. Each group was then assessed for spatial memory deficits with the Morris water maze at days 11–15 and 60–64 postinjury. Both acute and chronic MK-801 treatment in the combined insult group significantly reduced spatial memory deficits at 15 days postinjury relative to untreated injured cases (P < .01). This reduction appeared more robust at 15 days and persisted for up to 64 days in the chronically treated group (P < .05). By contrast, neither acute nor chronic MK-801 treatment affected memory performance with the BEC insult alone. Immunocytochemical localization of parvalbumin showed that chronic administration of MK-801 in the combined insult cases attenuated the injury-induced dendritic atrophy of inhibitory neurons in the dentate gyrus and area CA1. Synaptophysin immunobinding revealed that chronic MK-801 treatment of the BEC component of the combined insult normalized the distribution of presynaptic terminals within the dentate gyrus. These results suggest that cognitive deficits produced by head trauma involving both neuroexcitation and deafferentation can be attenuated with chronic application of glutamatergic antagonists during the period of deafferentation injury and that this attenuation is correlated with axo-dendritic integrity. Hippocampus 1998;8:390–401. © 1998 Wiley-Liss, Inc.     

Matrix metalloproteinase-3 expression profile differentiates adaptive and maladaptive synaptic plasticity induced by traumatic brain injury

The interaction between extracellular matrix (ECM) and regulatory matrix metalloproteinases (MMPs) is important in establishing and maintaining synaptic connectivity. By using fluid percussion traumatic brain injury (TBI) and combined TBI and bilateral entorhinal cortical lesion (TBI + BEC), we previously demonstrated that hippocampal stromelysin-1 (MMP-3) expression and activity increased during synaptic plasticity. We now report a temporal analysis of MMP-3 protein and mRNA response to TBI during both degenerative (2 day) and regenerative (7, 15 day) phases of reactive synaptogenesis. MMP-3 expression during successful synaptic reorganization (following unilateral entorhinal cortical lesion; UEC) was compared with MMP-3 expression when normal synaptogenesis fails (after combined TBI + BEC insult). Increased expression of MMP-3 protein and message was observed in both models at 2 days postinjury, and immuohistochemical (IHC) colocalization suggested that reactive astrocytes contribute to that increase. By 7 days postinjury, model differences in MMP-3 were observed. UEC MMP-3 mRNA was equivalent to control, and MMP-3 protein was reduced within the deafferented region. In contrast, enzyme mRNA remained elevated in the maladaptive TBI + BEC model, accompanied by persistent cellular labeling of MMP-3 protein. At 15 days survival, MMP-3 mRNA was normalized in each model, but enzyme protein remained higher than paired controls. When TBI + BEC recovery was enhanced by the N-methyl-D-aspartate antagonist MK-801, 7-day MMP-3 mRNA was significantly reduced. Similarly, MMP inhibition with FN-439 reduced the persistent spatial learning deficits associated with TBI + BEC insult. These results suggest that MMP-3 might differentially affect the sequential phases of reactive synaptogenesis and exhibit an altered pattern when recovery is perturbed.     

Dietary curcumin counteracts the outcome of traumatic brain injury on oxidative stress, synaptic plasticity, and cognition

The pervasive action of oxidative stress on neuronal function and plasticity after traumatic brain injury (TBI) is becoming increasingly recognized. Here, we evaluated the capacity of the powerful antioxidant curry spice curcumin ingested in the diet to counteract the oxidative damage encountered in the injured brain. In addition, we have examined the possibility that dietary curcumin may favor the injured brain by interacting with molecular mechanisms that maintain synaptic plasticity and cognition. The analysis was focused on the BDNF system based on its action on synaptic plasticity and cognition by modulating synapsin I and CREB. Rats were exposed to a regular diet or a diet high in saturated fat, with or without 500 ppm curcumin for 4 weeks (n = 8/group), before a mild fluid percussion injury (FPI) was performed. The high-fat diet has been shown to exacerbate the effects of TBI on synaptic plasticity and cognitive function. Supplementation of curcumin in the diet dramatically reduced oxidative damage and normalized levels of BDNF, synapsin I, and CREB that had been altered after TBI. Furthermore, curcumin supplementation counteracted the cognitive impairment caused by TBI. These results are in agreement with previous evidence, showing that oxidative stress can affect the injured brain by acting through the BDNF system to affect synaptic plasticity and cognition. The fact that oxidative stress is an intrinsic component of the neurological sequel of TBI and other insults indicates that dietary antioxidant therapy is a realistic approach to promote protective mechanisms in the injured brain.     

