RESUMO
BACKGROUND: After subarachnoid hemorrhage (SAH), neutrophils are deleterious and contribute to poor outcomes. Neutrophils can produce neutrophil extracellular traps (NETs) after ischemic stroke. Our hypothesis was that, after SAH, neutrophils contribute to delayed cerebral ischemia (DCI) and worse outcomes via cerebrovascular occlusion by NETs. METHODS: SAH was induced via endovascular perforation, and SAH mice were given either a neutrophil-depleting antibody, a PAD4 (peptidylarginine deiminase 4) inhibitor (to prevent NETosis), DNAse-I (to degrade NETs), or a vehicle control. Mice underwent daily neurological assessment until day 7 and then euthanized for quantification of intravascular brain NETs (iNETs). Subsets of mice were used to quantify neutrophil infiltration, NETosis potential, iNETs, cerebral perfusion, and infarction. In addition, NET markers were assessed in the blood of aneurysmal SAH patients. RESULTS: In mice, SAH led to brain neutrophil infiltration within 24 hours, induced a pro-NETosis phenotype selectively in skull neutrophils, and caused a significant increase in iNETs by day 1, which persisted until at least day 7. Neutrophil depletion significantly reduced iNETs, improving cerebral perfusion, leading to less neurological deficits and less incidence of DCI (16% versus 51.9%). Similarly, PAD4 inhibition reduced iNETs, improved neurological outcome, and reduced incidence of DCI (5% versus 30%), whereas degrading NETs marginally improved outcomes. Patients with aneurysmal SAH who developed DCI had elevated markers of NETs compared with non-DCI patients. CONCLUSIONS: After SAH, skull-derived neutrophils are primed for NETosis, and there are persistent brain iNETs, which correlated with delayed deficits. The findings from this study suggest that, after SAH, neutrophils and NETosis are therapeutic targets, which can prevent vascular occlusion by NETs in the brain, thereby lessening the risk of DCI. Finally, NET markers may be biomarkers, which can predict which patients with aneurysmal SAH are at risk for developing DCI.
Assuntos
Isquemia Encefálica , Transtornos Cerebrovasculares , Armadilhas Extracelulares , Hemorragia Subaracnóidea , Humanos , Camundongos , Animais , Hemorragia Subaracnóidea/complicações , Neutrófilos/metabolismo , Isquemia Encefálica/etiologia , Isquemia Encefálica/prevenção & controle , Transtornos Cerebrovasculares/complicaçõesRESUMO
BACKGROUND: Delayed cerebral ischemia (DCI) continues to be a significant contributor to morbidity and mortality following aneurysmal subarachnoid hemorrhage (aSAH). Subarachnoid blood and its degradation products have been implicated in DCI, and faster blood clearance has been hypothesized to confer better outcomes. This study evaluates the relationship between blood volume and its clearance on DCI (primary outcome) and location at 30 days (secondary outcome) after aSAH. METHODS: This is a retrospective review of adult patients presenting with aSAH. Hijdra sum scores (HSS) were assessed independently for each computed tomography (CT) scan of patients with available scans on post-bleed days 0-1 and 2-10. This cohort was used to evaluate the course of subarachnoid blood clearance (group 1). A subset of patients in the first cohort with available CT scans on both post-bleed days 0-1 and post-bleed days 3-4 composed the second cohort (group 2). This group was used to evaluate the association between initial subarachnoid blood (measured via HSS post-bleed days 0-1) and its clearance (measured via percentage reduction [HSS %Reduction] and absolute reduction [HSS-Abs-Reduction] in HSS between days 0-1 and 3-4) on outcomes. Univariable and multivariable logistic regression models were used to identify outcome predictors. RESULTS: One hundred fifty-six patients were in group 1, and 72 patients were in group 2. In this cohort, HSS %Reduction was associated with decreased risk of DCI in univariate (odds ratio [OR] = 0.700 [0.527-0.923], p = 0.011) and multivariable (OR = 0.700 [0.527-0.923], p = 0.012) analyses. Higher HSS %Reduction was significantly more likely to have better outcomes at 30 days in the multivariable analysis (OR = 0.703 [0.507-0.980], p = 0.036). Initial subarachnoid blood volume was associated with outcome location at 30 days (OR = 1.331 [1.040-1.701], p = 0.023) but not DCI (OR = 0.945 [0.780-1.145], p = 0.567). CONCLUSIONS: Early blood clearance after aSAH was associated with DCI (univariable and multivariable analyses) and outcome location at 30 days (multivariable analysis). Methods facilitating subarachnoid blood clearance warrant further investigation.
