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Neuraminidase-1 (NEU1) is the sialidase responsible for the catabolism of sialoglycoconjugates in lysosomes. Congenital NEU1 deficiency causes sialidosis, a severe lysosomal storage disease associated with a broad spectrum of clinical manifestations, which also include skeletal deformities, skeletal muscle hypotonia and weakness. Neu1(-/-) mice, a model of sialidosis, develop an atypical form of muscle degeneration caused by progressive expansion of the connective tissue that infiltrates the muscle bed, leading to fiber degeneration and atrophy. Here we investigated the role of Neu1 in the myogenic process that ensues during muscle regeneration after cardiotoxin-induced injury of limb muscles. A comparative analysis of cardiotoxin-treated muscles from Neu1(-/-) mice and Neu1(+/+) mice showed increased inflammatory and proliferative responses in the absence of Neu1 during the early stages of muscle regeneration. This was accompanied by significant and sequential upregulation of Pax7, MyoD, and myogenin mRNAs. The levels of both MyoD and myogenin proteins decreased during the late stages of regeneration, which most likely reflected an increased rate of degradation of the myogenic factors in the Neu1(-/-) muscle. We also observed a delay in muscle cell differentiation, which was characterized by prolonged expression of embryonic myosin heavy chain, as well as reduced myofiber cross-sectional area. At the end of the regenerative process, collagen type III deposition was increased compared to wild-type muscles and internal controls, indicating the initiation of fibrosis. Overall, these results point to a role of Neu1 throughout muscle regeneration.
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BACKGROUND: Mutations in GRN (progranulin) and MAPT (microtubule-associated protein tau) are among the most frequent causes of monogenic frontotemporal dementia (FTD), but data on the frequency of these mutations in regions such as Latin America are still lacking. OBJECTIVE: We aimed to investigate the frequencies of GRN and MAPT mutations in FTD cohorts from 2 Brazilian dementia research centers, the University of Sao Paulo and the Federal University of Minas Gerais medical schools. METHODS: We included 76 probands diagnosed with behavioral-variant FTD (n=55), semantic-variant Primary Progressive Aphasia (PPA) (n=11), or nonfluent-variant PPA (n=10). Twenty-five percent of the cohort had at least 1 relative affected with FTD. RESULTS: Mutations in GRN were identified in 7 probands, and in MAPT, in 2 probands. We identified 3 novel GRN mutations (p.Q130X, p.317Afs*12, and p.K259Afs*23) in patients diagnosed with nonfluent-variant PPA or behavioral-variant FTD. Plasma progranulin levels were measured and a cutoff value of 70 ng/mL was found, with 100% sensitivity and specificity to detect null GRN mutations. CONCLUSIONS: The frequency of GRN mutations was 9.6% and that of MAPT mutations was 7.1%. Among familial cases of FTD, the frequency of GRN mutations was 31.5% and that of MAPT mutations was 10.5%.
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Demência Frontotemporal/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas tau/genética , Idade de Início , Encéfalo/patologia , Brasil , Feminino , Demência Frontotemporal/diagnóstico por imagem , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Afasia Primária Progressiva não Fluente/diagnóstico por imagem , Afasia Primária Progressiva não Fluente/genética , ProgranulinasRESUMO
OBJECTIVE: To evaluate the effect of 2% lidocaine, 0.5% bupivacaine, and 0.75% ropivacaine on the release of substance P (SP) and calcitonin gene-related peptide (CGRP) in skin wounds. DESIGN: A primary, experimental, analytical, prospective, self-controlled, blinded study. SETTING: The study is set in a university research center. INTERVENTIONS: Twenty-eight Wistar rats were randomly divided into 4 groups: lidocaine, bupivacaine, ropivacaine, and the control. After general anesthesia, a local anesthetic or 0.9% saline (control) was injected subdermally along a 2-cm line on the dorsal midline of each rat; 30 minutes later, an incision (nociceptive stimulus) was made along this line. The animals were euthanized, and skin samples were collected from the center of the incision line and sent for CGRP and SP quantification. MAIN OUTCOME MEASURES: Quantification of CGRP and SP by Western blotting. RESULTS: Substance P levels were similar in the lidocaine and ropivacaine groups but were significantly lower than those of the control group (P = .002); no significant difference in SP levels was found between the bupivacaine and control groups. Procalcitonin gene-related peptide levels were significantly lower in the experimental groups than those in control subjects (P = .009), with no significant differences among the experimental groups. No significant differences in CGRP levels were found among all groups. Lidocaine and ropivacaine inhibited SP release. All 3 local anesthetics inhibited the release of procalcitonin gene-related peptide, but not the release of CGRP in rat skin. CONCLUSIONS: Lidocaine and ropivacaine may inhibit neurogenic inflammation by biochemical pathways activated by SP, whereas bupivacaine seems to have no influence on this process.
