RESUMEN
Extensive effort has been made to study the role of synaptic deficits in cognitive impairment after traumatic brain injury (TBI). Neurogranin (Ng) is a calcium-sensitive calmodulin (CaM)-binding protein essential for Ca2+/CaM-dependent kinase II (CaMKII) autophosphorylation which subsequently modulates synaptic plasticity. Given the loss of Ng expression after injury, additional research is warranted to discern changes in hippocampal post-synaptic signaling after TBI. Under isoflurane anesthesia, adult, male and female Sprague-Dawley rats received a sham/control or controlled cortical impact (CCI) injury. Ipsilateral hippocampal synaptosomes were isolated at 24 h and 1, 2, and 4 weeks post-injury, and western blot was used to evaluate protein expression of Ng-associated signaling proteins. Non-parametric Mann-Whitney tests were used to determine significance of injury for each sex at each time point. There were significant changes in the hippocampal synaptic expression of Ng and associated synaptic proteins such as phosphorylated Ng, CaMKII, and CaM up to 4 weeks post-CCI, demonstrating TBI alters hippocampal post-synaptic signaling. This study furthers our understanding of mechanisms of cognitive dysfunction within the synapse sub-acutely after TBI.
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RNA binding motif 5 (RBM5) is a tumor suppressor in cancer but its role in the brain is unclear. We used conditional gene knockout (KO) mice to test if RBM5 inhibition in the brain affects chronic cortical brain tissue survival or function after a controlled cortical impact (CCI) traumatic brain injury (TBI). RBM5 KO decreased baseline contralateral hemispheric volume (pâ¯<â¯0.0001) and exacerbated ipsilateral tissue loss at 21 d after CCI in male mice vs. wild type (WT) (pâ¯=â¯0.0019). CCI injury, but not RBM5 KO, impaired beam balance performance (0-5d post-injury) and swim speed on the Morris Water Maze (MWM) (19-20d) (pâ¯<â¯0.0001). RBM5 KO was associated with mild learning impairment in female mice (pâ¯=â¯0.0426), reflected as a modest increase in escape latency early in training (14-18d post-injury). However, KO did not affect spatial memory at 19d post-injury in male or in female mice but it was impaired by CCI in females (pâ¯=â¯0.0061). RBM5 KO was associated with impaired visual function in male mice on the visible platform test at 20d post-injury (pâ¯=â¯0.0256). To explore signaling disturbances in KOs related to behavior, we first cross-referenced known brain-specific RBM5-regulated gene targets with genes in the curated RetNet database that impact vision. We then performed a secondary literature search on RBM5-regulated genes with a putative role in hippocampal function. Regulating synaptic membrane exocytosis 2 (RIMS) 2 was identified as a gene of interest because it regulates both vision and hippocampal function. Immunoprecipitation and western blot confirmed protein expression of a novel ~170â¯kDa RIMS2 variant in the cerebellum, and in the hippocampus, it was significantly increased in KO vs WT (pâ¯<â¯0.0001), and in a sex-dependent manner (pâ¯=â¯0.0390). Furthermore, male KOs had decreased total canonical RIMS2 levels in the cerebellum (pâ¯=â¯0.0027) and hippocampus (pâ¯<â¯0.0001), whereas female KOs had increased total RIMS1 levels in the cerebellum (pâ¯=â¯0.0389). In summary, RBM5 modulates brain function in mammals. Future work is needed to test if RBM5 dependent regulation of RIMS2 splicing effects vision and cognition, and to verify potential sex differences on behavior in a larger cohort of mice.
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Lesiones Traumáticas del Encéfalo , Lesiones Encefálicas , Enfermedades del Sistema Nervioso , Proteínas Supresoras de Tumor , Animales , Femenino , Masculino , Ratones , Encéfalo/metabolismo , Lesiones Encefálicas/patología , Lesiones Traumáticas del Encéfalo/patología , Proteínas de Ciclo Celular/metabolismo , Cerebelo/patología , Proteínas de Unión al ADN/metabolismo , Técnicas de Inactivación de Genes , Hipocampo/metabolismo , Aprendizaje por Laberinto/fisiología , Ratones Noqueados , Enfermedades del Sistema Nervioso/patología , Proteostasis , Proteínas de Unión al ARN/metabolismoRESUMEN
Traumatic brain injury (TBI) is often associated with axonal injury that leads to significant motor and cognitive deficits. Ubiquitin carboxy terminal hydrolase L1 (UCHL1) is highly expressed in neurons and loss of its activity plays an important role in the pathogenesis of TBI. Fusion protein was constructed containing wild type (WT) UCHL1 and the HIV trans-activator of transcription capsid protein transduction domain (TAT-UCHL1) that facilitates transport of the protein into neurons after systemic administration. Additional mutant proteins bearing cysteine to alanine UCHL1 mutations at cysteine 152 (C152A TAT-UCHL1) that prevents nitric oxide and reactive lipid binding of C152, and at cysteine 220 (C220A TAT-UCHL1) that inhibits farnesylation of the C220 site were also constructed. WT, C152A, and C220A TAT-UCHL1 proteins administered to mice systemically after controlled cortical impact (CCI) were detectable in brain at 1 h, 4 h and 24 h after CCI by immunoblot. Mice treated with C152A or WT TAT-UCHL1 decreased axonal injury detected by NF200 immunohistochemistry 24 h after CCI, but C220A TAT-UCHL1 treatment had no significant effect. Further study indicated that WT TAT-UCHL1 treatment administered 24 h after CCI alleviated axonal injury as detected by SMI32 immunoreactivity 7 d after CCI, improved motor and cognitive deficits, reduced accumulation of total and K48-linked poly-Ub proteins, and attenuated the increase of the autophagy marker Beclin-1. These results suggest that UCHL1 activity contributes to the pathogenesis of white matter injury, and that restoration of UCHL1 activity by systemic treatment with WT TAT-UCHL1 after CCI may improve motor and cognitive deficits. These results also suggest that farnesylation of the C220 site may be required for the protective effects of UCHL1.
