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1.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199392

RESUMO

Coordination of four-limb movements during quadrupedal locomotion is controlled by supraspinal monoaminergic descending pathways, among which serotoninergic ones play a crucial role. Here we investigated the locomotor pattern during recovery from blockade of 5-HT7 or 5-HT2A receptors after intrathecal application of SB269970 or cyproheptadine in adult rats with chronic intrathecal cannula implanted in the lumbar spinal cord. The interlimb coordination was investigated based on electromyographic activity recorded from selected fore- and hindlimb muscles during rat locomotion on a treadmill. In the time of recovery after hindlimb transient paralysis, we noticed a presence of an unusual pattern of quadrupedal locomotion characterized by a doubling of forelimb stepping in relation to unaffected hindlimb stepping (2FL-1HL) after blockade of 5-HT7 receptors but not after blockade of 5-HT2A receptors. The 2FL-1HL pattern, although transient, was observed as a stable form of fore-hindlimb coupling during quadrupedal locomotion. We suggest that modulation of the 5-HT7 receptors on interneurons located in lamina VII with ascending projections to the forelimb spinal network can be responsible for the 2FL-1HL locomotor pattern. In support, our immunohistochemical analysis of the lumbar spinal cord demonstrated the presence of the 5-HT7 immunoreactive cells in the lamina VII, which were rarely 5-HT2A immunoreactive.


Assuntos
Locomoção/genética , Receptor 5-HT2A de Serotonina/genética , Receptores de Serotonina/genética , Traumatismos da Medula Espinal/genética , Animais , Ciproeptadina/farmacologia , Estimulação Elétrica , Eletromiografia , Membro Anterior/efeitos dos fármacos , Membro Anterior/fisiopatologia , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiopatologia , Humanos , Locomoção/efeitos dos fármacos , Região Lombossacral/fisiopatologia , Ratos , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Serotonina/genética , Serotonina/metabolismo , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Medula Espinal , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Coluna Vertebral/efeitos dos fármacos , Coluna Vertebral/fisiopatologia
2.
FASEB J ; 35(7): e21645, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34105824

RESUMO

Peripheral arterial disease (PAD) is one of the major complications of diabetes due to an impairment in angiogenesis. Since there is currently no drug with satisfactory efficacy to enhance blood vessel formation, discovering therapies to improve angiogenesis is critical. An imidazolinone metabolite of the metformin-methylglyoxal scavenging reaction, (E)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl) guanidine (IMZ), was recently characterized and identified in the urine of type-2 diabetic patients. Here, we report the pro-angiogenesis effect of IMZ (increased aortic sprouting, cell migration, network formation, and upregulated multiple pro-angiogenic factors) in human umbilical vein endothelial cells. Using genetic and pharmacological approaches, we showed that IMZ augmented angiogenesis by activating the endothelial nitric oxide synthase (eNOS)/hypoxia-inducible factor-1 alpha (HIF-1α) pathway. Furthermore, IMZ significantly promoted capillary density in the in vivo Matrigel plug angiogenesis model. Finally, the role of IMZ in post-ischemic angiogenesis was examined in a chronic hyperglycemia mouse model subjected to hind limb ischemia. We observed improved blood perfusion, increased capillary density, and reduced tissue necrosis in mice receiving IMZ compared to control mice. Our data demonstrate the pro-angiogenic effects of IMZ, its underlying mechanism, and provides a structural basis for the development of potential pro-angiogenic agents for the treatment of PAD.


Assuntos
Membro Posterior/fisiopatologia , Hiperglicemia/complicações , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/complicações , Metformina/metabolismo , Neovascularização Patológica/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Hipoglicemiantes/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imidazolinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Aldeído Pirúvico/metabolismo
3.
Am J Physiol Regul Integr Comp Physiol ; 320(6): R972-R983, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33949210

RESUMO

Peripheral artery disease (PAD) in the lower limb compromises oxygen supply due to arterial occlusion. Ischemic skeletal muscle is accompanied by capillary structural deformation. Therefore, using novel microscopy techniques, we tested the hypothesis that endothelial cell swelling temporally and quantitatively corresponds to enhanced microvascular permeability. Hindlimb ischemia was created in male Wistar rat's by iliac artery ligation (AL). The tibialis anterior (TA) muscle microcirculation was imaged using intravenously infused rhodamine B isothiocyanate dextran fluorescent dye via two-photon laser scanning microscopy (TPLSM) and dye extravasation at 3 and 7 days post-AL quantified to assess microvascular permeability. The TA microvascular endothelial ultrastructure was analyzed by transmission electron microscopy (TEM). Compared with control (0.40 ± 0.15 µm3 × 106), using TPLSM, the volumetrically determined interstitial leakage of fluorescent dye measured at 3 (3.0 ± 0.40 µm3 × 106) and 7 (2.5 ± 0.8 µm3 × 106) days was increased (both P < 0.05). Capillary wall thickness was also elevated at 3 (0.21 ± 0.06 µm) and 7 (0.21 ± 0.08 µm) days versus control (0.11 ± 0.03 µm, both P < 0.05). Capillary endothelial cell swelling was temporally and quantitatively associated with elevated vascular permeability in the AL model of PAD but these changes occurred in the absence of elevations in protein levels of vascular endothelial growth factor (VEGF) its receptor (VEGFR2 which decreased by AL-7 day) or matrix metalloproteinase. The temporal coherence of endothelial cell swelling and increased vascular permeability supports a common upstream mediator. TPLSM, in combination with TEM, provides a sensitive and spatially discrete technique to assess the mechanistic bases for, and efficacy of, therapeutic countermeasures to the pernicious sequelae of compromised peripheral arterial function.


