RESUMO
RNA-seq is widely used for studying gene expression, but commonly used sequencing platforms produce short reads that only span up to two exon junctions per read. This makes it difficult to accurately determine the composition and phasing of exons within transcripts. Although long-read sequencing improves this issue, it is not amenable to precise quantitation, which limits its utility for differential expression studies. We used long-read isoform sequencing combined with a novel analysis approach to compare alternative splicing of large, repetitive structural genes in muscles. Analysis of muscle structural genes that produce medium (Nrap: 5 kb), large (Neb: 22 kb), and very large (Ttn: 106 kb) transcripts in cardiac muscle, and fast and slow skeletal muscles identified unannotated exons for each of these ubiquitous muscle genes. This also identified differential exon usage and phasing for these genes between the different muscle types. By mapping the in-phase transcript structures to known annotations, we also identified and quantified previously unannotated transcripts. Results were confirmed by endpoint PCR and Sanger sequencing, which revealed muscle-type-specific differential expression of these novel transcripts. The improved transcript identification and quantification shown by our approach removes previous impediments to studies aimed at quantitative differential expression of ultralong transcripts.
Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , RNA Mensageiro , Análise de Sequência de RNA , Transcriptoma , Processamento Alternativo , Biologia Computacional/métodos , Éxons , Perfilação da Expressão Gênica/métodos , Humanos , Anotação de Sequência Molecular , Especificidade de Órgãos/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
OBJECTIVE AND DESIGN: The objective of this study was to assess the effect of vamorolone, a first-in-class dissociative steroidal compound, to inhibit inflammation when administered after disease onset in the murine collagen antibody-induced arthritis model of arthritis. ANIMALS: 84 DBA1/J mice were used in this study (n = 12 per treatment group). TREATMENT: Vamorolone or prednisolone was administered orally after disease onset for a duration of 7 days. METHODS: Disease score and bone erosion were assessed using previously described scoring systems. Cytokines were measured in joints via immunoassay, and joint cathepsin B activity (marker of inflammation) was assessed using optical imaging of joints on live mice. RESULTS: We found that vamorolone treatment led to a reduction of several disease parameters including disease score, joint inflammation, and the presence of pro-inflammatory mediators to a degree similar of that observed with prednisolone treatment. More importantly, histopathological analysis of affected joints showed that vamorolone treatment significantly reduced the degree of bone erosion while this bone-sparing property was not observed with prednisolone treatment at any of the tested doses. CONCLUSIONS: While many intervention regimens in other studies are administered prior to disease onset in animal models, the current study involves delivery of the potential therapeutic after disease onset. Based on the findings, vamorolone may offer an efficacious, yet safer alternative to conventional steroidal compounds in the treatment of rheumatoid arthritis and other inflammatory diseases.
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Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Pregnadienodiois/uso terapêutico , Animais , Anticorpos Monoclonais/imunologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Colágeno Tipo II/imunologia , Citocinas/imunologia , Articulações/efeitos dos fármacos , Articulações/imunologia , Articulações/patologia , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos DBARESUMO
Multiple sclerosis is a chronic disease of the central nervous system characterized by an autoimmune inflammatory reaction that leads to axonal demyelination and tissue damage. Glucocorticoids, such as prednisolone, are effective in the treatment of multiple sclerosis in large part due to their ability to inhibit pro-inflammatory pathways (e.g., NFκB). However, despite their effectiveness, long-term treatment is limited by adverse side effects. VBP15 is a recently described compound synthesized based on the lazeroid steroidal backbone that shows activity in acute and chronic inflammatory conditions, yet displays a much-reduced side effect profile compared to traditional glucocorticoids. The purpose of this study was to determine the effectiveness of VBP15 in inhibiting inflammation and disease progression in experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of multiple sclerosis. Our data show that VBP15 is effective at reducing both disease onset and severity. In parallel studies, we observed that VBP15 was able to inhibit the production of NFκB-regulated pro-inflammatory transcripts in human macrophages. Furthermore, treatment with prednisolone-but not VBP15-increased expression of genes associated with bone loss and muscle atrophy, suggesting lack of side effects of VBP15. These findings suggest that VBP15 may represent a potentially safer alternative to traditional glucocorticoids in the treatment of multiple sclerosis and other inflammatory diseases.