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Amidas/farmacologia , Anestésicos Locais/farmacologia , Bupivacaína/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Lidocaína/farmacologia , Pele/metabolismo , Substância P/metabolismo , Ferida Cirúrgica/metabolismo , Animais , Ratos , Ratos Wistar , Ropivacaina , Pele/efeitos dos fármacos , Pele/lesõesRESUMO
OBJECTIVE: To describe and standardize a protocol that overcomes the technical limitations of Western blot (WB) analysis in the quantification of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) following nociceptive stimuli in rat skin. DESIGN: Male Wistar rats (Rattus norvegicus albinus) weighing 250 to 350 g were used in this study. Elements of WB analysis were adapted by using specific manipulation of samples, repeated cycles of freezing and thawing, more thorough maceration, and a more potent homogenizer; increasing lytic reagents; promoting greater inhibition of protease activity; and using polyvinylidene fluoride membranes as transfer means for skin-specific protein. Other changes were also made to adapt the WB analysis to a rat model. SETTING: University research center. MAIN OUTCOME MEASURE: Western blot analysis adapted to a rat model. RESULTS: This research design has proven effective in collecting and preparing skin samples to quantify SP and CGRP using WB analysis in rat skin. CONCLUSION: This study described a research design that uses WB analysis as a reproducible, technically accessible, and cost-effective method for the quantification of SP and CGRP in rat skin that overcomes technical biases.
Assuntos
Western Blotting , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Dor Nociceptiva/metabolismo , Substância P/metabolismo , Animais , Modelos Animais de Doenças , Masculino , Dor Nociceptiva/etiologia , Ratos , Ratos Wistar , Projetos de PesquisaRESUMO
Centronuclear myopathy (CNM) is a rare congenital muscle disease characterized by fibers with prominent centralized nuclei in muscle biopsies. The disease is clinically heterogeneous, ranging from severe neonatal hypotonic phenotypes to adult-onset mild muscle weakness, and can have multiple modes of inheritance in association with various genes, including MTM1, DNM2, BIN1 and RYR1. Here we analyzed 18 sporadic patients with clinical and histological diagnosis of CNM and sequenced the DNM2 gene, which codes for the dynamin 2 protein. We found DNM2 missense mutations in two patients, both in exon 8, one known (p.E368K) and one novel (p.F372C), which is found in a position of presumed pathogenicity and appeared de novo. The patients had similar phenotypes characterized by neonatal signs followed by improvement and late childhood reemergence of slowly progressive generalized muscle weakness, elongated face with ptosis and ophthalmoparesis, and histology showing fibers with radiating sarcoplasmic strands (RSS). These patients were the only ones in the series to present this histological marker, which together with previous reports in the literature suggest that, when RSS are present, direct sequencing of DNM2 mutation hot spot regions should be the first step in the molecular diagnosis of CNM, even in sporadic cases.
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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of motor neurons. Familial ALS is strongly associated to dominant mutations in the gene for Cu/Zn superoxide dismutase (SOD1). Recent evidences point to skeletal muscle as a primary target in the ALS mouse model. Wnt/PI3 K signaling pathways and epithelial-mesenchymal transition (EMT) have important roles in maintenance and repair of skeletal muscle. Wnt/PI3 K pathways and EMT gene expression profile were investigated in gastrocnemius muscle from SOD1(G93A) mouse model and age-paired wild-type control in the presymptomatic ages of 40 and 80 days aiming the early neuromuscular abnormalities that precede motor neuron death in ALS. A customized cDNA microarray platform containing 326 genes of Wnt/PI3 K and EMT was used and results revealed eight up-regulated (Loxl2, Pik4ca, Fzd9, Cul1, Ctnnd1, Snf1lk, Prkx, Dner) and nine down-regulated (Pik3c2a, Ripk4, Id2, C1qdc1, Eif2ak2, Rac3, Cds1, Inppl1, Tbl1x) genes at 40 days, and also one up-regulated (Pik3ca) and five down-regulated (Cd44, Eef2 k, Fzd2, Crebbp, Piki3r1) genes at 80 days. Also, protein-protein interaction networks grown from the differentially expressed genes of 40 and 80 days old mice have identified Grb2 and Src genes in both presymptomatic ages, thus playing a potential central role in the disease mechanisms. mRNA and protein levels for Grb2 and Src were found to be increased in 80 days old ALS mice. Gene expression changes in the skeletal muscle of transgenic ALS mice at presymptomatic periods of disease gave further evidence of early neuromuscular abnormalities that precede motor neuron death. The results were discussed in terms of initial triggering for neuronal degeneration and muscle adaptation to keep function before the onset of symptoms.
