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BACKGROUND

Despite widespread searches, there are currently no validated biofluid markers for the detection of subclinical neuroinflammation in multiple sclerosis (MS). The dynamic nature of human metabolism in response to changes in homeostasis, as measured by metabolomics, may allow early identification of clinically silent neuroinflammation. Using the delayed-type hypersensitivity (DTH) MS rat model, we investigated the serum and cerebrospinal fluid (CSF) metabolomics profiles and neurofilament-light chain (NfL) levels, as a putative marker of neuroaxonal damage, arising from focal, clinically silent neuroinflammatory brain lesions and their discriminatory abilities to distinguish DTH animals from controls.

METHODS

H nuclear magnetic resonance (NMR) spectroscopy metabolomics and NfL measurements were performed on serum and CSF at days 12, 28 and 60 after DTH lesion initiation. Supervised multivariate analyses were used to determine metabolomics differences between DTH animals and controls. Immunohistochemistry was used to assess the extent of neuroinflammation and tissue damage.

RESULTS

Serum and CSF metabolomics perturbations were detectable in DTH animals (vs. controls) at all time points, with the greatest change occurring at the earliest time point (day 12) when the neuroinflammatory response was most intense (mean predictive accuracy [SD]-serum: 80.6 [10.7]%, p < 0.0001; CSF: 69.3 [13.5]%, p < 0.0001). The top discriminatory metabolites at day 12 (serum: allantoin, cytidine; CSF: glutamine, glucose) were all reduced in DTH animals compared to controls, and correlated with histological markers of neuroinflammation, particularly astrogliosis (Pearson coefficient, r-allantoin: r = - 0.562, p = 0.004; glutamine: r = - 0.528, p = 0.008). Serum and CSF NfL levels did not distinguish DTH animals from controls at day 12, rather, significant differences were observed at day 28 (mean [SEM]-serum: 38.5 [4.8] vs. 17.4 [2.6] pg/mL, p = 0.002; CSF: 1312.0 [379.1] vs. 475.8 [74.7] pg/mL, p = 0.027). Neither serum nor CSF NfL levels correlated with markers of neuroinflammation; serum NfL did, however, correlate strongly with axonal loss (r = 0.641, p = 0.001), but CSF NfL did not (p = 0.137).

CONCLUSIONS

While NfL levels were elevated later in the pathogenesis of the DTH lesion, serum and CSF metabolomics were able to detect early, clinically silent neuroinflammation and are likely to present sensitive biomarkers for the assessment of subclinical disease activity in patients.

背景

尽管进行了广泛的搜索，但目前仍没有经过验证的生物体液标志物可用于检测多发性硬化症（MS）中的亚临床神经炎症。代谢组学可测量人体代谢对体内平衡变化的动态反应，这可能有助于早期发现临床无症状的神经炎症。我们使用迟发型超敏反应（DTH）MS 大鼠模型，研究了血清和脑脊液（CSF）代谢组学特征以及神经丝轻链（NfL）水平（一种神经轴突损伤的推定标志物），以研究源自局灶性、临床无症状的神经炎症性脑损伤的这些标志物，及其区分 DTH 动物与对照组的能力。

方法

在 DTH 病变发生后 12、28 和 60 天，使用 H 核磁共振（NMR）光谱代谢组学和 NfL 测量方法，对血清和 CSF 进行检测。采用多元统计分析方法，确定 DTH 动物与对照组之间的代谢组学差异。采用免疫组织化学方法评估神经炎症和组织损伤的程度。

结果

在所有时间点，DTH 动物的血清和 CSF 代谢组学均发生改变（与对照组相比），在神经炎症反应最强烈的最早时间点（第 12 天）变化最大（血清平均预测准确性[标准差]：80.6[10.7]%，p<0.0001；CSF：69.3[13.5]%，p<0.0001）。第 12 天的最佳鉴别代谢物（血清：尿囊素、胞苷；CSF：谷氨酰胺、葡萄糖）在 DTH 动物中均较对照组降低，且与神经炎症的组织学标志物，尤其是星形胶质细胞增生相关（Pearson 系数，r-尿囊素：r=-0.562，p=0.004；r-谷氨酰胺：r=-0.528，p=0.008）。第 12 天，血清和 CSF NfL 水平不能区分 DTH 动物与对照组，而在第 28 天观察到显著差异（血清：38.5[4.8] vs. 17.4[2.6]pg/mL，p=0.002；CSF：1312.0[379.1] vs. 475.8[74.7]pg/mL，p=0.027）。血清和 CSF NfL 水平均与神经炎症标志物无相关性；但血清 NfL 与轴突丢失高度相关（r=0.641，p=0.001），而 CSF NfL 则没有（p=0.137）。