Moderate Parasagittal Fluid-percussion Brain Injury Research Articles

Enhancing cognitive recovery in chronic traumatic brain injury through simultaneous allosteric modulation of α7 nicotinic acetylcholine and α5 GABAA receptors

Traumatic brain injury (TBI) leads to changes in the neural circuitry of the hippocampus that result in chronic learning and memory deficits. However, effective therapeutic strategies to ameliorate these chronic learning and memory impairments after TBI are limited. Two pharmacological targets for enhancing cognition are nicotinic acetylcholine receptors (nAChRs) and GABAA receptors (GABAARs), both of which regulate hippocampal network activity to form declarative memories. A promising compound, 522–054, both allosterically enhances α7 nAChRs and inhibits α5 subunit-containing GABAARs. Administration of 522–054 enhances long-term potentiation (LTP) and cognitive functioning in non-injured animals. In this study, we assessed the effects of 522–054 on hippocampal synaptic plasticity and learning and memory deficits in the chronic post-TBI recovery period. Adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury or sham surgery. At 12 wk after injury, we assessed basal synaptic transmission and LTP at the Schaffer collateral-CA1 synapse of the hippocampus. Bath application of 522–054 to hippocampal slices reduced deficits in basal synaptic transmission and recovered TBI-induced impairments in LTP. Moreover, treatment of animals with 522–054 at 12 wk post-TBI improved cue and contextual fear memory and water maze acquisition and retention without a measurable effect on cortical or hippocampal atrophy. These results suggest that dual allosteric modulation of α7 nAChR and α5 GABAAR signaling may be a potential therapy for treating cognitive deficits during chronic recovery from TBI.

Early Life Stress Exacerbates Outcome after Traumatic Brain Injury.

The neurocognitive impairments associated with mild traumatic brain injury (TBI) often resolve within 1-2 weeks; however, a subset of people exhibit persistent cognitive dysfunction for weeks to months after injury. The factors that contribute to these persistent deficits are unknown. One potential risk factor for worsened outcome after TBI is a history of stress experienced by a person early in life. Early life stress (ELS) includes maltreatment such as neglect, and interferes with the normal construction of cortical and hippocampal circuits. We hypothesized that a history of ELS contributes to persistent learning and memory dysfunction following a TBI. To explore this interaction, we modeled ELS by separating Sprague Dawley pups from their nursing mothers from post-natal days 2-14 for 3 h daily. At 2 months of age, male rats received sham surgery or mild to moderate parasagittal fluid-percussion brain injury. We found that the combination of ELS with TBI in adulthood impaired hippocampal-dependent learning, as assessed with contextual fear conditioning, the water maze task, and spatial working memory. Cortical atrophy was significantly exacerbated in TBI animals exposed to ELS compared with normal-reared TBI animals. Changes in corticosterone in response to restraint stress were prolonged in TBI animals that received ELS compared with TBI animals that were normally reared or sham animals that received ELS. Our findings indicate that ELS is a risk factor for worsened outcome after TBI, and results in persistent learning and memory deficits, worsened cortical pathology, and an exacerbation of the hormonal stress response.

Positive allosteric modulation of the α7 nicotinic acetylcholine receptor as a treatment for cognitive deficits after traumatic brain injury.