Assuntos
Permeabilidade Capilar/fisiologia , Isquemia/fisiopatologia , Microscopia Confocal , Músculo Esquelético/irrigação sanguínea , Animais , Membro Posterior/fisiopatologia , Ligadura/métodos , Microcirculação/fisiologia , Microscopia Confocal/métodos , Microvasos/fisiopatologia , Neovascularização Fisiológica/fisiologia , Ratos Wistar
4.
Int J Mol Sci ; 22(8)2021 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-33920198

RESUMO

Sustained sarcolemma depolarization due to loss of the Na,K-ATPase function is characteristic for skeletal muscle motor dysfunction. Ouabain, a specific ligand of the Na,K-ATPase, has a circulating endogenous analogue. We hypothesized that the Na,K-ATPase targeted by the elevated level of circulating ouabain modulates skeletal muscle electrogenesis and prevents its disuse-induced disturbances. Isolated soleus muscles from rats intraperitoneally injected with ouabain alone or subsequently exposed to muscle disuse by 6-h hindlimb suspension (HS) were studied. Conventional electrophysiology, Western blotting, and confocal microscopy with cytochemistry were used. Acutely applied 10 nM ouabain hyperpolarized the membrane. However, a single injection of ouabain (1 µg/kg) prior HS was unable to prevent the HS-induced membrane depolarization. Chronic administration of ouabain for four days did not change the α1 and α2 Na,K-ATPase protein content, however it partially prevented the HS-induced loss of the Na,K-ATPase electrogenic activity and sarcolemma depolarization. These changes were associated with increased phosphorylation levels of AMP-activated protein kinase (AMPK), its substrate acetyl-CoA carboxylase and p70 protein, accompanied with increased mRNA expression of interleikin-6 (IL-6) and IL-6 receptor. Considering the role of AMPK in regulation of the Na,K-ATPase, we suggest an IL-6/AMPK contribution to prevent the effects of chronic ouabain under skeletal muscle disuse.


Assuntos
Interleucina-6/genética , Transtornos Musculares Atróficos/tratamento farmacológico , Ouabaína/farmacologia , Proteínas Quinases/genética , ATPase Trocadora de Sódio-Potássio/genética , Acetil-CoA Carboxilase/genética , Animais , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiopatologia , Elevação dos Membros Posteriores , Humanos , Interleucina-6/antagonistas & inibidores , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Transtornos Musculares Atróficos/genética , Transtornos Musculares Atróficos/patologia , Técnicas de Cultura de Órgãos , Proteínas Quinases/efeitos dos fármacos , Ratos , Ratos Wistar
5.
Nat Commun ; 12(1): 781, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536416

RESUMO

After complete spinal cord injuries (SCI), spinal segments below the lesion maintain inter-segmental communication via the intraspinal propriospinal network. However, it is unknown whether selective manipulation of these circuits can restore locomotor function in the absence of brain-derived inputs. By taking advantage of the compromised blood-spinal cord barrier following SCI, we optimized a set of procedures in which AAV9 vectors administered via the tail vein efficiently transduce neurons in lesion-adjacent spinal segments after a thoracic crush injury in adult mice. With this method, we used chemogenetic actuators to alter the excitability of propriospinal neurons in the thoracic cord of the adult mice with a complete thoracic crush injury. We showed that activating these thoracic neurons enables consistent and significant hindlimb stepping improvement, whereas direct manipulations of the neurons in the lumbar spinal cord led to muscle spasms without meaningful locomotion. Strikingly, manipulating either excitatory or inhibitory propriospinal neurons in the thoracic levels leads to distinct behavioural outcomes, with preferential effects on standing or stepping, two key elements of the locomotor function. These results demonstrate a strategy of engaging thoracic propriospinal neurons to improve hindlimb function and provide insights into optimizing neuromodulation-based strategies for treating SCI.


Assuntos
Dependovirus/genética , Membro Posterior/fisiopatologia , Locomoção/fisiologia , Neurônios/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Animais , Antipsicóticos/administração & dosagem , Clozapina/administração & dosagem , Clozapina/análogos & derivados , Vetores Genéticos/genética , Membro Posterior/inervação , Locomoção/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/terapia
6.
J Neurosci ; 41(4): 630-647, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33239399