Traumatic brain injury causes a long-lasting calcium (Ca2+)-plateau of elevated intracellular Ca levels and altered Ca2+ homeostatic mechanisms in hippocampal neurons surviving brain injury

Traumatic brain injury (TBI) survivors often suffer chronically from significant morbidity associated with cognitive deficits, behavioral difficulties and a post-traumatic syndrome and thus it is important to understand the pathophysiology of these long-term plasticity changes after TBI. Calcium (Ca2+) has been implicated in the pathophysiology of TBI-induced neuronal death and other forms of brain injury including stroke and status epilepticus. However, the potential role of long-term changes in neuronal Ca2+ dynamics after TBI has not been evaluated. In the present study, we measured basal free intracellular Ca2+ concentration ([Ca2+](i)) in acutely isolated CA3 hippocampal neurons from Sprague-Dawley rats at 1, 7 and 30 days after moderate central fluid percussion injury. Basal [Ca2+](i) was significantly elevated when measured 1 and 7 days post-TBI without evidence of neuronal death. Basal [Ca2+](i) returned to normal when measured 30 days post-TBI. In contrast, abnormalities in Ca2+ homeostasis were found for as long as 30 days after TBI. Studies evaluating the mechanisms underlying the altered Ca2+ homeostasis in TBI neurons indicated that necrotic or apoptotic cell death and abnormalities in Ca2+ influx and efflux mechanisms could not account for these changes and suggested that long-term changes in Ca2+ buffering or Ca2+ sequestration/release mechanisms underlie these changes in Ca2+ homeostasis after TBI. Further elucidation of the mechanisms of altered Ca2+ homeostasis in traumatized, surviving neurons in TBI may offer novel therapeutic interventions that may contribute to the treatment and relief of some of the morbidity associated with TBI.     

Injured Fluoro-Jade-positive hippocampal neurons contain high levels of zinc after traumatic brain injury

Hippocampal damage contributes to cognitive dysfunction after traumatic brain injury (TBI). We previously showed that Fluoro-Jade, a fluorescent stain that labels injured, degenerating brain neurons, quantifies the extent of hippocampal injury after experimental fluid percussion TBI in rats. Coincidentally, we observed that injured neurons in the rat hippocampus also stained with Newport Green, a fluorescent dye specific for free ionic zinc. Here, we show that, regardless of injury severity or therapeutic intervention, the post-TBI population of injured neurons in rat hippocampal subfields CA1, CA3 and dentate gyrus is indistinguishable, both in numbers and anatomical distribution, from the population of neurons containing high levels of zinc. Treatment with lamotrigine, which inhibits presynaptic release of glutamate and presumably zinc that is co-localized with glutamate, reduced numbers of Fluoro-Jade-positive and Newport Green-positive neurons equally as did treatment with nicardipine, which blocks voltage-gated calcium channels through which zinc enters neurons. To confirm using molecular techniques that Fluoro-Jade and Newport Green-positive neurons are equivalent populations, we isolated total RNA from 25 Fluoro-Jade-positive and 25 Newport Green-positive pyramidal neurons obtained by laser capture microdissection (LCM) from the CA3 subfield, linearly amplified the mRNA and used quantitative ribonuclease protection analysis to demonstrate similar expression of mRNA for selected TBI-induced genes. Our data suggest that therapeutic interventions aimed at reducing neurotoxic zinc levels after TBI may reduce hippocampal neuronal injury.     

Experience-dependent behavioral plasticity is disturbed following traumatic injury to the immature brain

Traumatic brain injury (TBI) is most prevalent in children and young adults. The long-term effects of pediatric TBI include cognitive and behavioral impairments; however, over time, it is difficult to distinguish individual variability in intellect and behavior from sequelae of early injury. Postnatal day (PND) 19 rats underwent lateral fluid percussion (FP) injury, followed by rearing in either standard (STD) or enriched environment (EE) conditions. The hypothesis was that the traditional enhancement of cognitive functioning following EE rearing would be attenuated when this rearing is preceded by TBI at PND19. Thirty days after injury, Morris water maze (MWM) acquisition and subsequent probe trial retention were used to assess the behavioral effects of injury on experience-dependent plasticity induced by housing in EE at two different time windows. MWM acquisition demonstrated improvements following early EE rearing in both sham and injured animals; however, the degree of improvement was greater for uninjured animals. When EE rearing was delayed for 2 weeks after injury, the injury effect was absent and the effect of rearing even stronger. Memory testing in the early EE groups using a delayed probe trial showed an effect of injury and housing, with the sham EE animals benefiting the most. After the delayed EE, sham EE animals again showed more probe target hits, while FPEE animals did not, demonstrating an enduring memory deficit. These data confirm that early TBI has effects on experience-dependent plasticity resulting in long-term neurobehavioral deficits. In addition, the ability to benefit from environmental stimulation following TBI is dependent upon time after injury.     