Cognitive impairments are a common consequence of traumatic brain injury (TBI). The hippocampus is a subcortical structure that plays a key role in the formation of declarative memories and is highly vulnerable to TBI. The α7 nicotinic acetylcholine receptor (nAChR) is highly expressed in the hippocampus and reduced expression and function of this receptor are linked with cognitive impairments in Alzheimer's disease and schizophrenia. Positive allosteric modulation of α7 nAChRs with AVL-3288 enhances receptor currents and improves cognitive functioning in naïve animals and healthy human subjects. Therefore, we hypothesized that targeting the α7 nAChR with the positive allosteric modulator AVL-3288 would enhance cognitive functioning in the chronic recovery period of TBI. To test this hypothesis, adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury or sham surgery. At 3 months after recovery, animals were treated with vehicle or AVL-3288 at 30 min prior to cue and contextual fear conditioning and the water maze task. Treatment of TBI animals with AVL-3288 rescued learning and memory deficits in water maze retention and working memory. AVL-3288 treatment also improved cue and contextual fear memory when tested at 24 hr and 1 month after training, when TBI animals were treated acutely just during fear conditioning at 3 months post-TBI. Hippocampal atrophy but not cortical atrophy was reduced with AVL-3288 treatment in the chronic recovery phase of TBI. AVL-3288 application to acute hippocampal slices from animals at 3 months after TBI rescued basal synaptic transmission deficits and long-term potentiation (LTP) in area CA1. Our results demonstrate that AVL-3288 improves hippocampal synaptic plasticity, and learning and memory performance after TBI in the chronic recovery period. Enhancing cholinergic transmission through positive allosteric modulation of the α7 nAChR may be a novel therapeutic to improve cognition after TBI.

A negative allosteric modulator of PDE4D enhances learning after traumatic brain injury.

Traumatic brain injury (TBI) significantly decreases cyclic AMP (cAMP) signaling which produces long-term synaptic plasticity deficits and chronic learning and memory impairments. Phosphodiesterase 4 (PDE4) is a major family of cAMP hydrolyzing enzymes in the brain and of the four PDE4 subtypes, PDE4D in particular has been found to be involved in memory formation. Although most PDE4 inhibitors target all PDE4 subtypes, PDE4D can be targeted with a selective, negative allosteric modulator, D159687. In this study, we hypothesized that treating animals with D159687 could reverse the cognitive deficits caused by TBI. To test this hypothesis, adult male Sprague Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury. After 3 months of recovery, animals were treated with D159687 (0.3 mg/kg, intraperitoneally) at 30 min prior to cue and contextual fear conditioning, acquisition in the water maze or during a spatial working memory task. Treatment with D159687 had no significant effect on these behavioral tasks in non-injured, sham animals, but did reverse the learning and memory deficits in chronic TBI animals. Assessment of hippocampal slices at 3 months post-TBI revealed that D159687 reversed both the depression in basal synaptic transmission in area CA1 as well as the late-phase of long-term potentiation. These results demonstrate that a negative allosteric modulator of PDE4D may be a potential therapeutic to improve chronic cognitive dysfunction following TBI.

Therapeutic benefits of phosphodiesterase 4B inhibition after traumatic brain injury.

Traumatic brain injury (TBI) initiates a deleterious inflammatory response that exacerbates pathology and worsens outcome. This inflammatory response is partially mediated by a reduction in cAMP and a concomitant upregulation of cAMP-hydrolyzing phosphodiesterases (PDEs) acutely after TBI. The PDE4B subfamily, specifically PDE4B2, has been found to regulate cAMP in inflammatory cells, such as neutrophils, macrophages and microglia. To determine if PDE4B regulates inflammation and subsequent pathology after TBI, adult male Sprague Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury (2 ± 0.2 atm) and were then treated with a PDE4B - selective inhibitor, A33, or vehicle for up to 3 days post-surgery. Treatment with A33 reduced markers of microglial activation and neutrophil infiltration at 3 and 24 hrs after TBI, respectively. A33 treatment also reduced cortical contusion volume at 3 days post-injury. To determine whether this treatment paradigm attenuated TBI-induced behavioral deficits, animals were evaluated over a period of 6 weeks after surgery for forelimb placement asymmetry, contextual fear conditioning, water maze performance and spatial working memory. A33 treatment significantly improved contextual fear conditioning and water maze retention at 24 hrs post-training. However, this treatment did not rescue sensorimotor or working memory deficits. At 2 months after surgery, atrophy and neuronal loss were measured. A33 treatment significantly reduced neuronal loss in the pericontusional cortex and hippocampal CA3 region. This treatment paradigm also reduced cortical, but not hippocampal, atrophy. Overall, these results suggest that acute PDE4B inhibition may be a viable treatment to reduce inflammation, pathology and memory deficits after TBI.

