RESUMO
Operation brain trauma therapy (OBTT) is a multi-center, pre-clinical drug and biomarker screening consortium for traumatic brain injury (TBI). Therapies are screened across three rat models (parasagittal fluid percussion injury, controlled cortical impact [CCI], and penetrating ballistic-like brain injury). Operation brain trauma therapy seeks to define therapies that show efficacy across models that should have the best chance in randomized clinical trials (RCTs) and/or to define model-dependent therapeutic effects, including TBI protein biomarker responses, to guide precision medicine-based clinical trials in targeted pathologies. The results of the first five therapies tested by OBTT (nicotinamide, erythropoietin, cyclosporine [CsA], simvastatin, and levetiracetam) were published in the Journal of Neurotrauma. Operation brain trauma therapy now describes preliminary results on four additional therapies (glibenclamide, kollidon-VA64, AER-271, and amantadine). To date, levetiracetam was beneficial on cognitive outcome, histology, and/or biomarkers in two models. The second most successful drug, glibenclamide, improved motor function and histology in CCI. Other therapies showed model-dependent effects (amantadine and CsA). Critically, glial fibrillary acidic protein levels predicted treatment effects. Operation brain trauma therapy suggests that levetiracetam merits additional pre-clinical and clinical evaluation and that glibenclamide and amantadine merit testing in specific TBI phenotypes. Operation brain trauma therapy has established that rigorous, multi-center consortia could revolutionize TBI therapy and biomarker development.
Assuntos
Lesões Encefálicas Traumáticas/tratamento farmacológico , Programas de Rastreamento/métodos , Animais , Biomarcadores/sangue , Cognição/efeitos dos fármacos , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/sangue , Programas de Rastreamento/tendências , Ratos , Ratos Sprague-Dawley/lesões , Recuperação de Função Fisiológica/efeitos dos fármacos , Ubiquitina Tiolesterase/análise , Ubiquitina Tiolesterase/sangueRESUMO
BACKGROUND: Posttraumatic seizures are a medical problem affecting patients with traumatic brain injury. Yet effective treatment is lacking owing to the limitations of antiepileptic drugs (AEDs) applicable to these patients. METHODS: In this study, we evaluated the dose-response efficacy of levetiracetam (12.5-100.0 mg/kg) and gabapentin (1.25-25.0 mg/kg) administered either individually or in pairs at fixed-dose ratios as a combination in mitigating posttraumatic nonconvulsive seizures induced by severe penetrating ballistic-like brain injury (PBBI) in rats. Seizures were detected by continuous electroencephalogram (EEG) monitoring for 72 hours postinjury. Animals were treated twice per day for 3 days by intravenous injections. RESULTS: Both levetiracetam (25-100 mg/kg) and gabapentin (6.25-25 mg/kg) significantly reduced PBBI-induced seizure frequency by 44% to 73% and 61% to 69%, and seizure duration by 45% to 64% and 70% to 78%, respectively. However, the two drugs manifested different dose-response profiles. Levetiracetam attenuated seizure activity in a dose-dependent fashion, whereas the beneficial effects of gabapentin plateaued across the three highest doses tested. Combined administration of levetiracetam and gabapentin mirrored the more classic dose-response profile of levetiracetam monotherapy. However, no additional benefit was derived from the addition of gabapentin. Furthermore, isobolographic analysis of the combination dose-response profile of levetiracetam and gabapentin failed to reach the expected level of additivity, suggesting an unlikelihood of favorable interactions between these two drugs against spontaneously occurring posttraumatic seizure activities at the particular set of dose ratios tested. CONCLUSION: This study was the first attempt to apply isobolographic approach to studying AED combination therapy in the context of spontaneously occurring posttraumatic seizures. Despite the failure to achieve additivity from levetiracetam and gabapentin combination, it is important to recognize the objectivity of the isobolographic approach in the evaluation of AED combination therapy against seizures directly associated with brain injuries.