Assuntos
Isquemia Encefálica , Hemorragia Subaracnóidea , Adulto , Humanos , Hemorragia Subaracnóidea/complicações , Estudos Retrospectivos , Infarto Cerebral/complicações , Isquemia Encefálica/complicações , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: After subarachnoid hemorrhage (SAH), early brain injury (EBI) and delayed cerebral ischemia (DCI) lead to poor outcomes. Discovery of biomarkers indicative of disease severity and predictive of DCI is important. We tested whether leucine-rich alpha-2-glycoprotein 1 (LRG1) is a marker of severity, DCI, and functional outcomes after SAH. METHODS: We performed untargeted proteomics using mass spectrometry in plasma samples collected at < 48 h of SAH in two independent discovery cohorts (n = 27 and n = 45) and identified LRG1 as a biomarker for DCI. To validate our findings, we used enzyme-linked immunosorbent assay and confirmed this finding in an internal validation cohort of plasma from 72 study participants with SAH (22 DCI and 50 non-DCI). Further, we investigated the relationship between LRG1 and markers of EBI, DCI, and poor functional outcomes (quantified by the modified Rankin Scale). We also measured cerebrospinal fluid (CSF) levels of LRG1 and investigated its relationship to EBI, DCI, and clinical outcomes. RESULTS: Untargeted proteomics revealed higher plasma LRG1 levels across EBI severity and DCI in both discovery cohorts. In the validation cohort, the levels of LRG1 were higher in the DCI group compared with the non-DCI group (mean (SD): 95 [44] vs. 72 [38] pg/ml, p < 0.05, Student's t-test) and in study participants who proceeded to have poor functional outcomes (84 [39.3] vs. 72 [43.2] pg/ml, p < 0.05). Elevated plasma LRG1 levels were also associated with markers of EBI. However, CSF levels of LRG1 were not associated with EBI severity or the occurrence of DCI. CONCLUSIONS: Plasma LRG1 is a biomarker for EBI, DCI, and functional outcomes after SAH. Further studies to elucidate the role of LRG1 in the pathophysiology of SAH are needed.
Assuntos
Lesões Encefálicas , Isquemia Encefálica , Hemorragia Subaracnóidea , Humanos , Biomarcadores , Lesões Encefálicas/complicações , Infarto Cerebral/complicações , Glicoproteínas , LeucinaRESUMO
Brain vascular staining is very important for understanding cerebrovascular pathologies. 4% paraformaldehyde is considered the gold standard fixation technique for immunohistochemistry and it revolutionized the examination of proteins in fixed tissues. However, this fixation technique produces inconsistent immunohistochemical staining results due to antigen masking. Here, we test a new fixation protocol using 3% glyoxal and demonstrate that this method improves the staining of the brain vasculature, pericytes, and tight junction proteins compared to 4% paraformaldehyde. Use of this new fixation technique will provide more detailed information about vascular protein expressions, their distributions, and colocalizations with other proteins at the molecular level in the brain vasculature.
Assuntos
Barreira Hematoencefálica , Pericitos , Barreira Hematoencefálica/patologia , Encéfalo/irrigação sanguínea , Glioxal/metabolismo , Imuno-Histoquímica , Pericitos/metabolismo , Junções Íntimas/metabolismoRESUMO
BACKGROUND: Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) plays an important role in mediating inflammatory responses during ischemic stroke. Bile acid receptor Takeda-G-protein-receptor-5 (TGR5) has been identified as an important component in regulating brain inflammatory responses. In this study, we investigated the mechanism of TGR5 in alleviating neuroinflammation after middle cerebral artery occlusion (MCAO). METHODS: Sprague-Dawley rats were subjected to MCAO and TGR5 agonist INT777 was administered intranasally 1 h after MCAO. Small interfering RNAs (siRNA) targeting TGR5 and Pellino3 were administered through intracerebroventricular injection 48 h before MCAO. Infarct volumes and neurologic scores were evaluated, and ELISA, flow cytometry, immunofluorescence staining, immunoblotting, and co-immunoprecipitation were used for the evaluations. RESULTS: Endogenous TGR5 and Pellino3 levels increased after MCAO. TGR5 activation by INT777 significantly decreased pro-inflammatory cytokine, cleaved caspase-8, and NLRP3 levels, thereby reducing brain infarctions; both short- and long-term neurobehavioral assessments showed improvements. Ischemic damage induced the interaction of TGR5 with Pellino3. Knockdown of either TGR5 or Pellino3 increased the accumulation of cleaved caspase-8 and NLRP3, aggravated cerebral impairments, and abolished the anti-inflammatory effects of INT777 after MCAO. CONCLUSIONS: TGR5 activation attenuated brain injury by inhibiting neuroinflammation after MCAO, which could be mediated by Pellino3 inhibition of caspase-8/NLRP3.