Assuntos
Anti-Inflamatórios/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Pregnadienodiois/uso terapêutico , Índice de Gravidade de Doença , Animais , Anti-Inflamatórios/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/patologia , Pregnadienodiois/farmacologia , Gravidez , Resultado do TratamentoRESUMO
BACKGROUND: Activity-based therapy (ABT) focuses on regaining motor and sensory function below the level of the lesion in persons with a spinal cord injury (SCI). This is accomplished through repetitive training of specific motor tasks. Research has shown that ABT may increase neuroplasticity in the rat and human spinal cord. OBJECTIVE: The primary aim of this study was to examine acute alterations in neuroplasticity-related proteins during ABT in persons with SCI. METHODS: Volunteers were current participants in an ABT program and consisted of 12 men and 3 women (age, 31.8 ± 10.9 years) with chronic SCI (injury duration, 63.9 ± 54.4 months). A single 2-hour bout of ABT consisted of standing load bearing, body weight-supported treadmill training, whole body vibration, and functional electrical stimulation. Blood samples were obtained at baseline and immediately after completion of each modality to determine serum levels of brain-derived neurotrophic factor (BDNF), prolactin, and cortisol. RESULTS: One-way analysis of variance (ANOVA) with repeated measures was used to examine differences in proteins over time. Results revealed baseline levels of BDNF (2.37 ± 1.41 ng/mL) that were lower than previous research has demonstrated in persons with SCI. No change in BDNF or cortisol was found, although prolactin was significantly reduced in response to ABT. CONCLUSION: Despite the length of the bout, acute changes in BDNF were not observed. Whether different intensities or modalities of ABT may promote acute increases in serum BDNF in individuals with SCI remains to be determined and further study is merited.
RESUMO
BACKGROUND: Spinal cord injury (SCI) induces secondary tissue damage that is associated with inflammation. We have previously demonstrated that inflammation-related gene expression after SCI occurs in two waves - an initial cluster that is acutely and transiently up-regulated within 24 hours, and a more delayed cluster that peaks between 72 hours and 7 days. Here we extend the microarray analysis of these gene clusters up to 6 months post-SCI. METHODS: Adult male rats were subjected to mild, moderate or severe spinal cord contusion injury at T9 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 hours, 24 hours, 7 days, 28 days, 3 months or 6 months post-injury and processed for microarray analysis and protein expression. RESULTS: Anchor gene analysis using C1qB revealed a cluster of genes that showed elevated expression through 6 months post-injury, including galectin-3, p22PHOX, gp91PHOX, CD53 and progranulin. The expression of these genes occurred primarily in microglia/macrophage cells and was confirmed at the protein level using both immunohistochemistry and western blotting. As p22PHOX and gp91PHOX are components of the NADPH oxidase enzyme, enzymatic activity and its role in SCI were assessed and NADPH oxidase activity was found to be significantly up-regulated through 6 months post-injury. Further, treating rats with the nonspecific, irreversible NADPH oxidase inhibitor diphenylene iodinium (DPI) reduced both lesion volume and expression of chronic gene cluster proteins one month after trauma. CONCLUSIONS: These data demonstrate that inflammation-related genes are chronically up-regulated after SCI and may contribute to further tissue loss.
Assuntos
Expressão Gênica , Inflamação/genética , Inflamação/imunologia , RNA Mensageiro/metabolismo , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Animais , Galectina 3/genética , Galectina 3/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Macrófagos/fisiologia , Imageamento por Ressonância Magnética , Masculino , Análise em Microsséries , Microglia/fisiologia , Família Multigênica , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/patologiaRESUMO
Aging adversely affects inflammatory processes in the brain, which has important implications in the progression of neurodegenerative disease. Following traumatic brain injury (TBI), aged animals exhibit worsened neurological function and exacerbated microglial-associated neuroinflammation. Type I Interferons (IFN-I) contribute to the development of TBI neuropathology. Further, the Cyclic GMP-AMP Synthase (cGAS) and Stimulator of Interferon Genes (STING) pathway, a key inducer of IFN-I responses, has been implicated in neuroinflammatory activity in several age-related neurodegenerative diseases. Here, we set out to investigate the effects of TBI on cGAS/STING activation, IFN-I signaling and neuroinflammation in young and aged C57Bl/6 male mice. Using a controlled cortical impact model, we evaluated transcriptomic changes in the injured cortex at 24 hours post-injury, and confirmed activation of key neuroinflammatory pathways in biochemical studies. TBI induced changes were highly enriched for transcripts that were involved in inflammatory responses to stress and host defense. Deeper analysis revealed that TBI increased expression of IFN-I related genes (e.g. Ifnb1, Irf7, Ifi204, Isg15) and IFN-I signaling in the injured cortex of aged compared to young mice. There was also a significant age-related increase in the activation of the DNA-recognition pathway, cGAS, which is a key mechanism to propagate IFN-I responses. Finally, enhanced IFN-I signaling in the aged TBI brain was confirmed by increased phosphorylation of STAT1, an important IFN-I effector molecule. This age-related activation of cGAS and IFN-I signaling may prove to be a mechanistic link between microglial-associated neuroinflammation and neurodegeneration in the aged TBI brain.