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Lesiones Traumáticas del Encéfalo , Ubiquitina Tiolesterasa , Ratones , Animales , Ubiquitina Tiolesterasa/genética , Productos del Gen tat/uso terapéutico , Cisteína , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/patología , Axones/patologíaRESUMEN
Under normal conditions, heat shock proteins work in unison through dynamic protein interactions collectively referred to as the "chaperome." Recent work revealed that during cellular stress, the functional interactions of the chaperome are modified to form the "epichaperome," which results in improper protein folding, degradation, aggregation, and transport. This study is the first to investigate this novel mechanism of protein dishomeostasis in traumatic brain injury (TBI). Male and female adult, Sprague-Dawley rats received a lateral controlled cortical impact (CCI) and the ipsilateral hippocampus was collected 24 h 1, 2, and 4 weeks after injury. The epichaperome complex was visualized by measuring HSP90, HSC70 and HOP expression in native-PAGE and normalized to monomeric protein expression. A two-way ANOVA examined the effect of injury and sex at each time-point. Native HSP90, HSC70 and HOP protein expression showed a significant effect of injury effect across all time-points. Additionally, HSC70 and HOP showed significant sex effects at 24 h and 4 weeks. Altogether, controlled cortical impact significantly increased formation of the epichaperome across all proteins measured. Further investigation of this pathological mechanism can lead to a greater understanding of the link between TBI and increased risk of neurodegenerative disease and targeting the epichaperome for therapeutics.
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Lesiones Traumáticas del Encéfalo , Enfermedades Neurodegenerativas , Femenino , Masculino , Ratas , Animales , Ratas Sprague-Dawley , Análisis de Varianza , HipocampoRESUMEN
Sepsis-associated brain injury (SABI) is characterized by an acute deterioration of mental status resulting in cognitive impairment and acquisition of new and persistent functional limitations in sepsis survivors. Previously, we reported that septic mice had evidence of axonal injury, robust microglial activation, and cytotoxic edema in the cerebral cortex, thalamus, and hippocampus in the absence of blood-brain barrier disruption. A key conceptual advance in the field was identification of sulfonylurea receptor 1 (SUR1), a member of the adenosine triphosphate (ATP)-binding cassette protein superfamily, that associates with the transient receptor potential melastatin 4 (TRPM4) cation channel to play a crucial role in cerebral edema development. Therefore, we hypothesized that knockout (KO) of Abcc8 (Sur1 gene) is associated with a decrease in microglial activation, cerebral edema, and improved neurobehavioral outcomes in a murine cecal ligation and puncture (CLP) model of sepsis. Sepsis was induced in 4-6-week-old Abcc8 KO and wild-type (WT) littermate control male mice by CLP. We used immunohistochemistry to define neuropathology and microglial activation along with parallel studies using magnetic resonance imaging, focusing on cerebral edema on days 1 and 4 after CLP. Abcc8 KO mice exhibited a decrease in axonal injury and cytotoxic edema vs. WT on day 1. Abcc8 KO mice also had decreased microglial activation in the cerebral cortex vs. WT. These findings were associated with improved spatial memory on days 7-8 after CLP. Our study challenges a key concept in sepsis and suggests that brain injury may not occur merely as an extension of systemic inflammation. We advance the field further and demonstrate that deletion of the SUR1 gene ameliorates CNS pathobiology in sepsis including edema, axonal injury, neuroinflammation, and behavioral deficits. Benefits conferred by Abcc8 KO in the murine CLP model warrant studies of pharmacological Abcc8 inhibition as a new potential therapeutic strategy for SABI.
