RESUMO
This paper presents the results of a study on the applicability of cerebrospinal fluid (CSF) collected from the spinal canal in the post-mortem determination of ethyl alcohol. The present study reviewed data of autopsy cases (n = 45), in which ethyl alcohol was detected in CSF using gas chromatography with a flame ionization detector (HS-GC-FID), to investigate ethyl alcohol concentrations in CSF, compared with blood. As a result of statistical analysis of the obtained data, a high positive correlation was found between blood ethanol concentration and cerebrospinal fluid collected from the spinal canal ethanol concentration. The Pearson correlation coefficient was statistically highly significant (p < 0.001) (r = 0.9503). The data obtained allowed us to conclude that cerebrospinal fluid collected from the spinal canal can be collected during an autopsy as an alternative biological specimen to assess the ethanol content. Cerebrospinal fluid collected from the spinal canal can corroborate and lend credibility to the results obtained for blood and, in special cases, when blood is drawn from putrefied bodies and may even be a superior specimen to blood for assessing ethyl alcohol intoxication status.
Assuntos
Etanol , Mudanças Depois da Morte , Humanos , Autopsia , Concentração Alcoólica no Sangue , Toxicologia Forense/métodos , Medula Espinal/químicaRESUMO
The in vivo chemogenetic property of mercuric ions (Hg2+) was investigated as a specific hypercalcemia actuator in snail's spinal cord cell manipulation by extracellular field potential biosensing analysis. For this purpose, a three-microelectrode system with working, counter, and pseudo reference electrodes was blindly implanted into the snail's spinal cord to electrically stimulate (triggering) the action potential with a staircase electrical voltage at a very low frequency level, along with measurement of the electrical current, as a detection system. Under optimum conditions, using the one-factor-at-a-time method, a wide linear range between 1.0 × 10-14 and 1.0 × 10-1 mol L-1 with correlation coefficients (R2) >0.98 and a response time (t90) of maximum 10.0 s were approximated. Percentages of relative standard deviation were estimated to be 3.08 (reproducibility, n = 50) and 7.31 (repeatability, n = 15). The detection limit was estimated to be sub 2.1 × 10-16 mol L-1 based on the Xb- + 3Sb definition. The reliability of this phenomenon was evidenced by the estimation of recovery percentages (between 95 and 107%) during spiking Hg2+ standard solutions. The probable mechanism behind this process could be attributed to the following: (i) the neuronal ephaptic coupling during electrical synchronization by a specific brain-triggered wave as a neuronal motor toolkit and (ii) chemical synchronization using a Hg2+ hypercalcemia actuator (biosensor). Linear correlation has been evidenced during interactions between Hg2+ and a calcium ionic channel's protein with a gram molecular weight of 66.2 ± 0.3 KCU. This process, therefore, caused an opening of the Ca2+ channel gates and majorly released the Ca2+ (hypercalcemia) that was detected as the main source of the measured electrical current. At this condition, ultratrace levels of Hg2+ ions not only were considered as nontoxic reagents but also had chemically regulating effects as ephaptic synchronizers to the neuron cells. This report may pave the way for using mercury ions at an ultratrace level for clinical controlling purposes during neuronal spinal cord cell manipulation.
Assuntos
Técnicas Biossensoriais , Hipercalcemia , Mercúrio , Técnicas Biossensoriais/métodos , Humanos , Íons , Mercúrio/toxicidade , Reprodutibilidade dos Testes , Medula Espinal/químicaRESUMO
Proprioception, the sense of limb and body position, generates a map of the body that is essential for proper motor control, yet we know little about precisely how neurons in proprioceptive pathways are wired. Defining the anatomy of secondary neurons in the spinal cord that integrate and relay proprioceptive and potentially cutaneous information from the periphery to the cerebellum is fundamental to understanding how proprioceptive circuits function. Here, we define the unique anatomic trajectories of long-range direct and indirect spinocerebellar pathways as well as local intersegmental spinal circuits using genetic tools in both male and female mice. We find that Clarke's column neurons, a major contributor to the direct spinocerebellar pathway, has mossy fiber terminals that diversify extensively in the cerebellar cortex with axons terminating bilaterally, but with no significant axon collaterals within the spinal cord, medulla, or cerebellar nuclei. By contrast, we find that two of the indirect pathways, the spino-lateral reticular nucleus and spino-olivary pathways, are in part, derived from cervical Atoh1-lineage neurons, whereas thoracolumbar Atoh1-lineage neurons project mostly locally within the spinal cord. Notably, while cervical and thoracolumbar Atoh1-lineage neurons connect locally with motor neurons, no Clarke's column to motor neuron connections were detected. Together, we define anatomic differences between long-range direct, indirect, and local proprioceptive subcircuits that likely mediate different components of proprioceptive-motor behaviors.SIGNIFICANCE STATEMENT We define the anatomy of long-range direct and indirect spinocerebellar pathways as well as local spinal proprioceptive circuits. We observe that mossy fiber axon terminals of Clarke's column neurons diversify proprioceptive information across granule cells in multiple lobules on both ipsilateral and contralateral sides, sending no significant collaterals within the spinal cord, medulla, or cerebellar nuclei. Strikingly, we find that cervical spinal cord Atoh1-lineage neurons form mainly the indirect spino-lateral reticular nucleus and spino-olivary tracts and thoracolumbar Atoh1-lineage neurons project locally within the spinal cord, whereas only a few Atoh1-lineage neurons form a direct spinocerebellar tract.