Assuntos
Envelhecimento/imunologia , Lesões Encefálicas Traumáticas/imunologia , Interferon Tipo I/fisiologia , Nucleotidiltransferases/metabolismo , Envelhecimento/metabolismo , Animais , Ativação Enzimática , Interferon Tipo I/genética , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/fisiologia , Doenças Neurodegenerativas/etiologia , Doenças Neuroinflamatórias/etiologia , Transdução de Sinais/fisiologiaRESUMO
PURPOSE: Immunotherapy promises unprecedented benefits to patients with cancer. However, the majority of cancer types, including high-risk neuroblastoma, remain immunologically unresponsive. High-intensity focused ultrasound (HIFU) is a noninvasive technique that can mechanically fractionate tumors, transforming immunologically "cold" tumors into responsive "hot" tumors. EXPERIMENTAL DESIGN: We treated <2% of tumor volume in previously unresponsive, large, refractory murine neuroblastoma tumors with mechanical HIFU and assessed systemic immune response using flow cytometry, ELISA, and gene sequencing. In addition, we combined this treatment with αCTLA-4 and αPD-L1 to study its effect on the immune response and long-term survival. RESULTS: Combining HIFU with αCTLA-4 and αPD-L1 significantly enhances antitumor response, improving survival from 0% to 62.5%. HIFU alone causes upregulation of splenic and lymph node NK cells and circulating IL2, IFNγ, and DAMPs, whereas immune regulators like CD4+Foxp3+, IL10, and VEGF-A are significantly reduced. HIFU combined with checkpoint inhibitors induced significant increases in intratumoral CD4+, CD8α+, and CD8α+CD11c+ cells, CD11c+ in regional lymph nodes, and decrease in circulating IL10 compared with untreated group. We also report significant abscopal effect following unilateral treatment of mice with large, established bilateral tumors using HIFU and checkpoint inhibitors compared with tumors treated with HIFU or checkpoint inhibitors alone (61.1% survival, P < 0.0001). This combination treatment significantly also induces CD4+CD44+hiCD62L+low and CD8α+CD44+hiCD62L+low population and is adoptively transferable, imparting immunity, slowing subsequent de novo tumor engraftment. CONCLUSIONS: Mechanical fractionation of tumors using HIFU can effectively induce immune sensitization in a previously unresponsive murine neuroblastoma model and promises a novel yet efficacious immunoadjuvant modality to overcome therapeutic resistance.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Ablação por Ultrassom Focalizado de Alta Intensidade/métodos , Imunidade Celular , Neuroblastoma/terapia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Terapia Combinada , Células Dendríticas/imunologia , Modelos Animais de Doenças , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos A , Neuroblastoma/imunologiaRESUMO
Changes in gene expression contribute to pathophysiological alterations following spinal cord injury (SCI). We examined gene expression over time (4 h, 24 h, 7 days) at the impact site, as well as rostral and caudal regions, following mild, moderate, or severe contusion SCI in rats. High-density oligonucleotide microarrays were used that included approximately 27,000 genes/ESTs (Affymetrix RG-U34; A, B and C arrays), together with multiple analyses (MAS 5.0, dChip). Alterations after mild injury were relatively rapid (4 and 24 h), whereas they were delayed and prolonged after severe injury (24 h and 7 days). The number and magnitude of gene expression changes were greatest at the injury site after moderate injury and increased in rostral and caudal regions as a function of injury severity. Sham surgery resulted in expression changes that were similar to mild injury, suggesting the importance of using time-linked surgical controls as well as naive animals for these kinds of studies. Expression of many genes and ESTs was altered; these were classified functionally based on ontology. Overall representation of these functional classes varied with distance from the site of injury and injury severity, as did the individual genes that contributed to each functional class. Different clustering approaches were used to identify changes in neuronal-specific genes and several transcription factors that have not previously been associated with SCI. This study represents the most comprehensive evaluation of gene expression changes after SCI to date. The results underscore the power of microarray approaches to reveal global genomic responses as well as changes in particular gene clusters and/or families that may be important in the secondary injury cascade.