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Antineoplásicos , Edema Encefálico , Lesiones Encefálicas , Disfunción Cognitiva , Sepsis , Canales Catiónicos TRPM , Ratones , Masculino , Animales , Ratones Noqueados , Receptores de Sulfonilureas/genética , Edema Encefálico/genética , Sepsis/complicaciones , Sepsis/genética , Sepsis/patología , Lesiones Encefálicas/complicaciones , Punciones , Edema , Ligadura , Ratones Endogámicos C57BLRESUMEN
Repetitive mild traumatic brain injury (rmTBI) is a prominent public health concern, with linkage to debilitating chronic sequelae. Developing reliable and well-characterized preclinical models of rmTBI is imperative in the investigation of the underlying pathophysiological mechanisms, as models can have varying parameters, affecting the overall pathology of the resulting injury. The lateral fluid percussion injury (FPI) model is a reliable and frequently used method of TBI replication in rodent subjects, though it is currently relatively underutilized in rmTBI research. In this study, we have performed a novel description of a variation of the lateral repetitive mild FPI (rmFPI) model, showing the graded acute behavioral impairment and histopathology occurring in response to one, two or four mild FPI (1.25 atm) or sham surgeries, implemented 24h apart. Beam walking performance revealed significant motor impairment in injured animals, with dysfunction increasing with additional injury. Based upon behavioral responses and histological observations, we further investigated the subacute pathophysiological outcomes of the dual FPI (dFPI). Immunoreactivity assessments showed that dFPI led to regionally-specific reductions in the post-synaptic protein neurogranin and increased subcortical white matter staining of the presynaptic protein synaptophysin at 2 weeks following dFPI. Immunohistochemical assessments of the microglial marker Iba-1 showed a striking increase in in several brain regions, and assessment of the astrocytic marker GFAP showed significantly increased immunoreactivity in the subcortical white matter and thalamus. With this study, we have provided a novel account of the subacute post injury outcomes occurring in response to a rmFPI utilizing these injury and frequency parameters, and thereby also demonstrating the reliability of the lateral FPI model in rmTBI replication.
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Traumatic brain injury (TBI) is known to impair synaptic function, and subsequently contribute to observed cognitive deficits. Retinoic Acid (RA) signaling modulates expression of synaptic plasticity proteins and is involved in hippocampal learning and memory. All trans-retinoic acid (ATRA), a metabolite of Vitamin A, has been identified as a potential pharmacotherapeutic for other neurological disorders due to this role. This study conducted an ATRA dose response to determine its therapeutic effects on cognitive behaviors and expression of hippocampal markers of synaptic plasticity and RA signaling proteins after experimental TBI. Under isoflurane anesthesia, adult male Sprague Dawley rats received either controlled cortical impact (CCI, 2.5 mm deformation, 4 m/s) or control surgery. Animals received daily intraperitoneal injection of 0.5, 1, 5, or 10 mg/kg of ATRA or vehicle for 2 weeks. Animals underwent motor and spatial learning and memory testing. Hippocampal expression of synaptic plasticity proteins neurogranin (Ng), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 sub-unit, as well as RA signaling proteins STRA6, ADLH1a1, CYP26A1 and CYP26B1 were evaluated by western blot at 2-weeks post-injury. ATRA treatment significantly recovered Ng synaptic protein expression, while having no effect on motor performance, spatial learning, and memory, and GluA1 expression after TBI. RA signaling protein expression is unchanged 2 weeks after TBI. Overall, ATRA administration after TBI showed limited therapeutic benefits compared to the vehicle.
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Lesiones Traumáticas del Encéfalo , Hipocampo , Animales , Lesiones Traumáticas del Encéfalo/metabolismo , Cognición , Hipocampo/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
Severe traumatic brain injury (TBI), such as that suffered by patients with cerebral contusion, is a major cause of death and disability in young persons. Effective therapeutics to treat or mitigate the effects of severe TBI are lacking, in part because drug delivery to the injured brain remains a challenge. Promising therapeutics targeting secondary injury mechanisms may have poor pharmacokinetics/pharmacodynamics, unwanted side effects, or high hydrophobicity. To address these challenges, we have developed a multi-lamellar vesicle nanoparticle (MLV-NP) formulation with a narrow size distribution (243 nm in diameter, 0.09 polydispersity index) and the capability of encapsulating hydrophobic small molecule drugs for delivery to the injured brain. To demonstrate the utility of these particles, we produced dual-fluorescent labeled nanoparticles containing the organic dyes, coumarin 153 and rhodamine B, that were delivered intravenously to Sprague-Dawley rats and C57Bl6/J mice at 1, 1 and 4, 24, or 48 h after controlled cortical impact injury. Distribution of particles was measured at 5, 25, 48, or 49 h post-injury by fluorescence microscopy of coronal brain sections. In all cases of MLV administration, a 1.2- to 1.9-fold enhancement of ipsilateral fluorescence signal was observed compared to the contralateral cortex. Enhanced fluorescence was also observed in the injured hippocampal tissue in these animals. MLV-NPs administered at 1 h were observed intracellularly in the injured hemisphere at 48 h, suggesting the possibility of concentrated drug delivery to injured cells. These results suggest that MLV-NP delivery of therapeutic agents may be a viable strategy for treating cerebral contusion TBI.