Chronic Cognitive Dysfunction after Traumatic Brain Injury Is Improved with a Phosphodiesterase 4B Inhibitor.

Learning and memory impairments are common in traumatic brain injury (TBI) survivors. However, there are no effective treatments to improve TBI-induced learning and memory impairments. TBI results in decreased cAMP signaling and reduced cAMP-response-element binding protein (CREB) activation, a critical pathway involved in learning and memory. TBI also acutely upregulates phosphodiesterase 4B2 (PDE4B2), which terminates cAMP signaling by hydrolyzing cAMP. We hypothesized that a subtype-selective PDE4B inhibitor could reverse the learning deficits induced by TBI. To test this hypothesis, adult male Sprague-Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury. At 3 months postsurgery, animals were administered a selective PDE4B inhibitor or vehicle before cue and contextual fear conditioning, water maze training and a spatial working memory task. Treatment with the PDE4B inhibitor significantly reversed the TBI-induced deficits in cue and contextual fear conditioning and water maze retention. To further understand the underlying mechanisms of these memory impairments, we examined hippocampal long-term potentiation (LTP). TBI resulted in a significant reduction in basal synaptic transmission and impaired expression of LTP. Treatment with the PDE4B inhibitor significantly reduced the deficits in basal synaptic transmission and rescued LTP expression. The PDE4B inhibitor reduced tumor necrosis factor-α levels and increased phosphorylated CREB levels after TBI, suggesting that this drug inhibited molecular pathways in the brain known to be regulated by PDE4B. These results suggest that a subtype-selective PDE4B inhibitor is a potential therapeutic to reverse chronic learning and memory dysfunction and deficits in hippocampal synaptic plasticity following TBI. Currently, there are an estimated 3.2-5.3 million individuals living with disabilities from traumatic brain injury (TBI) in the United States, and 8 of 10 of these individuals report cognitive disabilities (Thurman et al., 1999; Lew et al., 2006; Zaloshnja et al., 2008). One of the molecular mechanisms associated with chronic cognitive disabilities is impaired cAMP signaling in the hippocampus. In this study, we report that a selective phosphodiesterase 4B (PDE4B) inhibitor reduces chronic cognitive deficits after TBI and rescues deficits in hippocampal long-term potentiation. These results suggest that PDE4B inhibition has the potential to improve learning and memory ability and overall functioning for people living with TBI.

Traumatic Brain Injury Upregulates Phosphodiesterase Expression in the Hippocampus.

Traumatic brain injury (TBI) results in significant impairments in hippocampal synaptic plasticity. A molecule critically involved in hippocampal synaptic plasticity, 3′,5′-cyclic adenosine monophosphate, is downregulated in the hippocampus after TBI, but the mechanism that underlies this decrease is unknown. To address this question, we determined whether phosphodiesterase (PDE) expression in the hippocampus is altered by TBI. Young adult male Sprague Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury. Animals were analyzed by western blotting for changes in PDE expression levels in the hippocampus. We found that PDE1A levels were significantly increased at 30 min, 1 h and 6 h after TBI. PDE4B2 and 4D2 were also significantly increased at 1, 6, and 24 h after TBI. Additionally, phosphorylation of PDE4A was significantly increased at 6 and 24 h after TBI. No significant changes were observed in levels of PDE1B, 1C, 3A, 8A, or 8B between 30 min to 7 days after TBI. To determine the spatial profile of these increases, we used immunohistochemistry and flow cytometry at 24 h after TBI. PDE1A and phospho-PDE4A localized to neuronal cell bodies. PDE4B2 was expressed in neuronal dendrites, microglia and infiltrating CD11b+ immune cells. PDE4D was predominantly found in microglia and infiltrating CD11b+ immune cells. To determine if inhibition of PDE4 would improve hippocampal synaptic plasticity deficits after TBI, we treated hippocampal slices with rolipram, a pan-PDE4 inhibitor. Rolipram partially rescued the depression in basal synaptic transmission and converted a decaying form of long-term potentiation (LTP) into long-lasting LTP. Overall, these results identify several possible PDE targets for reducing hippocampal synaptic plasticity deficits and improving cognitive function acutely after TBI.

