RESUMO
This study aimed to investigate the galectin-3 and associated cytokines levels in the cerebrospinal fluid (CSF) of severe traumatic brain injury (sTBI) patients. Temporal CSF expression of galectin-3 and associated cytokines levels in sTBI patients within 1-week post-injury were studied using the multiplex bead array. STBI patient group was stratified using the Modified Rankin Score (mRS) into 3 groups: mRS 6 (died), mRS 5 (severely disabled) and mRS 1-4 (mild-to-moderately disabled) group. Analysis for bead array data using Kruskal-Wallis test with post hoc Dunn's multiple comparisons test, and temporal changes and correlation analysis using Spearman's correlation were carried out. At day 1 post-injury, CSF galectin-3 and interleukin-6 (IL-6), interleukin-10 (IL-10), cysteine-cysteine motif chemokine ligand-2 (CCL-2), and cysteine-cysteine motif chemokine ligand-20 (CCL-20), but not interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF-α) levels were significantly elevated in mRS 5 group compared to non-TBI controls. Temporal correlation analysis at 1-7 days showed decreased IL-10 level in the mRS 6 group, decreased IL-10 and CCL-2 levels in mRS 5 group, and decreased IL-6, CCL-2, and CCL-20 levels in the mRS 1-4 group. Receiver operating characteristic curve analyses revealed a significant area under the curve for comparison between mRS 6 and mRS 5 groups for galectin-3 and IL-6. No significant differences in sex, age, Glasgow Coma Scale score, C-reactive protein levels and types of TBI-induced hemorrhages were observed between the groups. CSF galectin-3 and associated cytokines, especially IL-6, CCL-2 and CCL-20 levels were different within sub-groups of sTBI patients, suggesting their potential use in sTBI prognostics.
Assuntos
Lesões Encefálicas Traumáticas , Citocinas , Galectina 3 , Humanos , Lesões Encefálicas Traumáticas/líquido cefalorraquidiano , Masculino , Feminino , Pessoa de Meia-Idade , Citocinas/líquido cefalorraquidiano , Adulto , Galectina 3/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Galectinas/líquido cefalorraquidiano , Idoso , Adulto Jovem , Proteínas SanguíneasRESUMO
Hypoxic-ischaemic encephalopathy (HIE) is an important cause of morbidity and mortality globally. Although mild therapeutic hypothermia (TH) may improve outcomes in selected babies, the mechanism of action is not fully understood. A proteomics discovery study was carried out to analyse proteins in the plasma of newborns with HIE. Proteomic analysis of plasma from 22 newborns with moderate-severe HIE that had initially undergone TH, and relative controls including 10 newborns with mild HIE who did not warrant TH and also cord blood from 10 normal births (non-HIE) were carried out using the isobaric Tandem Mass Tag (TMT®) 10plexTM labelling with tandem mass spectrometry. A total of 7818 unique peptides were identified in all TMT10plexTM samples, translating to 3457 peptides representing 405 proteins, after applying stringent filter criteria. Apart from the unique protein signature from normal cord blood, unsupervised analysis revealed several significantly regulated proteins in the TH-treated moderate-severe HIE group. GO annotation and functional clustering revealed various proteins associated with glucose metabolism: the enzymes fructose-bisphosphate aldolase A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase 1, phosphoglycerate kinase 1, and pyruvate kinase PKM were upregulated in newborns with favourable (sHIE+) outcomes compared to newborns with unfavourable (sHIE-) outcomes. Those with favourable outcomes had normal MR imaging or mild abnormalities not predictive of adverse outcomes. However, in comparison to mild HIE and the sHIE- groups, the sHIE+ group had the additional glucose metabolism-related enzymes upregulated, including triosephosphate isomerase, α-enolase, 6-phosphogluconate dehydrogenase, transaldolase, and mitochondrial glutathione reductase. In conclusion, our plasma proteomic study demonstrates that TH-treated newborns with favourable outcomes have an upregulation in glucose metabolism. These findings may open new avenues for more effective neuroprotective therapy.