RESUMEN
Ubiquitin carboxy terminal hydrolase L1 (UCHL1) is a protein highly expressed in neurons that may play important roles in the ubiquitin proteasome pathway (UPP) in neurons, axonal integrity, and motor function after traumatic brain injury (TBI). Binding of reactive lipid species to cysteine 152 of UCHL1 results in unfolding, aggregation, and inactivation of the enzyme. To test the role of this mechanism in TBI, mice bearing a cysteine to alanine mutation at site 152 (C152A mice) that renders UCHL1 resistant to inactivation by reactive lipids were subjected to the controlled cortical impact model (CCI) of TBI and compared to wild type (WT) controls. Alterations in protein ubiquitination and activation of autophagy pathway markers in traumatized brain were detected by immunoblotting. Cell death and axonal injury were determined by histological assessment and anti-amyloid precursor protein (APP) immunohistochemistry. Behavioral outcomes were determined using the beam balance and Morris water maze tests. C152A mice had reduced accumulation of ubiquitinated proteins, decreased activation of the autophagy markers Beclin-1 and LC3B, a decreased number of abnormal axons, decreased CA1 cell death, and improved motor and cognitive function compared to WT controls after CCI; no significant change in spared tissue volume was observed. These results suggest that binding of lipid substrates to cysteine 152 of UCHL1 is important in the pathogenesis of injury and recovery after TBI and may be a novel target for future therapeutic approaches.
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Lesiones Traumáticas del Encéfalo , Ubiquitina Tiolesterasa , Animales , Axones/metabolismo , Sitios de Unión , Muerte Celular , Lípidos , Ratones , Mutación/genética , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Traumatic brain injury (TBI) can produce lasting cognitive, emotional, and somatic difficulties that can impact quality of life for patients living with an injury. Impaired hippocampal function and synaptic alterations have been implicated in contributing to cognitive difficulties in experimental TBI models. In the synapse, neuronal communication is facilitated by the regulated release of neurotransmitters from docking presynaptic vesicles. The synaptic vesicle glycoprotein 2 (SV2) isoforms SV2A and SV2B play central roles in the maintenance of the readily releasable pool of vesicles and the coupling of calcium to the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex responsible for vesicle docking. Recently, we reported the findings of TBI-induced reductions in presynaptic vesicle density and SNARE complex formation; however, the effect of TBI on SV2 is unknown. To investigate this, rats were subjected to controlled cortical impact (CCI) or sham control surgery. Abundance of SV2A and SV2B were assessed at 1, 3, 7, and 14 days post-injury by immunoblot. SV2A and SV2B were reduced in the cortex at several time points and in the hippocampus at every time point assessed. Immunohistochemical staining and quantitative intensity measurements completed at 14 days post-injury revealed reduced SV2A immunoreactivity in all hippocampal subregions and reduced SV2B immunoreactivity in the molecular layer after CCI. Reductions in SV2A abundance and immunoreactivity occurred concomitantly with motor dysfunction and spatial learning and memory impairments in the 2 weeks post-injury. These findings provide novel evidence for the effect of TBI on SV2 with implications for impaired neurotransmission neurobehavioral dysfunction after TBI.
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Lesiones Traumáticas del Encéfalo/metabolismo , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Glicoproteínas de Membrana/deficiencia , Trastornos de la Memoria/etiología , Proteínas del Tejido Nervioso/deficiencia , Animales , Lesiones Traumáticas del Encéfalo/complicaciones , Reacción de Fuga , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Trastornos de la Memoria/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Equilibrio Postural , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Proteínas SNARE/metabolismo , Aprendizaje Espacial , Vesículas Sinápticas/metabolismo , Factores de TiempoRESUMEN
Civilian traumatic brain injury (TBI) guidelines recommend resuscitation of patients with hypotensive TBI with crystalloids. Increasing evidence, however, suggests that whole blood (WB) resuscitation may improve physiological and survival outcomes at lower resuscitation volumes, and potentially at a lower mean arterial blood pressure (MAP), than crystalloid after TBI and hemorrhagic shock (HS). The objective of this study was to assess whether WB resuscitation with two different MAP targets improved behavioral and histological outcomes compared with lactated Ringer's (LR) in a mouse model of TBI+HS. Anesthetized mice (n = 40) underwent controlled cortical impact (CCI) followed by HS (MAP = 25-27 mm Hg; 25 min) and were randomized to five groups for a 90 min resuscitation: LR with MAP target of 70 mm Hg (LR70), LR60, WB70, WB60, and monitored sham. Mice received a 20 mL/kg bolus of LR or autologous WB followed by LR boluses (10 mL/kg) every 5 min for MAP below target. Shed blood was reinfused after 90 min. Morris Water Maze testing was performed on days 14-20 post-injury. Mice were euthanized (21 d) to assess contusion and total brain volumes. Latency to find the hidden platform was greater versus sham for LR60 (p < 0.002) and WB70 (p < 0.007) but not LR70 or WB60. The WB resuscitation did not reduce contusion volume or brain tissue loss. The WB targeting a MAP of 60 mm Hg did not compromise function versus a 70 mm Hg target after CCI+HS, but further reduced fluid requirements (p < 0.03). Using LR, higher achieved MAP was associated with better behavioral performance (rho = -0.67, p = 0.028). Use of WB may allow lower MAP targets without compromising functional outcome, which could facilitate pre-hospital TBI resuscitation.