Impaired expression of long-term potentiation in hippocampal slices 4 and 48 h following mild fluid-percussion brain injury in vivo

The effect of fluid percussion brain injury on hippocampal long-term potentiation (LTP) was investigated in hippocampal slices in vitro. Mild to moderate (1.7–2.1 atm) lateral fluid percussion head injury or sham operation was produced in rats 4 or 48 h prior to harvesting brain slices from the ipsilateral hippocampus. Field excitatory post-synaptic potentials (fEPSPs) were recorded in stratum radiatum of hippocampal subfield CA1 in response to electrical stimulation of the Schaffer collaterals. The initial slope of fEPSPs was used to investigate changes in synaptic strength prior to and following 100 or 200 Hz (1 s) tetanic stimulation. TBI significantly inhibited expression of LTP in hippocampal slices in vitro. Post-tetanus fEPSP slopes increased more than 100% in hippocampal slices from sham-operated animals but less than 50% in slices from rats following TBI. The data suggest that changes in functional synaptic plasticity in the hippocampus may contribute to cognitive disorders associated with TBI (traumatic brain injury). The data also indicate that TBI-induced effects on hippocampal LTP are robust and may be investigated in the hippocampal slice preparation in vitro.     

Hydrogen-rich saline protects against oxidative damage and cognitive deficits after mild traumatic brain injury

Oxidative stress is the principal factor in traumatic brain injury (TBI) that initiates events that result in protracted neuronal dysfunction and remodeling. Importantly, antioxidants can protect the brain against oxidative damage and modulate the capacity of the brain to cope with synaptic dysfunction and cognitive impairment. However, no studies have investigated the effects of hydrogen-rich saline on cognitive deficits after TBI. In the present study, rats with fluid percussion injury (FPI) were used to investigate the protective effects of hydrogen-rich saline. The results showed that hydrogen-rich saline reduced the level of malondialdehyde (MDA) and elevated the level of silent information regulator 2 (Sir2). In addition, treatment with hydrogen-rich saline, which elevated the levels of molecules associated with brain-derived neurotropic factor (BDNF)-mediated synaptic plasticity, improved cognitive performance in the Morris water maze after mild TBI. These results suggest that hydrogen-rich saline can protect the brain against the deleterious effects of mild TBI on synaptic plasticity and cognition and that hydrogen-rich saline could be an effective therapeutic strategy for patients with cognitive deficits after TBI.     

Induction of tolerance against traumatic brain injury by ischemic preconditioning.

We tested the hypothesis that a transient non-lethal ischemic insult lasting 2 min would protect against subsequent moderate traumatic brain injury. Sprague-Dawley rats were randomized into three experimental groups, including sham ischemia procedures and ischemic preconditioning (IPC) followed 48 h later by moderate traumatic brain injury (TBI) provoked by parasagittal fluid percussion injury (1.8-2.1 atm) and IPC followed by 48 h sham TBI. Seven days after the secondary insult, animals were perfusion-fixed for quantitative histopathological analysis. The CA3 necrotic cell count was decreased by 63% in TBI animals that had undergone IPC as compared to TBI animals that underwent sham IPC. TBI animals that had undergone IPC demonstrated significantly smaller contusion volumes than the TBI alone group (6.44 +/- 1.51 vs 1.37 +/- 0.63 mm3, mean +/- s.e.m.) These data indicate that IPC applied 2 days before moderate fluid percussion brain injury increases the brain resistance to traumatic brain damage.     

Lubeluzole treatment does not attenuate neurobehavioral dysfunction or CA3 hippocampal neuronal loss following traumatic brain injury in rats

PURPOSE: One of the downstream consequences of glutamate-induced NMDA (N-methyl-D-aspartate) receptor activation following trau-matic brain injury (TBI) is production of nitric oxide (NO). In this study, we evaluated the ability of lubeluzole, a novel neuroprotective com-pound which downregulates the glutamate-activated nitric oxide pathway and blocks sodium and voltage-sensitive calcium channels, to improve behavioral and histological outcome in rats following TBI. METHODS: Rats were anesthetized and subjected to moderate lateral fluid percussion brain injury (2.4-2.6 atm) or were surgically prepared but not injured (sham). Fifteen minutes after injury, animals received a bolus of either vehicle (n = 12 injured, n = 14 uninjured) or lubeluzole (0.31 mg/kg, n = 12 injured, n = 8 uninjured) through the jugular vein followed by a one hour infusion of vehicle or lubeluzole (0.31 mg/kg). Animals were tested at 48 hours post-injury for cognitive performance in the Morris water maze, neuromotor function, and limb placing func-tion, and then sacrificed. RESULTS: While brain injury resulted in significant cognitive and motor deficits, injured animals treated with lubeluzole did not differ in spa-tial memory performance, neuromotor score, or limb placing function from injured, vehicle-treated animals. Furthermore, there was no differ-ence in the mean number of ipsilateral hippocampal CA3 neurons between injured rats treated with vehicle and those treated with lubeluzole. CONCLUSIONS: This single-dose study failed to demonstrate a beneficial effect of lubeluzole on the acute behavioral or histological sequelae following TBI.     