Assuntos
Asfixia , Proteômica , Lactente , Humanos , Recém-Nascido , Metabolismo dos Carboidratos , Espectrometria de Massas em Tandem , PeptídeosRESUMO
BACKGROUND: Initial tumor enlargement (or pseudoprogression) instead of true tumor progression is a common phenomenon in patients with acoustic neuromas who are treated with stereotactic radiosurgery (SRS). This phenomenon can affect clinical decision-making and patient management. This study assessed the correlation between initial tumor enlargement and magnetic resonance imaging characteristics in patients with acoustic neuromas who were treated with linear accelerator (LINAC)-based SRS. The long-term tumor control outcomes were also analyzed. MATERIALS AND METHODS: In total, 330 patients with sporadic acoustic neuromas who were treated with LINAC SRS between March 2006 and March 2020 were retrospectively evaluated to assess their initial tumor enlargement. The tumors were divided into homogeneously enhanced, heterogeneously enhanced, and cystic types based on the morphological characteristics noted on magnetic resonance images. Tumor control was assessed in 275 patients with a follow-up duration of more than 2 years. RESULTS: Initial enlargement was observed in 137 of 330 (41.5%) tumors as early as 3 months after LINAC SRS. Data analysis revealed that postoperative tumors with a residual volume lower than 2.5â¯cm3 had a lower incidence of initial enlargement (pâ¯= 0.039). No correlation was noted between the initial enlargement and morphological characteristics of tumors. In patients with a mean follow-up duration of 82.8⯱ 37.2 months, heterogeneously enhanced tumors exhibited a lower control rate than homogeneously enhanced and cystic tumors (pâ¯= 0.045). No correlation was noted between initial enlargement and tumor control. CONCLUSION: Initial enlargement can occur as early as 3 months after SRS. Postoperative residual tumors with a volume lower than 2.5â¯cm3 exhibit a lower incidence of initial enlargement. Heterogeneously enhanced tumors have a lower local control rate.
Assuntos
Neuroma Acústico , Radiocirurgia , Humanos , Radiocirurgia/métodos , Neuroma Acústico/diagnóstico por imagem , Neuroma Acústico/radioterapia , Neuroma Acústico/cirurgia , Estudos Retrospectivos , Imageamento por Ressonância Magnética , Aceleradores de Partículas , Seguimentos , Resultado do TratamentoRESUMO
Neuroinflammation has recently been identified as a fundamentally important pathological process in most, if not all, CNS diseases. The main contributor to neuroinflammation is the microglia, which constitute the innate immune response system. Accurate identification of microglia and their reactivity state is therefore essential to further our understanding of CNS pathophysiology. Many staining techniques have been used to visualise microglia in rodent and human tissue, and immunostaining is currently the most frequently used. Historically, identification of microglia was predominantly based on morphological structure, however, recently there has been a reliance on selective antigen expression, and microglia-specific markers have been identified providing increased certainty that the cells observed are in fact microglia, rather than the similar yet distinct macrophages. To date, the most microglia-specific markers are P2Y12 and TMEM119. However, other microglia-related markers can also be useful for demonstrating activation state, phagocytic state, and for neuroimaging purposes in longitudinal studies. Overall, it is important to be aware of the microglia-selectivity issues of the various stains and immunomarkers used by researchers to distinguish microglia in CNS tissue to avoid misinterpretation.
Assuntos
Sistema Nervoso Central , Microglia , Sistema Nervoso Central/metabolismo , Humanos , Imunidade Inata , Macrófagos/metabolismo , Microglia/metabolismoRESUMO
Traumatic brain injury (TBI) is an extremely complex disease and current systems classifying TBI as mild, moderate, and severe often fail to capture this complexity. Neuroimaging cannot resolve the cellular and molecular changes due to lack of resolution, and post-mortem tissue examination may not adequately represent acute disease. Therefore, we examined the cellular and molecular sequelae of TBI in fresh brain samples and related these to clinical outcomes. Brain biopsies, obtained shortly after injury from 25 living adult patients suffering severe TBI, underwent immunohistochemical analysis. There were no adverse events. Immunostaining revealed various qualitative cellular and biomolecular changes relating to neuronal injury, dendritic injury, neurovascular injury, and neuroinflammation, which we classified into 4 subgroups for each injury type using the newly devised Yip, Hasan and Uff (YHU) grading system. Based on the Glasgow Outcome Scale-Extended, a total YHU grade of ≤8 or ≥11 had a favourable and unfavourable outcome, respectively. Biomolecular changes observed in fresh brain samples enabled classification of this heterogeneous patient population into various injury severity categories based on the cellular and molecular pathophysiology according to the YHU grading system, which correlated with outcome. This is the first study investigating the acute biomolecular response to TBI.
