電磁場應用於治療糖尿病小鼠運動神經系統的神經損傷

Application of electromagnetic field in the treatment of neuron damage in the motor nervous system of the diabetic mice

高健瑋(JIAN-WEI GAO)

指導教授 ： 曾嘉儀

中原大學/工學院/生物醫學工程學系/碩士(2023年)

https://doi.org/10.6840/cycu202301011

摘要

糖尿病是一種全身性代謝異常疾病，與身體內的胰島素表達異常息息相關。糖尿病可被分為第一型及第二型糖尿病，其中第一型糖尿病占比10%，而第二型糖尿病占比90%。糖尿病患者自身高血糖的問題，也常伴隨著許多併發症的產生，像是會對神經、血管及視網膜的破壞。而糖尿病神經病變（Diabetic neuropathy, DN）則是糖尿病患者常見的一種併發症，糖尿病的症狀主要發生於四肢末梢，主要集中於神經疼痛以及感覺喪失的問題，使得感覺神經系統的損傷較運動神經損傷更受到關注。但根據研究顯示，糖尿病神經病變晚期，通常會表現出運動功能的障礙，像是易於跌倒、平衡感不佳或是步態的改變；患有糖尿病的大鼠產生運動障礙，且運動神經末梢的突觸數量減少，進而可以判斷糖尿病對運動神經的損傷與運動障礙有密切的關係。而考慮到糖尿病患者治療有許多副作用，像是藥物治療造成的身體不適或是中醫針灸治療而產生不易癒合的傷口，因此需要發展不具副作用的天然療法來改善糖尿病造成的運動神經病變損傷。電磁場（Electromagnetic Fields, MF），是一種非侵入式的物理輔助治療，適合糖尿病患者傷口不易癒合的特性，並具有促進骨頭癒合、緩解疼痛及改善傷口癒合等功效，欲透過電磁場非侵入式的物理治療，來改善糖尿病造成的運動神經系統損傷。目前電磁場對於肌肉與運動神經系統的影響尚未明確，故透過本實驗欲探討電磁場對於糖尿病運動神經系統損傷的影響。實驗使用C57BL/6JNart 小鼠及基因改殖之自發性第二型糖尿病小鼠BKS.Cg-Dock7m+/+Leprdb/JNarl（db/db）小鼠，並分組分為：（1）控制組（n=8）；（2）電磁場（MF）組（n=9）；（3）糖尿病（db/db）組（n=9）；（4）糖尿病電磁場治療（db/db+MF）組（n=10），固定每日將MF組及db/db+MF組小鼠放置於有電磁場的空筒中暴露30分鐘，而控制組及db/db組小鼠則是放置於無磁場的空筒中30分鐘。實驗期間會每週紀錄體重，而每個月紀錄血糖數值，並透過Rota-Rod Test來評估綜合運動能力，實驗結果顯示，電磁場並未改善糖尿病在高血糖及過重的問題，但小鼠的綜合運動能力及後肢運動表現有明顯的改善。透過蘇木精-伊紅染色（Hematoxylin eosin staining, H&E staining)對腓腸肌（Gastrocnemius）切片結果觀察到，MF的暴露確實可以防止後肢腓腸肌萎縮及破裂。免疫組織化學染色法（Immunohistochemistry, IHC）計算小鼠大腦運動皮質（Motor cortex, M1）第五層的厚度，結果顯示MF組V層有上升的趨勢，厚度相較於控制組上升20%，雖無顯著差異，但MF的暴露可以使V層厚度增加，增加運動神經訊號的傳遞。另外觀察小腦組織的III、IV/V、VI、VII、VIII、X葉上的浦金氏細胞 （Purkinje cell） 數量變化，其中於IV/V小葉，控制組、MF、db/db+MF組小鼠的Purkinje cell數量在120-130顆間，而db/db組小鼠的數量則下降至100顆；而VI小葉control、MF、db/db+MF組小鼠的Purkinje cell數量在90-120顆間，而db/db組小鼠的數量則下降至80顆，顯示db/db組小腦Purkinje cell數量下降，進而可能影響運動行為表現。此外，比較小鼠腓腸肌橫切面上的神經肌肉接合處 （Neuromuscular junction） 的表達差異。經重疊率計算後，控制組、MF、db/db+MF組小鼠的皮爾森相關係數在0.2-0.3間，db/db組則下降至0.07，顯示運動神經末梢與菸鹼型乙醯膽鹼受器重合率較低，表示其運動訊號的傳遞較為異常。以核振造影（Magnetic Resonance Imaging, MRI）去拍攝小鼠皮質脊髓束（Corticospinal tract, CST），並透過擴散張量核振造影（Diffusion Tensor Imaging, DTI）影像的呈現，觀察神經束的差異。 結果顯示控制組、MF、db/db+MF組小鼠的CST神經纖維數為4800-5700條間，而db/db組則減少至4300條，也顯示其CST神經纖維數下降，可能會造成運動行為的異常。總結，透過暴露電磁場，具有修復因糖尿病損傷的運動神經系統，並可以讓運動表現有改善的成效，因此電磁場的應用在未來治療糖尿病造成的運動神經損傷，是一種具有潛力的臨床療法。