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Esclerose Lateral Amiotrófica/enzimologia , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Músculo Esquelético/enzimologia , Superóxido Dismutase/biossíntese , Esclerose Lateral Amiotrófica/patologia , Animais , Linhagem Celular , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/patologiaRESUMO
Pigment epithelium derived factor (PEDF) exerts trophic actions to motoneurons and modulates nonneuronal restorative events, but its effects on neuroplasticity responses after spinal cord (SC) injury are unknown. Rats received a low thoracic SC photothrombotic ischemia and local injection of PEDF and were evaluated behaviorally six weeks later. PEDF actions were detailed in SC ventral horn (motor) in the levels of the lumbar central pattern generator (CPG), far from the injury site. Molecules related to neuroplasticity (MAP-2), those that are able to modulate such event, for instance, neurotrophic factors (NT-3, GDNF, BDNF, and FGF-2), chondroitin sulfate proteoglycans (CSPG), and those associated with angiogenesis and antiapoptosis (laminin and Bcl-2) and Eph (receptor)/ephrin system were evaluated at cellular or molecular levels. PEDF injection improved motor behavioral performance and increased MAP-2 levels and dendritic processes in the region of lumbar CPG. Treatment also elevated GDNF and decreased NT-3, laminin, and CSPG. Injury elevated EphA4 and ephrin-B1 levels, and PEDF treatment increased ephrin A2 and ephrins B1, B2, and B3. Eph receptors and ephrins were found in specific populations of neurons and astrocytes. PEDF treatment to SC injury triggered neuroplasticity in lumbar CPG and regulation of neurotrophic factors, extracellular matrix molecules, and ephrins.
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Proteínas do Olho/farmacologia , Atividade Motora/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Serpinas/farmacologia , Isquemia do Cordão Espinal/metabolismo , Animais , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas do Olho/uso terapêutico , Laminina/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/uso terapêutico , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/efeitos dos fármacos , Serpinas/uso terapêutico , Isquemia do Cordão Espinal/tratamento farmacológicoRESUMO
The phototherapy effects in the skin are related to biomodulation, usually to accelerate wound healing. However, there is no direct proof of the interrelation between the effects of low-level laser therapy (LLLT) and light-emitting diode (LED) in neuropeptide secretion, these substances being prematurely involved in the neurogenic inflammation phase of wound healing. This study therefore focused on investigating LLLT and LED in Calcitonin gene-related peptide (CGRP) and substance P (SP) secretion in healthy rat skin. Forty rats were randomly distributed into five groups with eight rats each: Control Group, Blue LED Group (470 nm, 350 mW power), Red LED Group (660 nm, 350 mW power), Red Laser Group (660 nm, 100 mW power), and Infrared Laser Group (808 nm, 100 mW power) (DMC® Equipamentos Ltda., São Carlos, São Paulo, Brazil). The skin of the animals in the experimental groups was irradiated using the punctual contact technique, with a total energy of 40 J, single dose, standardized at one point in the dorsal region. After 14 min of irradiation, the skin samples were collected for CGRP and SP quantification using western blot analysis. SP was released in Infrared Laser Group (p = 0.01); there was no difference in the CGRP secretion among groups. Infrared (808 nm) LLLT enhances neuropeptide SP secretion in healthy rat skin.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Eletrônica , Terapia com Luz de Baixa Intensidade , Pele/metabolismo , Substância P/metabolismo , Animais , Masculino , Ratos Wistar , Pele/efeitos da radiaçãoRESUMO
Neurogenic neuroprotection is a promising approach for treating patients with ischemic brain lesions. In rats, stimulation of the deep brain nuclei has been shown to reduce the volume of focal infarction. In this context, protection of neural tissue can be a rapid intervention that has a relatively long-lasting effect, making fastigial nucleus stimulation (FNS) a potentially valuable method for clinical application. Although the mechanisms of neuroprotection induced by FNS remain partially unclear, important data have been presented in the last two decades. A 1-h electrical FNS reduced, by 59%, infarctions triggered by permanent occlusion of the middle cerebral artery in Fisher rats. The acute effect of electrical FNS is likely mediated by a prolonged opening of potassium channels, and the sustained effect appears to be linked to inhibition of the apoptotic cascade. A better understanding of the neuronal circuitry underlying neurogenic neuroprotection may contribute to improving neurological outcomes in ischemic brain insults.