Assuntos
Caspase 8/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Mediadores da Inflamação/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Administração Intranasal , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ácidos Cólicos/administração & dosagem , Infarto da Artéria Cerebral Média/prevenção & controle , Mediadores da Inflamação/antagonistas & inibidores , Injeções Intraventriculares , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Ubiquitina-Proteína Ligases/antagonistas & inibidoresRESUMO
BACKGROUND AND PURPOSE: Delayed neurological deficits are a devastating consequence of subarachnoid hemorrhage (SAH), which affects about 30% of surviving patients. Although a very serious concern, delayed deficits are understudied in experimental SAH models; it is not known whether rodents recapitulate the delayed clinical decline seen in SAH patients. We hypothesized that mice with SAH develop delayed functional deficits and that microthrombi and infarction correlate with delayed decline. METHODS: Adult C57BL/6J mice of both sexes were subjected to endovascular perforation to induce SAH. Mice were allowed to survive for up to 1 week post-ictus and behavioral performance was assessed daily. Postmortem microthrombi, large artery diameters (to assess vasospasm), and infarct volume were measured. These measures were analyzed for differences between SAH mice that developed delayed deficits and SAH mice that did not get delayed deficits. Correlation analyses were performed to identify which measures correlated with delayed neurological deficits, sex, and infarction. RESULTS: Twenty-three percent of males and 47% of females developed delayed deficits 3 to 6 days post-SAH. Female mice subjected to SAH had a significantly higher incidence of delayed deficits than male mice with SAH. Mice that developed delayed deficits had significantly more microthrombi and larger infarct volumes than SAH mice that did not get delayed deficits. Microthrombi positively correlated with infarct volume, and both microthrombi and infarction correlated with delayed functional deficits. Vasospasm did not correlate with either infarction delayed functional deficits. CONCLUSIONS: We discovered that delayed functional deficits occur in mice following SAH. Sex differences were seen in the prevalence of delayed deficits. The mechanism by which microthrombi cause delayed deficits may be via formation of infarcts.
Assuntos
Comportamento Animal , Infarto Cerebral/etiologia , Trombose Intracraniana/etiologia , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/patologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Spontaneous intracerebral hemorrhage (ICH) is the deadliest stroke subtype and neuroinflammation is a critical component of the pathogenesis following ICH. Annexin A1-FPR2 signaling has been shown to play a protective role in animal stroke models. This study aimed to assess whether Annexin A1 attenuated neuroinflammation and brain edema after ICH and investigate the underlying mechanisms. Male CD-1 mice were subjected to collagenase-induced ICH. Annexin A1 was administered at 0.5 hr after ICH. Brain water content measurement, short-term and long-term neurobehavioral tests, Western blot and immnunofluorescence were performed. Results showed that Annexin A1 effectively attenuated brain edema, improved short-term neurological function and ameliorated microglia activation after ICH. Annexin A1 also improved memory function at 28 days after ICH. However, these beneficial effects were abolished with the administration of FPR2 antagonist Boc-2. Furthermore, AnxA1/FPR2 signaling may confer protective effects via inhibiting p38-associated inflammatory cascade. Our study demonstrated that Annexin A1/FPR2/p38 signaling pathway played an important role in attenuating neuroinflammation after ICH and that Annexin A1 could be a potential therapeutic strategy for ICH patients.