Assuntos
Aminas/farmacologia , Ácidos Cicloexanocarboxílicos/farmacologia , Traumatismos Cranianos Penetrantes/complicações , Piracetam/análogos & derivados , Convulsões/tratamento farmacológico , Convulsões/etiologia , Ácido gama-Aminobutírico/farmacologia , Animais , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Eletroencefalografia , Gabapentina , Levetiracetam , Masculino , Piracetam/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: MicroRNAs (miRNAs) are small stable RNAs that regulate translational degradation or repression of genes involved in brain trauma-mediated inflammation. More recently, miRNAs have emerged as potential novel TBI biomarkers. The aim of this study was to determine if a select set of miRNAs (miR-21, Let-7i, miR-124a, miR-146a, miR-107) that were previously associated with TBI models and clinical studies would be dysregulated and correlated to inflammatory cytokine abundance in the rat penetrating ballistic-like brain injury (PBBI) model. METHODS: Adult male Sprague-Dawley rats received a unilateral frontal 10% PBBI, which produces a temporary cavity. Sham animals received a craniotomy only. Ipsilateral brain tissue and serum were collected 4 hours to 7 days post-injury. Quantitation of miR-21, Let-7i, miR-124a, miR-146a, or miR-107 levels was conducted using Taqman PCR assays normalized to the endogenous reference, U6 snRNA. Brain tissue derived from matching cohorts was used to determine 1L-1beta and IL-6 levels by enzyme-linked immunosorbent assay. RESULTS: Brain tissue Let-7i and miR-21 increased at 4 hours and 1 day, whereas miR-124a and miR-107 were enhanced only 1 day post-injury. MiR-146a displayed a biphasic response and increased 1 day and 7 days, whereas elevation of miR-21 was sustained 1 day to 7 days after PBBI. Pathway analysis indicated that miRNAs were linked to inflammatory proteins, IL-6 and IL-1beta. Confirmation by enzyme-linked immunosorbent assay indicated that both cytokines were increased and peaked at 1 day, but fell at 3 days through 7 days after PBBI, indicating an inverse relationship with miRNA abundance. Serum Let-7i, alone, was differentially abundant 7 days after PBBI. CONCLUSION: Brain tissue-derived miRNAs linked to increased cytokine levels demonstrates a plausible therapeutic target of TBI-induced inflammation. Suppression of serum derived Let-7i may have utility as a biomarker of subacute injury progression or therapeutic responses.
Assuntos
Citocinas/metabolismo , Traumatismos Cranianos Penetrantes/metabolismo , MicroRNAs/metabolismo , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Masculino , Medicina Militar , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-DawleyRESUMO
Penetrating traumatic brain injury (PTBI) is one of the major cause of death and disability worldwide. Previous studies with penetrating ballistic-like brain injury (PBBI), a PTBI rat model revealed widespread perilesional neurodegeneration, similar to that seen in humans following gunshot wound to the head, which is unmitigated by any available therapies to date. Therefore, we evaluated human neural stem cell (hNSC) engraftment to putatively exploit the potential of cell therapy that has been seen in other central nervous system injury models. Toward this objective, green fluorescent protein (GFP) labeled hNSC (400,000 per animal) were transplanted in immunosuppressed Sprague-Dawley (SD), Fisher, and athymic (ATN) PBBI rats 1 week after injury. Tacrolimus (3 mg/kg 2 days prior to transplantation, then 1 mg/kg/day), methylprednisolone (10 mg/kg on the day of transplant, 1 mg/kg/week thereafter), and mycophenolate mofetil (30 mg/kg/day) for 7 days following transplantation were used to confer immunosuppression. Engraftment in SD and ATN was comparable at 8 weeks post-transplantation. Evaluation of hNSC differentiation and distribution revealed increased neuronal differentiation of transplanted cells with time. At 16 weeks post-transplantation, neither cell proliferation nor glial lineage markers were detected. Transplanted cell morphology was similar to that of neighboring host neurons, and there was relatively little migration of cells from the peritransplant site. By 16 weeks, GFP-positive processes extended both rostrocaudally and bilaterally into parenchyma, spreading along host white matter tracts, traversing the internal capsule, and extending â¼13 mm caudally from transplantation site reaching into the brainstem. In a Morris water maze test at 8 weeks post-transplantation, animals with transplants had shorter latency to platform than vehicle-treated animals. However, weak injury-induced cognitive deficits in the control group at the delayed time point confounded benefits of durable engraftment and neuronal differentiation. Therefore, these results justify further studies to progress towards clinical translation of hNSC therapy for PTBI.
Assuntos
Diferenciação Celular/fisiologia , Transtornos Cognitivos/terapia , Traumatismos Cranianos Penetrantes/terapia , Células-Tronco Neurais/transplante , Neurônios/fisiologia , Transplante de Células-Tronco/métodos , Animais , Lesões Encefálicas Traumáticas/diagnóstico , Lesões Encefálicas Traumáticas/terapia , Transtornos Cognitivos/diagnóstico , Traumatismos Cranianos Penetrantes/diagnóstico , Humanos , Distribuição Aleatória , Ratos , Ratos Endogâmicos F344 , Ratos Nus , Ratos Sprague-DawleyRESUMO
The tripeptide glycine-proline-glutamate analogue NNZ-2566 (Neuren Pharmaceuticals) demonstrates neuroprotective efficacy in models of traumatic brain injury. In penetrating ballistic-like brain injury (PBBI), it significantly decreases injury-induced upregulation of inflammatory cytokines including TNF-α, IFN-γ, and IL-6. However, the mechanism by which NNZ-2566 acts has yet to be determined. The activating transcription factor-3 (ATF3) is known to repress expression of these inflammatory cytokines and was increased at the mRNA and protein level 24-h post-PBBI. This study investigated whether 12 h of NNZ-2566 treatment following PBBI alters atf3 expression. PBBI alone significantly increased atf3 mRNA levels by 13-fold at 12 h and these levels were increased by an additional fourfold with NNZ-2566 treatment. To confirm that changes in mRNA translated to changes in protein expression, ATF3 expression levels were determined in vivo in microglia/macrophages, T cells, natural killer cells (NKCs), astrocytes, and neurons. PBBI alone significantly increased ATF3 in microglia/macrophages (820%), NKCs (58%), and astrocytes (51%), but decreased levels in T cells (48%). NNZ-2566 treatment further increased ATF3 protein expression in microglia/macrophages (102%), NKCs (308%), and astrocytes (13%), while reversing ATF3 decreases in T cells. Finally, PBBI increased ATF3 levels by 55% in neurons and NNZ-2566 treatment further increased these levels an additional 33%. Since increased ATF3 may be an innate protective mechanism to limit inflammation following injury, these results demonstrating that the anti-inflammatory and neuroprotective drug NNZ-2566 increase both mRNA and protein levels of ATF3 in multiple cell types provide a cellular mechanism for NNZ-2566 modulation of neuroinflammation following PBBI.