Temporal Profile of Cerebrospinal Fluid, Plasma, and Brain Interleukin-6 After Normothermic Fluid-Percussion Brain Injury: Effect of Secondary Hypoxia

Interleukin-6 (IL-6) is a proinflammatory cytokine that may play multiple roles in the pathogenesis of traumatic brain injury (TBI). The present study determined time-dependent changes in IL-6 concentrations in vulnerable brain regions, cerebrospinal fluid (CSF) samples, and plasma after normothermic TBI. Because secondary insults are common in head injured patients, we also assessed the consequences of a post-traumatic secondary hypoxic insult on this pleiotropic cytokine. Male Sprague-Dawley rats were intubated, anesthetized, and underwent a moderate parasagittal fluid-percussion brain injury (1.8-2.1 atm, 37°C) followed by either 30 minutes of normoxic or hypoxic (pO₂ = 30-40 mmHg) gas levels. Rats were sacrificed 3, 6, or 24 hours after TBI or shamoperated procedures. Brain samples, including the ipsilateral cerebral cortex and hippocampus were dissected and analyzed. Plasma and CSF samples were collected at similar times and stored at -80°C until analysis. IL-6 levels were significantly increased ( p < 0.05) at 3, 6, and 24 hours in the cerebral cortex and at 6 hours in the hippocampus after TBI. IL-6 levels in the TBI normoxic group for both structures returned to control levels by 24 hours. Plasma levels of IL-6 were elevated at all time points, while CSF levels were high at 3 and 6 hours, but normalized by 24 hours. Post-traumatic hypoxia led to significantly elevated ( p < 0.05) IL-6 protein levels in the cerebral cortex at 24 hours compared to sham-operated controls. These findings demonstrate that moderate TBI leads to an early increase in IL-6 brain, plasma, and CSF protein levels. Secondary post-traumatic hypoxia, a common secondary injury mechanism, led to prolonged elevations in plasma IL-6 levels that may participate in the pathophysiology of this complicated TBI model.

Phosphodiesterase isoform‐specific expression induced by traumatic brain injury

Traumatic brain injury (TBI) results in significant inflammation which contributes to the evolving pathology. Previously, we have demonstrated that cyclic AMP (cAMP), a molecule involved in inflammation, is down-regulated after TBI. To determine the mechanism by which cAMP is down-regulated after TBI, we determined whether TBI induces changes in phosphodiesterase (PDE) expression. Adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury (FPI) or sham injury, and the ipsilateral, parietal cortex was analyzed by western blotting. In the ipsilateral parietal cortex, expression of PDE1A, PDE4B2, and PDE4D2, significantly increased from 30 min to 24 h post-injury. PDE10A significantly increased at 6 and 24 h after TBI. Phosphorylation of PDE4A significantly increased from 6 h to 7 days post-injury. In contrast, PDE1B, PD4A5, and PDE4A8 significantly decreased after TBI. No changes were observed with PDE1C, PDE3A, PDE4B1/3, PDE4B4, PDE4D3, PDE4D4, PDE8A, or PDE8B. Co-localization studies showed that PDE1A, PDE4B2, and phospho-PDE4A were neuronally expressed, whereas PDE4D2 was expressed in neither neurons nor glia. These findings suggest that therapies to reduce inflammation after TBI could be facilitated with targeted therapies, in particular for PDE1A, PDE4B2, PDE4D2, or PDE10A.