Assuntos
Cerebelo/fisiologia , Rede Nervosa/fisiologia , Propriocepção/fisiologia , Medula Espinal/fisiologia , Tratos Espinocerebelares/fisiologia , Animais , Animais Recém-Nascidos , Cerebelo/química , Cerebelo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rede Nervosa/química , Rede Nervosa/citologia , Medula Espinal/química , Medula Espinal/citologia , Tratos Espinocerebelares/química , Tratos Espinocerebelares/citologiaRESUMO
Astrocytes are the most abundant glial cell in the brain and perform a wide range of tasks that support neuronal function and circuit activities. There is emerging evidence that astrocytes exhibit molecular and cellular heterogeneity; however, whether distinct subpopulations perform these diverse roles remains poorly defined. Here we show that the Lunatic Fringe-GFP (Lfng-GFP) bacteria artificial chromosome mouse line from both sexes specifically labels astrocyte populations within lamina III and IV of the dorsal spinal cord. Transcriptional profiling of Lfng-GFP+ astrocytes revealed unique molecular profiles, featuring an enriched expression of Notch- and Wnt- pathway components. Leveraging CRE-DOG viral tools, we ablated Lfng-GFP+ astrocytes, which decreased neuronal activity in lamina III and IV and impaired mechanosensation associated with light touch. Together, our findings identify Lfng-GFP+ astrocytes as a unique subpopulation that occupies a distinct anatomic location in the spinal cord and directly contributes to neuronal function and sensory responses.SIGNIFICANCE STATEMENT Astrocytes are the most abundant glial cell in the CNS, and their interactions with neurons are essential for brain function. However, understanding the functional diversity of astrocytes has been hindered because of the lack of reporters that mark subpopulations and genetic tools for accessing them. We discovered that the Lfng-GFP reporter mouse labels a laminae-specific subpopulation of astrocytes in the dorsal spinal cord and that ablation of these astrocytes reduces glutamatergic synapses. Further analysis revealed that these astrocytes have a role in maintaining sensory-processing circuity related to light touch.
Assuntos
Astrócitos/química , Astrócitos/fisiologia , Glicosiltransferases/análise , Proteínas de Fluorescência Verde/análise , Percepção/fisiologia , Animais , Feminino , Glicosiltransferases/deficiência , Glicosiltransferases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Medula Espinal/química , Medula Espinal/fisiologiaRESUMO
Kappa free light chain (KFLC) index, a measure for intrathecal production of free kappa chains, has been increasingly recognized for its diagnostic potential in multiple sclerosis (MS) as a quantitative alternative to IgG oligoclonal bands (OCBs). Our objective was to investigate the sensitivity, specificity, and overall diagnostic accuracy of KFLC index in MS. KFLC index was prospectively determined as part of the diagnostic workup in patients with suspected MS (n = 327) between May 2013 and February 2020. Patients with clinically isolated syndrome (CIS), radiologically isolated syndrome (RIS), and MS had markedly higher KFLC index (44.6, IQR 16-128) compared with subjects with other neuro-inflammatory disorders (ONID) and symptomatic controls (SC) (2.19, IQR 1.68-2.98, p < 0.001). KFLC index had a sensitivity of 0.93 (95% CI 0.88-0.95) and specificity of 0.87 (95% CI 0.8-0.92) to discriminate CIS/RIS/MS from ONID and SC (AUC 0.94, 95% CI 0.91-0.97, p < 0.001). KFLC index and intrathecal fraction (IF) KFLC had similar accuracies to detect MS. Treatment with disease-modifying therapy (DMT) did not influence the level of KFLC index and it was not affected by demographic factors or associated with degenerative or inflammatory biomarkers in cerebrospinal fluid (CSF). KFLC index in MS diagnostics has methodological advantages compared to OCB and is independent to subjective interpretation. Moreover, it is an attractive diagnostic tool since the diagnostic specificity and sensitivity of KFLC index are similar with that of OCBs and KFLCIF and better than for IgG index. We show that KFLC index was influenced neither by DMT nor by demographic factors or other inflammatory or degenerative processes in MS as determined by biomarkers in CSF.