RESUMO
Microglial proliferation and activation and macrophage accumulation are implicated in neuropathic pain development. In this study, we aim to suppress microgliosis and macrophage accumulation by over-expressing a non-functional soluble colony stimulating factor-1 receptor (sCSF1R) using an adeno-associated virus 9 vector (AAV9). AAV9/sCSF1R and the control vector AAV9/GFP were intrathecally administered into the lumbar spine of adult C57BL/6 mice. Two weeks later, these mice underwent partial sciatic nerve ligation to induce neuropathic pain. GFP and sCSF1R were highly expressed in lumbar dorsal root ganglia (DRG) and spinal cord of AAV9-injected mice. A significant increase in microglia densities in the dorsal and ventral horns of lumbar spinal cords and macrophage densities in DRG and sciatic nerves were observed in the mice with either ligation alone or pre-treated with AAV9/GFP. In nerve-ligated mice pre-treated with AAV9/sCSF1R the microglia densities in the dorsal and ventral horns and macrophage densities in DRG and sciatic nerves were significantly lower compared to nerve-ligated mice pre-treated with AAV9/GFP. Behavioral tests showed that nerve-ligated mice pre-treated with AAV9/sCSF1R had a significantly higher paw withdrawal threshold, indicating the alleviation of neuropathic pain. The results implicate that viral vector-mediated expression of sCSF1R may represent a novel strategy in the alleviation of neuropathic pain.
Assuntos
Fator Estimulador de Colônias de Macrófagos , Neuralgia , Animais , Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Neuralgia/metabolismo , Neuralgia/terapia , Receptor de Fator Estimulador de Colônias de Macrófagos , Medula Espinal/metabolismoRESUMO
There is conflicting results on whether prior antiplatelet therapy (APT) is associated with poor outcome in spontaneous intracerebral haemorrhage (ICH) patients. To determine whether prior APT is associated with spontaneous ICH, and whether there is a difference between the different types of APT, including cyclooxygenase inhibitor (COX-I), adenosine diphosphate receptor inhibitor (ADP-I) and phosphodiesterase inhibitor (PDE-I). A retrospective study of patients with ICH diagnosed between 2001 and 2013 in the National Health Insurance Research Database. Baseline unbalance between APT and non-APT groups was solved by multivariable adjustment (primary analysis) and propensity score matching (sensitivity analysis). Patients with prior APT had a higher rate of in-hospital death (odds ratio [OR], 1.16; 95% confidence interval [CI], 1.09-1.23) compared to non-APT group. Compared to non-APT group, there was a greater rate of in-hospital death with spontaneous ICH with ADP-I (OR, 1.49; 95% CI, 1.24-1.79) and COX-I (OR, 1.17; 95% CI, 1.09-1.25). PDE-I exhibited no difference in in-hospital death with spontaneous ICH (OR, 1.03; 95% CI, 0.91-1.16) compared to non-APT group. Remarkably, the in-hospital mortality rate was significantly higher in the ADP-I group than in the PDE-I group (hazard ratio, 1.45; 95% CI, 1.17-1.80). In this study, ADP-I and COX-1, but not PDE-I, are the most likely contributors to the association of APT with poor outcome with spontaneous ICH patients. These findings suggest that the complexity of the different mechanism of actions of prior APT can alter the outcome in spontaneous ICH.
Assuntos
Hemorragia Cerebral/induzido quimicamente , Inibidores de Fosfodiesterase/efeitos adversos , Inibidores da Agregação Plaquetária/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Hemorragia Cerebral/epidemiologia , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
Microglia are activated after spinal cord injury (SCI), but their phagocytic mechanisms and link to neuroprotection remain incompletely characterized. Docosahexaenoic acid (DHA) has been shown to have significant neuroprotective effects after hemisection and compression SCI and can directly affect microglia in these injury models. In rodent contusion SCI, we demonstrate that DHA (500 nmol/kg) administered acutely post-injury confers neuroprotection and enhances locomotor recovery, and also exerts a complex modulation of the microglial response to injury. In rodents, at 7 days after SCI, the level of phagocytosed myelin within Iba1-positive or P2Y12-positive cells was significantly lower after DHA treatment, and this occurred in parallel with an increase in intracellular miR-124 expression. Furthermore, intraspinal administration of a miR-124 inhibitor significantly reduced the DHA-induced decrease in myelin phagocytosis in mice at 7 days post-SCI. In rat spinal primary microglia cultures, DHA reduced the phagocytic response to myelin, which was associated with an increase in miR-124, but not miR-155. A similar response was observed in a microglia cell line (BV2) treated with DHA, and the effect was blocked by a miR-124 inhibitor. Furthermore, the phagocytic response of BV2 cells to stressed neurones was also reduced in the presence of DHA. In peripheral monocyte-derived macrophages, the expression of the M1, but not the M0 or M2 phenotype, was reduced by DHA, but the phagocytic activation was not altered. These findings show that DHA induces neuroprotection in contusion injury. Furthermore, the improved outcome is via a miR-124-dependent reduction in the phagocytic response of microglia.
