Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA; Department of Anesthesiology, University of the Ryukyus, 207 Uehara Nishihara-cho, Okinawa, Japan.
Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA.
Exp Neurol. 2018 Jul;305:66-75. doi: 10.1016/j.expneurol.2018.03.013. Epub 2018 Mar 30.
The loss of local spinal glycine-ergic tone has been postulated as one of the mechanisms contributing to the development of spinal injury-induced spasticity. In our present study using a model of spinal transection-induced muscle spasticity, we characterize the effect of spinally-targeted GlyT2 downregulation once initiated at chronic stages after induction of spasticity in rats. In animals with identified hyper-reflexia, the anti-spasticity effect was studied after intrathecal treatment with: i) glycine, ii) GlyT2 inhibitor (ALX 1393), and iii) GlyT2 antisense oligonucleotide (GlyT2-ASO). Administration of glycine and GlyT2 inhibitor led to significant suppression of spasticity lasting for a minimum of 45-60 min. Treatment with GlyT2-ASO led to progressive suppression of muscle spasticity seen at 2-3 weeks after treatment. Over the subsequent 4-12 weeks, however, the gradual appearance of profound spinal hyper-reflexia was seen. This was presented as spontaneous or slight-tactile stimulus-evoked muscle oscillations in the hind limbs (but not in upper limbs) with individual hyper-reflexive episodes lasting between 3 and 5 min. Chronic hyper-reflexia induced by GlyT2-ASO treatment was effectively blocked by intrathecal glycine. Immunofluorescence staining and Q-PCR analysis of the lumbar spinal cord region showed a significant (>90%) decrease in GlyT2 mRNA and GlyT2 protein. These data demonstrate that spinal GlyT2 downregulation provides only a time-limited therapeutic benefit and that subsequent loss of glycine vesicular synthesis resulting from chronic GlyT2 downregulation near completely eliminates the tonic glycine-ergic activity and is functionally expressed as profound spinal hyper-reflexia. These characteristics also suggest that chronic spinal GlyT2 silencing may be associated with pro-nociceptive activity.
局部脊髓甘氨酸能张力丧失被认为是导致脊髓损伤后痉挛性发生的机制之一。在我们目前的研究中，使用脊髓横切诱导的肌肉痉挛模型，我们描述了在诱导痉挛后慢性阶段开始时靶向脊髓下调 GlyT2 对大鼠痉挛的影响。在确定存在反射亢进的动物中，在鞘内给予以下物质后研究抗痉挛作用：i）甘氨酸，ii）GlyT2 抑制剂（ALX 1393），和 iii）GlyT2 反义寡核苷酸（GlyT2-ASO）。甘氨酸和 GlyT2 抑制剂的给药导致痉挛的显著抑制，持续至少 45-60 分钟。GlyT2-ASO 的治疗导致在治疗后 2-3 周内观察到肌肉痉挛的逐渐抑制。然而，在随后的 4-12 周内，逐渐出现严重的脊髓反射亢进。这表现为下肢（而不是上肢）的自发性或轻微触觉刺激诱发的肌肉振荡，每个反射亢进发作持续 3 到 5 分钟。GlyT2-ASO 治疗引起的慢性反射亢进被鞘内甘氨酸有效阻断。腰椎脊髓区域的免疫荧光染色和 Q-PCR 分析显示 GlyT2 mRNA 和 GlyT2 蛋白显著减少（>90%）。这些数据表明，脊髓 GlyT2 下调仅提供有限的治疗益处，并且随后由于慢性 GlyT2 下调导致的甘氨酸囊泡合成丧失几乎完全消除了紧张性甘氨酸能活性，并在功能上表现为严重的脊髓反射亢进。这些特征还表明，慢性脊髓 GlyT2 沉默可能与伤害感受性活动有关。