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Presión Sanguínea/efectos de los fármacos , Transfusión Sanguínea/métodos , Lesiones Traumáticas del Encéfalo/terapia , Lactato de Ringer/uso terapéutico , Choque Hemorrágico/terapia , Animales , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/psicología , Servicios Médicos de Urgencia , Fluidoterapia , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos C57BL , Desempeño Psicomotor , Resucitación , Choque Hemorrágico/complicaciones , Choque Hemorrágico/psicología , Resultado del TratamientoRESUMEN
Ubiquitin (Ub) C-terminal hydrolase L1 (UCHL1) is a multifunctional protein that is expressed in neurons throughout brain at high levels. UCHL1 deletion is associated with axonal degeneration, progressive sensory motor ataxia, and premature death in mice. UCHL1 has been hypothesized to play a role in the pathogenesis of neurodegenerative diseases and recovery after neuronal injury. UCHL1 hydrolyzes Ub from polyubiquitinated (poly-Ub) proteins, but also may ligate Ub to select neuronal proteins, and interact with cytoskeletal proteins. These and other mechanisms have been hypothesized to underlie UCHL1's role in neurodegeneration and response to brain injury. A UCHL1 knockin mouse containing a C90A mutation (C90A) devoid of hydrolase activity was constructed. The C90A mouse did not develop the sensory and motor deficits, degeneration of the gracile nucleus and tract, or premature death as seen in UCHL1 deficient mice. C90A and wild type (WT) mice were subjected to the controlled cortical impact (CCI) model of traumatic brain injury (TBI), and cell death, axonal injury and behavioral outcome were assessed. C90A mice exhibited decreased spared tissue volume, greater loss of CA1 hippocampal neurons and greater axonal injury as detected using anti-amyloid precursor protein (APP) antibody and anti- non-phosphorylated neurofilament H (SMI-32) antibody immunohistochemistry after CCI compared to WT controls. Poly-Ub proteins and Beclin-1 were elevated after CCI in C90A mice compared to WT controls. Vestibular motor deficits assessed using the beam balance test resolved by day 5 after CCI in WT mice but not in C90A mice. These results suggest that the hydrolase activity of UCHL1 does not account for the progressive neurodegeneration and premature death seen in mice that do not express full length UCHL1. The hydrolase activity of UCHL1 contributes to the function of the ubiquitin proteasome pathway (UPP), ameliorates activation of autophagy, and improves motor recovery after CCI. Thus, UCHL1 hydrolase activity plays an important role in acute injury response after TBI.
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Axones/patología , Lesiones Traumáticas del Encéfalo/patología , Muerte Celular/efectos de los fármacos , Neuronas/patología , Ubiquitina Tiolesterasa/genética , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Animales , Autofagia , Beclina-1/metabolismo , Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/psicología , Región CA1 Hipocampal/patología , Muerte Celular/genética , Técnicas de Sustitución del Gen , Ratones , Mutación/genética , Desempeño Psicomotor , Transducción de Señal/genética , UbiquitinaciónRESUMEN
Traumatic brain injury (TBI) is known to cause short- and long-term synaptic changes in the brain, possibly underlying downstream cognitive impairments. Neuronal levels of neurogranin, a calcium-sensitive calmodulin-binding protein essential for synaptic plasticity and postsynaptic signaling, are correlated with cognitive function. This study aims to understand the effect of TBI on neurogranin by characterizing changes in protein expression at various time points after injury. Adult, male rats were subjected to either controlled cortical impact (CCI) or control surgery. Expression of neurogranin and post-synaptic density 95 (PSD-95) were evaluated by Western blot in the cortex and hippocampus at 24 h and 1, 2, and 4 weeks post-injury. We hypothesized that CCI reduces neurogranin levels in the cortex and hippocampus, and demonstrate different expression patterns from PSD-95. Neurogranin levels were reduced in the ipsilateral cortex and hippocampus up to 2 weeks after injury but recovered to sham levels by 4 weeks. The contralateral cortex and hippocampus were relatively resistant to changes in neurogranin expression post-injury. Qualitative immunohistochemical assessment corroborated the immunoblot findings. Particularly, the pericontusional cortex and ipsilateral Cornu Ammonis (CA)3 region showed marked reduction in immunoreactivity. PSD-95 demonstrated similar expression patterns to neurogranin in the cortex; however, in the hippocampus, protein expression was increased compared with sham at the 2 and 4 week time points. Our results indicate that CCI lowers neurogranin expression with temporal and regional specificity and that this occurs independently of dendritic loss. Further understanding of the role of neurogranin in synaptic biology after TBI will elucidate pathological mechanisms contributing to cognitive dysfunction.