關鍵字

第二型糖尿病 ； 電磁場 ； 運動神經損傷 ； 行為學

並列摘要

Diabetes is a common metabolic disease closely linked to the body's insulin levels. Diabetes can be divided into Type 1 and Type 2 diabetes, with Type 1 diabetes accounting for 10% and Type 2 diabetes accounting for 90%. Many complications, such as damage to nerves, blood vessels, and retina, often accompany the problem of high blood sugar in diabetic patients. The most common complication for people with diabetes is diabetic neuropathy (DN), in which many difficulties, such as damage to nerves, blood vessels, and retina, often accompany the problem of high blood sugar in diabetic patients. However, studies have shown that in advanced stages of diabetic neuropathy, there are often motor impairments, such as the risk of falls, poor balance, or changes in gait. Literature has shown that diabetic rats have movement disorders, and the number of synapses at the motor nerve endings is reduced. As a result, we can conclude that motor neuron damage and motor dysfunctions were associated with diabetes. Given that pharmacological therapy can have unpleasant side effects, the physical discomfort caused by drug treatment, or difficult-to-heal wounds caused by traditional Chinese medicine acupuncture. We need to develop a natural remedy free of side effects to help people with diabetes with their neuropathy. For diabetic patients with slow-healing wounds, magnetic fields are an effective non-invasive physical therapy that speeds up bone healing, lessens pain, and improves wound healing. The influence of electromagnetic fields on the muscle and motor nervous system is not yet evident. Therefore, this study explores the impact of electromagnetic fields on motor nervous system damage in diabetes. C57BL/6JNart mice and genetically modified spontaneous type 2 diabetic mice BKS.Cg-Dock7m +/+ Leprdb /JNarl (db/db) were used in the experiment and divided into four groups: (1) Control group (n=8) (2) Electromagnetic field (MF) group (n=9) (3) Diabetic (db/db) group (n=9) (4) Diabetic electromagnetic field treatment (db/db+MF ) group (n=10). The mice in the MF group and db/db+MF group were placed in an electromagnetic cylinder (1.8 mT) for 30 minutes every day, while the mice in the control group and db/db group were placed in a non-magnetic empty can 30 minutes. During the experiment, the body weight was recorded weekly, and the blood sugar value was recorded monthly. The Rota-Rod Test was used to evaluate the total exercise capacity, and the Tight Rope Test and Rearing Count Test were used to assess the muscle strength of the front and rear limbs. The experimental results showed that MF exposure did not reduce obesity and hyperglycemia in diabetic mice. Based on the behavioral test, it was found that MF exposure can enhance diabetic mice's capacity for general exercise and hindlimb exercise. Through the results of Hematoxylin eosin staining (H&E staining) on gastrocnemius (Gastrocnemius) section, the exposure of MF can indeed prevent the atrophy and rupture of the gastrocnemius of the hindlimb. Immunohistochemistry (IHC) was used to calculate the thickness of the fifth layer of the mouse brain motor cortex (Motor cortex, M1). No significant difference, but MF exposure can increase the thickness of the V layer and increase the transmission of motor nerve signals. In addition, among which in lobes IV/V of the cerebellum, The number of Purkinje cells in control group, MF, db/db+MF group mice were between 120-130, while the number of mice in the db/db group dropped to 100. The number of Purkinje cells in the VI lobule control, MF, db/db+MF group mice was between 90-120, while the number of mice in the db/db group dropped to 80, indicating that the db/db group had fewer cells, which had an impact on their exercise behavior. Moreover, the expression difference of the neuromuscular junction (Neuromuscular junction) in the transverse section of mouse gastrocnemius muscle was compared. The pearson correlation results for the control, MF, and db/db+MF groups ranged from 0.2 to 0.3, while those in. The db/db group's decrease to 0.07, revealing a lower rate of acetylcholine receptor and motor neuron terminal overlap that the transmission of motor signals may be relatively abnormal. Magnetic Resonance Imaging (MRI) was used to photograph the corticospinal tract (CST) of mice, and the differences in nerve bundles were observed through the presentation of Diffusion Tensor Imaging (DTI) images. The results showed that the number of CST nerve fibers in mice in the control, MF, and db/db+MF groups was 4800-5700, while that in the db/db group was reduced to 4300. It implies that the number of CST nerve fibers decreased, which may cause abnormalities in motor behavior. In conclusion, a magnetic field can repair diabetes's damage to the motor system and enhance expression of movement. Therefore, using a magnetic field to treat the diabetes-related damage to the motor system will be a potential clinical therapy.

並列關鍵字

Type 2 diabetes ； Electromagnetic field ； Motor system damage ； Behavioral tests

參考文獻

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