RESUMO

Animal locomotion requires changing direction, from forward to backward. Here, we tested the hypothesis that sensorimotor circuits within the spinal cord generate backward locomotion and adjust it to task demands. We collected kinematic and electromyography (EMG) data during forward and backward locomotion at different treadmill speeds before and after complete spinal transection in six adult cats (three males and three females). After spinal transection, five/six cats performed backward locomotion, which required tonic somatosensory input in the form of perineal stimulation. One spinal cat performed forward locomotion but not backward locomotion while two others stepped backward but not forward. Spatiotemporal adjustments to increasing speed were similar in intact and spinal cats during backward locomotion and strategies were similar to forward locomotion, with shorter cycle and stance durations and longer stride lengths. Patterns of muscle activations, including muscle synergies, were similar for forward and backward locomotion in spinal cats. Indeed, we identified five muscle synergies that were similar during forward and backward locomotion. Lastly, spinal cats also stepped backward on a split-belt treadmill, with the left and right hindlimbs stepping at different speeds. Therefore, our results show that spinal sensorimotor circuits generate backward locomotion but require additional excitability compared with forward locomotion. Similar strategies for speed modulation and similar patterns of muscle activations and muscle synergies during forward and backward locomotion are consistent with a shared spinal locomotor network, with sensory feedback from the limbs controlling the direction.SIGNIFICANCE STATEMENT Animal locomotion requires changing direction, including forward, sideways and backward. This paper shows that the center controlling locomotion within the spinal cord can produce a backward pattern when instructed by sensory signals from the limbs. However, the spinal locomotor network requires greater excitability to produce backward locomotion compared with forward locomotion. The paper also shows that the spinal network controlling locomotion in the forward direction also controls locomotion in the backward direction.


Assuntos
Locomoção/fisiologia , Medula Espinal/fisiologia , Animais , Fenômenos Biomecânicos , Gatos , Estimulação Elétrica , Eletromiografia , Retroalimentação Sensorial , Feminino , Membro Posterior/fisiopatologia , Masculino , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Períneo/fisiologia , Recuperação de Função Fisiológica/fisiologia
7.
J Vis Exp ; (164)2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33104075

RESUMO

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats' paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method's limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.


Assuntos
Análise da Marcha/métodos , Traumatismos dos Nervos Periféricos/fisiopatologia , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Autoenxertos , Automação , Modelos Animais de Doenças , Nervo Femoral/patologia , Nervo Femoral/fisiopatologia , Marcha/fisiologia , Membro Posterior/fisiopatologia , Abrigo para Animais , Masculino , Regeneração Nervosa/fisiologia , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia , Neuropatia Ciática/fisiopatologia , Vértebras Torácicas/patologia , Vértebras Torácicas/fisiopatologia
8.
Int J Mol Sci ; 21(21)2020 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-33113794

RESUMO

Women with polycystic ovary syndrome (PCOS) are reported to have greater lean mass and insulin resistance. To examine muscular changes in a prenatally androgenized (PNA) rat model for PCOS, Sprague-Dawley rats were exposed to 5 mg testosterone or vehicle daily on gestational days 16-19. At 15 weeks of age, endurance on a rota-rod treadmill was measured. At 16 weeks of age, fasting blood glucose and insulin, hindlimb skeletal muscle mass, muscle fiber cross-sectional area (CSA) and composition, and intra- and peri-muscular lipid droplets were examined. Expression of mitochondrial marker ATP synthase and insulin signaling proteins were also investigated. Compared with controls, PNA female rats demonstrated greater total body and hindlimb muscle weights, greater muscle fiber CSA, and trending reduced time on the rota-rod. An increase in fibers co-expressing the slow and fast isoforms of myosin (90 vs. 86%, p < 0.05) and greater expression of ATP synthase (6-fold, p < 0.005) were observed in the gastrocnemius (GN) muscle. More lipid content was observed in GN and tibialis anterior (TA) muscles. PNA rats had elevated fasting serum insulin (1.9 vs. 1.2 ng/mL, p < 0.005) but comparable fasting glucose. Expression of total and Ser636/9-phosphorylated IRS1 were altered in PNA rat hindlimb muscles. Together, skeletal muscle alterations in hindlimb muscles of a PNA rat model for PCOS may represent consequences of, or adaptations to, insulin resistance in this model.


Assuntos
Membro Posterior/fisiopatologia , Resistência à Insulina/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiopatologia , Síndrome do Ovário Policístico/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Animais , Peso Corporal/fisiologia , Modelos Animais de Doenças , Feminino , Membro Posterior/metabolismo , Humanos , Lipídeos/análise , Atividade Motora/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Fenótipo , Síndrome do Ovário Policístico/induzido quimicamente , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Testosterona
9.
J Neurosci ; 40(43): 8292-8305, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-32978289