Chronic failure in the maintenance of long-term potentiation following fluid percussion injury in the rat

Traumatic brain injury (TBI) can produce chronic cognitive learning/memory deficits that are thought to be mediated, in part, by impaired hippocampal function. Experimentally induced TBI is associated with deficits in hippocampal synaptic plasticity (long-term potentiation, or LTP) at acute post-injury intervals but plasticity has not been examined at long-term survival periods. The present study was conducted to assess the temporal profile of LTP after injury and to evaluate the effects of injury severity on plasticity. Separate groups of rats were subjected to mild (1.1-1.4 atm), moderate (1.8-2.1 atm), or severe (2.2-2.7 atm) fluid percussion (FP) injury (or sham surgery) and processed for hippocampal electrophysiology in the first or eighth week after injury. LTP was defined as a lasting increase in field excitatory post-synaptic potential (fEPSP) slope in area CA1 following tetanic stimulation of the Schaffer collaterals. The fEPSP slope was measured for 60 min after tetanus. Assessment of LTP at the acute interval (6 days) revealed modest peak slope potentiation values (129-139%), which declined in each group (including sham) over the hour-long recording session and did not differ between groups. Eight weeks following injury, slices from all groups exhibited robust maximal potentiation (134-147%). Levels of potentiation among groups were similar at the 5-min test interval but differed significantly at the 30- and 60-min test intervals. Whereas sham slices showed stable potentiation for the entire 60-min assessment period, slices in all of the injury groups exhibited a significant decline in potentiation over this period. These experiments reveal a previously unknown effect of TBI whereby experimentally induced injury results in a chronic inability of the CA1 hippocampus to maintain synaptic plasticity. They also provide evidence that sham surgical procedures can significantly influence hippocampal physiology at the acute post-TBI intervals. The results have implications for the mechanisms underlying the impaired synaptic plasticity following TBI.     

The effects of combined fluid percussion traumatic brain injury and unilateral entorhinal deafferentation on the juvenile rat brain

The current study was designed to address the effects of traumatic brain injury (TBI) on plasticity and reorganization in the juvenile brain. Given that two of the major pathological sequelae of TBI involve a generalized neuroexcitation insult and diffuse axonal injury, we have employed models of these pathologies, delivered either independently or in combination, to examine their effects on injury-induced synaptic reorganization of the dentate gyrus in the developing rat. Postnatal day 28 rats received either sham, central fluid percussion traumatic brain injury (TBI), unilateral entorhinal cortical lesion (UEC), or TBI+UEC (TUEC) injury. Cognitive performance was assessed in the Morris water maze (MWM) between 11 and 15 days post-injury and the brains were processed for synaptophysin immunohistochemistry and routine electron microscopy. The MWM results revealed that TBI or UEC lesions delivered independently do not produce significant morbidity in P28 rats. However, when these injuries are combined, they reveal significant deficits in the MWM, accompanied by measurable changes in the distribution of presynaptic synaptophysin immunoreactivity over the deafferented dentate molecular layer. These observations are further supported by qualitative ultrastructural alterations in synaptic architecture in the same subregions of the dentate neuropil. The present findings show that the resilience of the immature brain following TBI is reduced when neuroexcitatory insult is combined with deafferentation. Moreover, when deafferented tissue is assessed morphologically, evidence exists for aberrant plasticity and abnormal synaptic reorganization in the juvenile brain.     