Assuntos
Anexina A1/farmacologia , Edema Encefálico/tratamento farmacológico , Hemorragia Cerebral/tratamento farmacológico , Ciclo-Oxigenase 2/metabolismo , Receptores de Formil Peptídeo/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Anexina A1/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Edema Encefálico/etiologia , Edema Encefálico/metabolismo , Hemorragia Cerebral/induzido quimicamente , Hemorragia Cerebral/complicações , Hemorragia Cerebral/metabolismo , Colagenases , Modelos Animais de Doenças , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
Hemorrhagic transformation after ischemic stroke is an independent predictor for poor outcome and is characterized by blood vessel rupture leading to brain edema. To date, no therapies for preventing hemorrhagic transformation exist. Disintegrins from the venom of Crotalus atrox have targets within the coagulation cascade, including receptors on platelets. We hypothesized that disintegrins from C. atrox venom can attenuate hemorrhagic transformation by preventing activation of matrix metalloproteinase after middle cerebral artery occlusion (MCAO) in hyperglycemic rats. We subjected 48 male Sprague-Dawley rats weighing 240-260 g to MCAO and hyperglycemia to induce hemorrhagic transformation of the infarction. At reperfusion, we administered either saline (vehicle), whole C. atrox venom (two doses were used), or fractionated C. atrox venom (HPLC Fraction 2). Rats were euthanized 24 hr post-ictus for measurement of infarction and hemoglobin volume. Reversed-phase HPLC was performed to fractionate the whole venom and peaks were combined to form Fraction 2, which contained the disintegrin Crotatroxin. Fraction 2 protected against hemorrhagic transformation after MCAO, and attenuated activation of matrix metalloproteinase-9. Administering matrix metalloproteinase antagonists prevented the protection by Fraction 2. The results of this study indicate that disintegrins found in C. atrox venom may have therapeutic potential for reducing hemorrhagic transformation after ischemic stroke. Moreover, the RP-HPLC fractions retained sufficient protein activity to suggest that gentler and less efficient orthogonal chromatographic methods may be unnecessary to isolate proteins and explore their function.
Assuntos
Desintegrinas/farmacologia , Hiperglicemia/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Hemorragias Intracranianas/prevenção & controle , Metaloproteinase 9 da Matriz/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Animais , Modelos Animais de Doenças , Desintegrinas/uso terapêutico , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Hemorragias Intracranianas/etiologia , Hemorragias Intracranianas/metabolismo , Hemorragias Intracranianas/patologia , Masculino , Inibidores da Agregação Plaquetária/uso terapêutico , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The disruption of the blood-brain barrier (BBB) plays a critical event in the pathogenesis of ischemia stroke. TGR5 is recognized as a potential target for the treatment for neurologic disorders. METHODS: This study investigated the roles of TGR5 activation in attenuating BBB damage and underlying mechanisms after middle cerebral artery occlusion (MCAO). Sprague-Dawley rats were subjected to model of MCAO and TGR5 agonist, INT777, was administered intranasally. Small interfering RNA (siRNA) for TGR5 and BRCA1 were administered through intracerebroventricular injection 48 h before MCAO. Infarct volumes, brain water content, BBB permeability, neurological scores, Western blot, immunofluorescence staining and co- immunoprecipitation were evaluated. RESULTS: Endogenous TGR5 and BRCA1 were upregulated in the injured hemisphere after MCAO and TGR5 expressed in endothelial cells. Treatment with INT777 alleviated brain water content and BBB permeability, reduced infarction volume and improved neurological scores at 24 h and 72 h after ischemia. INT777 administration increased BRCA1 and Sirt1 expression, as well as upregulated expressions of tight junction proteins. Ischemic damage induced interaction of TGR5 with BRCA1. TGR5 siRNA and BRCA1 siRNA significantly inhibited expressions of BRCA1 and Sirt1, aggravated BBB permeability and exacerbated stroke outcomes after MCAO. The protective effects of INT777 at 24 h after MCAO were also abolished by TGR5 siRNA or BRCA1 siRNA. CONCLUSIONS: Our findings demonstrate that activating TGR5 could reduce BBB breakdown and improve neurological functions through BRCA1/Sirt1 signaling pathway after MCAO. TGR5 may serve as a potential new candidate to relieve brain injury after MCAO.
Assuntos
Barreira Hematoencefálica/fisiologia , Infarto da Artéria Cerebral Média/patologia , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via the original article.
