糖尿病性疼痛大小鼠模型的差异性研究

Comparative Analysis of Diabetic Neuropathic Pain in Rats and Mice

黄瑞紫

11749073

硕士

生物学

宋学军

2019-05-31

2019-06-20

哈尔滨工业大学

深圳

糖尿病周围神经病变（Diabetic Peripheral Neuropathy, DPN）是糖尿病最常见的并发症之一，糖尿病性疼痛（Diabetic Neuropathic Pain, DNP）是DPN的主要表现症状之一，包括自发性疼痛、痛觉超敏（Allodynia）和痛觉过敏（Hyperalgesia），约有40%至60%的DPN患者遭受DNP带来的痛苦，占总体糖尿病患者10%至20%。目前对于DNP的机制理解不清楚、临床治疗效果不理想，因此迫切需要研究DNP的发生发展机制并开发新的治疗方法。目前主要用于DNP研究的动物模型是在大鼠和小鼠上用链脲佐菌素（STZ）腹腔注射诱导的糖尿病性疼痛。但是研究模型存在DNP成功率低和不稳定等严重问题。本研究在最常使用的实验动物Sprague Dawley (SD)大鼠和C57BL/6小鼠上，研究STZ诱导的血糖变化、胰岛功能、糖尿病发生率和糖尿病性疼痛发生率等关键指标，分析对比它们之间的关联和差异。本研究的目标旨在建立一个稳定可靠的研究糖尿病性疼痛的动物模型，为进一步研究糖尿病性疼痛的发生发展机制、寻找有效的镇痛药物提供可靠的动物研究模型。本研究在大鼠和小鼠上建立STZ诱导的糖尿病模型。SD大鼠单次大剂量腹腔注射STZ 70mg/kg；C57BL/6小鼠腹腔注射STZ 40mg/kg（连续注射5天）。糖尿病模型成功的判断标准：以大鼠血糖高于16.6mmol/L，小鼠血糖高于13.8mmol/L为高血糖指标，再根据胰腺切片HE和胰岛素染色结果综合判断糖尿病造模。糖尿病性疼痛：在糖尿病模型大鼠和小鼠上，检测动物对机械刺激敏感性的变化（使用Von Frey Filament检测）以判断动物是否存在机械痛过敏，进而确定动物出现糖尿病性神经病理性疼痛（DNP）。 结果显示，注射STZ后6周内，SD大鼠糖尿病发病率为86.4%（19/22），死亡率为13.6%（3/22），DNP发病率为56.3%（9/16）；C57BL/6小鼠糖尿病发病率为75.9%（41/54），死亡率为0，DNP率为85.4%（35/41）。这一结果对长期用于DNP研究STZ模型产生的最大挑战是STZ在SD大鼠DNP的成模率只有大约56％。在此基础上，我们研究继续探讨该模型动物产生痛敏和非痛敏差异的可能原因。Luxol Fast Blue (LFB)染色结果显示，与对照组相比，痛敏组与非痛敏组SD大鼠和C57BL/6小鼠坐骨神经均出现鞘磷脂和磷脂表达显著下降、雪旺氏细胞的完整性被破坏等脱髓鞘现象；并且，痛敏组动物髓鞘畸形脱落病变情况比非痛敏组显著。蛋白质免疫印迹实验（Western blot）和免疫组化实验结果显示，痛敏组C57BL/6小鼠脊髓中GFAP表达水平显著高于对照组和非痛敏组，而非痛敏组未出现统计学显著性变化。此外，脊髓切片染色结果显示，与对照组相比，痛敏组和非痛敏组SD大鼠和C57BL/6小鼠脊髓中星形胶质细胞和小胶质细胞均出现胞体增大、数目增多、信号变强等变化，并且痛敏组比非痛敏组的激活情况更为显著。以上结果说明，高血糖会导致坐骨神经脱髓鞘病变以及脊髓星形胶质细胞和小胶质细胞的激活；这种变化在痛敏组中更为显著，与DNP发生发展有明显关联。结论：STZ诱导的糖尿病的发病率和死亡率在SD大鼠中均高于C57BL/6小鼠；但是，SD大鼠只有约半数（56.3%）表现出糖尿病性疼痛症状，远低于C57BL/6小鼠的85.4%。高血糖会导致外周神经脱髓鞘病变和脊髓神经胶质细胞激活，这些变化与DNP的发生发展呈显著正相关。因此，在利用STZ诱导研究大、小鼠糖尿病性疼痛时，必须严格区分这种差异。特别是SD大鼠有近半数不能被STZ诱导产生DNP，这与临床DNP发生率的报道（只有10％-20%）的趋势是一致的。前人的很大部分动物实验研究直接采用全部的STZ SD大鼠来研究DNP，而不对齐动物是否确实产生长时程的高血糖以及显著性的疼痛进行一对一的严格鉴定，因此得出的结果和结论很可能存在巨大偏差甚至错误。本研究鉴定区分并提供了一个稳定可靠的研究糖尿病性疼痛的大鼠和小鼠动物模型，对进一步研究糖尿病性疼痛的发生发展机制、寻找有效的镇痛药物具有重要价值。

