Section of Infectious Diseases, Department of Medicine, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan and National Yang-Ming University, Taipei, Taiwan, R.O.C.
Department of Parasitology and Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, R.O.C.
PLoS One. 2019 Mar 7;14(3):e0213244. doi: 10.1371/journal.pone.0213244. eCollection 2019.
The 14-3-3 proteins are cerebrospinal fluid (CSF) markers of neuronal damage during infectious meningitis and Creutzfeldt-Jakob disease. Little is known about dynamic changes in the individual isoforms in response to parasitic eosinophilic meningitis. The purposes of this study were to determine the 14-3-3 protein isoform patterns, examine the kinetics and correlate the severity of blood brain barrier (BBB) damage with the expressions of these markers in mice with eosinophilic meningitis. Mice were orally infected with 50 A. cantonensis L3 via an oro-gastric tube and sacrificed every week for 3 consecutive weeks after infection. The Evans blue method and BBB junctional protein expressions were used to measure changes in the BBB. Hematoxylin and eosin staining was used to analyze pathological changes in the mice brains following 1-3 weeks of infection with A. cantonensis. The levels of 14-3-3 protein isoforms in serum/CSF and brain homogenates were analyzed by Western blot, and immunohistochemistry (IHC) was used to explore the different isoform distributions of 14-3-3 proteins and changes in BBB junctional proteins in the mice brain meninges. Dexamethasone was injected intraperitoneally from the seventh day post infection (dpi) until the end of the study (21 dpi) to study the changes in BBB junctional proteins. The amounts of Evans blue, tight junction and 14-3-3 protein isoforms in the different groups of mice were compared using the nonparametric Kruskal-Wallis test. There were significant increases in 14-3-3 protein isoforms β and γ in the CSF in the second and third weeks after infection compared to the controls and first week of infection, which were correlated with the severity of BBB damage in brain histology, and Evans blue extravasation. Using IHC to assess the distribution of 14-3-3 protein isoforms and changes in BBB junctional proteins in the mice brain meninges, the expressions of isoforms β, γ, ε, and θ and junctional proteins occludin and claudin-5 in the brain meninges increased over a 3-week period after infection compared to the controls and 1 week after infection. The administration of dexamethasone decreased the expressions of BBB junctional proteins occludin and claudin-5 in the mice brain meninges. Our findings support that 14-3-3 proteins β and γ can potentially be used as a CSF marker of neuronal damage in parasitic eosinophilic meningitis caused by A. cantonensis.
14-3-3 蛋白是感染性脑膜炎和克雅氏病期间神经元损伤的脑脊液（CSF）标志物。关于寄生虫嗜酸性脑膜炎时个别同工型的动态变化知之甚少。本研究旨在确定 14-3-3 蛋白同工型模式，检查动力学并将血脑屏障（BBB）损伤的严重程度与这些标志物在感染嗜酸性脑膜炎的小鼠中的表达相关联。通过经口胃管将 50 条广州管圆线虫 L3 感染给小鼠，并在感染后连续 3 周每周进行一次处死。使用 Evans 蓝法和 BBB 连接蛋白表达来测量 BBB 的变化。苏木精和伊红染色用于分析感染广州管圆线虫后 1-3 周小鼠大脑的病理变化。通过 Western blot 分析血清/CSF 和脑匀浆中 14-3-3 蛋白同工型的水平，并通过免疫组化（IHC）来探索 14-3-3 蛋白的不同同工型分布和小鼠脑膜中 BBB 连接蛋白的变化。从感染后第 7 天（dpi）开始到研究结束（21 dpi），通过腹腔内注射地塞米松来研究 BBB 连接蛋白的变化。使用非参数 Kruskal-Wallis 检验比较不同组小鼠的 Evans 蓝、紧密连接和 14-3-3 蛋白同工型的量。与对照组和感染第 1 周相比，感染后第 2 和第 3 周 CSF 中 14-3-3 蛋白同工型 β 和 γ 的量显著增加，与脑组织学中 BBB 损伤的严重程度和 Evans 蓝外渗相关。使用 IHC 评估小鼠脑膜中 14-3-3 蛋白同工型的分布和 BBB 连接蛋白的变化，与对照组和感染第 1 周相比，感染后 3 周内脑膜中同工型 β、γ、ε和θ以及连接蛋白 occludin 和 claudin-5 的表达增加。地塞米松的给药降低了小鼠脑膜中 BBB 连接蛋白 occludin 和 claudin-5 的表达。我们的研究结果支持 14-3-3 蛋白 β 和 γ 可以作为由广州管圆线虫引起的寄生虫嗜酸性脑膜炎中神经元损伤的 CSF 标志物。