RESUMO

Traditionally, the brainstem has been seen as hardwired and poorly capable of plastic adaptations following spinal cord injury (SCI). Data acquired over the past decades, however, suggest differently: following SCI in various animal models (lamprey, chick, rodents, nonhuman primates), different forms of spontaneous anatomic plasticity of reticulospinal projections, many of them originating from the gigantocellular reticular nucleus (NRG), have been observed. In line with these anatomic observations, animals and humans with incomplete SCI often show various degrees of spontaneous motor recovery of hindlimb/leg function. Here, we investigated the functional relevance of two different modes of reticulospinal fiber growth after cervical hemisection, local rewiring of axotomized projections at the lesion site versus compensatory outgrowth of spared axons, using projection-specific, adeno-associated virus-mediated chemogenetic neuronal silencing. Detailed assessment of joint movements and limb kinetics during overground locomotion in female adult rats showed that locally rewired as well as compensatory NRG fibers were responsible for different aspects of recovered forelimb and hindlimb functions (i.e., stability, strength, coordination, speed, or timing). During walking and swimming, both locally rewired as well as compensatory NRG plasticity were crucial for recovered function, while the contribution of locally rewired NRG plasticity to wading performance was limited. Our data demonstrate comprehensively that locally rewired as well as compensatory plasticity of reticulospinal axons functionally contribute to the observed spontaneous improvement of stepping performance after incomplete SCI and are at least partially causative to the observed recovery of function, which can also be observed in human patients with spinal hemisection lesions.SIGNIFICANCE STATEMENT Following unilateral hemisection of the spinal cord, reticulospinal projections are destroyed on the injured side, resulting in impaired locomotion. Over time, a high degree of recovery can be observed in lesioned animals, like in human hemicord patients. In the rat, recovery is accompanied by pronounced spontaneous plasticity of axotomized and spared reticulospinal axons. We demonstrate the causative relevance of locally rewired as well as compensatory reticulospinal plasticity for the recovery of locomotor functions following spinal hemisection, using chemogenetic tools to selectively silence newly formed connections in behaviorally recovered animals. Moving from a correlative to a causative understanding of the role of neuroanatomical plasticity for functional recovery is fundamental for successful translation of treatment approaches from experimental studies to the clinics.


Assuntos
Locomoção , Formação Reticular/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Axônios , Axotomia , Fenômenos Biomecânicos , Feminino , Membro Anterior/fisiopatologia , Membro Posterior/fisiopatologia , Fibras Nervosas , Regeneração Nervosa , Plasticidade Neuronal , Ratos , Ratos Endogâmicos Lew , Recuperação de Função Fisiológica , Natação , Caminhada
10.
Elife ; 92020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32880575

RESUMO

Genetic ablation or pharmacological inhibition of the heat-activated cation channel TRPM3 alleviates inflammatory heat hyperalgesia, but the underlying mechanisms are unknown. We induced unilateral inflammation of the hind paw in mice, and directly compared expression and function of TRPM3 and two other heat-activated TRP channels (TRPV1 and TRPA1) in sensory neurons innervating the ipsilateral and contralateral paw. We detected increased Trpm3 mRNA levels in dorsal root ganglion neurons innervating the inflamed paw, and augmented TRP channel-mediated calcium responses, both in the cell bodies and the intact peripheral endings of nociceptors. In particular, inflammation provoked a pronounced increase in nociceptors with functional co-expression of TRPM3, TRPV1 and TRPA1. Finally, pharmacological inhibition of TRPM3 dampened TRPV1- and TRPA1-mediated responses in nociceptors innervating the inflamed paw, but not in those innervating healthy tissue. These insights into the mechanisms underlying inflammatory heat hypersensitivity provide a rationale for developing TRPM3 antagonists to treat pathological pain.


Assuntos
Inflamação/metabolismo , Nociceptores/metabolismo , Canais de Cátion TRPM/metabolismo , Regulação para Cima/fisiologia , Animais , Feminino , Gânglios Espinais/metabolismo , Membro Posterior/metabolismo , Membro Posterior/fisiopatologia , Temperatura Alta , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/metabolismo
11.
J Orthop Surg Res ; 15(1): 427, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948213

RESUMO

BACKGROUND: The prognostic and therapeutic potential of microRNAs (miRNAs) in spinal cord injury (SCI) has aroused increasing concerns. This study aims to research the functions of miR-29a/199B in the neurological function recovery after SCI and the mechanical mechanism. METHODS: A rat model with SCI was induced with sham-operated ones as control. The locomotor function and coordination of rat hindlimbs were determined by a Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and a ladder-climbing test, respectively. Expression of a neurofilament protein NF-200 and synaptophysin in gray matter of rats was determined to evaluate neuronal recovery in a cellular perspective. Binding relationships between miR-29a/199B with RGMA were predicted and validated using luciferase assays. Altered expression of miR-29a/199B and RGMA was introduced to explore their functions in rat neurological functions. The protein level and phosphorylation of STAT3 in gray matter were measured by western blot analysis. RESULTS: miR-29a and miR-199B were poorly expressed, while RGMA was abundantly expressed in gray matter at injury sites. Either miR-29a or miR-199B could bind to RGMA. Overexpression of miR-29a/199B or silencing of RGMA led to an increase in BBB locomotor scores, hindlimb coordination ability, and the expression of NF-200 and synaptophysin in gray matter. Further inhibition in miR-29a/199B blocked the promoting roles of RGMA silencing in neurological recovery. Upregulation of miR-29a/199B or downregulation of RGMA suppressed the phosphorylation of STAT3. CONCLUSION: This study evidenced that miR-29a and miR-199B negatively regulated RGMA to suppress STAT3 phosphorylation, therefore promoting the neurological function recovery in rats following SCI.