Mechanisms underlying the inability to induce area CA1 LTP in the mouse after traumatic brain injury

Traumatic brain injury (TBI) is a significant health issue that often causes enduring cognitive deficits, in particular memory dysfunction. The hippocampus, a structure crucial in learning and memory, is frequently damaged during TBI. Since long-term potentiation (LTP) is the leading cellular model underlying learning and memory, this study was undertaken to examine how injury affects area CA1 LTP in mice using lateral fluid percussion injury (FPI). Brain slices derived from FPI animals demonstrated an inability to induce LTP in area CA1 7 days postinjury. However, area CA1 long-term depression could be induced in neurons 7 days postinjury, demonstrating that some forms of synaptic plasticity can still be elicited. Using a multi-disciplined approach, potential mechanisms underlying the inability to induce and maintain area CA1 LTP were investigated. This study demonstrates that injury leads to significantly smaller N-methyl-D-aspartate potentials and glutamate-induced excitatory currents, increased dendritic spine size, and decreased expression of alpha-calcium calmodulin kinase II. These findings may underlie the injury-induced lack of LTP and thus, contribute to cognitive impairments often associated with TBI. Furthermore, these results provide attractive sites for potential therapeutic intervention directed toward alleviating the devastating consequences of human TBI.     

Effects of diazepam on glutamatergic synaptic transmission in the hippocampal CA1 area of rats with traumatic brain injury

The activity of the Schaffer collaterals of hippocampal CA3 neurons and hippocampal CA1 neurons has been shown to increase after fluid percussion injury. Diazepam can inhibit the hyperexcitability of rat hippocampal neurons after injury, but the mechanism by which it affects excitatory synaptic transmission remains poorly understood. Our results showed that diazepam treatment significantly increased the slope of input-output curves in rat neurons after fluid percussion injury. Diazepam significantly decreased the numbers of spikes evoked by super stimuli in the presence of 15 μmol/L bicuculline, indicating the existence of inhibitory pathways in the injured rat hippocampus. Diazepam effectively increased the paired-pulse facilitation ratio in the hippocampal CA1 region following fluid percussion injury, reduced miniature excitatory postsynaptic potentials, decreased action-potential-dependent glutamine release, and reversed spontaneous glutamine release. These data suggest that diazepam could decrease the fluid percussion injury-induced enhancement of excitatory synaptic transmission in the rat hippocampal CA1 area.     

脑创伤后bcl—2蛋白的神经保护作用

目的 探讨液压脑损伤后凋亡抑制基因bcl—2的变化规律及bcl—2基因在创伤性脑损伤后细胞凋亡中的作用。方法 应用免疫组化观察大鼠中型液压脑损伤伤前及伤后6h、12h、1d、3d、7dbcl—2蛋白表达情况,应用TUNEIL和电镜观察伤后细胞死亡的形态。结果 免疫反应阳性细胞主要位于伤侧大脑半球皮质、皮层下白质、海马CAl、CA3及齿状回的神经元和神经胶质细胞,以海马CA3区最为显。在高倍镜下,表达Bcl—2蛋白的神经细胞胞核形态正常,很少见到凋亡或坏死的形态特征。伤后早期(6h),打击侧海马CA3区Bcl—2蛋白表达显下降;Bcl—2早期改变出现在伤后6h,比细胞凋亡提前表现;伤后l—3h,Bcl—2的表达下降相对缓慢。结论 bcl—2蛋白在抑制脑创伤后细胞凋亡中起重要作用,bcl—2可能是一种可诱导的神经保护因子。     

Hippocampal vulnerability following traumatic brain injury: a potential role for neurotrophin-4/5 in pyramidal cell neuroprotection

Traumatic brain injury (TBI) causes selective hippocampal cell death, which is believed to be associated with cognitive impairment observed both in clinical and experimental settings. Although neurotrophin administration has been tested as a strategy to prevent cell death following TBI, the potential neuroprotective role of neurotrophin-4/5 (NT-4/5) in TBI remains unknown. We hypothesized that NT-4/5 would offer neuroprotection for selectively vulnerable hippocampal neurons following TBI. Measurements of NT-4/5 in rats subjected to lateral fluid percussion (LFP) TBI revealed two-threefold increases in the injured cortex and hippocampus in the acute period (1-3 days) following brain injury. Subsequently, the response of NT-4/5 knockout (NT-4/5(-/-)) mice to controlled-cortical impact TBI was investigated. NT-4/5(-/-) mice were more susceptible to selective pyramidal cell loss in Ahmon's corn (CA) subfields of the hippocampus following TBI, and showed impaired motor recovery when compared with their brain-injured wild-type controls (NT-4/5(wt)). Additionally, we show that acute, prolonged administration of recombinant NT-4/5 (5 microg/kg/day) prevented up to 50% of the hippocampal CA pyramidal cell death following LFP TBI in rats. These results suggest that post-traumatic increases in endogenous NT-4/5 may be part of an adaptive neuroprotective response in the injured brain, and that administration of this neurotrophic factor may be useful as a therapeutic strategy following TBI.