Assuntos
Fator 3 Ativador da Transcrição/biossíntese , Anti-Inflamatórios não Esteroides/uso terapêutico , Traumatismos Cranianos Penetrantes/tratamento farmacológico , Proteínas do Tecido Nervoso/biossíntese , Fármacos Neuroprotetores/uso terapêutico , Oligopeptídeos/uso terapêutico , Fator 3 Ativador da Transcrição/genética , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Traumatismos Cranianos Penetrantes/metabolismo , Traumatismos Cranianos Penetrantes/patologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Oligopeptídeos/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Previous work has shown that human amnion-derived progenitor (AMP) cell therapy is neuroprotective in a penetrating ballistic-like brain injury (PBBI) model. However, the neuroprotective capacity of AMP cells seemed to be mediated by the sustained secretion of AMP cell-derived neurotrophic factors, which are abundant in the amnion-derived cellular cytokine suspension (ACCS). To test this theory, the current study assessed the neuroprotective efficacy of long-term ACCS delivery in the PBBI model. METHODS: Experiment 1 assessed the bioactive stability and neuroprotective capacity of ACCS in an in vitro model of neurodegeneration. Experiment 2 evaluated the therapeutic effects of ACCS delivery initiated 15 minutes after PBBI and continued for 2 weeks after injury. Experiment 3 was designed to identify the therapeutic window for long-term ACCS delivery in the PBBI model. Outcome metrics included neurobehavioral assessments and neuropathologic measures of neuroinflammation and axonal/neuronal degeneration. RESULTS: Experiment 1 demonstrated that ACCS is thermally stable for 1 week at 37°C and that ACCS treatment protected neurite against staurosporine toxicity. Experiment 2 identified the optimal infusion rate of ACCS (1 µL/h) and demonstrated that long-term infusion of ACCS was capable of promoting significant protection against PBBI-induced neuropathology and motor abnormalities, but was not sufficient for reducing cognitive deficits. Finally, the results of Experiment 3 showed that ACCS is effective in promoting significant neuroprotection even when onset of treatment is delayed out to 24 hours (but not 48 hours) after PBBI. CONCLUSIONS: Collectively, our results support the hypothesis that the neuroprotective effects of AMP cells are mediated through a sustained delivery of ACCS, which implicates ACCS as a promising neuroprotection agent for clinical study.
Assuntos
Âmnio/citologia , Citocinas/uso terapêutico , Traumatismos Cranianos Penetrantes/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Âmnio/fisiologia , Animais , Técnicas In Vitro , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Destreza Motora/efeitos dos fármacos , Doenças Neurodegenerativas/tratamento farmacológico , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: This study compared early serum levels of ubiquitin C-terminal hydrolase (UCH-L1) from patients with mild and moderate traumatic brain injury (TBI) with uninjured and injured controls and examined their association with traumatic intracranial lesions on computed tomography (CT) scan (CT positive) and the need for neurosurgical intervention (NSI). METHODS: This prospective cohort study enrolled adult patients presenting to three tertiary care Level I trauma centers after blunt head trauma with loss of consciousness, amnesia, or disorientation and a Glasgow Coma Scale (GCS) score 9 to 15. Control groups included normal uninjured controls and nonhead injured trauma controls presenting to the emergency department with orthopedic injuries or motor vehicle crash without TBI. Blood samples were obtained in all trauma patients within 4 hours of injury and measured by enzyme-linked immunosorbent assay for UCH-L1 (ng/mL ± standard error of the mean). RESULTS: There were 295 patients enrolled, 96 TBI patients (86 with GCS score 13-15 and 10 with GCS score 9-12), and 199 controls (176 uninjured, 16 motor vehicle crash controls, and 7 orthopedic controls). The AUC for distinguishing TBI from uninjured controls was 0.87 (95% confidence interval [CI], 0.82-0.92) and for distinguishing those TBIs with GCS score 15 from controls was AUC 0.87 (95% CI, 0.81-0.93). Mean UCH-L1 levels in patients with CT negative versus CT positive were 0.620 (± 0.254) and 1.618 (± 0.474), respectively (p < 0.001), and the AUC was 0.73 (95% CI, 0.62-0.84). For patients without and with NSI, levels were 0.627 (0.218) versus 2.568 (0.854; p < 0.001), and the AUC was 0.85 (95% CI, 0.76-0.94). CONCLUSION: UCH-L1 is detectable in serum within an hour of injury and is associated with measures of injury severity including the GCS score, CT lesions, and NSI. Further study is required to validate these findings before clinical application. LEVEL OF EVIDENCE: II, prognostic study.