Assuntos
Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica/genética , MicroRNAs/genética , Atividade Motora/genética , Proteínas do Tecido Nervoso/metabolismo , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/terapia , Animais , Modelos Animais de Doenças , Proteínas Ligadas por GPI/fisiologia , Expressão Gênica , Substância Cinzenta/metabolismo , Membro Posterior/fisiopatologia , Masculino , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Fosforilação , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Sinaptofisina/genética , Sinaptofisina/metabolismo
12.
Biochem Biophys Res Commun ; 530(1): 95-99, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32828321

RESUMO

BACKGROUND: The circulating level of trimethylamine N-oxide (TMAO) has been reported to be associated with the prognosis of of peripheral arterial disease (PAD) patients. However, the effects of TMAO on neovascularization and perfusion recovery after PAD are not known. METHODS: Unilateral hindlimb ischemia was generated in mice as experimental PAD model, TMAO or 3,3-dimethyl-1-butanol (DMB) were added to the drinking water for these mice. In cultured endothelial cells, TMAO was added to culture medium to assess the effects on cell viability and tube formation under simulated ischemic conditions. RESULTS: In experimental PAD, TMAO treatment increased malondialdehyde (MDA), interleukin (IL)-1ß and IL-6 in the ischemic muscle, impaired perfusion recovery, and decreased capillary density. On the other hand, mice fed with DMB drinking water showed lower TMAO level, interleukin (IL)-1ß and IL-6, and higher vascular endothelial growth factor in the ischemic muscle, and better perfusion recovery after experimental PAD. In cultured endothelial cell, TMAO decreased intracellular nitric oxide, cell viability and tube formation, and increased intracellular reactive oxygen species levels. CONCLUSIONS: TMAO increases oxidative stress and inflammation, and impairs perfusion recovery and angiogenesis in experimental PAD.


Assuntos
Membro Posterior/irrigação sanguínea , Isquemia/sangue , Metilaminas/sangue , Doença Arterial Periférica/sangue , Animais , Circulação Sanguínea , Modelos Animais de Doenças , Membro Posterior/metabolismo , Membro Posterior/fisiopatologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Isquemia/diagnóstico , Isquemia/metabolismo , Isquemia/fisiopatologia , Masculino , Metilaminas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Estresse Oxidativo , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/metabolismo , Doença Arterial Periférica/fisiopatologia , Prognóstico
13.
Int J Mol Sci ; 21(15)2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32752261

RESUMO

Intraspinal grafting of serotonergic (5-HT) neurons was shown to restore plantar stepping in paraplegic rats. Here we asked whether neurons of other phenotypes contribute to the recovery. The experiments were performed on adult rats after spinal cord total transection. Grafts were injected into the sub-lesional spinal cord. Two months later, locomotor performance was tested with electromyographic recordings from hindlimb muscles. The role of noradrenergic (NA) innervation was investigated during locomotor performance of spinal grafted and non-grafted rats using intraperitoneal application of α2 adrenergic receptor agonist (clonidine) or antagonist (yohimbine). Morphological analysis of the host spinal cords demonstrated the presence of tyrosine hydroxylase positive (NA) neurons in addition to 5-HT neurons. 5-HT fibers innervated caudal spinal cord areas in the dorsal and ventral horns, central canal, and intermediolateral zone, while the NA fiber distribution was limited to the central canal and intermediolateral zone. 5-HT and NA neurons were surrounded by each other's axons. Locomotor abilities of the spinal grafted rats, but not in control spinal rats, were facilitated by yohimbine and suppressed by clonidine. Thus, noradrenergic innervation, in addition to 5-HT innervation, plays a potent role in hindlimb movement enhanced by intraspinal grafting of brainstem embryonic tissue in paraplegic rats.


Assuntos
Tronco Encefálico/transplante , Transplante de Tecido Encefálico/métodos , Regeneração Nervosa/fisiologia , Paraplegia/cirurgia , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/cirurgia , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Animais , Tronco Encefálico/embriologia , Clonidina/farmacologia , Feminino , Membro Posterior/efeitos dos fármacos , Membro Posterior/inervação , Membro Posterior/fisiopatologia , Locomoção/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Paraplegia/fisiopatologia , Ratos Wistar , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/fisiopatologia , Ioimbina/farmacologia
14.
Mol Neurobiol ; 57(10): 4143-4155, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32676989

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron degenerative disease that is without effective treatment. The receptor for advanced glycation end products (RAGE) is a major component of the innate immune system that has been implicated in ALS pathogenesis. However, the contribution of RAGE signalling to the neuroinflammation that underlies ALS neurodegeneration remains unknown. The present study therefore generated SOD1G93A mice lacking RAGE and compared them with SOD1G93A transgenic ALS mice in respect to disease progression (i.e. body weight, survival and muscle strength), neuroinflammation and denervation markers in the spinal cord and tibialis anterior muscle. We found that complete absence of RAGE signalling exerted a protective effect on SOD1G93A pathology, slowing disease progression and significantly extending survival by ~ 3 weeks and improving motor function (rotarod and grip strength). This was associated with reduced microgliosis, cytokines, innate immune factors (complement, TLRs, inflammasomes), and oxidative stress in the spinal cord, and a reduction of denervation markers in the tibialis anterior muscle. We also documented that RAGE mRNA expression was significantly increased in the spinal cord and muscles of preclinical SOD1 and TDP43 models of ALS, supporting a widespread involvement for RAGE in ALS pathology. In summary, our results indicate that RAGE signalling drives neuroinflammation and contributes to neurodegeneration in ALS and highlights RAGE as a potential immune therapeutic target for ALS.