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Infarto Encefálico/prevenção & controle , Núcleos Cerebelares/fisiologia , Estimulação Encefálica Profunda/métodos , Animais , Infarto Encefálico/etiologia , Núcleos Cerebelares/anatomia & histologia , Núcleos Cerebelares/metabolismo , Circulação Cerebrovascular/fisiologia , Modelos Animais de Doenças , Encefalite/etiologia , Encefalite/prevenção & controle , Humanos , Infarto da Artéria Cerebral Média/complicações , Vias Neurais/fisiologia , Lobo Parietal/fisiologiaRESUMO
BACKGROUND/AIM: Chagas' disease is caused by Trypanosoma cruzi and occurs in most Latin American countries. The protozoan may colonize the central nervous system (CNS) of immune-compromised human hosts, thus causing neuronal disorders. Systemic control of the intracellular forms of the parasite greatly depends on the establishment of a TH1 response and subsequent nitric oxide (NO) release. At the CNS, it is known that low concentrations of NO promote neuronal survival and growth, while high concentrations exert toxic effects and neuron death. Accounting for NO production by astrocytes is the glia-derived factor S100beta, which is overproduced in some neurodegenerative diseases. In the current work, we studied the expression of NO, interferon (IFN)-gamma and S100beta in the spinal cord tissue of IL-12p40KO mice infected with T. cruzi, a model of neurodegenerative process. METHODS: IL-12p40KO and wild-type (WT) female mice infected with T. cruzi Sylvio X10/4 (10(5) trypomastigotes, intraperitoneally) were euthanized when IL-12p40KO individuals presented limb paralysis. Spinal cord sections were submitted to immunohistochemical procedures for localization of neurofilament, laminin, nitrotyrosine, NO synthases (NOS), IFN-gamma and S100beta. The total number of neurons was estimated by stereological analysis and the area and intensity of immunoreactivities were assessed by microdensitometric/morphometric image analysis. RESULTS: No lesion was found in the spinal cord sections of WT mice, while morphological disarrangements, many inflammatory foci, enlarged vessels, amastigote nests and dying neurons were seen at various levels of IL-12p40KO spinal cord. Compared to WT mice, IL-12p40KO mice presented a decrement on total number of neurons (46.4%, p < 0.05) and showed increased values of immunoreactive area for nitrotyrosine (239%, p < 0.01) and NOS (544%, p < 0.001). Moreover, the intensity of nitrotyrosine (16%, p < 0.01), NOS (38%, p < 0.05) and S100beta (21%, p < 0.001) immunoreactivities were also augmented. No IFN-gamma-labeled cells were seen in WT spinal cord tissue, contrary to IL-12p40KO tissue that displayed inflammatory infiltrating cells and also some parenchymal cells positively labeled. CONCLUSION: We suggest that overproduction of NO may account for neuronal death at the spinal cord of T. cruzi-infected IL-12p40KO mice and that IFN-gamma and S100beta may contribute to NOS activation in the absence of IL-12.
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Subunidade p40 da Interleucina-12/genética , Mielite/metabolismo , Degeneração Neural/metabolismo , Óxido Nítrico/biossíntese , Medula Espinal/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Células Cultivadas , Doença de Chagas/metabolismo , Doença de Chagas/fisiopatologia , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Parasita , Imunidade Inata/imunologia , Mediadores da Inflamação/metabolismo , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mielite/parasitologia , Mielite/fisiopatologia , Degeneração Neural/parasitologia , Degeneração Neural/fisiopatologia , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Neurônios/parasitologia , Neurônios/patologia , Óxido Nítrico Sintase Tipo I/metabolismo , Paraplegia/metabolismo , Paraplegia/parasitologia , Paraplegia/fisiopatologia , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Medula Espinal/parasitologia , Medula Espinal/fisiopatologia , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration and loss of lower motor neurons in the spinal cord and brainstem. Clinically, SMA has been classified into four types, according to the maximum function attained. The disease is caused by deletion or mutation of the telomeric copy of the SMN gene (SMN1), and the clinical severity is in part determined by the copy number of the centromeric SMN gene (SMN2). The SMN2 mRNA lacks exon 7, resulting in reduced production of the full-length SMN protein. Treatment of SMA consists of supportive care, although many drugs have been demonstrated to improve muscle strength and motor function of patients. The development of animal models of SMA has led to better interpretation of the physiopathology of the disease and testing of potential drug targets. Several mechanisms have been targeted in SMA drug trials, including neuroprotection, neurogenesis, energy metabolism improvement, anabolic stimulation and increment of SMN2 transcripts. Gene therapy and cell transplantation have also been tested in murine SMA.