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Lesiones Traumáticas del Encéfalo/metabolismo , Corteza Cerebral/metabolismo , Neurogranina/biosíntesis , Animales , Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/patología , Corteza Cerebral/patología , Expresión Génica , Masculino , Neurogranina/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Traumatic brain injury (TBI) and the activation of secondary injury mechanisms have been linked to impaired cognitive function, which, as observed in TBI patients and animal models, can persist for months and years following the initial injury. Impairments in neurotransmission have been well documented in experimental models of TBI, but the mechanisms underlying this dysfunction are poorly understood. Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex facilitates vesicular docking and neurotransmitter release in the synaptic cleft. Published studies highlight a direct link between reduced SNARE complex formation and impairments in neurotransmitter release. While alterations in the SNARE complex have been described following severe focal TBI, it is not known if deficits in SNARE complex formation manifest in a model with reduced severity. We hypothesized that lateral fluid percussion injury (lFPI) reduces the abundance of SNARE proteins, impairs SNARE complex formation, and contributes to impaired neurobehavioral function. To this end, rats were subjected to lFPI or sham injury and tested for acute motor performance and cognitive function at 3 weeks post-injury. lFPI resulted in motor impairment between 1 and 5 days post-injury. Spatial acquisition and spatial memory, as assessed by the Morris water maze, were significantly impaired at 3 weeks after lFPI. To examine the effect of lFPI on synaptic SNARE complex formation in the injured hippocampus, a separate cohort of rats was generated and brains processed to evaluate hippocampal synaptosomal-enriched lysates at 1 week post-injury. lFPI resulted in a significant reduction in multiple monomeric SNARE proteins, including VAMP2, and α-synuclein, and SNARE complex abundance. The findings in this study are consistent with our previously published observations suggesting that impairments in hippocampal SNARE complex formation may contribute to neurobehavioral dysfunction associated with TBI.
RESUMEN
Traumatic brain injury (TBI) impairs neuronal function and can culminate in lasting cognitive impairment. While impaired neurotransmitter release has been well established after experimental TBI, little is understood about the mechanisms underlying this consequence. In the synapse, vesicular docking and neurotransmitter release requires the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Impairments in vesicle docking, and alterations in SNARE complex formation are associated with impaired neurotransmitter release. We hypothesized that TBI reduces SNARE complex formation and disrupts synaptic vesicle distribution in the hippocampus. To examine the effect of TBI on the SNARE complex, rats were subjected to controlled cortical impact (CCI) or sham injury, and the brains were assessed at 6 h, 1 d, one week, two weeks, or four weeks post-injury. Immunoblotting of hippocampal homogenates revealed significantly reduced SNARE complex formation at one week and two weeks post-injury. To assess synaptic vesicles distribution, rats received CCI or sham injury and the brains were processed for transmission electron microscopy at one week post-injury. Synapses in the hippocampus were imaged at 100k magnification, and vesicle distribution was assessed in pre-synaptic terminals at the active zone. CCI resulted in a significant reduction in vesicle number within 150 nm of the active zone. These findings provide the first evidence of TBI-induced impairments in synaptic vesicle docking, and suggest that reductions in the pool of readily releasable vesicles and impaired SNARE complex formation are two novel mechanisms contributing to impaired neurotransmission after TBI.
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Lesiones Encefálicas/metabolismo , Hipocampo/metabolismo , Proteínas SNARE/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Animales , Masculino , Unión Proteica , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
PURPOSE: In previous studies, collagen based matrices have been implanted into the site of lesion in different models of brain injury. We hypothesized that semisynthetic collagen matrix can have neuroprotective function in the setting of traumatic brain injury. METHODS: Rats were subjected to sham injury or controlled cortical impact. They either received extracellular matrix graft (DuraGen) over the injury site or did not receive any graft and underwent beam balance/beam walking test at post injury days 1-5 and Morris water maze at post injury days 14-18. Animals were sacrificed at day 18 for tissue analysis. RESULTS: Collagen matrix implantation in injured rats did not affect motor function (beam balance test: p = 0.627, beam walking test: p = 0.921). However, injured group with collagen matrix had significantly better spatial memory acquisition (p < 0.05). There was a significant reduction in lesion volume, as well as neuronal loss in CA1 (p < 0.001) and CA3 (p < 0.05) regions of the hippocampus in injured group with collagen matrix (p < 0.05). CONCLUSIONS: Collagen matrix reduces contusional lesion volume, neuronal loss, and cognitive deficit after traumatic brain injury. Further studies are needed to demonstrate the mechanisms of neuroprotection by collagen matrix.