Trauma-induced expression of astrocytic thrombospondin-1 is regulated by P2 receptors coupled to protein kinase cascades

Thrombospondin-1 (TSP-1) is an extracellular matrix protein produced by astrocytes, which can promote synaptogenesis. The regulation of astrocytic TSP-1 involves extracellular ATP through the activation of P2Y receptors coupled to various protein kinase signaling pathways. However, not much is known about the mechanisms regulating TSP-1 expression in primary cortical astrocytes after a traumatic brain injury. Using an in-vitro model of central nervous system trauma that stimulates the release of ATP, we found that trauma-induced expression and release of TSP-1 involved purinergic signaling as both expression and release were significantly attenuated by pyridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid, a P2 receptor antagonist. Further antagonist studies with reactive blue 2 point to a role for P2Y4, as reactive blue 2 is a potent antagonist for rat P2Y4 receptors. In addition, the injury-induced expression of TSP-1 was significantly attenuated by the inhibition of extracellular signal-regulated kinase and p38/mitogen-activated protein kinase, whereas injury-induced release of TSP-1 was significantly blocked by the inhibition of extracellular signal-regulated kinase and Akt. Using an in-vivo model of a moderate parasagittal fluid-percussion brain injury, we found that TSP-1 levels were increased when compared with those in sham animals in the cortex, thalamus, and hippocampus. We conclude that TSP-1 expression after injury can be regulated by the activation of P2 receptors coupled with protein kinase signaling pathways and suggest that purinergic signaling, by regulating TSP expression, may play an important role in cell-matrix and cell-cell interactions such as those occurring during central nervous system repair.

STAT3 signaling after traumatic brain injury

Astrocytes respond to trauma by stimulating inflammatory signaling. In studies of cerebral ischemia and spinal cord injury, astrocytic signaling is mediated by the cytokine receptor glycoprotein 130 (gp130) and Janus kinase (Jak) which phosphorylates the transcription factor signal transducer and activator of transcription-3 (STAT3). To determine if STAT3 is activated after traumatic brain injury (TBI), adult male Sprague-Dawley rats received moderate parasagittal fluid-percussion brain injury or sham surgery, and then the ipsilateral cortex and hippocampus were analyzed at various post-traumatic time periods for up to 7 days. Western blot analyses indicated that STAT3 phosphorylation significantly increased at 30 min and lasted for 24 h post-TBI. A significant increase in gp130 and Jak2 phosphorylation was also observed. Confocal microscopy revealed that STAT3 was localized primarily within astrocytic nuclei. At 6 and 24 h post-TBI, there was also an increased expression of STAT3 pathway-related genes: suppressor of cytokine signaling 3, nitric oxide synthase 2, colony stimulating factor 2 receptor β, oncostatin M, matrix metalloproteinase 3, cyclin-dependent kinase inhibitor 1A, CCAAT/enhancer-binding protein β, interleukin-2 receptor γ, interleukin-4 receptor α, and α-2-macroglobulin. These results clarify some of the signaling pathways operative in astrocytes after TBI and demonstrate that the gp130-Jak2-STAT3 signaling pathway is activated after TBI in astrocytes.

Fluid-percussion brain injury induces changes in aquaporin channel expression

Edema, the accumulation of excess fluid, is a major pathological change in the brain that contributes significantly to pathology and mortality after moderate to severe brain injury. Edema is regulated by aquaporin (AQP) channels which transport water across cellular membranes. Six AQPs are found in the brain (1, 3, 4, 5, 8, and 9), and previous studies have found that AQP4 is regulated after traumatic brain injury (TBI). To further understand how AQPs contribute to brain edema, we investigated whether expression of AQP1, 3, and 9 are also regulated after TBI. Adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury (FPI) or sham surgery. After induction of FPI, the injured, ipsilateral parietal cortex and hippocampus were dissected and analyzed by Western blotting. We observed a small decrease in AQP3 and 4 levels at 7 days after FPI in the ipsilateral, parietal cortex. Both AQP1 and 9 significantly increased within 30 min post-injury and remained elevated for up to 6 h in the ipsilateral, parietal cortex. Aqp1 and 9 mRNA levels were also significantly increased at 30 min post-FPI. Administration of an AQP1 and 4 antagonist, AqB013, non-significantly increased brain water content in sham, non-injured animals, and did not prevent edema formation 24 h after trauma in either the parietal cortex or hippocampus. These results indicate that Aqp1 and 9 mRNA and protein levels increase after moderate parasagittal FPI and that an inhibitor of AQP1 and 4 does not decrease edema after moderate parasagittal FPI.