Assuntos
Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Atrofia Muscular Espinal/terapia , Animais , Transplante de Células/métodos , Eletromiografia , Terapia Genética/métodos , Humanos , Atrofia Muscular Espinal/classificação , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
PURPOSE: The aversive nature of regenerative milieu is the main problem related to the failure of neuronal restoration in the injured spinal cord which however might be addressed with an adequate repair intervention. We evaluated whether glial cell line-derived neurotrophic factor (GDNF) may increase the ability of sciatic nerve graft, placed in a gap promoted by complete transections of the spinal cord, to enhance motor recovery and local fiber growth. METHODS: Rats received a 4 mm-long gap at low thoracic level and were repaired with a fragment of the sciatic nerve. GDNF was added (NERVE+/-GDNF) or not to the grafts (NERVE--GDNF). Motor behavior score (BBB) and sensorimotor tests-linked to the combined behavior score (CBS), which indicate the degree of the motor improvement and the percentage of functional deficit, respectively, and also the spontaneous motor behavior in an open field by means of an infrared motion sensor activity monitor were analyzed. At the end of the third month post surgery, the tissue composed by the graft and the adjacent regions of the spinal cord was removed and submitted to the immunohistochemistry of the neurofilament-200 (NF-200), growth associated protein-43 (GAP-43), microtubule associated protein-2 (MAP-2), 5-hidroxytryptamine (serotonin, 5-HT) and calcitonin gene related peptide (CGRP). The immunoreactive fibers were quantified at the epicenter of the graft by means of stereological procedures. RESULTS: Higher BBB and lower CBS levels (p < 0.001) were found in NERVE+/-GDNF rats. GDNF added to the graft increased the levels of individual sensorimotor tests mainly at the third month. Analysis of the spontaneous motor behavior showed decreases in the time and number of small movement events by the third month without changes in time and number of large movement events in the NERVE$+$GDNF rats. Immunoreactive fibers were encountered inside the grafts and higher amounts of NF-200, GAP-43 and MAP-2 fibers were found in the epicenter of the graft when GDNF was added. A small amount of descending 5-HT fibers was seen reentering in the adjacent caudal levels of the spinal cords which were grafted in the presence of GDNF, event that has not occurred without the neurotrophic factor. GDNF in the graft also led to a large amount of MAP-2 perikarya and fibers in the caudal levels of the cord gray matter, as determined by the microdensitometric image analysis. CONCLUSIONS: GDNF added to the nerve graft favored the motor recovery, local neuronal fiber growth and neuroplasticity in the adjacent spinal cord.
Assuntos
Axônios/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/uso terapêutico , Transtornos dos Movimentos , Nervo Isquiático/transplante , Traumatismos da Medula Espinal/complicações , Animais , Axônios/fisiologia , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Laminectomia/métodos , Masculino , Transtornos dos Movimentos/tratamento farmacológico , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/cirurgia , Proteínas do Tecido Nervoso/metabolismo , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Ratos , Ratos Wistar , Reflexo/efeitos dos fármacos , Reflexo/fisiologia , Índice de Gravidade de Doença , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/cirurgia , Fatores de TempoRESUMO
We have observed in previous studies that 6-hydroxydopamine (6-OHDA)-induced lesions in the nigrostriatal dopamine (DA) system promote increases of the astroglial basic fibroblast growth factor (FGF-2, bFGF) synthesis in the ascending DA pathways, event that could be modified by adrenosteroid hormones. Here, we first evaluated the changes of microglial reactivity in relation to the FGF-2-mediated trophic responses in the lesioned nigrostriatal DA system. 6-OHDA was injected into the left side of the rat substantia nigra. The OX42 immunohistochemistry combined with stereology showed the time course of the microglial activation. The OX42 immunoreactivity (IR) was already increased in the pars compacta of the substantia nigra (SNc) and ventral tegmental area (VTA) 2 h after the 6-OHDA injection, peaked on day 7, and remained increased on the 14th day time-interval. In the neostriatum, OX42 immunoreactive (ir) microglial profiles increased at 24 h, peaked at 72 h, was still increased at 7 days but not 14 days after the 6-OHDA injection. Two-colour immunofluorescence analysis of the tyrosine hydroxylase (TH) and OX42 IRs revealed the presence of small patches of TH IR within the activated microglia. A decreased FGF-2 IR was seen in the cytoplasm of DA neurons of the SNc and VTA as soon as 2 h after 6-OHDA injection. The majority of the DA FGF-2 ir cells of these regions had disappeared 72 h after neurotoxin. The astroglial FGF-2 IR increased in the SNc and VTA, which peaked on day 7. Two-colour immunofluorescence and immunoperoxidase analyses of the FGF-2 and OX42 IRs revealed no FGF-2 IR within the reactive or resting microglia. Second, we have evaluated in a series of biochemical experiments whether adrenocortical manipulation can interfere with the nigral lesion and the state of local astroglial reaction, looking at the TH and GFAP levels respectively. Rats were adrenalectomized (ADX) and received a nigral 6-OHDA stereotaxical injection 2 days later and sacrificed up to 3 weeks after the DA lesion. Western blot