Assuntos
Perfilação da Expressão Gênica , Traumatismos da Medula Espinal/metabolismo , Algoritmos , Animais , Sequência de Bases , Sistema Nervoso Central/patologia , Análise por Conglomerados , Etiquetas de Sequências Expressas , Expressão Gênica , Regulação da Expressão Gênica , Dados de Sequência Molecular , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/química , Canais de Potássio/química , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição GênicaRESUMO
Thyrotropin-releasing hormone (TRH) and TRH analogues have been reported to be neuroprotective in experimental models of spinal cord injury and head injury. We have previously shown that a diketopiperazine structurally related to the TRH metabolite cyclo-his-pro reduces neuronal cell death in vitro and in vivo. Here we report the neuroprotective activity of other cyclic dipeptides in multiple in vitro models of neuronal injury and after controlled cortical impact (CCI) in mice. Using primary neuronal cultures, three novel dipeptides were compared to the previously reported diketopiperazine as well as to vehicle controls; each of the compounds reduced cell death after direct physical trauma or trophic withdrawal. Two of these peptides also protected against glutamate toxicity and beta-amyloid-induced injury; the latter also strongly inhibited glutamate-induced increases in intracellular calcium. Treatment with each of the test compounds resulted in highly significant improvement of motor and cognitive recovery after CCI, as well as markedly reducing lesion volumes as shown by high field magnetic resonance imaging. DNA microarray studies following fluid percussion induced traumatic brain injury (TBI) in rats showed that treatment with one of these dipeptides after injury significantly down-regulated expression of mRNAs for cell cycle proteins, aquaporins, cathepsins and calpain in ipsilateral cortex and/or hippocampus, while up-regulating expression of brain-derived neurotrophic factor, hypoxia-inducible factor and several heat-shock proteins. Many of these mRNA expression changes were paralleled at the protein level. The fact that these small peptides modulate multiple mechanisms favoring neuronal cell survival, as well as their ability to improve functional outcome and reduce posttraumatic lesion size, suggests that they may have potential utility in clinical head injury.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Peptídeos/farmacologia , Peptídeos beta-Amiloides/toxicidade , Animais , Lesões Encefálicas/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Interpretação de Imagem Assistida por Computador , Immunoblotting , Imuno-Histoquímica , L-Lactato Desidrogenase/metabolismo , Imageamento por Ressonância Magnética , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/uso terapêutico , Gravidez , RNA/biossíntese , RNA/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
It has long been recognized that thyrotropin-releasing hormone (TRH) and certain TRH analogues are neuroprotective in a variety of animal models of CNS trauma. In addition to these neuroprotective actions, TRH and most TRH analogues have other physiological actions that may not be desirable for treatment of acute injury, such as analeptic, autonomic, and endocrine effects. We have developed a series of dual-substituted TRH analogues that have strong neuroprotective actions, but are largely devoid of these other physiological actions. In addition, we have developed a family of cyclized dipeptides (diketopiperazines), structurally somewhat related to a metabolic product of TRH, that appear even more effective as neuroprotective agents in vitro and in vivo, and may have nootropic properties. Here, we review these novel tripeptide and dipeptide compounds.