Asunto(s)
Lesiones Encefálicas/terapia , Colágeno/farmacología , Fármacos Neuroprotectores/farmacología , Andamios del Tejido , Animales , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/patología , Región CA1 Hipocampal/fisiopatología , Región CA3 Hipocampal/efectos de los fármacos , Región CA3 Hipocampal/patología , Región CA3 Hipocampal/fisiopatología , Modelos Animales de Enfermedad , Masculino , Aprendizaje por Laberinto , Actividad Motora , Neuronas/efectos de los fármacos , Neuronas/fisiología , Procedimientos Neuroquirúrgicos , Ratas Sprague-Dawley , Memoria Espacial , Resultado del TratamientoRESUMEN
Buspirone, a 5-HT1A receptor agonist, and environmental enrichment (EE) enhance cognition and reduce histopathology after traumatic brain injury (TBI) in adult rats, but have not been fully evaluated after pediatric TBI, which is the leading cause of death in children. Hence, the aims of this study were to assess the efficacy of buspirone alone (Experiment 1) and in combination with EE (Experiment 2) in TBI postnatal day-17 male rats. The hypothesis was that both therapies would confer cognitive and histological benefits when provided singly, but their combination would be more efficacious. Anesthetized rats received a cortical impact or sham injury and then were randomly assigned to receive intraperitoneal injections of buspirone (0.08 mg/kg, 0.1 mg/kg, and 0.3 mg/kg) or saline vehicle (1.0 mL/kg) 24 h after surgery and once daily for 16 days (Experiment 1). Spatial learning and memory were assessed using the Morris water maze (MWM) on post-operative days 11-16, and cortical lesion volume was quantified on day 17. Sham controls for each condition were significantly better than all TBI groups. In the TBI groups, buspirone (0.1 mg/kg) enhanced MWM performance versus vehicle and buspirone (0.08 mg/kg and 0.3 mg/kg) (p<0.05) and reduced lesion volume relative to vehicle (p=0.038). In Experiment 2, buspirone (0.1 mg/kg) or vehicle was combined with EE after TBI, and the data were compared to the standard (STD)-housed groups from Experiment 1. EE lead to a significant enhancement of spatial learning and a reduction in lesion size versus STD. Moreover, the combined treatment group (buspirone+EE) performed markedly better than the buspirone+STD and vehicle+EE groups, which suggests an additive effect and supports the hypothesis. The data replicate previous studies assessing these therapies in adult rats. These novel findings may have important rehabilitation-relevant implications for clinical pediatric TBI.
Asunto(s)
Lesiones Encefálicas/rehabilitación , Buspirona/farmacología , Ambiente , Aprendizaje por Laberinto/fisiología , Recuperación de la Función/fisiología , Agonistas de Receptores de Serotonina/farmacología , Animales , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/psicología , Buspirona/uso terapéutico , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Agonistas de Receptores de Serotonina/uso terapéuticoRESUMEN
We have used a rapid induction of profound hypothermia (<10 degrees C) with delayed resuscitation using cardiopulmonary bypass (CPB) as a novel approach for resuscitation from exsanguination cardiac arrest (ExCA). We have defined this approach as emergency preservation and resuscitation (EPR). We observed that 2 h but not 3 h of preservation could be achieved with favorable outcome using ice-cold normal saline flush to induce profound hypothermia. We tested the hypothesis that adding energy substrates to saline during induction of EPR would allow intact recovery after 3 h CA. Dogs underwent rapid ExCA. Two minutes after CA, EPR was induced with arterial ice-cold flush. Four treatments (n=6/group) were defined by a flush solution with or without 2.5% glucose (G+ or G-) and with either oxygen or nitrogen (O+ or O-) rapidly targeting tympanic temperature of 8 degrees C. At 3 h after CA onset, delayed resuscitation was initiated with CPB, followed by intensive care to 72 h. At 72 h, all dogs in the O+G+ group regained consciousness, and the group had better neurological deficit scores and overall performance categories than the O-groups (both P<0.05). In the O+G- group, four of the six dogs regained consciousness. All but one dog in the O-groups remained comatose. Brain histopathology in the O-G+ was worse than the other three groups (P<0.05). We conclude that EPR induced with a flush solution containing oxygen and glucose allowed satisfactory recovery of neurological function after a 3 h of CA, suggesting benefit from substrate delivery during induction or maintenance of a profound hypothermic CA.