Assuntos
Ácidos Docosa-Hexaenoicos/uso terapêutico , MicroRNAs/metabolismo , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Contusões/tratamento farmacológico , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/farmacologia , Feminino , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/metabolismo , Bainha de Mielina/metabolismo , Neurônios/metabolismo , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Células PC12 , Ratos , Ratos Sprague-DawleyRESUMO
Docosahexaenoic acid (DHA) is an ω-3 polyunsaturated fatty acid that is essential in brain development and has structural and signaling roles. Acute DHA administration is neuroprotective and promotes functional recovery in animal models of adult spinal cord injury (SCI). However, the mechanisms underlying this recovery have not been fully characterized. Here we investigated the effects of an acute intravenous bolus of DHA delivered after SCI and characterized DHA-induced neuroplasticity within the adult injured spinal cord. We found robust sprouting of uninjured corticospinal and serotonergic fibers in a rat cervical hemisection SCI model. A mouse pyramidotomy model was used to confirm that this robust sprouting was not species or injury model specific. Furthermore, we demonstrated that corticospinal fibers sprouting to the denervated side of the cord following pyramidotomy contact V2a interneurons. We also demonstrated increased serotonin fibers and synaptophysin in direct contact with motor neurons. DHA also increased synaptophysin in rat cortical cell cultures. A reduction in phosphatase and tensin homolog (PTEN) has been shown to be involved in axonal regeneration and synaptic plasticity. We showed that DHA significantly upregulates miR-21 and downregulates PTEN in corticospinal neurons. Downregulation of PTEN and upregulation of phosphorylated AKT by DHA were also seen in primary cortical neuron cultures and were accompanied by increased neurite outgrowth. In summary, acute DHA induces anatomical and synaptic plasticity in adult injured spinal cord. This study shows that DHA has therapeutic potential in cervical SCI and provides evidence that DHA could exert its beneficial effects in SCI via enhancement of neuroplasticity. SIGNIFICANCE STATEMENT: In this study, we show that an acute intravenous injection of docosahexaenoic acid (DHA) 30 min after spinal cord injury induces neuroplasticity. We found robust sprouting of uninjured corticospinal and serotonergic fibers in a rat hemisection spinal cord injury model. A mouse pyramidotomy model was used to confirm that the robust sprouting involved V2a interneurons. We show that DHA significantly upregulates miR-21 and phosphorylated AKT, and downregulates phosphatase and tensin homolog (PTEN), which is involved in suppressing anatomical plasticity, in corticospinal neurons and in primary cortical neuron cultures. We conclude that acute DHA can induce anatomical and synaptic plasticity. This provides direct evidence that DHA could exert its beneficial effects in spinal cord injury via neuroplasticity enhancement.