analysis showed time-dependent decrease and elevation of TH and GFAP levels, respectively, in the lesioned versus contralateral midbrain sides, events potentiated by ADX and worsened by corticosterone replacement. ADX decreased the levels of FGF-2 protein (23 kDa isoform) in the lesioned side of the ventral midbrain compared contralaterally. The results indicate that reactive astroglia, but not reactive microglia, showed an increased FGF-2 IR in the process of DA cell degeneration induced by 6-OHDA. However, interactions between these glial cells may be relevant to the mechanisms which trigger the increased astroglial FGF-2 synthesis and thus may be related to the trophic state of DA neurons and the repair processes following DA lesion. The findings also gave further evidence that adrenocortical hormones may regulate astroglial-mediated trophic mechanisms and wound repair events in the lesioned DA system that may be relevant to the progression of Parkinson's disease.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Glucocorticoides/metabolismo , Microglia/metabolismo , Transtornos Parkinsonianos/patologia , Cicatrização/fisiologia , Adrenalectomia/métodos , Animais , Antígeno CD11b/metabolismo , Corpo Estriado/citologia , Modelos Animais de Doenças , Fator 2 de Crescimento de Fibroblastos/genética , Lateralidade Funcional , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Microglia/efeitos dos fármacos , Oxidopamina , Transtornos Parkinsonianos/induzido quimicamente , Ratos , Ratos Wistar , Estatísticas não Paramétricas , Substância Negra/patologia , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismo , Área Tegmentar Ventral/patologia , Cicatrização/efeitos dos fármacosRESUMO
This paper analyzes the astroglial and neuronal responses in subtelencephalic structures, following a bilateral ablation of the telencephalon in the Columba livia pigeons. Control birds received a sham operation. Four months later the birds were sacrificed and their brains processed for glial fibrillary acid protein (GFAP) and neurofilament immunohistochemistry, markers for astrocytes and neurons, respectively. Computer-assisted image analysis was employed for quantification of the immunoreactive labeling in the nucleus rotundus (N.Rt) and the optic tectum (OT) of the birds. An increased number of GFAP immunoreactive astrocytes were found in several subregions of the N.Rt (p< .001), as well as in layers 1, 2cd, 3, and 6 of the OT (p< .001) of the lesioned animals. Neurofilament immunoreactivity decreased massively in the entire N.Rt of the lesioned birds; however, remaining neurons with healthy aspect showing large cytoplasm and ramified branches were detected mainly in the periphery of the nucleus. In view of the recently described paracrine neurotrophic properties of the activated astrocytes, the data of the present study may suggest a long-lasting neuroglial interaction in regions of the lesioned bird brain far from injury. Such events may trigger neuronal plasticity in remaining brain structures that may lead spontaneous behavior recovery as the one promoted here even after a massive injury.
Assuntos
Astrócitos/metabolismo , Columbidae/fisiologia , Gliose/fisiopatologia , Plasticidade Neuronal/fisiologia , Telencéfalo/fisiologia , Vias Visuais/fisiologia , Animais , Astrócitos/citologia , Biomarcadores/análise , Biomarcadores/metabolismo , Mapeamento Encefálico , Columbidae/anatomia & histologia , Denervação , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/metabolismo , Gliose/patologia , Citometria por Imagem , Imuno-Histoquímica , Masculino , Proteínas de Neurofilamentos/análise , Proteínas de Neurofilamentos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Especificidade da Espécie , Coloração e Rotulagem , Colículos Superiores/anatomia & histologia , Colículos Superiores/fisiologia , Telencéfalo/anatomia & histologia , Tálamo/anatomia & histologia , Tálamo/fisiologia , Fatores de Tempo , Vias Visuais/anatomia & histologiaRESUMO
Neuropeptide Y (NPY) is an important neuromodulator found in central and peripheral neurons. NPY was investigated in the peripheral auditory pathway of conventional housed rats and after nontraumatic sound stimulation in order to localize the molecule and also to describe its response to sound stimulus. Rats from the stimulation experiment were housed in monitored sound-proofed rooms. Stimulated animals received sound stimuli (pure tone bursts of 8 kHz, 50 ms duration presented at a rate of 2 per second) at an intensity of 80 dB sound pressure level for 1 hr per day during 7 days. After euthanizing, rat cochleae were processed for one-color immunohistochemistry. The NPY immunoreactivity was detected in inner hair cells (IHC) and also in pillar and Deiters' cells of organ of Corti, and in the spiral ganglion putative type I (> or = 1,009 microm(3)) and type II (< or = 225 microm(3)) neurons. Outer hair cells (OHC) showed light immunoreaction product. Quantitative microdensitometry showed strong and moderate immunoreactions in IHC and spiral ganglion neurons, respectively, without differences among cochlear turns. One week of acoustic stimulation was not able to induce changes in the NPY immunoreactivity intensity in the IHC of cochlea. However, stimulated rats showed an overall increase in the number of putative type I and type II NPY immunoreactive spiral ganglion neurons with strong, moderate, and weak immunolabeling. Localization and responses of NPY to acoustic stimulus suggest an involvement of the neuropeptide in the neuromodulation of afferent transmission in the rat peripheral auditory pathway.