RESUMO
BACKGROUND AND PURPOSE: Ischemic stroke activates Toll-like receptors (TLRs), triggering rapid inflammatory cytokine production. Axl signaling has multiple roles, including regulating cytokine secretion, clearing apoptotic cells, and maintaining cell survival, however, its role in inflammation after ischemic stroke has not been examined. We hypothesized that activation of Axl by recombinant Growth-arrest-specific protein 6 (rGas6) attenuates neuroinflammation by inhibiting the TLR/TRAF/NF-κB pathway after middle cerebral artery occlusion (MCAO) in rats. METH: Sprague-Dawley rats were subjected to 2h of MCAO. One hour after reperfusion, the rats were given an intranasal injection of rGas6, vehicle, or R428 (Axl receptor inhibitor). Neurological scores, infarct volumes, immunofluorescence staining, Morris Water Maze, rotarod test and histology alterations were analyzed. The expressions of proinflammatory cytokines, including IL-1ß, IL-6, TNF-α, and Gas6, Axl, STAT1, SOCS1, SOCS3 and the TLR/TRAF/NF-κB pathway were quantified using Western blot. RESULTS: Endogenous expressions of Gas6 and Axl decreased significantly by 24h after MCAO. rGas6 reduced brain infarction and improved neurologic deficits scores, and increased expression of Axl and decreased the expressions of TRAF3, TRAF6 and inflammatory factors IL-1ß, IL-6, and TNF-α. Four weeks after MCAO, rGas6 improved long-term neurological behavior and memory. Inhibition of the Axl/TLR/TRAF/NF-κB pathway reversed the brain protection by rGas6. CONCLUSION: rGas6 reduced the neurological deficits by inhibiting neuroinflammation via the TLR/TRAF/NF-κB signaling pathway. rGas6 can be used as potential treatment to ischemic stroke.
Assuntos
Encéfalo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/patologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Fármacos Neuroprotetores/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Encéfalo/metabolismo , Encéfalo/patologia , Ativação Enzimática/efeitos dos fármacos , Infarto da Artéria Cerebral Média/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Receptores Toll-Like/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Germinal matrix hemorrhage is induced by stereotaxic injection of collagenase into the germinal matrix of P7 Sprague-Dawley rats. Hemoglobin assay, western blot, immunofluorescence and neurobehavioral tests were used to test the effects of BLVRA on hematoma resolution and anti-inflammatory response. We showed that BLVRA triggered a signaling cascade that ameliorated post-hemorrhagic neurological deficits in both short-term and long-term neurobehavioral tests in a GMH rat model. Specifically, BLVRA inhibited toll-like receptor 4 (TLR4) expression by translocating to the nucleus in an endothelial nitric oxide (eNOS)/nitric oxide (NO)-dependent manner. BLVRA also induced the upregulation of CD36 scavenger receptor level in microglia/microphages, of which the prominent role is to enhance hematoma resolution. However, the beneficial effects of BLVRA were abolished with the knockdown of eNOS, indicating that the eNOS/NO system is an important downstream factor of BLVRA. Our results demonstrate a mechanism of BLVRA modulating hematoma resolution and suppressing inflammation through eNOS/NO/TLR4 pathway in the GMH rat model.
Assuntos
Hemorragia Cerebral/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/farmacologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Ventrículos Laterais/efeitos dos fármacos , Ventrículos Laterais/patologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Germinal matrix hemorrhage (GMH) is a common neurologic event with high morbidity and mortality in preterm infants. Spleen has been reported to play a critical role in inflammatory responses by regulating peripheral immune cells which contributes to secondary brain injury. METHODS: The current study investigated the mechanistic role of biliverdin reductase-A (BLVRA) in the splenic response and brain damage in neonates following a collagenase GMH model. Neurological outcomes and splenic weights were assessed. Neutrophil production and infiltration were quantitated in the spleen and brain, respectively. Western blot was performed in both splenic and brain tissues to measure protein levels of toll-like receptor 4 and proinflammatory cytokines. RESULTS: BLVRA treatment alleviated GMH-induced developmental delay and attenuated splenic atrophy at 1 and 3 days after GMH. Quantification analysis showed that spleen-stored peripheral immune cells mobilized into circulation and infiltrated in the brain following GMH, which was abrogated by BLVRA administration, resulting in reduced splenic inflammatory response. Furthermore, we showed that regulation of eNOS/NO signaling by BLVRA stimulation blunted toll-like receptor-4 (TLR4) signal. The eNOS-generated NO, in part, translocated BLVRA into the nucleus, where BLVRA inhibited TLR4 expression. CONCLUSION: We revealed a BLVRA-dependent signaling pathway in modulating the splenic inflammation in response to GMH via the eNOS/NO/TLR4 pathway.