Post‐traumatic seizure susceptibility is attenuated by hypothermia therapy

Traumatic brain injury (TBI) is a major risk factor for the subsequent development of epilepsy. Currently, chronic seizures after brain injury are often poorly controlled by available antiepileptic drugs. Hypothermia treatment, a modest reduction in brain temperature, reduces inflammation, activates pro-survival signaling pathways, and improves cognitive outcome after TBI. Given the well-known effect of therapeutic hypothermia to ameliorate pathological changes in the brain after TBI, we hypothesized that hypothermia therapy may attenuate the development of post-traumatic epilepsy and some of the pathomechanisms that underlie seizure formation. To test this hypothesis, adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury, and were then maintained at normothermic or moderate hypothermic temperatures for 4 h. At 12 weeks after recovery, seizure susceptibility was assessed by challenging the animals with pentylenetetrazole, a GABA(A) receptor antagonist. Pentylenetetrazole elicited a significant increase in seizure frequency in TBI normothermic animals as compared with sham surgery animals and this was significantly reduced in TBI hypothermic animals. Early hypothermia treatment did not rescue chronic dentate hilar neuronal loss nor did it improve loss of doublecortin-labeled cells in the dentate gyrus post-seizures. However, mossy fiber sprouting was significantly attenuated by hypothermia therapy. These findings demonstrate that reductions in seizure susceptibility after TBI are improved with post-traumatic hypothermia and provide a new therapeutic avenue for the treatment of post-traumatic epilepsy.

Post-Traumatic Seizures Exacerbate Histopathological Damage after Fluid-Percussion Brain Injury

The purpose of this study was to investigate the effects of an induced period of post-traumatic epilepsy (PTE) on the histopathological damage caused by traumatic brain injury (TBI). Male Sprague Dawley rats were given a moderate parasagittal fluid-percussion brain injury (1.9-2.1 atm) or sham surgery. At 2 weeks after surgery, seizures were induced by administration of a GABA(A) receptor antagonist, pentylenetetrazole (PTZ, 30 mg/kg). Seizures were then assessed over a 1-h period using the Racine clinical rating scale. To evaluate whether TBI-induced pathology was exacerbated by the seizures, contusion volume and cortical and hippocampal CA3 neuronal cell loss were measured 3 days after seizures. Nearly all TBI rats showed clinical signs of PTE following the decrease in inhibitory activity. In contrast, clinically evident seizures were not observed in TBI rats given saline or sham-operated rats given PTZ. Contusions in TBI-PTZ-treated rats were significantly increased compared to the TBI-saline-treated group (p < 0.001). In addition, the TBI-PTZ rats showed less NeuN-immunoreactive cells within the ipsilateral parietal cerebral cortex (p < 0.05) and there was a trend for decreased hippocampal CA3 neurons in TBI-PTZ rats compared with TBI-saline or sham-operated rats. These results demonstrate that an induced period of post-traumatic seizures significantly exacerbates the structural damage caused by TBI. These findings emphasize the need to control seizures after TBI to limit even further damage to the injured brain.