Assuntos
Cadeias Leves de Imunoglobulina/análise , Esclerose Múltipla/diagnóstico , Adulto , Biomarcadores , Doenças Desmielinizantes/diagnóstico , Diagnóstico Diferencial , Progressão da Doença , Feminino , Humanos , Imunoglobulina G/análise , Inflamação/diagnóstico , Imageamento por Ressonância Magnética , Masculino , Esclerose Múltipla/diagnóstico por imagem , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Medula Espinal/química , Medula Espinal/metabolismoRESUMO
In the spinal cord, classes of interneurons have been studied in vitro to determine their role in producing or regulating locomotion. It is unclear whether all locomotor behaviors are produced by the same circuitry or engage different subsets of neurons. Here, in neonatal mice of either sex, we test this idea by comparing the actions of a class of spinal, inhibitory interneuron (V1) expressing channelrhodopsin driven by the engrailed-1 transcription factor on the rhythms elicited by different methods. We find that, although the overall locomotor activities in vitro are similar, V1 interneuron depolarization produces opposite effects depending of the mode of activation of the locomotor circuitry. The differential behavior of V1 neurons suggests that their function depends on how the locomotor rhythm is activated and is consistent with the idea that the functional organization of the corresponding locomotor networks also differs.SIGNIFICANCE STATEMENT The neural networks dictating the execution of fictive locomotion are located in the spinal cord. It is generally assumed that the mode of activation of these spinal networks should not change the recruitment or function of neurons. Here, we manipulated the activity of a class of interneuron (V1), which targets these networks and found that their activation induces opposite effects depending on the mode of activation. This suggests that the mode of activation of the spinal networks differentially recruits either V1 interneurons or other interneurons, or both.
Assuntos
Interneurônios/fisiologia , Locomoção/fisiologia , Rede Nervosa/fisiologia , Optogenética/métodos , Medula Espinal/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Interneurônios/química , Masculino , Camundongos , Camundongos Transgênicos , Rede Nervosa/química , Técnicas de Cultura de Órgãos , Medula Espinal/químicaRESUMO
The pharmacokinetic profile of AAV particles following intrathecal delivery has not yet been clearly defined. The present study evaluated the distribution profile of adeno-associated virus serotype 5 (AAV5) viral vectors following lumbar intrathecal injection in mice. After a single bolus intrathecal injection, viral DNA concentrations in mouse whole blood, spinal cord, and peripheral tissues were determined using quantitative polymerase chain reaction (qPCR). The kinetics of AAV5 vector in whole blood and the concentration over time in spinal and peripheral tissues were analyzed. Distribution of the AAV5 vector to all levels of the spinal cord, dorsal root ganglia, and into systemic circulation occurred rapidly within 30 min following injection. Vector concentration in whole blood reached a maximum 6 h postinjection with a half-life of approximately 12 h. Area under the curve data revealed the highest concentration of vector distributed to dorsal root ganglia tissue. Immunohistochemical analysis revealed AAV5 particle colocalization with the pia mater at the spinal cord and macrophages in the dorsal root ganglia (DRG) 30 min after injection. These results demonstrate the widespread distribution of AAV5 particles through cerebrospinal fluid and preferential targeting of DRG tissue with possible clearance mechanisms via DRG macrophages.