Assuntos
Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Baço/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Hemorragia/complicações , Marcação In Situ das Extremidades Cortadas , Inflamação/etiologia , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/etiologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Sprague-Dawley , Baço/efeitos dos fármacos , Fatores de TempoRESUMO
INTRODUCTION: Postoperative cerebral edema is a devastating complication in neurosurgical patients. Loss of blood-brain barrier integrity has been shown to lead to the development of brain edema following neurosurgical procedures. The aim of this study was to evaluate preconditioning with Crotalus helleri venom (Cv-PC) as a potential preventive therapy for reducing postoperative brain edema in the rodent SBI model. C. helleri venom is known to contain phospholipase A2 (PLA2), an enzyme upstream to cyclooxygenase-2 (COX-2) in the inflammatory cascade, acts to increase the production of inflammatory mediators, such as prostaglandins. We hypothesize that Cv-PC will downregulate the response of the COX-2 pathway to injury, thereby reducing the inflammatory response and the development of brain edema after SBI. MATERIALS AND METHODS: 75 male Sprague Dawley rats (280-330g) were divided to the following groups-naïve+vehicle, naïve+Cv-PC, sham, vehicle, Cv-PC, Cv-PC+NS398 (COX-2 inhibitor). Vehicle preconditioned and Cv-PC animals received either three daily subcutaneous doses of saline or C. helleri venom at 72h, 48h, and 24h prior to surgery. In Cv-PC+NS398 animals, NS398 was administered intraperitoneally 1h prior to each Cv-PC injection. Sham-operated animals received craniotomy only, whereas SBI animals received a partial right frontal lobectomy. Neurological testing and brain water content were assessed at 24h and 72h after SBI; COX-2 and PGE2 expression was assessed at 24h postoperatively by Western blot and immunohistochemistry, respectively. RESULTS: At 24h after SBI, the vehicle-treated animals were observed to have increased brain water content (83.1±0.2%) compared to that of sham animals (80.2±0.1%). The brain water content of vehicle-treated animals at 72h post-SBI was elevated at 83.3±0.2%. Cv-PC-treated animals with doses of 10% LD50 had significantly reduced brain water content of 81.92±0.7% and 81.82±0.3% at 24h and 72h, respectively, after SBI compared to that of vehicle-treated animals, while Cv-PC with 5% LD50 doses showed brain water content that trended lower but did not reach statistical significance. At 24h and 72h post-SBI, Cv-PC-treated animals had significantly higher neurological score than vehicle-treated animals. The COX-2 over-expression characterized in SBI was attenuated in Cv-PC-treated animals; NS398 reversed the protective effect of Cv-PC on COX-2 expression. Cv-PC tempered the over-expression of the inflammatory marker PGE2. CONCLUSION: Our findings indicate that Cv-PC may provide a promising therapy for reducing postoperative edema and improving neurological function after neurosurgical procedures.