Assuntos
Fármacos Neuroprotetores , Oligopeptídeos/farmacologia , Animais , Ciclização , Humanos , Oligopeptídeos/química , Hormônio Liberador de Tireotropina/análogos & derivadosRESUMO
In studies designed to evaluate the therapeutic window for treatment of traumatic brain injury, the caspase 3 inhibitor z-DEVD-fmk improved neurologic function and reduced lesion volumes when administered at 1 but not at 4, 8, or 24 hours after injury. Moreover, neither caspase 3 nor PARP, a caspase 3 substrate, were cleaved in injured, untreated cortex from 1 to 72 hours after injury. Few cortical neurons expressed active caspase 3 or were TUNEL positive from 6 to 24 hours after injury, and TUNEL staining was primarily Type I (necrotic). Nissl staining revealed extensive neuronal necrosis in the injured cortex from 6 to 24 hours after impact. Considered together, these data suggested that z-DEVD-fmk may reduce neuronal necrosis, so we used an in vitro model of necrotic cell death induced by maitotoxin to test this further and explore the potential mechanism(s) involved. Z-DEVD-fmk (1 nM-100 microM) significantly attenuated maitotoxin induced neuronal cell death and markedly reduced expression of the 145 kD calpain-mediated alpha-spectrin breakdown product after maitotoxin injury. Neither the 120 kD caspase-mediated alpha-spectrin cleavage product nor cathepsin B were expressed after maitotoxin injury. In a cell free assay, z-DEVD-fmk reduced hydrolysis of casein by purified calpain I. Finally, z-DEVD-fmk reduced expression of the 145 kD calpain-mediated alpha-spectrin cleavage fragment after traumatic brain injury in vivo. These data suggest that neuroprotection by z-DEVD-fmk may, in part, reflect inhibition of calpain-related necrotic cell death.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/metabolismo , Calpaína/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Oligopeptídeos/farmacologia , Animais , Lesões Encefálicas/patologia , Calpaína/metabolismo , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Sistema Livre de Células , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Degeneração Neural/tratamento farmacológico , Degeneração Neural/metabolismo , Degeneração Neural/patologiaRESUMO
Endocannabinoids, including 2-arachidonoylglycerol and anandamide (N-arachidonoylethanolamine; AEA), have neuroprotective effects in the brain through actions at CB1 receptors. However, AEA also binds to vanilloid (VR1) receptors and induces cell death in several cell lines. Here we show that anandamide causes neuronal cell death in vitro and exacerbates cell loss caused by stretch-induced axonal injury or trophic withdrawal in rat primary neuronal cultures. Administered intracerebroventricularly, AEA causes sustained cerebral edema, as reflected by diffusion-weighted magnetic resonance imaging, regional cell loss, and impairment in long-term cognitive function. These effects are mediated, in part, through VR1 as well as through calpain-dependent mechanisms, but not through CB1 receptors or caspases. Central administration of AEA also significantly upregulates genes involved in pro-inflammatory/microglial-related responses. Thus, anandamide produces neurotoxic effects both in vitro and in vivo through multiple mechanisms independent of the CB1 receptor.
Assuntos
Ácidos Araquidônicos/metabolismo , Moduladores de Receptores de Canabinoides/metabolismo , Morte Celular/fisiologia , Endocanabinoides , Neurônios/metabolismo , Animais , Ácidos Araquidônicos/administração & dosagem , Ácidos Araquidônicos/toxicidade , Calpaína/metabolismo , Moduladores de Receptores de Canabinoides/administração & dosagem , Moduladores de Receptores de Canabinoides/toxicidade , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Córtex Cerebral/anatomia & histologia , Córtex Cerebral/metabolismo , Embrião de Mamíferos/anatomia & histologia , Ativação Enzimática , Perfilação da Expressão Gênica , Hipocampo/anatomia & histologia , Hipocampo/metabolismo , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Aprendizagem em Labirinto , Transtornos da Memória , Camundongos , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Alcamidas Poli-Insaturadas , RatosRESUMO
The authors developed a novel diketopiperazine that shows neuroprotective activity in a variety of in vitro models, as well as in a clinically relevant experimental model of traumatic brain injury (TBI) in rats. Treatment with 1-ARA-35b (35b), a cyclized dipeptide derived from a modified thyrotropin-releasing hormone (TRH) analog, significantly reduced cell death associated with necrosis (maitotoxin), apoptosis (staurosporine), or mechanical injury in neuronal-glial cocultures. Rats subjected to lateral fluid percussion-induced TBI and then treated with 1 mg/kg intravenous 35b thirty minutes after trauma showed significantly improved motor recovery and spatial learning compared with vehicle-treated controls. Treatment also significantly reduced lesion volumes as shown by magnetic resonance imaging, and decreased the number of TUNEL-positive neurons observed in ipsilateral hippocampus. Unlike TRH or traditional TRH analogs, 35b treatment did not change mean arterial pressure, body temperature, or thyroid-stimulating hormone release, and did not have analeptic activity. Moreover, in contrast to TRH or typical TRH analogs, 35b administration after TBI did not alter free-magnesium concentration or cellular bioenergetic state. Receptor-binding studies showed that 35b did not act with high affinity at 50 classical receptors, channels, or transporters. Thus, 35b shows none of the typical physiologic actions associated with TRH, but possesses neuroprotective actions in vivo and in vitro, and appears to attenuate both necrotic and apoptotic cell death.
