Goldshmit Yona, Banyas Evgeni, Bens Nicole, Yakovchuk Alex, Ruban Angela
1Steyer School of Health Professions, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
2Australian Regenerative Medicine Institute, Monash Biotechnology, Clayton, Victoria, Australia; and.
J Neurosurg Spine. 2020 Jul 3;33(5):692-704. doi: 10.3171/2020.4.SPINE20302. Print 2020 Nov 1.
Excitotoxicity due to neuronal damage and glutamate release is one of the first events that leads to the progression of neuronal degeneration and functional impairment. This study is based on a paradigm shift in the therapeutic approach for treating spinal cord injury (SCI). The authors tested a new treatment targeting removal of CNS glutamate into the blood circulation by injection of the blood glutamate scavengers (BGSs) recombinant enzyme glutamate-oxaloacetate transaminase (rGOT1) and its cosubstrate oxaloacetic acid (OxAc). Their primary objective was to investigate whether BGS treatment, followed by treadmill exercises in mice with SCI, could attenuate excitotoxicity, inflammation, scarring, and axonal degeneration and, at a later time point, improve functional recovery.
A pharmacokinetic experiment was done in C57BL/6 naive mice to verify rGOT1/OxAc blood activity and to characterize the time curve of glutamate reduction in the blood up to 24 hours. The reduction of glutamate in CSF after BGS administration in mice with SCI was confirmed by high-performance liquid chromatography. Next, SCI (left hemisection) was induced in the mice, and the mice were randomly assigned to one of the following groups at 1 hour postinjury: control (underwent SCI and received PBS), treadmill exercises, rGOT1/OxAc treatment, or rGOT1/OxAc treatment followed by treadmill exercises. Treatment started 1 hour postinjury with an injection of rGOT1/OxAc and continued for 5 consecutive days. Starting 1 week after SCI, the exercises and the combined treatment groups recommenced the treadmill exercise regimen 5 days a week for 3 months. Locomotor function was assessed for 3 months using the horizontal grid walking test and CatWalk. Axonal anterograde and wallerian degenerations were evaluated using tetramethylrhodamine dextran. Tissue sections were immunofluorescently stained for Iba1, GFAP, GAP-43, synaptophysin, and NeuN.
BGS treatment decreased the CSF glutamate level up to 50%, reduced axonal wallerian degeneration, and increased axonal survival and GAP-43 expression in neuronal cells. Combined treatment reduced inflammation, scarring, and lesion size. Additionally, the combination of BGS treatment and exercises increased synapses around motor neurons and enhanced axonal regeneration through the lesion site. This resulted in motor function improvement 3 months post-SCI.
As shown by biochemical, immunohistochemical, and functional analysis, BGSs exhibit a substantial neuroprotective effect by reducing excitotoxicity and secondary damage after SCI. Furthermore, in combination with exercises, they reduced axonal degeneration and scarring and resulted in improved functional recovery.
神经元损伤和谷氨酸释放所致的兴奋毒性是导致神经元变性进展和功能障碍的早期事件之一。本研究基于脊髓损伤（SCI）治疗方法的范式转变。作者测试了一种新的治疗方法，即通过注射血液谷氨酸清除剂（BGSs）重组酶谷氨酸草酰乙酸转氨酶（rGOT1）及其辅酶草酰乙酸（OxAc），将中枢神经系统谷氨酸清除到血液循环中。他们的主要目的是研究在SCI小鼠中，BGS治疗后进行跑步机运动是否能减轻兴奋毒性、炎症、瘢痕形成和轴突变性，并在稍后时间点改善功能恢复。
在C57BL/6未处理小鼠中进行药代动力学实验，以验证rGOT1/OxAc的血液活性，并描绘直至24小时血液中谷氨酸减少的时间曲线。通过高效液相色谱法证实了在SCI小鼠中给予BGS后脑脊液中谷氨酸的减少。接下来，在小鼠中诱导SCI（左侧半横断），并在损伤后1小时将小鼠随机分配至以下组之一：对照组（接受SCI并给予PBS）、跑步机运动组、rGOT1/OxAc治疗组或rGOT1/OxAc治疗后进行跑步机运动组。损伤后1小时开始治疗，注射rGOT1/OxAc，并连续进行5天。从SCI后1周开始，运动组和联合治疗组每周5天重新开始跑步机运动方案，持续3个月。使用水平网格行走试验和CatWalk评估3个月的运动功能。使用四甲基罗丹明葡聚糖评估轴突顺行性和沃勒变性。组织切片用Iba1、GFAP、GAP-43、突触素和NeuN进行免疫荧光染色。
BGS治疗使脑脊液谷氨酸水平降低达50%，减少了轴突沃勒变性，增加了神经元细胞中轴突存活和GAP-43表达。联合治疗减少了炎症、瘢痕形成和损伤大小。此外，BGS治疗与运动的联合增加了运动神经元周围的突触，并增强了通过损伤部位的轴突再生。这导致SCI后3个月运动功能改善。
生化、免疫组化和功能分析表明，BGSs通过减少SCI后的兴奋毒性和继发性损伤表现出显著的神经保护作用。此外，与运动相结合，它们减少了轴突变性和瘢痕形成，并导致功能恢复改善。