Deficits in ERK and CREB activation in the hippocampus after traumatic brain injury

Traumatic brain injury (TBI) activates several protein kinase signaling pathways in the hippocampus that are critical for hippocampal-dependent memory formation. In particular, extracellular signal-regulated kinase (ERK), a protein kinase activated during and necessary for hippocampal-dependent learning, is transiently activated after TBI. However, TBI patients experience hippocampal-dependent cognitive deficits that occur for several months to years after the initial injury. Although basal activation levels of ERK return to sham levels within hours after TBI, we hypothesized that activation of ERK may be impaired after TBI. Adult male Sprague–Dawley rats received either sham surgery or moderate parasagittal fluid-percussion brain injury. At 2, 8, or 12 weeks after surgery, the ipsilateral hippocampi of sham surgery and TBI animals were sectioned into transverse slices. After 2 h of recovery in oxygenated artificial cerebrospinal fluid, the hippocampal slices were stimulated with glutamate or KCl depolarization, then analyzed by western blotting for phosphorylated, activated ERK and one of its downstream effectors, the transcription factor cAMP response element-binding protein (CREB). We found that activation of ERK ( p < 0.05) and CREB ( p < 0.05) after 30 s of glutamate stimulation or KCl depolarization was decreased in hippocampal slices from animals at 2, 8, or 12 weeks after TBI as compared to sham animals. Basal levels of phosphorylated or total ERK were not significantly altered at 2, 8, or 12 weeks after TBI, although basal levels of phosphorylated CREB were decreased 12 weeks post-trauma. These results suggest that TBI results in chronic signaling deficits through the ERK–CREB pathway in the hippocampus.

Hypothermia treatment potentiates ERK1/2 activation after traumatic brain injury

Traumatic brain injury (TBI) results in significant hippocampal pathology and hippocampal-dependent memory loss, both of which are alleviated by hypothermia treatment. To elucidate the molecular mechanisms regulated by hypothermia after TBI, rats underwent moderate parasagittal fluid-percussion brain injury. Brain temperature was maintained at normothermic or hypothermic temperatures for 30 min prior and up to 4 h after TBI. The ipsilateral hippocampus was assayed with Western blotting. We found that hypothermia potentiated extracellular signal-regulated kinase 1/2 (ERK1/2) activation and its downstream effectors, p90 ribosomal S6 kinase (p90RSK) and the transcription factor cAMP response element-binding protein. Phosphorylation of another p90RSK substrate, Bad, also increased with hypothermia after TBI. ERK1/2 regulates mRNA translation through phosphorylation of mitogen-activated protein kinase-interacting kinase 1 (Mnk1) and the translation factor eukaryotic initiation factor 4E (eIF4E). Hypothermia also potentiated the phosphorylation of both Mnk1 and eIF4E. Augmentation of ERK1/2 activation and its downstream signalling components may be one molecular mechanism that hypothermia treatment elicits to improve functional outcome after TBI.

Alterations in Mammalian Target of Rapamycin Signaling Pathways after Traumatic Brain Injury

In response to traumatic brain injury (TBI), neurons initiate neuroplastic processes through the activation of intracellular signaling pathways. However, the molecular mechanisms underlying neuroplasticity after TBI are poorly understood. To study this, we utilized the fluid-percussion brain injury (FPI) model to investigate alterations in the mammalian target of rapamycin (mTOR) signaling pathways in response to TBI. Mammalian target of rapamycin stimulates mRNA translation through phosphorylation of eukaryotic initiation factor 4E binding protein-1 (4E-BP1), p70 ribosomal S6 kinase (p70S6K), and ribosomal protein S6 (rpS6). These pathways coordinate cell growth and neuroplasticity via dendritic protein synthesis. Rats received sham surgery or moderate parasagittal FPI on the right side of the parietal cortex, followed by 15 mins, 30 mins, 4 h, 24 h, or 72 h of recovery. Using Western blot analysis, we found that mTOR, p70S6K, rpS6, and 4E-BP1 phosphorylation levels were significantly increased in the ipsilateral parietal cortex and hippocampus from 30 mins to 24 h after TBI, whereas total protein levels were unchanged. Using confocal microscopy to localize these changes, we found that rpS6 phosphorylation was increased in the parietal cortex and all subregions of the hippocampus. In accordance with these results, eIF4E, a key, rate-limiting mRNA translation factor, was also phosphorylated by mitogen-activated protein kinase-interacting kinase 1 (Mnk1) 15 mins after TBI. Together, these results suggest that changes in mRNA translation may be one mechanism that neurons use to respond to trauma and may contribute to the neuroplastic changes observed after TBI.