Assuntos
Lesões Encefálicas/fisiopatologia , Lesões Encefálicas/psicologia , Cognição/efeitos dos fármacos , Dipeptídeos/farmacologia , Atividade Motora/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Peptídeos Cíclicos/farmacologia , Piperazinas/farmacologia , Animais , Sistema Nervoso Autônomo/efeitos dos fármacos , Sistema Nervoso Autônomo/fisiopatologia , Lesões Encefálicas/diagnóstico , Células Cultivadas , Estimulantes do Sistema Nervoso Central/farmacologia , Técnicas de Cocultura , Dipeptídeos/metabolismo , Marcação In Situ das Extremidades Cortadas , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Peptídeos Cíclicos/metabolismo , Piperazinas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/metabolismo , Recuperação de Função Fisiológica , Ferimentos não Penetrantes/diagnóstico , Ferimentos não Penetrantes/fisiopatologia , Ferimentos não Penetrantes/psicologiaRESUMO
1-ARA-35b (35b) is a cyclized dipeptide that shows considerable neuroprotective activity in vitro and improves neurologic recovery after fluid percussion-induced traumatic brain injury in rats. The authors evaluated the effects of treatment with 35b in mice subjected to controlled cortical impact brain injury. Animals treated with intravenous 35b after traumatic injury showed significantly enhanced recovery of beam walking and place learning functions compared with vehicle-treated controls, in addition to reduced lesion volumes. Beneficial effects were dose related and showed an inverted U-shaped dose-response curve between 0.1 and 10 mg/kg. Protective actions were found when the drug was administered initially at 30 minutes or 1, 4, or 8 hours, but not at 24 hours, after trauma. In separate experiments, rats treated with 35b on days 7 through 10 after injury showed remarkably improved place learning in comparison with injured controls. These studies confirm and extend the neuroprotective effects of this diketopiperazine in traumatic brain injury. In addition, they show that 35b has a relatively wide therapeutic window and improves cognitive function after both acute and chronic injury.
Assuntos
Lesões Encefálicas/fisiopatologia , Lesões Encefálicas/psicologia , Córtex Cerebral/patologia , Dipeptídeos/farmacologia , Fármacos Neuroprotetores/farmacologia , Nootrópicos/farmacologia , Peptídeos Cíclicos/farmacologia , Piperazinas/farmacologia , Ferimentos não Penetrantes/fisiopatologia , Ferimentos não Penetrantes/psicologia , Animais , Lesões Encefálicas/patologia , Córtex Cerebral/lesões , Córtex Cerebral/fisiopatologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Ferimentos não Penetrantes/patologiaRESUMO
The tripeptide thyrotropin-releasing hormone (TRH) and/or related analogues have shown neuroprotective activity across multiple animal trauma models as well as in a small clinical trial of spinal cord injury. The metabolic product of TRH (cyclo-his-pro) retains physiological activity. We have developed a number of novel cyclic dipeptides that are structurally similar to cyclo-his-pro, and have examined their neuroprotective activity across multiple in vitro models of neuronal injury and after traumatic brain injury (TBI) in rodents. Four such compounds were found to reduce cell death after trophic withdrawal or traumatic injury in primary neuronal cultures; two also protected against glutamate or beta-amyloid neurotoxicity. All compounds significantly improved motor and cognitive recovery after controlled cortical impact injury in mice, and markedly reduced lesion volumes as shown by high field magnetic resonance imaging. Further, compound 35b, which is being developed for clinical trials, also showed considerable neuroprotection after fluid percussion induced TBI in rats, and improved cognitive function after daily administration in chronically brain injured rats. At a mechanistic level, the drugs attenuate both apoptotic and necrotic cell death in primary neuronal cultures, markedly reduce intracellular calcium accumulation after injury, and limit changes in mitochondrial membrane potential and associated cytochrome c release. In addition, microarray studies show that 35b reduces transcriptional changes after injury for a number of genes (and proteins) that may be associated with secondary injury, including cell cycle genes, aquaporins and cathepsins. It also upregulates brain-derived neurotrophic factor (BDNF), heat shock proteins (HSP) and hypoxia inducible factor (HIF). Thus, these novel dipeptides have multipotential actions that make them candidates for the treatment of both acute and chronic neurodegeneration.