Assuntos
Lesões Encefálicas/enzimologia , Procedimentos Neurocirúrgicos/métodos , Ubiquitina Tiolesterase/sangue , Ferimentos não Penetrantes/enzimologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Lesões Encefálicas/diagnóstico por imagem , Lesões Encefálicas/cirurgia , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Tomografia Computadorizada por Raios X , Centros de Traumatologia , Índices de Gravidade do Trauma , Ferimentos não Penetrantes/diagnóstico por imagem , Ferimentos não Penetrantes/cirurgia , Adulto JovemRESUMO
This study evaluated the injury severity profile of unilateral, frontal penetrating ballistic-like brain injury (PBBI) on neurofunctional outcome, blood-brain barrier (BBB) permeability, and brain edema formation. The degree of injury severity was determined by the delivery of a water-pressure pulse designed to produce a temporary cavity by rapid (<40 ms) expansion of the probe's elastic balloon calibrated to equal 5%, 10%, 12.5%, or 15% of total rat brain volume (control groups consisted of sham surgery or insertion of the probe only). Neurofunctional assessments revealed motor and cognitive deficits related to the degree of injury severity, with the most clear-cut profile of PBBI injury severity depicted by the Morris water maze (MWM) results. A biphasic pattern of BBB leakage was detected in the injured hemisphere at all injury severity levels at 4 h post-injury, and again at 48-72 h post-injury, which remained evident out to 7 days post-PBBI in the 10% and 12.5% PBBI groups. Likewise, significant brain edema was detected in the injured hemisphere by 4 h post-injury and remained elevated out to 7 days post-injury in the 10% and 12.5% PBBI groups. However, following 5% PBBI, significant levels of edema were only detected from 24 h to 48h post-injury. These results identify an injury severity profile of BBB permeability, brain edema, and neurofunctional impairment that provides sensitive and clinically relevant outcome metrics for studying potential therapeutics.
Assuntos
Barreira Hematoencefálica/fisiologia , Edema Encefálico/etiologia , Traumatismos Cranianos Penetrantes/patologia , Doenças do Sistema Nervoso/etiologia , Animais , Comportamento Animal/fisiologia , Barreira Hematoencefálica/patologia , Encéfalo/patologia , Edema Encefálico/patologia , Extravasamento de Materiais Terapêuticos e Diagnósticos , Membro Anterior/fisiologia , Lateralidade Funcional , Traumatismos Cranianos Penetrantes/complicações , Traumatismos Cranianos Penetrantes/cirurgia , Pressão Intracraniana/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , Doenças do Sistema Nervoso/patologia , Procedimentos Neurocirúrgicos , Permeabilidade , Equilíbrio Postural/fisiologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função FisiológicaRESUMO
One of the histopathological consequences of a penetrating ballistic brain injury is the formation of a permanent cavity. In a previous study using the penetrating ballistic-like brain injury (PBBI) model, engrafted human amnion-derived multipotent progenitor (AMP) cells failed to survive when injected directly in the injury tract, suggesting that the cell survival requires a supportive matrix. In this study, we seated AMP cells in a collagen-based scaffold, injected into the injury core, and investigated cell survival and neuroprotection following PBBI. AMP cells suspended in AMP cell conditioned medium (ACCS) or in a liquefied collagen matrix were injected immediately after a PBBI along the penetrating injury tract. Injured control rats received only liquefied collagen matrix. All animals were allowed to survive two weeks. Consistent with our previous results, AMP cells suspended in ACCS failed to survive; likewise, no collagen was identified at the injury site when injected alone. In contrast, both AMP cells and the collagen were preserved in the injury cavity when injected together. In addition, AMP cells/collagen treatment preserved some apparent brain tissue in the injury cavity, and there was measurable infiltration of endogenous neural progenitor cells and astrocytes into the preserved brain tissue. AMP cells were also found to have migrated into the subventricular zone and the corpus callosum. Moreover, the AMP cell/collagen treatment significantly attenuated the PBBI-induced axonal degeneration in the corpus callosum and ipsilateral thalamus and improved motor impairment on rotarod performance. Overall, collagen-based scaffold provided a supportive matrix for AMP cell survival, migration, and neuroprotection.