Assuntos
Estimulação Acústica , Gânglios Espinais/metabolismo , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Órgão Espiral/metabolismo , Análise de Variância , Animais , Densitometria , Imuno-Histoquímica , Masculino , Ratos , Ratos WistarRESUMO
Muscle atrophy occurs in many conditions, including use of glucocorticoids. N-3 (omega-3) is widely consumed due its healthy properties; however, concomitant use with glucocorticoids can increase its side effects. We evaluated the influences of N-3 on glucocorticoid atrophy considering IGF-1, Myostatin, MEK/ERK, AMPK pathways besides the ubiquitin-proteasome system (UPS) and autophagic/lysosomal systems. Sixty animals constituted six groups: CT, N-3 (EPA 100 mg/kg/day for 40 days), DEXA 1.25 (DEXA 1.25 mg/kg/day for 10 days), DEXA 1.25 + N3 (EPA for 40 days + DEXA 1.25 mg/kg/day for the last 10 days), DEXA 2.5 (DEXA 2.5 mg/kg/day for 10 days), and DEXA 2.5 + N3 (EPA for 40 days + DEXA 2.5 mg/kg/day for 10 days). Results: N-3 associated with DEXA increases atrophy (fibers 1 and 2A), FOXO3a, P-SMAD2/3, Atrogin-1/MAFbx (mRNA) expression, and autophagic protein markers (LC3II, LC3II/LC3I, LAMP-1 and acid phosphatase). Additionally, N-3 supplementation alone decreased P-FOXO3a, PGC1-alpha, and type 1 muscle fiber area. Conclusion: N-3 supplementation increases muscle atrophy caused by DEXA in an autophagic, AMPK and UPS process.
Assuntos
Autofagia , Dexametasona/efeitos adversos , Ácidos Graxos Ômega-3/efeitos adversos , Glucocorticoides/efeitos adversos , Atrofia Muscular/etiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinas/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Ácidos Graxos Ômega-3/farmacologia , Proteína Forkhead Box O3/metabolismo , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Quinases/metabolismo , Ratos , Ratos Wistar , Proteínas Smad/metabolismoRESUMO
Glucocorticoids represent some of the most prescribed drugs that are widely used in the treatment of neuromuscular diseases, but their usage leads to side effects such as muscle atrophy. However, different synthetic glucocorticoids can lead to different muscle effects, depending upon its chemical formulation. Here, we intended to demonstrate the muscle histologic and molecular effects of administering different glucocorticoids in equivalency and different dosages. Methods: Seventy male Wistar rats distributed into seven groups received different glucocorticoids in equivalency for ten days or saline solution. The study groups were: Control group (CT) saline solution; dexamethasone (DX) 1.25 or 2.5 mg/kg/day; methylprednisolone (MP) 6.7 or 13.3mg/kg/day; and deflazacort (DC) 10 or 20 mg/kg/day. At the end of the study, the animals were euthanized, and the tibialis anterior and gastrocnemius muscles were collected for metachromatic ATPase (Cross-sectional area (CSA) measurement), Western blotting (protein expression of IGF-1 and Ras/Raf/MEK/ERK pathways) and RT-PCR (MYOSTATIN, MuRF-1, Atrogin-1, REDD-1, REDD-2, MYOD, MYOG and IRS1/2 genes expression) experiments. Results: Muscle atrophy occurred preferentially in type 2B fibers in all glucocorticoid treated groups. DC on 10 mg/kg/day was less harmful to type 2B fibers CSA than other doses and types of synthetic glucocorticoids. In type 1 fibers CSA, lower doses of DC and DX were more harmful than high doses. DX had a greater effect on the IGF-1 pathway than other glucocorticoids. MP more significantly affected P-ERK1/2 expression, muscle fiber switching (fast-to-slow), and expression of REDD1 and MyoD genes than other glucocorticoids. Compared to DX and MP, DC had less of an effect on the expression of atrogenes (MURF-1 and Atrogin-1) despite increased MYOSTATIN and decreased IRS-2 genes expression. Conclusions: Different glucocorticoids appears to cause muscle atrophy affecting secondarily different signaling mechanisms. MP is more likely to affect body/muscles mass, MEK/ERK pathway and fiber type transition, DX the IGF-1 pathway and IRS1/2 expression. DC had the smallest effect on muscle atrophic response possibly due a delayed timing on atrogenes response.