Assuntos
Dipeptídeos/farmacologia , Fármacos Neuroprotetores/farmacologia , Nootrópicos/farmacologia , Peptídeos Cíclicos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cálcio/metabolismo , Técnicas de Cultura de Células , Cognição/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Expressão Gênica/genética , Genes bcl-2/efeitos dos fármacos , Genes bcl-2/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Imageamento por Ressonância Magnética , Aprendizagem em Labirinto/efeitos dos fármacos , Necrose , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Caspase-3 is a cysteine protease that is strongly implicated in neuronal apoptosis. Activation of caspase-3 may be induced by at least two major initiator pathways: a caspase-8-mediated pathway activated through cell surface death receptors (extrinsic pathway), and a caspase-9-mediated pathway activated by signals from the mitochondria that lead to formation of an apoptosomal complex (intrinsic pathway). In the present studies, we compare the activation of caspases-3, -8, and -9 after lateral fluid-percussion traumatic brain injury (TBI) in rats. Immunoblot analysis identified cleaved forms of caspases-3 and -9, but not caspase-8, at 1, 12, and 48 h after injury. Immunocytochemistry specific for cleaved caspases-3 and -9 revealed their expression primarily in neurons. These caspases were also frequently localized in TUNEL-positive cells, some of which demonstrated morphological features of apoptosis. However, caspases-3 and -9 were also found in neurons that were not TUNEL-positive, and other TUNEL-positive cells did not show activated caspases. In contrast to caspases-3 or -9, caspase-8 expression was only minimally changed by injury. An increase in expression of this caspase was undetectable by immunoblotting methods, and appeared as positive immunostaining restricted to a few cells within the injured cortex. Treatment with the pan-caspase inhibitor z-VAD-fmk at 15 min after TBI improved performance on motor and spatial learning tests. These data suggest that several caspases may be involved in the pathophysiology of TBI and that pan-caspase inhibition strategies may improve neurological outcomes.
Assuntos
Lesões Encefálicas/metabolismo , Encéfalo/enzimologia , Caspases/metabolismo , Animais , Especificidade de Anticorpos , Encéfalo/patologia , Lesões Encefálicas/patologia , Caspases/análise , Caspases/imunologia , Fragmentação do DNA , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Mitocôndrias/enzimologia , Neurônios/enzimologia , Neurônios/patologia , Ratos , Recuperação de Função FisiológicaRESUMO
Inflammation has long been implicated in secondary tissue damage after spinal cord injury (SCI). Our previous studies of inflammatory gene expression in rats after SCI revealed two temporally correlated clusters: the first was expressed early after injury and the second was up-regulated later, with peak expression at 1-2 weeks and persistent up-regulation through 6 months. To further address the role of inflammation after SCI, we examined inflammatory genes in a second species, mice, through 28 days after SCI. Using anchor gene clustering analysis, we found similar expression patterns for both the acute and chronic gene clusters previously identified after rat SCI. The acute group returned to normal expression levels by 7 days post injury. The chronic group, which included C1qB, p22(phox) and galectin-3, showed peak expression at 7 days and remained up-regulated through 28 days. Immunohistochemistry and western blot analysis showed that the protein expression of these genes was consistent with the mRNA expression. Further exploration of the role of one of these genes, galectin-3, suggests that galectin-3 may contribute to secondary injury. In summary, our findings extend our prior gene profiling data by demonstrating the chronic expression of a cluster of microglial associated inflammatory genes after SCI in mice. Moreover, by demonstrating that inhibition of one such factor improves recovery, the findings suggest that such chronic up-regulation of inflammatory processes may contribute to secondary tissue damage after SCI, and that there may be a broader therapeutic window for neuroprotection than generally accepted.