Assuntos
Colágeno , Matriz Extracelular/transplante , Traumatismos Cranianos Penetrantes/cirurgia , Células-Tronco Multipotentes/transplante , Degeneração Neural/patologia , Recuperação de Função Fisiológica , Âmnio , Animais , Movimento Celular , Sobrevivência Celular , Corpo Caloso/patologia , Modelos Animais de Doenças , Traumatismos Cranianos Penetrantes/patologia , Traumatismos Cranianos Penetrantes/fisiopatologia , Humanos , Masculino , Microinjeções , Atividade Motora , Degeneração Neural/cirurgia , Ratos , Ratos Sprague-Dawley , Teste de Desempenho do Rota-Rod , Transplante de Células-Tronco , Tálamo/patologia , Alicerces Teciduais , Resultado do TratamentoRESUMO
To identify a viable cell source with potential neuroprotective effects, we studied amnion-derived multipotent progenitor (AMP) cells in a rat model of penetrating ballistic-like brain injury (PBBI). AMP cells were labeled with fluorescent dye PKH26 and injected in rats immediately following right hemispheric PBBI or sham PBBI surgery by ipsilateral i.c.v. administration. At 2 weeks post-injury, severe necrosis developed along the PBBI tract and axonal degeneration was prominent along the corpus callosum (cc) and in the ipsilateral thalamus. Injected AMP cells first entered the subventricular zone (SVZ) in both sham and PBBI rats. Further AMP cell migration along the cc only occurred in PBBI animals. No significant difference in injury volume was observed across all treatment groups. In contrast, treatment with AMP cells significantly attenuated axonal degeneration in both the thalamus and the cc. Interestingly, PKH26-labeled AMP cells were detected only in the SVZ and the cc (in parallel with the axonal degeneration), but not in the thalamus. None of the labeled AMP cells appeared to express neural differentiation, as evidenced by the lack of double labeling with nestin, S-100, GFAP, and MAP-2 immunostaining. In conclusion, AMP cell migration was specifically induced by PBBI and requires SVZ homing, yet the neuroprotective effect of intracerebral ventrical treatment using AMP cells was not limited to the area where the cells were present. This suggests that the attenuation of the secondary brain injury following PBBI was likely to be mediated by mechanisms other than cell replacement, possibly through delivery or sustained secretion of neurotrophic factors.
Assuntos
Lesões Encefálicas/patologia , Lesões Encefálicas/cirurgia , Células-Tronco Multipotentes/transplante , Degeneração Neural/cirurgia , Âmnio/citologia , Animais , Axônios/patologia , Diferenciação Celular , Movimento Celular , Humanos , Imuno-Histoquímica , Masculino , Células-Tronco Multipotentes/citologia , Degeneração Neural/patologia , Ratos , Ratos Sprague-DawleyRESUMO
To gain additional insights into the pathogenic cellular and molecular mechanisms underlying different types of brain injury (e.g., trauma versus ischemia), recently attention has focused on the discovery and study of protein biomarkers. In previous studies, using a high-throughput immunoblotting (HTPI) technique, we reported changes in 29 out of 998 proteins following acute injuries to the rat brain (penetrating traumatic versus focal ischemic). Importantly, we discovered that one protein, endothelial monocyte-activating polypeptide II precursor (p43/pro-EMAPII), was differentially expressed between these two types of brain injury. Among other functions, p43/pro-EMAPII is a known pro-inflammatory cytokine involved in the progression of apoptotic cell death. Our current objective was to verify the changes in p43/pro-EMAPII expression, and to evaluate the potentially important implications that the differential regulation of this protein has on injury development. At multiple time points following either a penetrating ballistic-like brain injury (PBBI), or a transient middle cerebral artery occlusion (MCAo) brain injury, tissue samples (6-72 h), CSF samples (24 h), and blood samples (24 h) were collected from rats for analysis. Changes in protein expression were assessed by Western blot analysis and immunohistochemistry. Our results indicated that p43/pro-EMAPII was significantly increased in brain tissues, CSF, and plasma following PBBI, but decreased after MCAo injury compared to their respective sham control samples. This differential expression of p43/pro-EMAPII may be a useful injury-specific biomarker associated with the underlying pathologies of traumatic versus ischemic brain injury, and provide valuable information for directing injury-specific therapeutics.