Assuntos
Esclerose Amiotrófica Lateral/genética , Inflamação/patologia , Receptor para Produtos Finais de Glicação Avançada/deficiência , Superóxido Dismutase-1/genética , Animais , Astrócitos/patologia , Biomarcadores/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Denervação , Modelos Animais de Doenças , Progressão da Doença , Deleção de Genes , Força da Mão , Membro Posterior/fisiopatologia , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/patologia , Músculos/inervação , Músculos/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Teste de Desempenho do Rota-Rod , Índice de Gravidade de Doença , Medula Espinal/patologia , Análise de Sobrevida , Regulação para Cima
15.
Poult Sci ; 99(7): 3301-3311, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32616223

RESUMO

Stress and leg weakness are detrimental to broiler production, health, and welfare. Traditional methods to evaluate stress may be stressful to the bird because they are invasive and require handling and restraint. Two studies examined the effects of light intensity and flooring on the following in broilers: 1) traditional methods for assessing stress using heterophil-to-lymphocyte ratios and serum corticosterone (CORT) concentrations, 2) noninvasive measures of stress from infrared thermography (IRT) eye and beak surface temperatures, and 3) latency-to-lie (LTL) test times of birds tested individually and in groups of 5. Day-of-hatch male broiler chicks were placed into 6 pens (N = 120 chicks/pen). At 1 wk, pens were allocated to 3 light intensity treatments (2, 5, or 10 lux). At 4 wk, half of the birds from each pen were moved to a pen with wire flooring and the same light intensity. At 1, 4, 5, and 8 wk, blood samples were collected and IRT images of the heads of 5 clinically healthy broilers from each pen were captured. In study 2, IRT images of the heads of birds that became lame in the wire flooring pens were taken. There were no treatment effects on the LTL times of birds tested in groups or individually (P > 0.05). On day 56 in study 1, birds on wire flooring had elevated heterophil-to-lymphocyte ratios and CORT concentrations (P ≤ 0.002) and depressed IRT eye and beak temperatures (P < 0.0001). In both studies, there were negative correlations between CORT concentrations and IRT beak surface temperatures (P < 0.05). Lame birds had lower IRT eye and beak surface temperatures than sound birds (P ≤ 0.004), and the IRT beak surface temperatures of lame birds were lower than their eye surface temperatures (P = 0.004) in study 2. These studies indicate that the IRT surface temperatures of the eye, and more distinctly of the beak, can be used as sensitive noninvasive indicators of stress.


Assuntos
Galinhas/fisiologia , Abrigo para Animais/classificação , Coxeadura Animal/fisiopatologia , Iluminação , Doenças das Aves Domésticas/fisiopatologia , Animais , Anticorpos Heterófilos/sangue , Corticosterona/sangue , Pisos e Cobertura de Pisos/classificação , Membro Posterior/fisiopatologia , Luz , Linfócitos/metabolismo , Masculino , Estresse Fisiológico , Termografia/veterinária
16.
Ulus Travma Acil Cerrahi Derg ; 26(3): 351-360, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32436980

RESUMO

BACKGROUND: Acute ischemia/reperfusion (I/R) injury of skeletal muscle, an important mortality and morbidity cause, is associated with oxidative stress. Lycopene is a carotenoid pigment with potent antioxidant activity and is found in vegetables and fruits. This study aims to investigate the protective effects of lycopene against I/R injury in rat hind limb muscle model. METHODS: Thirty-two Wistar-albino rats were randomly allocated to control, lycopene, I/R and I/R+lycopene groups. In lycopene and I/R+lycopene groups, the rats received 10 mg/kg/day lycopene orally for 15 days before the experiment. Dissection around abdominal aorta at the infrarenal level was performed in all rats under general anesthesia. The aorta was clamped at the infrarenal level in the I/R and I/R+lycopene groups for two hours. Then, reperfusion was allowed for two hours in these groups. Samples were obtained from the hind limb muscles of rats after sacrifice for biochemical and histopathological analyses. RESULTS: Serum and tissue malondialdehyde and ischemia-modified albumin levels were significantly lower in the I/R+lycopene group compared to I/R group (p<0.001). Serum glutathione peroxidase (GSH-Px) levels were significantly lower in the I/R group compared to those in control and I/R+lycopene groups (p<0.05). Tissue GSH-Px levels were significantly lower in the I/R group compared to the Lycopene group (p=0.003). Serum superoxide dismutase (SOD) levels were significantly lower in the I/R group compared to three groups (p<0.001). Tissue SOD levels were significantly lower in the I/R group compared to those in control and Lycopene groups (p=0.005). Histopathological assessments revealed that inflammatory changes following I/R injury were significantly reduced in the I/R+lycopene group. CONCLUSION: The findings obtained in this study show lycopene's cytoprotective activity against I/R injury in rat skeletal muscle model.


