■脑炎是一种具有高发病率和死亡率的破坏性神经系统疾病。自身免疫原因大致与传染性原因一样常见。N-甲基-D-天冬氨酸受体(NMDAR)脑炎(NMDARE),以血清和/或脊髓液NMDAR抗体为特征,是最常见的自身免疫性脑炎(AE)。翻译啮齿动物NMDARE模型将允许AE的病理生理学研究,导致这种使人衰弱的神经精神疾病的诊断和治疗的进展。这项工作的主要目的是确定小鼠中NMDARE的最佳主动免疫条件。
■将8周龄的雌性C57BL/6J小鼠皮下注射完全弗氏佐剂的乳剂,杀死和干燥的结核分枝杆菌,和NMDARE患者抗体靶向的NMDARGluN1亚基N368/G369残基侧翼的30个氨基酸肽。使用将肽乳液混合物皮下注射到小鼠中检查了三种不同的诱导方法1)腹面,2)背侧表面,或3)在4周和8周再次免疫的背侧表面(加强)。小鼠每两周抽血一次,并在第2、4、6、8和14周处死。血清和CSFNMDAR抗体滴度,老鼠的行为,海马细胞表面和突触后NMDAR簇密度,并检查了脑免疫细胞进入和细胞因子含量。
■所有免疫小鼠均产生血清和CSFNMDAR抗体,在血清中6周和在CSF免疫后6周(腹侧和背侧增强)或8周(背侧未增强)达到峰值,免疫后6周海马NMDAR簇密度降低。与背侧免疫的小鼠相反,腹侧诱导的小鼠表现出翻译相关的表型,包括记忆缺陷和抑郁行为,脑细胞素的变化,在4周的时间点,T细胞进入大脑。仅在连续增强后,在背侧免疫的小鼠中观察到类似的记忆功能障碍和焦虑表型,这也导致更高的抗体滴度。
■我们的研究揭示了NMDARE疾病主动免疫小鼠模型中诱导方法依赖性的差异。与背侧诱导的动物相比,一种新型的腹侧诱导的NMDARE模型更早地证明了AE的特征,并且可能适用于大多数短期研究。然而,在长期的纵向研究中,增强和改善免疫反应的持久性可能是首选.
Encephalitis is a devastating neurologic disorder with high morbidity and mortality. Autoimmune causes are roughly as common as infectious ones. N-methyl-D-aspartic acid receptor (NMDAR) encephalitis (NMDARE), characterized by serum and/or spinal fluid NMDAR antibodies, is the most common form of autoimmune encephalitis (AE). A translational rodent NMDARE model would allow for pathophysiologic studies of AE, leading to advances in the diagnosis and treatment of this debilitating neuropsychiatric disorder. The main objective of this work was to identify optimal active immunization conditions for NMDARE in mice.
Female C57BL/6J mice aged 8 weeks old were injected subcutaneously with an emulsion of complete Freund\'s adjuvant, killed and dessicated Mycobacterium tuberculosis, and a 30 amino acid peptide flanking the NMDAR GluN1 subunit N368/G369 residue targeted by NMDARE patients\' antibodies. Three different induction methods were examined using subcutaneous injection of the peptide emulsion mixture into mice in 1) the ventral surface, 2) the dorsal surface, or 3) the dorsal surface with reimmunization at 4 and 8 weeks (boosted). Mice were bled biweekly and sacrificed at 2, 4, 6, 8, and 14 weeks. Serum and CSF NMDAR antibody titer, mouse behavior, hippocampal cell surface and postsynaptic NMDAR cluster density, and brain immune cell entry and cytokine content were examined.
All immunized mice produced serum and CSF NMDAR antibodies, which peaked at 6 weeks in the serum and at 6 (ventral and dorsal boosted) or 8 weeks (dorsal unboosted) post-immunization in the CSF, and demonstrated decreased hippocampal NMDAR cluster density by 6 weeks post-immunization. In contrast to dorsally-immunized mice, ventrally-induced mice displayed a translationally-relevant phenotype including memory deficits and depressive behavior, changes in cerebral cytokines, and entry of T-cells into the brain at the 4-week timepoint. A similar phenotype of memory dysfunction and anxiety was seen in dorsally-immunized mice only when they were serially boosted, which also resulted in higher antibody titers.
Our study revealed induction method-dependent differences in active immunization mouse models of NMDARE disease. A novel ventrally-induced NMDARE model demonstrated characteristics of AE earlier compared to dorsally-induced animals and is likely suitable for most short-term studies. However, boosting and improving the durability of the immune response might be preferred in prolonged longitudinal studies.