Activation of Calcium/Calmodulin-Dependent Protein Kinases after Traumatic Brain Injury

A prominent cognitive impairment after traumatic brain injury (TBI) is hippocampal-dependent memory loss. Although the histopathologic changes in the brain are well documented after TBI, the underlying biochemical mechanisms that contribute to memory loss have yet to be thoroughly delineated. Thus, we determined if calcium/calmodulin-dependent protein kinases (CaMKs), known to be necessary for the formation of hippocampal-dependent memories, are regulated after TBI. Sprague-Dawley rats underwent moderate parasagittal fluid-percussion brain injury on the right side of the parietal cortex. The ipsilateral hippocampus and parietal cortex were Western blotted for phosphorylated, activated alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaMKII), CaMKIV, and CaMKI. alpha-Calcium/calmodulin-dependent protein kinase II was activated in membrane subcellular fractions from the hippocampus and parietal cortex 30 mins after TBI. CaMKI and CaMKIV were activated in a more delayed manner, increasing in phosphorylation 1 h after TBI. The increase in activated alpha-CaMKII in membrane fractions was accompanied by a decrease in cytosolic total alpha-CaMKII, suggesting redistribution to the membrane. Using confocal microscopy, we observed that alpha-CaMKII was activated within hippocampal neurons of the dentate gyrus, CA3, and CA1 regions. Two downstream substrates of alpha-CaMKII, the AMPA-type glutamate receptor GluR1, and cytoplasmic polyadenylation element-binding protein, concomitantly increased in phosphorylation in the hippocampus and cortex 1 h after TBI. These results demonstrate that several of the biochemical cascades that subserve memory formation are activated unselectively in neurons after TBI. As memory formation requires activation of CaMKII signaling pathways at specific neuronal synapses, unselective activation of CaMKII signaling in all synapses may disrupt the machinery for memory formation, resulting in memory loss after TBI.

Inducible nitric oxide synthase expression after traumatic brain injury and neuroprotection with aminoguanidine treatment in rats.

We investigated the time course of inducible nitric oxide synthase (iNOS) enzymatic activity and immunocytochemical localization of iNOS expression after traumatic brain injury (TBI), as well as the possible role of iNOS in the pathogenesis of TBI. Male Sprague-Dawley rats were anesthetized and underwent moderate parasagittal fluid-percussion brain injury. Rats were decapitated 5 minutes, 6 hours, 1 day, 3 days, 7 days, or 14 days later, and iNOS enzymatic activities were measured (n = 6-8). To determine whether nitric oxide produced by iNOS contributed to the histopathological consequences of TBI, inhibition of iNOS activity using aminoguanidine (intraperitoneal injections of 100 mg/kg aminoguanidine [n = 9] or vehicle [n = 8], twice each day) was conducted for 3 days. Significantly elevated iNOS activity was detected at 3 days (276.8+/-72.3% of contralateral value, means +/- standard errors; P < 0.05), and the most robust increase occurred 7 days after TBI (608.0+/-127.0%, P < 0.01) in the injured parietal cerebral cortex. Immunostaining for iNOS and glial fibrillary acidic protein, at 3 and 7 days after TBI, revealed that the major cellular sources of iNOS expression were cortical Layer 1 astrocytes and macrophages within the subarachnoid space. Administration of aminoguanidine did not reduce contusion volume significantly; however, treatment reduced total cortical necrotic neuron counts (1367.6+/-210.3; P < 0.01, compared with vehicle, 2808.5+/-325.1). These data indicate that iNOS is expressed after moderate parasagittal fluid-percussion brain injury, in a time-dependent manner, and that inhibition of iNOS synthesis improves histopathological outcomes. Thus, inhibition of iNOS activation may represent a potential therapeutic strategy for the treatment of TBI.