Diabetic neuropathic pain (DNP) is the main symptom of diabetic peripheral neuropathy (DPN), which is one of the most common complications of diabetes. Symptoms of DNP exhibited as spontaneous pain, allodynia, and hyperalgesia. The incidence of DNP in diabetic patients is about 10%-20% and 40%- 60% in diabetic patients with documented neuropathy. The mechanisms underlying DNP remain elusive and the treatment drugs and other approaches are very limited. It is important in theory and clinical management to understand the development of DNP, however, based on the data and experiences reported by our previous study and many other labs in the filed in the whole world, the mostly used animal models of DNP in rats and mice are very unstable in incidence of development of neuroathy and neuropathic diabetic pain. Therefore, this study was to study and compare, in the most commonly used models in Sprague Dawley (SD) rats and C57BL/6 mice, the changes of the key indicators including blood glucose, pancreas function, incidence of diabetes and diabetic pain and accompanied neurochemical alterations. The purpose of this study was to provide a reliable models of diabetic neuropathic pain in rats and mice for studying mechanisms underlying DNP and developing new drug for treatment of DNP. We established STZ-induced diabetes models in SD rats and C57BL/6 mice. STZ was intraperitoneally injected in SD rats (single dose, 70 mg/kg) and in C57BL/6 mice (40 mg/kg, daily for 5 consecutive days). The success model of diabetes was evaluated by the blood glucose (rat: >16.6mmol/L; mouse: >13.8mmol/L) combined with the pancreas damage examined by HE staining of pancreas and peripheral nerve neuropathy. DNP was evaluated by testing the mechanical sensitivity of the hind paw to the Von Frey Filament stimulation in the STZ rats and STZ mice. The significantly increased mechanical sensitivity ( mechanical allodynia) was used as the behavioral sign of DNP. The results showed that, six weeks after STZ injection, incidences of diabetes, mortality and DNP in rats and mice, respectively, were as follows: diabetes, 86.4% and 75.9%; mortality, 13.6% and 0%; and DNP, 56.3% and 85.4%. The most prominent phenomenon is that there were approximately half of the STZ rats did not show painful mechanical allodynia and such an incidence was greatly lower than that in mice (85.4%). These findings indicate that the previously reported studies on DNP using the STZ model in SD rats may have severe defects in their results and conclusions.On the basis of these observations, we continued to investigate the possible reasons underlying such difference in the mechanical sensitivity in the animals. Western blotting and immunohistochemical analyses showed that expression of GFAP and IBA1 in the spinal cord in painful SD rats and C57BL/6 mice were significantly greater than those in groups of naïve control and non-pain STZ animals. There were no significant alterations in the signs of neurochemical alterations between control naïve and non-pain STZ-animals. These results suggest that activation of the astrocytes and microglial cells is strongly associated with the development of DNP and may play important roles in production and persistence of DNP. In addition, Luxol Fast Blue staining showed a significance demyelination occurring in the sciatic nerves in both STZ-SD rats and STZ-C57BL/6 mice with mechanical allodynia, one of the behaviorally expressed symptoms of DNP.In conclusion, this study demonstrates the differences in incidences of diabetes, mortality, and DNP in SD rats and C57BL/6 mice following STZ treatment. The incidence of diabetes and mortality in those rats are significantly higher than that in C57BL/6 mice, while the incidence of DNP in STZ-rats is significantly lower than that in STZ-mice. Meanwhile, the high blood glucose can cause activation of astrocytes and peripheral nerve demyelination, which are positively related to the development of DNP. Considering that there are approximately half of the SD rats that previously received STZ treatment do not exhibit mechanical allodynia, thus the behaviorally expressed painful conditions has to be identified in each of animals to make sure the animal is with DNP. Actually, there should be no surprise for this low incidence of DNP because the low incidence of DNP is true in diabetic patients. It is thus clear that the previous publications focusing on STZ-induced diabetic pain in SD rats without identifying their behaviorally expressed painful symptoms in each of the included animals may result in inaccurate or incorrect results and conclusions. This study has provided reliable models of DNP in SD rats and C57BL/6 mice and is valuable for further studying mechanisms underlying DNP and developing new drug for treatment of DNP.

糖尿病 神经病理性疼痛 链脲佐菌素 大鼠 小鼠 胶质细胞 外周神经脱髓鞘

Diabetes Neuropathic Pain Streptozotocin Sprague Dawley rats C57BL/6 mice Glia cells peripheral nerve demyelination

中文

联合培养

南方科技大学

黄瑞紫. 糖尿病性疼痛大小鼠模型的差异性研究[D]. 深圳. 哈尔滨工业大学,2019.