Assuntos
Dependovirus , Vetores Genéticos/farmacocinética , Animais , DNA Viral/análise , DNA Viral/sangue , Feminino , Vetores Genéticos/administração & dosagem , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos ICR , Reação em Cadeia da Polimerase em Tempo Real , Medula Espinal/química , Distribuição Tecidual , Transdução Genética/métodosRESUMO
Corticotropin-releasing factor (CRF) orchestrates our body's response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. Thus, the aim of the present study was to examine CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund's complete adjuvant-induced unilateral inflammation of the hind paw using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, the antinociceptive effects of intrathecal (i.t.) CRF were measured by paw pressure algesiometer and their possible antagonism by selective antagonists for CRF-R1 and/or CRF-R2 as well as for opioid receptors. Our results demonstrated a preference for the expression of CRF-R2 over CRF-R1 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonists elicited potent dose-dependent antinociceptive effects which were antagonized by the i.t. CRF-R2 selective antagonist K41498, but not by the CRF-R1 selective antagonist NBI35965. In addition, i.t. applied opioid antagonist naloxone dose-dependently abolished the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Importantly, double immunofluorescence confocal microscopy of the spinal dorsal horn showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonists inhibit somatic inflammatory pain predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons which finally results in endogenous opioid-mediated pain inhibition.
Assuntos
Dor/fisiopatologia , Receptores de Hormônio Liberador da Corticotropina/fisiologia , Medula Espinal/química , Acenaftenos/farmacologia , Proteínas de Anfíbios/farmacologia , Animais , Artrite Experimental/fisiopatologia , Hormônio Liberador da Corticotropina/farmacologia , Encefalinas/fisiologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/fisiopatologia , Interneurônios/fisiologia , Masculino , Naloxona/farmacologia , Nociceptividade/fisiologia , Hormônios Peptídicos/farmacologia , Células do Corno Posterior/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Receptores de Hormônio Liberador da Corticotropina/biossíntese , Receptores de Hormônio Liberador da Corticotropina/genética , Medula Espinal/fisiopatologia , Urocortinas/farmacologiaRESUMO
Trigeminal neuralgia pain remains a challenge to treat. Natural compounds may be promising options for relieving pain. This study was aimed at investigating the effects of aconitine in a rat model of trigeminal neuralgia pain. Infraorbital nerve chronic constriction injury was performed in adult Wistar Albino rats. After the neuropathic pain developed, the rats were assigned to one of the treatment groups: carbamazepine 40 or 80 mg/kg; aconitine 0.25, 0.50, or 0.75 mg/kg; or saline injection (control group). Behavioral testing with von Frey filaments and the rotarod test were carried out before the surgical procedure and on the 24th to 29th postoperative days. Following the completion of tests, ipsilateral and contralateral spinal cords were harvested for Western blot analyses to assess NR-1 protein expression. ANOVA followed by Mann-Whitney U test was performed for the statistical analyses. P values of <0.05 were considered significant. Aconitine significantly reduced mechanical sensitivity in a dose-dependent manner. A significant reduction in motor coordination was noted for the higher doses of aconitine which was similar with the 40 and 80 mg/kg doses of carbamazepine. NR-1 expression was reduced in the ipsilateral spinal cord, whereas no significant difference was noted between the groups in the expression of NR-1 in the contralateral spinal cord. Aconitine had a significant pain relieving effect, which was similar to carbamazepine, in a dose-dependent manner. Aconitine may be an alternative pharmacological agent for the control of trigeminal neuralgia pain.