Assuntos
Edema Encefálico/prevenção & controle , Encéfalo/cirurgia , Lobo Frontal/lesões , Complicações Intraoperatórias/tratamento farmacológico , Fármacos Neuroprotetores/administração & dosagem , Complicações Pós-Operatórias/tratamento farmacológico , Venenos de Serpentes/administração & dosagem , Animais , Água Corporal/efeitos dos fármacos , Edema Encefálico/etiologia , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Crotalus , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dinoprostona/metabolismo , Modelos Animais de Doenças , Epiderme/efeitos dos fármacos , Epiderme/imunologia , Epiderme/patologia , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/patologia , Lobo Frontal/cirurgia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Complicações Intraoperatórias/metabolismo , Complicações Intraoperatórias/patologia , Masculino , Procedimentos Neurocirúrgicos , Nitrobenzenos/farmacologia , Complicações Pós-Operatórias/metabolismo , Complicações Pós-Operatórias/patologia , Ratos Sprague-Dawley , Sulfonamidas/farmacologiaRESUMO
The primary measure for experimental stroke studies, infarct volume, can be affected by brain swelling. The algorithm by Lin et al. was developed to correct for brain swelling, however, the correction is not adequate. This chapter presents a new infarct volume algorithm that more appropriately corrects for brain hemisphere volume changes (swelling and stunted growth). Fifty-one adult rats were sacrificed 24 h after middle cerebral artery occlusion (MCAO). Forty-four P10 rat pups were sacrificed 48 h after hypoxia-ischemia (HI). Infarct volumes for 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) stained brains were calculated using our algorithm and that of Lin and colleagues. For MCAO animals, the algorithm of Lin et al. computed smaller infarct volumes than those of our algorithm. For HI animals, Lin et al.'s algorithm's infarct volumes were greater than those of our algorithm. For sham animals, Lin et al.'s algorithm's computed infarct volumes were significantly different from those of our algorithm. Our algorithm produces a more robust estimation of infarct volume than Lin et al.'s algorithm because the effects of ipsilesional hemisphere volume changes are minimized. Herein, our algorithm yields an infarct volume that better corrects for brain swelling and stunted brain growth compared with the algorithm of Lin et al.
Assuntos
Algoritmos , Edema Encefálico/patologia , Infarto Encefálico/patologia , Encéfalo/patologia , Hipóxia-Isquemia Encefálica/patologia , Infarto da Artéria Cerebral Média/patologia , Animais , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-Dawley , Acidente Vascular CerebralRESUMO
Brain edema is routinely measured using the wet-dry method. Volume, however, is the sum total of all cerebral tissues, including water. Therefore, volumetric change following injury may not be adequately quantified using percentage of edema. We thus tested the hypothesis that dried brains can be reconstituted with water and then re-measured to determine the actual volume. Subarachnoid hemorrhage (SAH) was induced by endovascular perforation in adult male Sprague-Dawley rats (n = 30). Animals were euthanized at 24 and 72 h after evaluation of neurobehavior for determination of brain water content. Dried brains were thereafter reconstituted with equal parts of water (lost from brain edema) and centrifuged to remove air bubbles. The total volume was quantified using hydrostatic (underwater) physics principles that 1 ml water (mass) = 1 cm(3) (volume). The amount of additional water needed to reach a preset level marked on 2-ml test tubes was added to that lost from brain edema, and from the brain itself, to determine the final volume. SAH significantly increased both brain water and volume while worsening neurological function in affected rats. Volumetric measurements demonstrated significant brain swelling after SAH, in addition to the brain edema approach. This modification of the "wet-dry" method permits brain volume determination using valuable post hoc dried brain tissue.
Assuntos
Edema Encefálico/patologia , Encéfalo/patologia , Hemorragia Subaracnóidea/patologia , Animais , Comportamento Animal , Edema Encefálico/fisiopatologia , Modelos Animais de Doenças , Procedimentos Endovasculares , Masculino , Tamanho do Órgão , Punções , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/fisiopatologiaRESUMO
Hemorrhagic transformation occurs in as many as 48 % of stroke patients and is a major contributor to post-insult morbidity and mortality. Experimental models of hemorrhagic transformation are utilized for understanding the mechanisms behind its development, as well as for investigating potential therapeutics for prevention and reduction of bleeding. Thoroughly studying animal models of hemorrhagic transformation is critically important for testing novel treatments. Thus far, no study has examined the progression of brain swelling and hemorrhagic transformation after transient middle cerebral artery occlusion (MCAO). Herein, we investigate the development of infarction, brain swelling, and hemorrhagic transformation following MCAO in hyperglycemic rats. Twenty-five Sprague-Dawley rats were subjected to either 1.5 h of MCAO or sham surgery 15 min after induction of hyperglycemia. Animals were sacrificed at 0.25, 1, 3, or 24 h after reperfusion for measurement of infarct volume, brain swelling, and hemoglobin volume. Within 15 min of reperfusion, the infarct volume was significantly larger than in sham animals and did not increase in size over the 24 h. However, both brain swelling and hemorrhagic transformation, which began immediately after reperfusion, increase over 24 h after reperfusion.