Assuntos
Ácidos Docosa-Hexaenoicos/uso terapêutico , Interneurônios/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Tratos Piramidais/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Medula Espinal/efeitos dos fármacos , Animais , Células Cultivadas , Vértebras Cervicais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Comportamento Exploratório/efeitos dos fármacos , Feminino , Transtornos Neurológicos da Marcha/tratamento farmacológico , Transtornos Neurológicos da Marcha/etiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Intravenosas , Interneurônios/fisiologia , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , Neurônios Motores/fisiologia , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/ultraestrutura , Plasticidade Neuronal/fisiologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , PTEN Fosfo-Hidrolase/biossíntese , PTEN Fosfo-Hidrolase/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tratos Piramidais/lesões , Tratos Piramidais/patologia , Tratos Piramidais/fisiologia , Ratos , Ratos Sprague-Dawley , Neurônios Serotoninérgicos/fisiologia , Neurônios Serotoninérgicos/ultraestrutura , Medula Espinal/fisiologia , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
OBJECTIVE: The objective of the present study was to determine whether selective inflammatory cytokine concentrations within cerebrospinal fluid are useful markers for the differential diagnosis of aseptic and bacterial meningitis within neurosurgical patients. DESIGN: Prospective, open-label, observational, cohort study. SETTING: Neurosurgical ICU, Chang Gung Memorial Hospital. PATIENTS: Thirty-two consecutive neurosurgical patients who had postoperative fever following external ventricular drain insertion for the treatment of brain injury underwent serial cerebrospinal fluid cytokine analysis pre and post fever to determine the value of such markers in ascertaining the differential diagnosis of meningitis. INTERVENTION: Cerebrospinal fluid samples were collected on the day of fever onset, as well as on day 2 and 4 pre and post fever development. Tumor necrosis factor-α, interleukin-1ß, interleukin-6, interleukin-8, transforming growth factor-ß, and procalcitonin were subsequently analyzed using enzyme-linked immunosorbent assay analysis techniques. MEASUREMENT AND MAIN RESULTS: Inflammatory marker levels were compared among febrile aseptic, bacterial, and nonmeningitis patients to determine cerebrospinal fluid inflammatory changes over time. Significant increases in cerebrospinal fluid tumor necrosis factor -α, interleukin-1ß, interleukin-6, and interleukin-8 levels were observed within patients with bacterial meningitis at fever onset, which was not evident in aseptic or nonmeningitis patients. Furthermore, significant increases in cerebrospinal fluid tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interleukin-8 levels were detected as early as 4 days prior to fever onset within patients with bacterial meningitis when compared with both aseptic and nonmeningitis groups. Interestingly, procalcitonin was only significantly increased in patients with bacterial meningitis on the fourth day post fever. CONCLUSION: The present study suggests that raised cerebrospinal fluid tumor necrosis factor -α, interleukin-1ß, and interleukin-8 in a temporal manner may indicate early bacterial meningitis development in neurosurgical patients, enabling earlier diagnostic certainty and improved patient outcomes.
Assuntos
Citocinas/sangue , Meningite Asséptica/líquido cefalorraquidiano , Meningites Bacterianas/líquido cefalorraquidiano , Procedimentos Neurocirúrgicos/efeitos adversos , Adulto , Idoso , Área Sob a Curva , Calcitonina/líquido cefalorraquidiano , Peptídeo Relacionado com Gene de Calcitonina , Estudos de Coortes , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Feminino , Febre/líquido cefalorraquidiano , Febre/etiologia , Humanos , Mediadores da Inflamação/líquido cefalorraquidiano , Interleucina-6/análise , Interleucina-8/análise , Masculino , Meningite Asséptica/diagnóstico , Meningite Asséptica/etiologia , Meningite Asséptica/mortalidade , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/etiologia , Meningites Bacterianas/mortalidade , Pessoa de Meia-Idade , Procedimentos Neurocirúrgicos/métodos , Complicações Pós-Operatórias/líquido cefalorraquidiano , Complicações Pós-Operatórias/fisiopatologia , Prognóstico , Estudos Prospectivos , Precursores de Proteínas/líquido cefalorraquidiano , Curva ROC , Medição de Risco , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/análiseRESUMO
Chronic visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage-gated sodium channel subtype NaV1.9 is expressed in half of gut-projecting rodent dorsal root ganglia sensory neurons. We show that NaV1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE2 were substantially reduced in NaV1.9(-/-) mice. Deletion of NaV1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE2, and 5HT) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of NaV1.9, and there was a rightward shift in stimulus-response function to ramp colonic distension. By contrast, responses to rapid, high-intensity phasic distension of the colon are initially unaffected; however, run-down of responses to repeat phasic distension were exacerbated in NaV1.9(-/-) afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in NaV1.9(-/-) mice. These results demonstrate that NaV1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that NaV1.9 represents a high-value target for development of visceral analgesics.