Assuntos
Aconitina/uso terapêutico , Neuralgia do Trigêmeo/tratamento farmacológico , Aconitina/farmacologia , Animais , Masculino , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/análise , Medula Espinal/química , Neuralgia do Trigêmeo/metabolismoRESUMO
Multiple sclerosis (MS) is known as a chronic neuroinflammatory disorder typified by an immune-mediated demyelination process with ensuing axonal damage and loss. Sinomenine is a natural alkaloid with different therapeutic benefits, including anti-inflammatory and immunosuppressive activities. In this study, possible beneficial effects of sinomenine in an MOG-induced model of MS were determined. Sinomenine was given to MOG35-55-immunized C57BL/6 mice at doses of 25 or 100 mg/kg/day after onset of MS clinical signs till day 30 post-immunization. Analyzed data showed that sinomenine reduces severity of the clinical signs and to some extent decreases tissue level of pro-inflammatory cytokines IL-1ß, IL-6, IL-18, TNFα, IL-17A, and increases level of anti-inflammatory IL-10. In addition, sinomenine successfully attenuated tissue levels of inflammasome NLRP3, ASC, and caspase 1 besides its reduction of intensity of neuroinflammation, demyelination, and axonal damage and loss in lumbar spinal cord specimens. Furthermore, immunoreactivity for MBP decreased and increased for GFAP and Iba1 after MOG-immunization, which was in part reversed upon sinomenine administration. Overall, sinomenine decreases EAE severity, which is attributed to its alleviation of microglial and astrocytic mobilization, demyelination, and axonal damage along with its suppression of neuroinflammation, and its beneficial effect is also associated with its inhibitory effects on inflammasome and pyroptotic pathways; this may be of potential benefit for the primary progressive phenotype of MS.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Inflamassomos/antagonistas & inibidores , Morfinanos/uso terapêutico , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Animais , Astrócitos/efeitos dos fármacos , Peso Corporal , Citocinas/análise , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Morfinanos/administração & dosagem , Morfinanos/farmacologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Piroptose/efeitos dos fármacos , Distribuição Aleatória , Organismos Livres de Patógenos Específicos , Medula Espinal/químicaRESUMO
Storage and voiding of urine from the lower urinary tract (LUT) must be timed precisely to occur in appropriate behavioral contexts. A major part of the CNS circuit that coordinates this activity is found in the lumbosacral spinal cord. Immediate early gene (IEG) activity mapping has been widely used to investigate the lumbosacral LUT-related circuit, but most reports focus on the effects of noxious stimulation in anesthetized female rats. Here we use c-Fos and EGR-1 (Zif268) activity mapping of lumbosacral spinal cord to investigate cystometry-induced micturition in awake female and male rats. In females, after cystometry c-Fos neurons in spinal cord segments L5-S2 were concentrated in the sacral parasympathetic nucleus (SPN), dorsal horn laminae II-IV, and dorsal commissural nucleus (SDCom). Comparisons of cystometry and control groups in male and female revealed sex differences. Activity mapping suggested dorsal horn laminae II-IV was activated in females but showed net inhibition in males. However, inhibition in male rats was not detected by EGR-1 activity mapping, which showed low coexpression with c-Fos. A class of catecholamine neurons in SPN and SDCom neurons were also more strongly activated by micturition in females. In both sexes, most c-Fos neurons were identified as excitatory by their absence of Pax2 expression. In conclusion, IEG mapping in awake male and female rats has extended our understanding of the functional molecular anatomy of the LUT-related circuit in spinal cord. Using this approach, we have identified sex differences that were not detected by previous studies in anesthetized rats.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Caracteres Sexuais , Medula Espinal/metabolismo , Micção/fisiologia , Animais , Proteína 1 de Resposta de Crescimento Precoce/análise , Feminino , Masculino , Proteínas Proto-Oncogênicas c-fos/análise , Ratos , Ratos Sprague-Dawley , Sacro/inervação , Sacro/metabolismo , Medula Espinal/química , Bexiga Urinária/química , Bexiga Urinária/inervação , Bexiga Urinária/metabolismoRESUMO
Polysialic acid (polySia), a homopolymer of α2,8-linked glycans, is a posttranslational modification on a few glycoproteins, most commonly in the brain, on the neural cell adhesion molecule. Most research in the adult central nervous system has focused on its expression in higher brain regions, where its distribution coincides with regions known to exhibit high levels of synaptic plasticity. In contrast, scant attention has been paid to the expression of polySia in the hindbrain. The main aims of the study were to examine the distribution of polySia immunoreactivity in the brainstem and thoracolumbar spinal cord, to compare the distribution of polySia revealed by two commercial antibodies commonly used for its investigation, and to compare labeling in the rat and mouse. We present a comprehensive atlas of polySia immunoreactivity: we report that polySia labeling is particularly dense in the dorsal tegmentum, medial vestibular nuclei and lateral parabrachial nucleus, and in brainstem regions associated with autonomic function, including the dorsal vagal complex, A5, rostral ventral medulla, A1, and midline raphe, as well as sympathetic preganglionic neurons in the spinal cord and central targets of primary sensory afferents (nucleus of the solitary tract, spinal trigeminal nucleus, and dorsal horn [DH]). Ultrastructural examination showed labeling was present predominantly on the plasma membrane/within the extracellular space/in or on astrocytes. Labeling throughout the brainstem and spinal cord were very similar for the two antibodies and was eliminated by the polySia-specific sialidase, Endo-NF. Similar patterns of distribution were found in rat and mouse brainstem with differences evident in DH.