Assuntos
Glicemia/metabolismo , Edema Encefálico/metabolismo , Hemorragia Cerebral/metabolismo , Hiperglicemia/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Animais , Edema Encefálico/etiologia , Hemorragia Cerebral/etiologia , Modelos Animais de Doenças , Glucose/farmacologia , Hiperglicemia/induzido quimicamente , Infarto da Artéria Cerebral Média/complicações , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de Risco , Edulcorantes/farmacologia , Fatores de TempoRESUMO
Traumatic brain injury (TBI), the foremost cause of morbidity and mortality in persons under 45 years of age worldwide, leads to about 200,000 victims requiring hospitalization and approximately 52,000 deaths per year in the United States. TBI is characterized by cerebral edema leading to raised intracranial pressure, brain herniation, and subsequent death. Current therapies for TBI treatment are often ineffective, thus novel therapies are needed. Recent studies have shown that an osmotic transport device (OTD) is capable of reducing brain water content and improving survival in mice with severe cerebral edema. Here we compare the effects of a craniectomy and an OTD plus craniectomy on neurological function in mice after TBI. Animals treated with a craniectomy plus an OTD had significantly better neurological function 2 days after TBI compared with those treated with craniectomy only. This study suggests that an OTD for severe brain swelling may improve patient functional outcome. Future studies include a more comprehensive neurological examination, including long-term memory tests.
Assuntos
Edema Encefálico/terapia , Lesões Encefálicas Traumáticas/terapia , Craniectomia Descompressiva , Equipamentos e Provisões , Osmose , Animais , Comportamento Animal , Edema Encefálico/etiologia , Edema Encefálico/fisiopatologia , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/fisiopatologia , Modelos Animais de Doenças , Feminino , CamundongosRESUMO
No matter how carefully a neurosurgical procedure is performed, it is intrinsically linked to postoperative deficits resulting in delayed healing caused by direct trauma, hemorrhage, and brain edema, termed surgical brain injury (SBI). Cerebral edema occurs several hours after SBI and is a major contributor to patient morbidity, resulting in increased postoperative care. Currently, the correlation between functional recovery and brain edema after SBI remains unknown. Here we examine the correlation between neurological function and brain water content in rats 42 h after SBI. SBI was induced in male Sprague-Dawley rats via frontal lobectomy. Twenty-four hours post-ictus animals were subjected to four neurobehavior tests: composite Garcia neuroscore, beam walking test, corner turn test, and beam balance test. Animals were then sacrificed for right-frontal brain water content measurement via the wet-dry method. Right-frontal lobe brain water content was found to significantly correlate with neurobehavioral deficits in the corner turn and beam balance tests: the number of left turns (percentage of total turns) for the corner turn test and distance traveled for the beam balance test were both inversely proportional with brain water content. No correlation was observed for the composite Garcia neuroscore or the beam walking test.
Assuntos
Edema Encefálico/fisiopatologia , Lesões Encefálicas/fisiopatologia , Procedimentos Neurocirúrgicos , Animais , Comportamento Animal , Edema Encefálico/etiologia , Lesões Encefálicas/complicações , Modelos Animais de Doenças , Progressão da Doença , Lobo Frontal/cirurgia , Complicações Intraoperatórias , Masculino , Ratos , Ratos Sprague-Dawley , Transtornos de Sensação/etiologia , Transtornos de Sensação/fisiopatologiaRESUMO
The leading cause of morbidity and mortality in infants is hypoxia-ischemia (HI). The current therapies for HI have limited success, in part due to a lack of understanding of HI pathophysiology and underlying mechanisms. Herein, a neonatal rat model of HI was used to examine the changes in brain swelling and infarct volume over 4 days after HI. Forty-four P10 rat pups were sacrificed at 2, 3, or 4 days post-HI. After sacrifice, the brains were removed, sliced, and stained with TTC (2,3,5-triphenyl-2H-tetrazolium chloride). Images of TTC-stained brains were used for measurement of the ipsilateral hemisphere brain volumes and infarct volumes, calculated using standard equations. The hemispheric brain volumes of HI animals in all groups was lower than that of sham animals and decreased as the post-HI sacrifice time increased. The infarct volume of HI animals was larger than that of sham animals. Infarct volumes tended to decrease over the days post-HI. The change in infarct volume is likely the result of a combination of brain growth and repair mechanisms. However, changes in the hemispheric brain volume may include tissue growth and repair mechanism, so also may be a limitation of the current algorithm used for calculating ipsilateral hemisphere brain volume.