Assuntos
Lesões Encefálicas/diagnóstico , Isquemia Encefálica/diagnóstico , Citocinas/metabolismo , Proteínas de Neoplasias/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Biomarcadores/metabolismo , Lesões Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Contagem de Células , Immunoblotting , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE AND IMPORTANCE: Cortical spreading depolarizations (CSD) are waves of mass tissue depolarization that mediate progressive development of cortical infarction in animal models and occur in approximately 50% of patients with acute brain injury. Here we performed multi-modal cerebral monitoring to investigate pathologies associated with CSD occurrence in a case of severe traumatic brain injury. CLINICAL PRESENTATION: A 20 years old male suffering severe traumatic brain injury from a fall had extensive frontal subdural and intraparenchymal hemorrhage with mass effect. Craniectomy was performed for hematoma evacuation and decompression. INTERVENTION: During surgery, a subdural electrocorticography (ECoG) electrode strip, along with microdialysis and PtiO2 probes, was placed beside injured cortex for CSD monitoring. Within 13-81 hours post-injury, 34 CSD occurred. CSD incidence increased during spontaneous hyperthermia and decreased during induced normothermia. Periods of CSD activity were also associated with low brain glucose (<0.10 mmol/l), elevated glutamate (>40 mmol/l) and lactate/pyruvate (>40), and PtiO2<10 mmHg. CSD caused progressive deterioration of ECoG activity only in regions with infarction at follow-up on day 27. CONCLUSION: Repetitive mass tissue depolarizations accompanied a negative course of hemorrhagic lesion progression in the presence of ischemic conditions after traumatic brain injury. Whether as cause or effect, CSD may represent an inherent component of progressive metabolic failure leading to tissue death, and temperature appears to be an important factor influencing their occurrence. Continuous ECoG is a valuable tool for monitoring subclinical events such as CSD and seizures and for translational research in acute brain injury mechanisms and therapeutics.
Assuntos
Lesões Encefálicas/fisiopatologia , Córtex Cerebral/lesões , Depressão Alastrante da Atividade Elétrica Cortical , Hemorragia Intracraniana Traumática/fisiopatologia , Adulto , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Progressão da Doença , Eletroencefalografia , Febre/etiologia , Febre/fisiopatologia , Humanos , Hemorragia Intracraniana Traumática/etiologia , Hemorragia Intracraniana Traumática/patologia , Masculino , Recuperação de Função Fisiológica , Fatores de TempoRESUMO
In an earlier study, we demonstrated that PAN-811 (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a novel neuroprotectant, provides protection against glutamate, staurosporine, veratridine, or hypoxia/hypoglycemia toxicities in primary cortical neuronal cultures by upregulating Bcl-2 expression [R.-W. Chen, C. Yao, X.C. Lu, Z.-G. Jiang, R. Whipple, Z. Liao, H.A. Ghanbari, B. Almassian, F.C. Tortella, J.R. Dave. PAN-811 (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a novel neuroprotectant, elicits its function in primary neuronal cultures by upregulating Bcl-2 expression. Neuroscience 135 (2005) 191-201]. Both JNK (c-Jun N-terminal kinase) and p38 MAP (mitogen-activated protein) kinase activation have a direct inhibitory action on Bcl-2 by phosphorylation. In the present study, we continued to explore the mechanism of PAN-811 neuroprotection. Our results indicate that treatment of cultured cortical neurons with glutamate (100 microM) induces phosphorylation of both JNK and p38 MAPK. Specifically, pretreatment of neurons with 10 microM PAN-811 (an optimal neuroprotective concentration) for 1h, 4h, or 24h significantly suppresses glutamate-mediated activation of both JNK and p38 MAPK. Furthermore, the p38 MAPK-specific inhibitor SB203580 and the JNK-specific inhibitor SP600125 prevented glutamate-induced neuronal death in these primary cultures. Our results demonstrate that glutamate-induced phosphorylation of JNK and p38 MAPK is suppressed by PAN-811, which might contribute to Bcl-2 upregulation and PAN-811 neuroprotection.