Assuntos
Tronco Encefálico/química , Vértebras Lombares , Ácidos Siálicos/análise , Medula Espinal/química , Vértebras Torácicas , Animais , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Ácidos Siálicos/biossíntese , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
Chronic inflammation pain is a debilitating disease, and its mechanism still remains poorly understood. This study attempted to illuminate the metabolic mechanism of chronic inflammation pain induced by complete Freund's adjuvant (CFA) injection, especially at spinal level. The chronic inflammation pain model was established by CFA administration. Behavioral testing including mechanical allodynia and thermal hyperalgesia was performed. Meanwhile, a liquid chromatography-mass spectrometry-based metabolomics approach was applied to analyze potential metabolic biomarkers. The orthogonal partial least squares discrimination analysis mode was employed for determining metabolic changes, and a western blot was performed to detect the protein expression change. The results showed that 27 metabolites showed obviously abnormal expression and seven metabolic pathways were significantly enriched, comprising aminoacyl-tRNA biosynthesis, arginine and proline metabolism, histidine metabolism, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, glutathione metabolism, and phenylalanine metabolism. Meanwhile, the results showed that the expression of arginase I and nitric oxide levels were elevated in the CFA group compared with the control group, while the argininosuccinate synthetase and argininosuccinatelyase proteins were not significantly different between the groups. These findings demonstrate that metabolic changes of the spinal cord may be implicated in neurotransmitter release and pain conductivity following CFA administration.
Assuntos
Adjuvante de Freund , Inflamação , Metaboloma/efeitos dos fármacos , Metabolômica/métodos , Dor , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Adjuvante de Freund/efeitos adversos , Adjuvante de Freund/farmacologia , Hiperalgesia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/análise , Óxido Nítrico/metabolismo , Dor/induzido quimicamente , Dor/metabolismo , Medula Espinal/química , Medula Espinal/metabolismoRESUMO
Cutaneous somatosensory modalities play pivotal roles in generating a wide range of sensorimotor behaviors, including protective and corrective reflexes that dynamically adapt ongoing movement and posture. How interneurons (INs) in the dorsal horn encode these modalities and transform them into stimulus-appropriate motor behaviors is not known. Here, we use an intersectional genetic approach to functionally assess the contribution that eight classes of dorsal excitatory INs make to sensorimotor reflex responses. We demonstrate that the dorsal horn is organized into spatially restricted excitatory modules composed of molecularly heterogeneous cell types. Laminae I/II INs drive chemical itch-induced scratching, laminae II/III INs generate paw withdrawal movements, and laminae III/IV INs modulate dynamic corrective reflexes. These data reveal a key principle in spinal somatosensory processing, namely, sensorimotor reflexes are driven by the differential spatial recruitment of excitatory neurons.
Assuntos
Medição da Dor/métodos , Desempenho Psicomotor/fisiologia , Reflexo/fisiologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Animais , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Estimulação Física/efeitos adversos , Medula Espinal/químicaRESUMO
BACKGROUND: Workplace risk factors, such as repetitive tasks, can cause work-related musculoskeletal disorders. In a rat model, decreased grip strength and median nerve injury develop following repetitive reaching and grasping tasks, involving negligible force. OBJECTIVE: We investigated whether median nerve injury is involved in the early onset of decreased grip strength due to such tasks METHODS: Sprague-Dawley rats were divided into: non-task-performing (0-week) and task-performing (1-, 2-, and 3-week) groups. After an initial training period, the task-performing groups continued to perform the task for 2 h/day, 3 days/week, for 1-3 weeks. Grip strength and relative muscle weight of the flexor digitorum superficialis (FDS) muscle were measured. Median nerve injury was evaluated by histopathology and immunohistochemistry. RESULTS: Grip strength of the reach limb (forelimb used in tasks) was significantly lower in the 3-week group compared with the other groups and was significantly lower than that of the non-reach limb in all groups. There were no significant differences in the relative FDS muscle weights of either limb among groups. No evidence of median nerve demyelination was observed and no cells expressed activating transcription factor-3, a specific marker of peripheral nerve injury, in the anterior horn of the spinal cord. CONCLUSION: Median nerve injury does not contribute to the decreased grip strength caused by 3 weeks of repetitive reaching and grasping tasks, involving negligible force, in rats.