Assuntos
Colo/inervação , Hiperalgesia/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.9/metabolismo , Fibras Aferentes Viscerais/metabolismo , Potenciais de Ação/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Adolescente , Adulto , Idoso , Animais , Colo/metabolismo , Colo/fisiopatologia , Dinoprostona/farmacologia , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Humanos , Hiperalgesia/fisiopatologia , Inflamação/metabolismo , Inflamação/fisiopatologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.9/genética , Estimulação Física , Fibras Aferentes Viscerais/efeitos dos fármacos , Fibras Aferentes Viscerais/fisiopatologia , Adulto JovemRESUMO
Pain remains an area of considerable unmet clinical need, and this is particularly true of pain associated with bone metastases, in part because existing analgesic drugs show only limited efficacy in many patients and in part because of the adverse side effects associated with these agents. An important issue is that the nature and roles of the algogens produced in bone that drive pain-signalling systems remain unknown. Here, we tested the hypothesis that adenosine triphosphate is one such key mediator through actions on P2X3 and P2X2/3 receptors, which are expressed selectively on primary afferent nocioceptors, including those innervating the bone. Using a well-established rat model of bone cancer pain, AF-353, a recently described potent and selective P2X3 and P2X2/3 receptor antagonist, was administered orally to rats and found to produce highly significant prevention and reversal of bone cancer pain behaviour. This attenuation occurred without apparent modification of the disease, since bone destruction induced by rat MRMT-1 carcinoma cells was not significantly altered by AF-353. Using in vivo electrophysiology, evidence for a central site of action was provided by dose-dependent reductions in electrical, mechanical and thermal stimuli-evoked dorsal horn neuronal hyperexcitability following direct AF-353 administration onto the spinal cord of bone cancer animals. A peripheral site of action was also suggested by studies on the extracellular release of adenosine triphosphate from MRMT-1 carcinoma cells. Moreover, elevated phosphorylated-extracellular signal-regulated kinase expression in dorsal root ganglion neurons, induced by co-cultured MRMT-1 carcinoma cells, was significantly reduced in the presence of AF-353. These data suggest that blockade of P2X3 and P2X2/3 receptors on both the peripheral and central terminals of nocioceptors contributes to analgesic efficacy in a model of bone cancer pain. Thus, systemic P2X3 and P2X2/3 receptor antagonists with central nervous system penetration may offer a promising therapeutic tool in treating bone cancer pain.
Assuntos
Dor/tratamento farmacológico , Dor/psicologia , Antagonistas do Receptor Purinérgico P2 , Pirimidinas/uso terapêutico , Trifosfato de Adenosina/metabolismo , Administração Oral , Amidinas , Animais , Neoplasias Ósseas/complicações , Neoplasias Ósseas/patologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Carcinoma/complicações , Carcinoma/patologia , Células Cultivadas , Técnicas de Cocultura/métodos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gânglios Espinais/citologia , Hiperalgesia/tratamento farmacológico , Dor/diagnóstico por imagem , Dor/etiologia , Medição da Dor , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3 , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Microtomografia por Raio-X/métodosRESUMO
P2X(3) and P2X(2/3) receptors are localized on sensory afferents both peripherally and centrally and have been implicated in various sensory functions. However, the physiological role of these receptors expressed presynaptically in the spinal cord in regulating sensory transmission remains to be elucidated. Here, a novel selective P2X(3) and P2X(2/3) antagonist, AF-792 [5-(5-ethynyl-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine, previously known as RO-5], in addition to less selective purinoceptor ligands, was applied intrathecally in vivo. Cystometry recordings were made to assess changes in the micturition reflex contractions after drug treatments. We found that AF-792 inhibited micturition reflex activity significantly (300 nmol; from baseline contraction intervals of 1.18 +/- 0.07 to 9.33 +/- 2.50 min). Furthermore, inhibition of P2X(3) and P2X(2/3) receptors in the spinal cord significantly attenuated spinal activation of extracellular-signal regulated kinases induced by acute peripheral stimulation of the bladder with 1% acetic acid by 46.4 +/- 12.0% on average. Hence, the data suggest that afferent signals originating from the bladder are regulated by spinal P2X(3) and P2X(2/3) receptors and establish directly an endogenous central presynaptic purinergic mechanism to regulate visceral sensory transmission. Identification of this spinal purinergic control in visceral activities may help the development of P2X(3) and P2X(2/3) antagonist to treat urological dysfunction, such as overactive bladder, and possibly other debilitating sensory disorders, including chronic pain states.