Assuntos
Antagonistas de Aminoácidos Excitatórios , Ácido Glutâmico/toxicidade , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Piridinas/farmacologia , Tiossemicarbazonas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Antracenos/farmacologia , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Feminino , Genes bcl-2/genética , Gravidez , Ratos , Ratos Sprague-Dawley , Sais de Tetrazólio , TiazóisRESUMO
Cellular injury can involve the aberrant stimulation of cell cycle proteins in part through activation of phosphodiesterases (PDEs) and downstream expression of cell-cycle components such as cyclin D1. In mature non-proliferating cells activation of the cell cycle can lead to the induction of programmed cell death. In the present study, we investigated the in vitro neuroprotective efficacy and mechanism of action of vinpocetine (PDE1 inhibitor), trequinsin (PDE3 inhibitor), and rolipram (PDE4 inhibitor) in four mechanistically-distinct models of injury to primary rat cortical neurons as related to cell cycle regulation and apoptosis. Cellular injury was induced by hypoxia/hypoglycemia, veratridine (10 microM), staurosporine (1 microM), or glutamate (100 microM), resulting in average neuronal cell death rates of 43-48% as determined by MTT assay. Treatment with each PDE inhibitor (PDEI) resulted in a similar concentration-dependent neuroprotection profile with maximal effective concentrations of 5-10 microM (55-77% neuroprotection) in all four neurotoxicity models. Direct cytotoxicity due to PDE inhibition alone was not observed at concentrations below 100 microM. Further studies indicated that PDEIs can suppress the excitotoxic upregulation of cyclin D1 similar to the effects of flavopiridol, a cyclin-dependent kinase inhibitor, including suppression of pro-apoptotic caspase-3 activity. Overall, these data indicate that PDEIs are broad-spectrum neuroprotective agents acting through modulation of cell cycle elements and may offer a novel mode of therapy against acute injury to the brain.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Proteínas de Ciclo Celular/efeitos dos fármacos , Degeneração Neural/tratamento farmacológico , Neurônios/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Animais , Apoptose/fisiologia , Caspase 3/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/fisiopatologia , Ciclina D1/efeitos dos fármacos , Ciclina D1/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Degeneração Neural/enzimologia , Degeneração Neural/fisiopatologia , Neurônios/enzimologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/metabolismo , Inibidores de Fosfodiesterase/uso terapêutico , Diester Fosfórico Hidrolases/efeitos dos fármacos , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The ubiquitin proteasome system (UPS) is a major cellular protein degradation pathway that involves the modulation of key proteins controlling inflammation, cell cycle regulation and gene expression. Modulation of the UPS with proteasome inhibitors has indicated efficacy in the treatment of several disease states including cancer and neuro-inflammatory disorders. In particular, a series of recent reports have evaluated the pre-clinical efficacy of the proteasome inhibitor MLN519 for the treatment of focal ischemic/reperfusion brain injury in rats. Evidence from these studies indicate that the neuroprotection provided by MLN519 is related to an anti-inflammatory effect linked to the modulation of nuclear factor kappaB (NF-kappaB) activity, attenuation of cytokine (TNF-alpha, IL-1beta, and IL-6) and cellular adhesion molecule (ICAM-1 and E-selectin) expression, and reduction of neutrophil and macrophage infiltration into the injured rat brain. It is the aim of this paper to review the experimental neuroprotection data reported using MLN519 with a focus on the molecular and cellular mechanisms of anti-inflammatory action.
Assuntos
Acetilcisteína/análogos & derivados , Infarto Encefálico/tratamento farmacológico , Isquemia Encefálica/tratamento farmacológico , Encefalite/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Animais , Infarto Encefálico/metabolismo , Infarto Encefálico/fisiopatologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/fisiologia , Encefalite/fisiopatologia , Encefalite/prevenção & controle , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de ProteassomaRESUMO
In the present study, we evaluated delayed treatment effects of the proteasome inhibitor and anti-inflammatory agent MLN519 (initiated 10 h post-injury) to improve recovery following ischemic brain injury in rodents. Male rats were exposed to 2 h of middle cerebral artery occlusion (MCAo) and treated with MLN519 (1.0 mg/kg, i.v. @ 10, 24, and 48 h post-occlusion) or vehicle. By 2 weeks post-injury, 60% (6/10) of vehicle animals survived, which was improved (although non-significantly) to 78% (7/9) following MLN519 treatment. The percent loss of tissue in the ipsilateral brain hemisphere (at 2 weeks) was significantly reduced from 27+/-4% (vehicle) to 15+/-4% (MLN519). MLN519 treated animals also lost significantly less body weight (39%) and showed significant improvement in overall neurological function across the 2-week recovery period. However, no significant treatment effects were observed to reduce foot-fault deficits (balance beam) or improve recovery of operant performance (active avoidance test). Overall, delayed treatment with MLN519 provided significant improvement in 3 of 6 test metrics (histopathology, body weight, and neurological dysfunction) supporting improved outcome for brain-injured subjects.
Assuntos
Acetilcisteína/análogos & derivados , Isquemia Encefálica/tratamento farmacológico , Recuperação de Função Fisiológica/efeitos dos fármacos , Acetilcisteína/administração & dosagem , Animais , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Injeções Intraventriculares , Masculino , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Fatores de TempoRESUMO
BACKGROUND AND PURPOSE: Clinical development of novel neuroprotection therapies for the treatment of brain injury has been unsuccessful. One critical limitation is the lack of a viable therapeutic treatment window (TW). In this study, we evaluated the neuroprotection TW for the proteosome inhibitor MLN519 after ischemia/reperfusion brain injury in rats as related to its antiinflammatory mechanism. METHODS: Male Sprague-Dawley rats were subjected to 2 hours of middle cerebral artery occlusion (MCAo), followed by 70 hours of reperfusion and recovery. MLN519 was administered after injury (starting 6 to 12 hours after MCAo) to evaluate the full TW. Brain infarction, neuronal degeneration, neurological recovery, leukocyte infiltration, and inflammatory gene mRNA levels were assessed. RESULTS: Core infarct volume in vehicle-treated rats (216+/-25 mm3) was reduced with delayed MLN519 treatments of 6, 8, or 10 hours after injury (45+/-13, 86+/-28, and 150+/-27 mm3, respectively, P<0.05) and was associated with reductions in neuronal and axonal degeneration. MLN519-treated rats had reduced brain mRNA levels of TNF-alpha (46%, P<0.05), ICAM-1 (58%, P<0.05), IL-6 (58%, P<0.05), and E-selectin (72%, P<0.05) at 24 hours after injury. Furthermore, MLN519 treatment reduced leukocyte infiltration by 32% to 80% (P<0.05) in ischemic brain regions. CONCLUSIONS: Neuroprotection treatment with MLN519 provides an extended TW of up to 10 hours after ischemia/reperfusion brain injury, in part by attenuating the inflammatory response. As such, the delayed onset of brain inflammation after an ischemic injury offers a prime target for extending the neuroprotective TW with compounds such as MLN519, used either alone or possibly as an adjunctive therapy with thrombolytic agents.
