Table_3_Measurement of Neutrophil Gelatinase-Associated Lipocalin Concentration in Canine Cerebrospinal Fluid and Serum and Its Involvement in Neuroinflammation.XLSX

Neutrophil gelatinase-associated Lipocalin (NGAL) is a glycoprotein involved in inflammation acting as an acute phase protein and chemokine as well as a regulator of iron homeostasis. NGAL has been shown to be upregulated in experimental autoimmune encephalomyelitis (EAE) in mice. Increased NGAL concentration in cerebrospinal fluid (CSF) and expression in central nervous system (CNS) has been described in human neuroinflammatory disease such as multiple sclerosis and neuropsychiatric lupus as well as in bacterial meningitis. We aimed to investigate involvement of NGAL in spontaneous canine neuroinflammation as a potential large animal model for immune- mediated neurological disorders. A commercially available Enzyme-linked Immunosorbent Assay (ELISA) for detection of canine NGAL was validated for use in canine CSF. Concentration in CSF and serum of canine patients suffering from steroid- responsive meningitis- arteriitis (SRMA), Meningoencephalitis of unknown origin (MUO), different non- inflammatory CNS disease and control dogs were compared. Relationship between NGAL concentration in CSF and serum and inflammatory parameters in CSF and blood (IgA concentration, total nucleated cell count (TNCC), protein content) as well as association with erythrocytes in CSF, duration of illness, plasma creatinine and urinary leucocytes were evaluated. In dogs with SRMA and MUO, CSF concentration of NGAL was significantly higher than in dogs with idiopathic epilepsy, compressive myelopathy, intracranial neoplasia and SRMA in remission (p < 0.0001). Patients with acute SRMA had significantly higher levels of NGAL in CSF than neurologically normal controls (p < 0.0001). Serum NGAL concentrations were significantly higher in dogs with SRMA than in patients with myelopathy and intracranial neoplasia (p < 0.0001). NGAL levels in CSF were strongly positively associated with IgA concentration (rSpear= 0.60116, p < 0.0001), TNCC (rSpear= 0.65746, p < 0.0001) and protein content (rSpear= 0.73353, p < 0.0001) in CSF. It can be measured in CSF of healthy and diseased dogs. Higher concentrations in canine patients with SRMA as well as positive association with TNCC in CSF suggest an involvement in pro-inflammatory pathways and chemotaxis in SRMA. High serum levels of NGAL in serum of SRMA patients in different stages of disease might reflect the systemic character of the disease.

中性粒细胞明胶酶相关脂质运载蛋白（Neutrophil gelatinase-associated Lipocalin, NGAL）是一种参与炎症反应的糖蛋白，兼具急性期蛋白、趋化因子的功能，同时可调控铁稳态。已有研究证实，小鼠实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）模型中NGAL的表达会上调。在人类多发性硬化、神经精神性狼疮等神经炎症性疾病以及细菌性脑膜炎患者中，均已观察到脑脊液（cerebrospinal fluid, CSF）中NGAL浓度升高，且中枢神经系统（central nervous system, CNS）内NGAL表达异常。本研究旨在探究NGAL在自发性犬类神经炎症中的作用，将其作为免疫介导性神经系统疾病的潜在大型动物模型。本研究对商业化犬NGAL检测酶联免疫吸附试验（Enzyme-linked Immunosorbent Assay, ELISA）进行了方法学验证，使其可适用于犬脑脊液样本检测。本研究比较了类固醇反应性脑膜炎-动脉炎（steroid-responsive meningitis-arteriitis, SRMA）、未知病因性脑膜脑炎（Meningoencephalitis of unknown origin, MUO）、各类非炎症性中枢神经系统疾病患犬以及健康对照犬的脑脊液与血清NGAL浓度，并评估了脑脊液及血清中NGAL浓度与脑脊液、血液炎症指标（IgA浓度、有核细胞总数total nucleated cell count, TNCC、蛋白含量）的相关性，以及NGAL与脑脊液红细胞数、病程、血浆肌酐及尿白细胞的关联。结果显示，SRMA及MUO患犬的脑脊液NGAL浓度显著高于特发性癫痫、压迫性脊髓病、颅内肿瘤患犬以及处于缓解期的SRMA患犬（p < 0.0001）。急性SRMA患犬的脑脊液NGAL水平显著高于神经学正常的对照犬（p < 0.0001）。SRMA患犬的血清NGAL浓度也显著高于脊髓病及颅内肿瘤患犬（p < 0.0001）。脑脊液NGAL水平与脑脊液内IgA浓度（斯皮尔曼相关系数rSpear=0.60116, p < 0.0001）、有核细胞总数（rSpear=0.65746, p < 0.0001）及蛋白含量（rSpear=0.73353, p < 0.0001）呈强正相关。NGAL可在健康及患病犬的脑脊液中被检测到。SRMA患犬NGAL浓度升高，且与脑脊液有核细胞总数呈正相关，提示NGAL可能参与SRMA的促炎症通路及趋化过程。不同疾病阶段的SRMA患者血清中高NGAL水平，或可反映该疾病的全身性特征。