Assuntos
Receptores Purinérgicos P2/metabolismo , Medula Espinal/metabolismo , Bexiga Urinária/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Feminino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Pressão , Antagonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Medula Espinal/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacosRESUMO
Microglia are important in homeostasis and widely considered to have roles in the pathogenesis of conditions such as neuropathic pain and multiple sclerosis. The need to study microglia from the adult spinal cord is essential to further understand the role of these cells in disease pathology. Primary microglia are often prepared from brain tissues obtained from embryonic or perinatal age rodents and the process can take over a week to complete. The protocol in this study provides rapid isolation of microglia from adult spinal cord, allowing immediate availability for experimentation of both ex vivo and in vitro within a few hours. A purity of 99% with little or no neuronal or astrocytic contamination can be achieved. Between 70% and 85% of these adult microglia were in a relatively non-activated state. Functionally, these microglia respond to lipopolysaccharide incubation with increases in both phospho-p38 MAPK and OX42 immunostaining, as well as release of ATP, as compared to un-stimulated microglia. This technique provides a protocol to achieve rapid and efficient extraction of high purity, quiescent and functionally active microglia from adult mouse spinal cord, allowing greater study of adult spinal microglia in physiological and pathophysiological states.
Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Microglia/fisiologia , Medula Espinal/citologia , Trifosfato de Adenosina/metabolismo , Análise de Variância , Animais , Antígeno CD11b/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Contagem de Células/métodos , Proteína Glial Fibrilar Ácida/metabolismo , Indóis , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos , Microglia/efeitos dos fármacos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12 , Fatores de Tempo , Tubulina (Proteína)/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
A recent major conceptual advance has been the recognition of the importance of immune system-neuronal interactions in the modulation of brain function, one example of which is spinal pain processing in neuropathic states. Here, we report that in peripheral nerve-injured rats, the lysosomal cysteine protease cathepsin S (CatS) is critical for the maintenance of neuropathic pain and spinal microglia activation. After injury, CatS was exclusively expressed by activated microglia in the ipsilateral dorsal horn, where expression peaked at day 7, remaining high on day 14. Intrathecal delivery of an irreversible CatS inhibitor, morpholinurea-leucine-homophenylalanine-vinyl phenyl sulfone (LHVS), was antihyperalgesic and antiallodynic in neuropathic rats and attenuated spinal microglia activation. Consistent with a pronociceptive role of endogenous CatS, spinal intrathecal delivery of rat recombinant CatS (rrCatS) induced hyperalgesia and allodynia in naïve rats and activated p38 mitogen-activated protein kinase (MAPK) in spinal cord microglia. A bioinformatics approach revealed that the transmembrane chemokine fractalkine (FKN) is a potential substrate for CatS cleavage. We show that rrCatS incubation reduced the levels of cell-associated FKN in cultured sensory neurons and that a neutralizing antibody against FKN prevented both FKN- and CatS-induced allodynia, hyperalgesia, and p38 MAPK activation. Furthermore, rrCatS induced allodynia in wild-type but not CX3CR1-knockout mice. We suggest that under conditions of increased nociception, microglial CatS is responsible for the liberation of neuronal FKN, which stimulates p38 MAPK phosphorylation in microglia, thereby activating neurons via the release of pronociceptive mediators.
Assuntos
Catepsinas/antagonistas & inibidores , Microglia/enzimologia , Dor/tratamento farmacológico , Dor/etiologia , Medula Espinal/enzimologia , Animais , Catepsinas/genética , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Injeções Espinhais , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Medição da Dor , Ratos , Ratos Wistar , Proteínas Recombinantes/antagonistas & inibidores , Nervo Isquiático/lesões , Fatores de TempoRESUMO
We have developed a pyramidotomy model in mice to lesion the corticospinal tract at the level of the brainstem pyramidal tract, and evaluated the resultant impairments in motor function in a series of behavioural tests. Adult C57BL/6 mice received a unilateral pyramidotomy and a control group of mice underwent sham surgery. We studied the effects of this lesion on forepaw function using five behavioural paradigms, some of which have been widely used in rat studies but have not been fully explored in mice. The tests used were: a rearing test, which assesses forepaw use for weight support during spontaneous vertical exploration of a cylinder; a grid walking test, which assesses the ability to accurately place the forepaws during exploration of an elevated grid; a tape-removal test, which measures both sensory and motor function of the forepaw; a CatWalk automated gait analysis, which provides a number of quantitative measures including stride length and stride width during locomotion; and a staircase reaching task, which assesses skilled independent forepaw use. All tests revealed lesion effects on forepaw function with the tape removal, grid walking, rearing and CatWalk tests demonstrating robust effects throughout the testing period. The development of a pyramidotomy lesion model in mice, together with behavioural tests which can reliably measure functional impairments, will provide a valuable tool for assessing therapeutic strategies to promote regeneration and plasticity.