Assuntos
Antioxidantes/farmacologia , Licopeno/farmacologia , Músculo Esquelético/efeitos dos fármacos , Traumatismo por Reperfusão , Animais , Membro Posterior/fisiopatologia , Ratos , Superóxido Dismutase
17.
J Neuroinflammation ; 17(1): 101, 2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-32248810

RESUMO

BACKGROUND: Cannabinoid-2 receptor (CB2R) plays an important role in the cascading inflammation following ischemic injury. The toll-like receptors 4 (TLR4)/matrix metalloproteinase 9 (MMP9) signal pathway is involved in blood-brain barrier dysfunction induced by ischemia stroke. The aim of this study is to investigate the roles of exogenous activation of CB2R on attenuating neurological deficit and blood-spinal cord barrier (BSCB) disruption during rat spinal cord ischemia reperfusion (I/R) injury, through modulation of the TLR4/MMP9 axis. METHODS: Animals were intraperitoneally pretreated with TLR4 inhibitor TAK-242, CB2R agonist JWH-133 with or without CB2R antagonist AM630, or equivalent volume of vehicle 1 h before undergoing 14-min occlusion of descending aorta or sham operation. One, two, three, and 7 days after reperfusion, hindlimb locomotor function was evaluated with Basso, Beattie, and Bresnahan (BBB) Locomotor Scale, BSCB integrity was detected by measurement of Evans blue (EB) extravasation and spinal cord edema. The protein expression levels of CB2R, tight junction protein Zonula occluden-1 (ZO-1), TLR4, MMP9, MyD88, NF-κB p65, and NF-κB p-p65 were determined by western blot. The MMP9 activity was analyzed by gelatin zymography. Double immunofluorescence staining was used to identify the perivascular localization of CB2R, TLR4, MMP9, and reactive astrocytes, as well as the colocalization of CB2R, TLR4, and MMP9 with reactive astrocytes. RESULTS: JWH-133 pretreatment attenuated hindlimb motor functional deficit and BSCB leakage, along with preventing downregulation of ZO-1 and upregulation of TLR4/MMP9, similar to the effects of TAK-242 preconditioning. JWH-133 or TAK-242 pretreatment reduced the perivascular expression of TLR4/MMP9 and reactive astrocytes following injury. JWH-133 pretreatment also downregulated MyD88/NF-κB level, MMP9 activity, and the astrocytic TLR4/MMP9 after I/R injury. CONCLUSIONS: Exogenous activation of CB2R by JWH-133 attenuated neurological deficit and BSCB disruption after spinal cord I/R injury via inhibition of TLR4/MMP9 expression.


Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Receptor CB2 de Canabinoide/agonistas , Traumatismo por Reperfusão/metabolismo , Isquemia do Cordão Espinal/metabolismo , Medula Espinal/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Canabinoides/farmacologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismo por Reperfusão/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiopatologia , Isquemia do Cordão Espinal/fisiopatologia , Sulfonamidas/farmacologia , Regulação para Cima/fisiologia , Regulação para Cima/efeitos da radiação
18.
J Neurosci ; 40(20): 3882-3895, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32291327

RESUMO

Neonatal tissue damage induces long-term deficits in inhibitory synaptic transmission within the spinal superficial dorsal horn (SDH) that include a reduction in primary afferent-evoked, feedforward inhibition onto adult projection neurons. However, the subpopulations of mature GABAergic interneurons which are compromised by early-life injury have yet to be identified. The present research illuminates the persistent effects of neonatal surgical injury on the function of inhibitory SDH interneurons derived from the prodynorphin (DYN) lineage, a population that synapses directly onto lamina I spinoparabrachial neurons and is known to suppress mechanical pain and itch in adults. The results demonstrate that hindpaw incision at postnatal day 3 (P3) significantly decreased the strength of primary afferent-evoked glutamatergic drive onto DYN neurons within the adult mouse SDH while increasing the appearance of afferent-evoked inhibition onto the same population. Neonatal injury also dampened the intrinsic membrane excitability of mature DYN neurons, and reduced their action potential discharge in response to sensory input, compared with naive littermate controls. Furthermore, P3 incision decreased the efficacy of inhibitory DYN synapses onto adult spinoparabrachial neurons, which reflected a prolonged reduction in the probability of GABA release. Collectively, the data suggest that early-life tissue damage may persistently constrain the ability of spinal DYN interneurons to limit ascending nociceptive transmission to the adult brain. This is predicted to contribute to the loss of feedforward inhibition onto mature projection neurons, and the "priming" of nociceptive circuits in the developing spinal cord, following injuries during the neonatal period.SIGNIFICANCE STATEMENT Neonatal injury has lasting effects on pain processing in the adult CNS, including a reduction in feedforward inhibition onto ascending projection neurons in the spinal dorsal horn. While it is clear that spinal GABAergic interneurons are comprised of multiple subpopulations that play distinct roles in somatosensation, the identity of those interneurons which are compromised by tissue damage during early life remains unknown. Here we document persistent deficits in spinal inhibitory circuits involving dynorphin-lineage interneurons previously implicated in gating mechanical pain and itch. Notably, neonatal injury reduced the strength of dynorphin-lineage inhibitory synapses onto mature lamina I spinoparabrachial neurons, a major output of the spinal nociceptive network, which could contribute to the priming of pain pathways by early tissue damage.