Assuntos
Galectina 3/fisiologia , Regulação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Galectina 3/biossíntese , Galectina 3/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologiaRESUMO
Galectins are pleiotropic carbohydrate-binding lectins involved in inflammation, growth/differentiation, and tissue remodeling. The functional role of galectins in amyotrophic lateral sclerosis (ALS) is unknown. Expression studies revealed increases in galectin-1 mRNA and protein in spinal cords from SOD1(G93A) mice, and in galectin-3 and -9 mRNAs and proteins in spinal cords of both SOD1(G93A) mice and sporadic ALS patients. As the increase in galectin-3 appeared in early presymptomatic stages and increased progressively through to end stage of disease in the mouse, it was selected for additional study, where it was found to be mainly expressed by microglia. Galectin-3 antagonists are not selective and do not readily cross the blood-brain barrier; therefore, we generated SOD1(G93A)/Gal-3(-/-) transgenic mice to evaluate galectin-3 deletion in a widely used mouse model of ALS. Disease progression, neurological symptoms, survival, and inflammation were assessed to determine the effect of galectin-3 deletion on the SOD1(G93A) disease phenotype. Galectin-3 deletion did not change disease onset, but resulted in more rapid progression through functionally defined disease stages, more severely impaired neurological symptoms at all stages of disease, and expiration, on average, 25 days earlier than SOD1(G93A)/Gal-3(+/+) cohorts. In addition, microglial staining, as well as TNF-α, and oxidative injury were increased in SOD1(G93A)/Gal-3(-/-) mice compared with SOD1(G93A)/Gal-3(+/+) cohorts. These data support an important functional role for microglial galectin-3 in neuroinflammation during chronic neurodegenerative disease. We suggest that elevations in galectin-3 by microglia as disease progresses may represent a protective, anti-inflammatory innate immune response to chronic motor neuron degeneration.
RESUMO
The dysferlin deficient SJL/J mouse strain is commonly used to study dysferlin deficient myopathies. Therefore, we systematically evaluated behavior in relatively young (9-25 weeks) SJL/J mice and compared them to C57BL6 mice to determine which functional end points may be the most effective to use for preclinical studies in the SJL/J strain. SJL/J mice had reduced body weight, lower open field scores, higher creatine kinase levels, and less muscle force than did C57BL6 mice. Power calculations for expected effect sizes indicated that grip strength normalized to body weight and open field activity were the most sensitive indicators of functional status in SJL/J mice. Weight and open field scores of SJL/J mice deteriorated over the course of the study, indicating that progressive myopathy was ongoing even in relatively young (<6 months old) SJL/J mice. To further characterize SJL/J mice within the context of treatment, we assessed the effect of fasudil, a rho-kinase inhibitor, on disease phenotype. Fasudil was evaluated based on previous observations that Rho signaling may be overly activated as part of the inflammatory cascade in SJL/J mice. Fasudil treated SJL/J mice showed increased body weight, but decreased grip strength, horizontal activity, and soleus muscle force, compared to untreated SJL/J controls. Fasudil either improved or had no effect on these outcomes in C57BL6 mice. Fasudil also reduced the number of infiltrating macrophages/monocytes in SJL/J muscle tissue, but had no effect on muscle fiber degeneration/regeneration. These studies provide a basis for standardization of preclinical drug testing trials in the dysferlin deficient SJL/J mice, and identify measures of functional status that are potentially translatable to clinical trial outcomes. In addition, the data provide pharmacological evidence suggesting that activation of rho-kinase, at least in part, may represent a beneficial compensatory response in dysferlin deficient myopathies.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Proteínas de Membrana/deficiência , Distrofia Muscular do Cíngulo dos Membros/tratamento farmacológico , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/administração & dosagem , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Disferlina , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/fisiopatologiaRESUMO
The dual role of microglia in cytotoxicity and neuroprotection is believed to depend on the specific, temporal expression of microglial-related genes. To better clarify this issue, we used high-density oligonucleotide microarrays to examine microglial gene expression after spinal cord injury (SCI) in rats. We compared expression changes at the lesion site, as well as in rostral and caudal regions after mild, moderate, or severe SCI. Using microglial-associated anchor genes, we identified two clusters with different temporal profiles. The first, induced by 4 h postinjury to peak between 4 and 24 h, included interleukin-1beta, interleukin-6, osteopontin, and calgranulin, among others. The second was induced 24 h after SCI, and peaked between 72 h and 7 days; it included C1qB, Galectin-3, and p22(phox). These two clusters showed similar expression profiles regardless of injury severity, albeit with slight decreases in expression in mild or severe injury vs. moderate injury. Expression was also decreased rostral and caudal to the lesion site. We validated the expression of selected cluster members at the mRNA and protein levels. In addition, we demonstrated that stimulation of purified microglia in culture induces expression of C1qB, Galectin-3, and p22(phox). Finally, inhibition of p22(phox) activity within microglial cultures significantly suppressed proliferation in response to stimulation, confirming that this gene is involved in microglial activation. Because microglial-related factors have been implicated both in secondary injury and recovery, identification of temporally distinct clusters of genes related to microglial activation may suggest distinct roles for these groups of factors.