Assuntos
Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Isquemia Encefálica/prevenção & controle , Infarto da Artéria Cerebral Média/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Acetilcisteína/uso terapêutico , Animais , Biomarcadores/sangue , Química Encefálica/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Quimioterapia Adjuvante , Modelos Animais de Doenças , Selectina E/análise , Selectina E/efeitos dos fármacos , Fibrinolíticos/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Inflamação/sangue , Inflamação/imunologia , Inflamação/prevenção & controle , Molécula 1 de Adesão Intercelular/análise , Masculino , Fármacos Neuroprotetores/uso terapêutico , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/imunologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Fator de Necrose Tumoral alfa/análiseRESUMO
Secondary brain injury due to ischemia includes the infiltration of leukocytes into the brain parenchyma mediated by activation of nuclear factor-kappaB (NF-kappaB), which is activated by proteasome degradation. Neuroprotection with the proteasome inhibitor MLN519 has previously been reported to decrease ischemic brain injury in rats. The authors used higher doses of MLN519 to evaluate the neuroprotection therapeutic window after 24 hours of brain injury in rats as correlated to proteasome levels, activated NF-kappaB immunoreactivity, and leukocyte infiltration. Male Sprague-Dawley rats were subjected to 2-hour middle cerebral artery occlusion (MCAO) and recovery. MLN519 or vehicle was administered after injury with a single injection given in delayed increments of 2 hours (i.e., 4, 6, or 8 hours after MCAO). Treatment with MLN519 up to 6 hours after MCAO (4 hours after reperfusion) effectively reduced neuronal and astrocytic degeneration, decreased cortical infarct volume, and increased neurologic recovery. These effects were related to >80% reductions in blood proteasome levels, reduced neutrophil infiltration, and a decrease in activated NF-kappaB immunoreactivity. This improved neuroprotection profile and antiinflammatory effect of MLN519 provides an exciting avenue for potential treatment of focal ischemic brain injury in humans.
Assuntos
Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Isquemia Encefálica/complicações , Infarto da Artéria Cerebral Média/etiologia , Infarto da Artéria Cerebral Média/patologia , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/fisiopatologia , Animais , Astrócitos/patologia , Isquemia Encefálica/metabolismo , Movimento Celular/fisiologia , Cisteína Endopeptidases/metabolismo , Gliose/patologia , Mediadores da Inflamação/metabolismo , Leucócitos/fisiologia , Masculino , Complexos Multienzimáticos/metabolismo , NF-kappa B/fisiologia , Degeneração Neural/etiologia , Complexo de Endopeptidases do Proteassoma , Ratos , Ratos Sprague-Dawley , Recuperação de Função FisiológicaRESUMO
Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules and cytokines, some of which are regulated by the nuclear transcription factor NF-kappaB. In this study the authors examined mRNA expression levels for several important genes associated with inflammation at five time points (3, 6, 12, 24, and 72 hours) after transient middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. A sensitive and quantitative technique (TaqMan real-time QRT-PCR) was used to simultaneously measure mRNA levels for key cell adhesion molecules and inflammatory cytokines. Gene expression increased significantly in the injured hemisphere for interleukin (IL)-1beta (12-fold increase at 24 hours), IL-6 (25-fold increase at 6 hours) and ICAM-1 (4-fold increase at 24 hours), and the interhemispheric differences for these genes were significant for every time point examined (P < 0.05 for all values). Tumor necrosis factor-alpha mRNA was upregulated in the injured versus uninjured hemisphere from 3 to 24 hours (5-fold increase at 6 hours), while E-selectin showed a significant increase in mRNA levels from 6 to 24 hours after MCAO (10-fold increase at 6 hours) (P < 0.05 for all values). VCAM-1 mRNA levels did not respond differentially to injury at any time point between the two brain hemispheres. At all time points examined, activated NF-kappaB immunoreactivity was observed in cells throughout the infarct-damaged tissue. These results are consistent with the proinflammatory properties of the induced molecules, which are involved in the initiation of the inflammatory cascade, and may thus contribute to secondary cellular responses that lead to further brain damage.