Assuntos
Dinorfinas , Membro Posterior/lesões , Inibição Neural , Vias Neurais/fisiopatologia , Corno Dorsal da Medula Espinal/lesões , Potenciais de Ação , Animais , Animais Recém-Nascidos , Análise por Conglomerados , Feminino , Glutamatos/fisiologia , Membro Posterior/inervação , Membro Posterior/fisiopatologia , Interneurônios , Camundongos , Neurônios Aferentes , Nociceptividade , Técnicas de Patch-Clamp , Medula Espinal/fisiopatologia , Corno Dorsal da Medula Espinal/fisiopatologia
19.
PLoS One ; 15(4): e0231904, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320449

RESUMO

Detection of lameness in cats can be very time-consuming and frustrating. Feline studies have shown that the success of treatment can be evaluated by measurement of the ground reaction force (GRF). However, the possibility of multiple limb involvement or the presence of a compensatory mechanism has not been investigated. Furthermore, there has been no research in cats on possible differences in GRFs between those with stifle problems and those with hip problems, as reported in dogs. In this study, we compared temporospatial parameters and GRFs in 20 lame cats after femoral head and neck ostectomy (FHO) or stifle disease to those in 15 healthy cats. An orthopedic examination was performed in all cats and radiographs were obtained to confirm the disease. GRFs, including peak vertical force (PFz), vertical impulse (IFz), time to PFz, and temporospatial parameters, including step length, paw contact area, and stance phase duration, were calculated. We also calculated the symmetry index (SI) in the forelimbs and hind limbs. The GRFs were normalized to total force (% TF). We found that the IFz (% TF) and PFz (% TF) were lower in the affected limb than in the other limbs in the lame cats. When the lame cats were compared with the sound cats, this difference was only significant for IFz (% TF). The SI values for the PFz and IFz were significantly higher in the hind limbs than in the forelimbs in the lame cats group but there was no difference in the SI according to whether the problem was in the hip or stifle. There were also differences in stance phase duration and paw contact area in both the forelimbs and hind limbs between the sound group and the lame group. There was no difference in PFZ (% TF) or IFZ (% TF) in the affected limb between the lame cats with stifle and those after FHO; however, there were changes in time to PFz and step length. In conclusion, mild to moderate lameness can be detected and measured in cats using pressure plates. The compensatory mechanisms in cats at a walk appear to involve shifting the weight to the other three legs without favoring either the contralateral or the diagonal limb.


Assuntos
Membro Posterior , Coxeadura Animal/diagnóstico , Pressão , Animais , Fenômenos Biomecânicos , Gatos , Feminino , Membro Posterior/patologia , Membro Posterior/fisiopatologia , Articulações/patologia , Articulações/fisiopatologia , Coxeadura Animal/patologia , Coxeadura Animal/fisiopatologia , Masculino
20.
Vet Surg ; 49(5): 860-869, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32166788

RESUMO

OBJECTIVE: To determine the effectiveness of durotomy as an adjunct to surgical decompression in dogs with thoracolumbar intervertebral disc herniation (TL-IVDH) and loss of deep pain perception (DPP) in the hind limbs. STUDY DESIGN: Retrospective study. ANIMALS: Dogs (n = 116) with TL-IVDH and loss of DPP treated with hemilaminectomy. METHODS: Signalment, surgical site, recovery rate, incidence of progressive myelomalacia (PMM), time elapsed from onset of paraplegia of the hind limbs to surgery (TPS), and the length of area of hyperintensity of the spinal cord on magnetic resonance T2-weighted images compared with L2 vertebral body length (LHT2) were compared between dogs treated with hemilaminectomy alone and those treated with adjunct durotomy. Multivariate logistic regression analyses were used to test the association between outcomes and the external view of the spinal cord parenchyma after durotomy. RESULTS: The percentage of dogs regaining ambulation was greater when durotomy was performed (56.9%) than when dogs were treated with hemilaminectomy alone (38.5%; P = .04). In the hemilaminectomy group, 14 dogs died of suspected PMM, while no PMM was detected in the durotomy group. Durotomy, breed, surgical site, and LHT2 influenced recovery. No association was detected between age, sex, body weight, and TPS and recovery. CONCLUSION: Performing a durotomy in combination with decompression improved the return to function and prevented PMM in our clinical setting. CLINICAL SIGNIFICANCE: Surgeons should consider durotomy in dogs with TL-IVDH and loss of DPP in hind limbs to improve surgical outcome.


Assuntos
Doenças do Cão/cirurgia , Dura-Máter/cirurgia , Membro Posterior/fisiopatologia , Deslocamento do Disco Intervertebral/veterinária , Percepção da Dor , Animais , Descompressão Cirúrgica/veterinária , Cães , Feminino , Deslocamento do Disco Intervertebral/cirurgia , Masculino , Estudos Retrospectivos
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