Asunto(s)
Reanimación Cardiopulmonar , Glucosa/uso terapéutico , Paro Cardíaco/complicaciones , Paro Cardíaco/terapia , Hipotermia Inducida , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/terapia , Oxígeno/uso terapéutico , Animales , Temperatura Corporal/fisiología , Encéfalo/patología , Puente Cardiopulmonar , Estado de Conciencia/fisiología , Cuidados Críticos , Perros , Servicios Médicos de Urgencia , Glucosa/administración & dosificación , Masculino , Enfermedades del Sistema Nervioso/patología , Oxígeno/administración & dosificación , Resultado del TratamientoRESUMEN
Emergency preservation and resuscitation (EPR) is a new approach for resuscitation of exsanguination cardiac arrest (CA) victims to buy time for surgical hemostasis. EPR uses a cold aortic flush to induce deep hypothermic preservation, followed by resuscitation with cardiopulmonary bypass (CPB). We previously reported that 20 min of EPR was feasible with intact outcome. In this report, we tested the limits for EPR in rats. Adult male isoflurane-anesthetized rats were subjected to rapid hemorrhage (12.5 ml over 5 min), followed by esmolol/KCl-induced CA and 1 min of no-flow. EPR was then induced by perfusion with 270 ml of ice-cold Plasma-Lyte to decrease body temperature to 15 degrees C. After 60 min (n=7) or 75 min (n=7) of EPR, resuscitation was attempted with CPB over 60 min, blood transfusion, correction of acid-base balance and electrolyte disturbances, and mechanical ventilation for 2h. Survival, overall performance category (OPC: 1=normal, 5=death), neurological deficit score (NDS), and histological damage score (HDS) were assessed in survivors on day 3. While all rats after 60 min EPR survived, only two out of seven rats after 75 min EPR survived (p<0.05). All rats after 60 min EPR achieved OPC 1 and normal NDS by day 3. Survivors after 75 min EPR achieved best OPC 3 (p<0.05 vs. 60 min EPR). HDS of either brain or individual viscera was not statistically different after 60 versus 75 min EPR, except for kidneys (0+/-0 vs. 1.9+/-1.3, respectively; p<0.05), with a strong trend toward greater injury in all extracerebral organs in the 75-min EPR group (p<0.06). Histological findings were dominated by cardiac lesions observed in both groups and acute renal tubular and liver necrosis in the 75-min EPR group. In conclusion, we have shown that 60 min of EPR after exsanguination CA is associated with survival and favorable neurological outcome, while 75 min of EPR results in significant mortality and neurological damage in survivors. Surprisingly, extracerebral lesions predominated at 75-min EPR group. This model should serve as a screening model both for testing new pharmacological adjuncts to improve survival after exsanguination CA, and for elucidating the underlying mechanisms of ischemia/reperfusion injury.
Asunto(s)
Puente Cardiopulmonar , Reanimación Cardiopulmonar/métodos , Paro Cardíaco/terapia , Hipotermia Inducida/métodos , Animales , Esquema de Medicación , Electrólitos/administración & dosificación , Paro Cardíaco/complicaciones , Paro Cardíaco/patología , Masculino , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/prevención & control , Ratas , Factores de TiempoRESUMEN
BACKGROUND: In a previous study, titration of a hypertonic saline (HTS) solution during severe uncontrolled hemorrhagic shock (UHS) failed to reduce mortality. In a separate study, a novel antioxidant, polynitroxylated albumin (PNA) plus tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), infused during shock increased long-term survival. We hypothesized that combining potent antioxidants with a hypertonic solution during UHS would preserve the logistical advantage of small volume resuscitation and improve survival. METHODS: An UHS outcome model in rats was used. UHS phase I (90 min) included blood withdrawal of 30 ml/kg over 15 min, followed by tail amputation for uncontrolled bleeding. At 20 min, rats were randomized to four groups (n=10 each) for hypotensive resuscitation from 20 to 90 min (mean arterial pressure [MAP] > or = 40 mmHg): HTS/starch group received 7.2% NaCl/10% hydroxyethyl starch; HTS/albumin group received 7.5% NaCl/20% albumin; HTS/PNA group received 7.5% NaCl/20% PNA; HTS/albumin+tempol group received 7.5% NaCl/20% albumin plus tempol. Resuscitation phase II (180 min) included hemostasis, return of shed blood and administration of fluids to restore MAP > or = 80 mmHg. Observation phase III was to 72 h. RESULTS: The total amount of fluid required to maintain hypotensive MAP during HS was low and did not differ between groups (range: 3.4+/-1.9 to 5.3+/-2.5 ml/kg). The rate of fluid administration required was higher in the HTS/albumin+tempol group compared to all other groups (p=0.006). Additional uncontrolled blood loss was highest in the HTS/PNA group (16.2+/-5.7 ml/kg [p=0.01] versus 10.4+/-7.9 ml/kg in the HTS/starch group, 7.7+/-5.2 ml/kg in the HTS/albumin group and 8.2+/-7.1 ml/kg in the HTS/albumin+tempol group). MAP after start of resuscitation in phase I was lower in the HTS/albumin+tempol group than the HTS/albumin or HTS/PNA groups (p<0.01). This group was also less tachycardic. Long-term survival was low in all groups (2 of 10 after HTS/starch and 1 of 10 after HTS/albumin, 3 of 10 after HTS/PNA, 1 of 10 after HTS/albumin+tempol). Median survival time was shortest in the HTS/albumin+tempol group (72 min [CI 34-190]) compared to all other groups (p=0.01). CONCLUSIONS: Despite its benefits in other model systems, free tempol is potentially hazardous when combined with hypertonic fluids. PNA abrogates these deleterious effects on acute mortality but may lead to increased blood loss in the setting of UHS.