Assuntos
Dexametasona/farmacologia , Metilprednisolona/farmacologia , Músculo Esquelético/efeitos dos fármacos , Pregnenodionas/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Dexametasona/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/administração & dosagem , Glucocorticoides/farmacologia , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Metilprednisolona/administração & dosagem , Desenvolvimento Muscular/efeitos dos fármacos , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Pregnenodionas/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos WistarRESUMO
Glutamatergic transmission through metabotropic and ionotropic receptors, including kainate receptors, plays an important role in the nucleus of the solitary tract (NTS) functions. Glutamate system may interact with several other neurotransmitter systems which might also be influenced by steroid hormones. In the present study we analyzed the ability of systemic kainate to stimulate rat NTS neurons, which was evaluated by c-Fos as a marker of neuronal activation, and also to change the levels of NTS neurotransmitters such as GABA, NPY, CGRP, GAL, NT and NO by means of quantitative immunohistichemistry combined with image analysis. The analysis was also performed in adrenalectomized and kainate stimulated rats in order to evaluate a possible role of adrenal hormones on NTS neurotransmission. Male Wistar rats (3 month-old) were used in the present study. A group of 15 rats was submitted either to bilateral adrenalectomy or sham operation. Forty-eight hours after the surgeries, adrenalectomized rats received a single intraperitoneal injection of kainate (12 mg/kg) and the sham-operated rats were injected either with saline or kainate and sacrificed 8 hours later. The same experimental design was applied in a group of rats in order to register the arterial blood pressure. Systemic kainate decreased the basal values of mean arterial blood pressure (35%) and heart rate (22%) of sham-operated rats, reduction that were maintained in adrenalectomized rats. Kainate triggered a marked elevation of c-Fos positive neurons in the NTS which was 54% counteracted by adrenalectomy. The kainate activated NTS showed changes in the immunoreactive levels of GABA (143% of elevation) and NPY (36% of decrease), which were not modified by previous ablation of adrenal glands. Modulation in the levels of CGRP, GAL and NT immunoreactivities were only observed after kainate in the adrenalectomized rats. Treatments did not alter NOS labeling. It is possible that modulatory function among neurotransmitter systems in the NTS might be influenced by steroid hormones and the implications for central regulation of blood pressure or other visceral regulatory mechanisms control should be further investigated.
Assuntos
Adrenalectomia , Pressão Sanguínea/efeitos dos fármacos , Ácido Caínico/farmacologia , Neurotransmissores/metabolismo , Núcleo Solitário/efeitos dos fármacos , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Galanina/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Masculino , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neuropeptídeo Y/metabolismo , Neurotensina/metabolismo , Óxido Nítrico Sintase/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Wistar , Núcleo Solitário/citologia , Núcleo Solitário/enzimologia , Ácido gama-Aminobutírico/metabolismoRESUMO
The present study investigated the effects of bilateral adrenalectomy (ADX) on the synthesis of basic fibroblast growth factor (bFGF, FGF-2) mRNA and on the expression of its FGF receptor subtype-2 (FGFR2) mRNA after a 6-hydroxydopamine (6-OHDA)-induced lesion of nigrostriatal dopamine system. In previous papers we have demonstrated that corticosterone increases FGF-2 immunoreactivity mainly in the astrocytes of the substantia nigra [Chadi, G., Rosen, L., Cintra, A., Tinner, B., Zoli, M., Pettersson, R.F., Fuxe, K., 1993b. Corticosterone increases FGF-2 (bFGF) immunoreactivity in the substantia nigra of the rat. Neuroreport 4, 783-786.] and that 6-OHDA injected in the ventral midbrain upregulates FGF-2 synthesis in reactive astrocytes in the ascending dopamine pathways [Chadi, G., Cao, Y., Pettersson, R.F., Fuxe, K., 1994. Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons. Neuroscience 61, 891-910.]. Rats were adrenalectomized and received a 6-OHDA stereotaxical injection in the ventral midbrain 2 days later. Seven days after the dopamine lesion, Western blot analysis showed a decreased level of tyrosine hydroxylase in the lesioned side of the midbrain, an event that was not altered by ADX or corticosterone replacement. Moreover, the degeneration of nigral dopamine neurons, which was confirmed by the disappearance of acidic FGF (FGF-1) mRNA and the decrement of tyrosine hydroxylase mRNA labeled nigral neurons, was not altered by ADX. The FGF-2 protein (23 kDa isoform but not 21 kDa fraction) levels increased in the lesioned side of the ventral midbrain. This elevation was counteracted by ADX, an effect that was fully reversed by corticosterone replacement. In situ hybridization revealed that ADX counteracted the elevated FGF-2 mRNA levels in putative glial cells of the ipsilateral pars compacta of the substantia nigra and in the ventral tegmental area. The ADX also counteracted the increased density and intensity of the astroglial FGF-2 immunoreactive profiles within the lesioned pars compacta of the substantia nigra and the ventral tegmental area as determined by stereology. The stereotaxical mechanical needle insertion triggered the expression of FGFR2 mRNA in putative glial cells, spreading to the entire ipsilateral ventral midbrain from the region of needle track, an occurrence that was partially reversed by ADX. In conclusion, bilateral ADX counteracted the increased astroglial FGF-2 synthesis in the dopamine regions of the ventral midbrain following a 6-OHDA-induced local lesion and interfered with FGF receptor regulation around injury. These findings give further evidence that adrenocortical hormones may regulate the astroglial FGF-2-mediated trophic mechanisms and wound repair events in the lesioned central nervous system.