Assuntos
Força da Mão , Nervo Mediano/lesões , Traumatismos dos Nervos Periféricos/fisiopatologia , Fator 3 Ativador da Transcrição/metabolismo , Animais , Feminino , Nervo Mediano/fisiopatologia , Músculo Esquelético/patologia , Traumatismos dos Nervos Periféricos/patologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/químicaRESUMO
Destruction of myelin, or demyelination, is a characteristic of traumatic spinal cord injury and pathognomonic for primary demyelinating pathologies such as multiple sclerosis (MS). The regenerative process known as remyelination, which can occur following demyelination, fails as MS progresses. Models of focal demyelination by local injection of gliotoxins have provided important biological insights into the demyelination/remyelination process. Here, injection of lysolecithin to induce spinal cord demyelination is investigated using matrix-assisted laser desorption/ionization mass spectrometry imaging. A segmentation analysis revealed changes to the lipid composition during lysolecithin-induced demyelination at the lesion site and subsequent remyelination over time. The results of this study can be utilized to identify potential myelin-repair mechanisms and in the design of therapeutic strategies to enhance myelin repair.
Assuntos
Doenças Desmielinizantes/patologia , Bainha de Mielina/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Medula Espinal/patologia , Animais , Doenças Desmielinizantes/induzido quimicamente , Modelos Animais de Doenças , Feminino , Lipídeos/análise , Lisofosfatidilcolinas/efeitos adversos , Camundongos Endogâmicos BALB C , Bainha de Mielina/química , Remielinização , Medula Espinal/químicaRESUMO
Mechanisms of pruritus are implicated in the dysregulation of the metabolites in the spinal cord. We investigated pruritus behavioral testing in three groups of young adult male C57Bl/6 mice, including one group treated with normal saline, while the other groups intradermally injected with α-Me-5-HT (histamine-independent pruritogen), compound 48/80 (histamine-dependent pruritogen) at the nape skin of the neck, respectively. Proton nuclear magnetic resonance spectroscopy (MRS) was used to compare spinal metabolites from the vertebral cervical among three groups, and to study the association of spinal metabolite ratio and pruritus intensity. The MRS-measured N-acetylaspartate-to-myoinositol ratio (NAA/Ins) was significantly correlated with the number of scratches between normal saline group and 48/80 group or α-Me-5-HT group (both P<0.0001), indicating that NAA/Ins may be a robust surrogate marker of histamine-independent/dependent pruritogen. There was significant difference in Glu/Ins between normal saline group and 48/80 group (P=0.017), indicating that Glu/Ins may be a surrogate marker of histamine-dependent pruritogen, while GABA/Ins was highly significantly different between normal saline group and α-Me-5-HT group (P=0.008), suggesting that GABA/Ins may be a surrogate marker of histamine-independent pruritogen. MRS may reflect the extent of pruritus intensity elicited by α-Me-5-HT and compound 48/80 with sensitivity similar to the number of scratches, and above potential markers need to be further validated in pre-clinical and clinical treatment trials.
Assuntos
Ácido Aspártico/análogos & derivados , Inositol/análise , Prurido/diagnóstico por imagem , Serotonina/análogos & derivados , Medula Espinal/diagnóstico por imagem , p-Metoxi-N-metilfenetilamina/efeitos adversos , Animais , Ácido Aspártico/análise , Biomarcadores/análise , Injeções Intradérmicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espectroscopia de Prótons por Ressonância Magnética , Prurido/induzido quimicamente , Serotonina/efeitos adversos , Medula Espinal/químicaRESUMO
Proper glutamatergic neurotransmission requires a balance between glutamate release and removal. The removal is mainly catalyzed by the glutamate transporters EAAT1-3, while the glutamate-cystine exchanger (system xc- with specific subunit xCT) represents one of the release mechanisms. Previous studies of the spinal cord have focused on the cellular distribution of EAAT1-3 with special reference to the dorsal horn, but have not provided quantitative data and have not systematically compared multiple segments. Here we have studied the distribution of EAAT1-3 and xCT in sections of multiple spinal cord segments using knockout tissue as negative controls. EAAT2 and EAAT3 were evenly expressed in all gray matter areas at all segmental levels, albeit with slightly higher levels in laminae 1-4 (dorsal horn). Somewhat higher levels of EAAT2 were also seen in lamina 9 (ventral horn), while EAAT3 was also detected in the lateral spinal nucleus. EAAT1 was concentrated in laminae 1-3, lamina 10, the intermediolateral nucleus and the sacral parasympathetic nucleus, while xCT was concentrated in laminae 1-3, lamina 10 and the leptomeninges. The levels of these four transporters were low in white matter, which represents 42% of the spinal cord volume. Quantitative immunoblotting revealed that the average level of EAAT1 in the whole spinal cord was 0.6 ± 0.1% of that in the cerebellum, while the levels of EAAT2, EAAT3 and xCT were, respectively, 41.6 ± 12%, 39.8 ± 7.6%, and 30.8 ± 4.3% of the levels in the hippocampus (mean values ± SEM). Conclusions: Because the hippocampal tissue content of EAAT2 protein is two orders of magnitude higher than the content of the EAAT3, it follows that most of the gray matter in the spinal cord depends almost exclusively on EAAT2 for glutamate removal, while the lamina involved in the processing of autonomic and nociceptive information rely on a complex system of transporters.
Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 3 de Aminoácido Excitatório/metabolismo , Medula Espinal/metabolismo , Sistema y+ de Transporte de Aminoácidos/análise , Animais , Transportador 1 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/análise , Transportador 3 de Aminoácido Excitatório/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Medula Espinal/químicaRESUMO
Two patients with familial amyotrophic lateral sclerosis (ALS) and mutations in the gene encoding superoxide dismutase 1 (SOD1) were treated with a single intrathecal infusion of adeno-associated virus encoding a microRNA targeting SOD1. In Patient 1, SOD1 levels in spinal cord tissue as analyzed on autopsy were lower than corresponding levels in untreated patients with SOD1-mediated ALS and in healthy controls. Levels of SOD1 in cerebrospinal fluid were transiently and only slightly lower in Patient 1 but were not affected in Patient 2. In Patient 1, meningoradiculitis developed after the infusion; Patient 2 was pretreated with immunosuppressive drugs and did not have this complication. Patient 1 had transient improvement in the strength of his right leg, a measure that had been relatively stable throughout his disease course, but there was no change in his vital capacity. Patient 2 had stable scores on a composite measure of ALS function and a stable vital capacity during a 12-month period. This study showed that intrathecal microRNA can be used as a potential treatment for SOD1-mediated ALS.
Assuntos
Esclerose Lateral Amiotrófica/terapia , MicroRNAs/uso terapêutico , Superóxido Dismutase-1/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/genética , Dependovirus , Evolução Fatal , Inativação Gênica , Terapia Genética , Vetores Genéticos , Humanos , Injeções Espinhais , Masculino , Meningoencefalite , Pessoa de Meia-Idade , Mutação , Estudo de Prova de Conceito , Medula Espinal/química , Medula Espinal/patologia , Superóxido Dismutase-1/análise , Superóxido Dismutase-1/genética , Capacidade Vital , Adulto JovemRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting motor neurons which shows sexual dimorphism in its incidence, age of onset, and progression rate. All steroid hormones, including androgens, estrogens, and progestogens, have been implicated in modulating ALS. Increasing evidence suggests that steroid hormones provide neuroprotective and neurotrophic support to motor neurons, either directly or via surrounding glial cell interactions, by activating their respective nuclear hormone receptors and initiating transcriptional regulatory responses. The SOD1G93A transgenic mouse also shows sex-specific differences in age of onset and progression, and remains the most widely used model in ALS research. To provide a more comprehensive understanding of the influences of steroid hormone signaling in ALS, we systemically characterized sex hormone receptor expression at transcript and protein levels, cellular localization, and the impact of disease course in lumbar spinal cords of male and female SOD1G93A mice. We found that spinal motor neurons highly express nuclear androgen receptor (AR), estrogen receptor (ER)α, ERß, and progesterone receptor with variations in glial cell expression. AR showed the most robust sex-specific difference in expression and was downregulated in male SOD1G93A mouse spinal cord, in association with depletion in 5α-reductase type 2 isoform, which primarily metabolizes testosterone to 5α-dihydrotestosterone. ERα was highly enriched in reactive astrocytes of SOD1G93A mice and ERß was strongly upregulated. The 5α-reductase type 1 isoform was upregulated with disease progression and may influence local spinal cord hormone levels. In conclusion, steroid hormone receptor expression is dynamic and cell-type specific in SOD1G93A mice which may provide targets to modulate progression in ALS.