罗娅,谭明亮,唐渝,等.低强度聚焦超声调控星形胶质细胞表型转化对脊髓损伤后大鼠运动功能的影响[J].中华物理医学与康复杂志,2026,48(4):289-297
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| 低强度聚焦超声调控星形胶质细胞表型转化对脊髓损伤后大鼠运动功能的影响 |
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| DOI：10.3760/cma.j.cn421666-20250425-00362 |
| 中文关键词: 脊髓损伤 低强度聚焦超声 星形胶质细胞 运动功能 神经保护 |
| 英文关键词: Spinal cord injury Low-intensity focused ultrasound Astrocytes Motor function Neuroprotection |
| 基金项目:重庆市自然科学基金项目 (cstc2020jcyj-msxmX0029) |
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| 中文摘要: |
| 目的 观察低强度聚焦超声（LIFU）调控星形胶质细胞表型转化对脊髓损伤（SCI）后大鼠运动功能的影响。 方法 选取健康成年雌性清洁级Sprague-Dawley（SD）大鼠88只。实验分为两个部分进行，第一部分筛选安全干预时间窗，将12只大鼠按随机数字表分为第1天假刺激组、第1天LIFU组、第3天假刺激组和第3天LIFU组，每组3只大鼠；第二部分为机制验证实验，将76只大鼠按随机数字表分为假手术组13只、模型组13只、假刺激组25只和LIFU组25只。第一部分实验中的第1天假刺激组、第1天LIFU组、第3天假刺激组和第3天LIFU组，以及第二部分中的假手术组、模型组、假刺激组和LIFU组均基于标准化脊髓钳夹伤模型和方法进行SCI造模。第一部分实验，第1天LIFU组于造模成功后第1天开始LIFU干预，第3天LIFU组于造模成功后第3天开始LIFU干预，第1天假刺激组和第3天假刺激组仅接受假LIFU干预。第二部分实验，依据第一部分的实验结果，将造模成功后第3天设为LIFU组开始LIFU干预的最佳时间，LIFU干预的参数与第一部分实验相同，每周干预4次，每次20 min，连续干预4周。假手术组、模型组均常规饲养不接受LIFU干预，假刺激组仅接受假LIFU干预。采用大鼠脊髓损伤行为学评分（BBB）、斜板试验、足迹分析和运动诱发电位（MEP）于相应时间点评估第二部分实验中4组大鼠的运动和神经传导功能。第二部分实验，4组大鼠造模成功后第28天，采用免疫荧光染色检测重链神经丝蛋白（NF-H）的表达，采用TUNEL法检测神经元凋亡情况，采用ELISA法检测脑源性神经营养因子（BDNF）和神经生长因子（NGF）的表达。第二部分实验，于4组大鼠造模成功后第7天和第14天进行免疫荧光染色，评估其星形胶质细胞神经毒性表型（C3）和神经保护性表型（S100A10）标志物的表达。 结果 第一部分实验的病理染色结果表明，第1天LIFU组经LIFU干预后组织损伤较第1天假刺激组明显加重，而第3天LIFU组的组织形态与第3天假刺激组无明显差异，即造模成功后第3天为LIFU干预的安全窗口期。第二部分实验，造模成功后第14和28天，LIFU组的BBB评分显著高于假刺激组（P＜0.01）；造模成功后第28天，LIFU组大鼠的斜板度数显著高于假刺激组（P＜0.05）；造模成功后第28天，LIFU组大鼠的步幅和步宽均显著优于假刺激组，差异均有统计学意义（P＜0.01）。造模成功后第28天，LIFU组MEP的潜伏期较假刺激组显著缩短，波幅则明显升高，差异均有统计学意义（P＜0.01）。造模成功后第28天，LIFU组NF-H的阳性面积占比、BDNF和NGF的蛋白表达量较假刺激组均显著增加（P＜0.05），其神经细胞凋亡数量亦显著低于假刺激组（P＜0.01）。造模成功后第7天，LIFU组神经毒性标记物C3的表达显著低于假刺激组（P＜0.05），造模成功后第14天，LIFU组神经保护性标记物S100A10的表达显著高于假刺激组（P＜0.01）。 结论 LIFU可通过调控星形胶质细胞表型的转化来增强局部神经的营养支持，从而抑制神经元凋亡，促进轴突再生，最终改善SCI后大鼠的运动功能。 |
| 英文摘要: |
| Objective To observe any effect of low-intensity focused ultrasound(LIFU) on the motor function of rats after a spinal cord injury(SCI). Methods In the study′s first phase(safety window screening), twelve healthy, adult, female Sprague-Dawley rats were randomly assigned to either a 1-day sham, 1-day LIFU, 3-day sham or 3-day LIFU group with 3 rats in each. Those groups received the intervention at the respective time points post-modeling to determine the best safety window. In the mechanism verification phase seventy-six similar rats were randomly divided into a sham operation group(n=13), a model group(n=13), a sham stimulation group(n=25), and an LIFU group(n=25). Based on the phase I results, the LIFU group's intervention in phase II began on day 3 post-injury. Treatments were administered four times weekly for 4 weeks(20min/ session). The sham operation and model groups received standard care without LIFU, while the sham stimulation group received sham intervention. Motor and electrophysiological functioning were assessed using the Basso, Beattie and Bresnahan(BBB) scale, an inclined plane test, footprint analysis, and motor evoked potentials. On day 28, immunofluorescence staining was used to detect neurofilament heavy chains(NF-Hs) and TUNEL assays detected neuron apoptosis. Enzyme-linked immunosorbent assay(ELISA) was employed to quantify the levels of brain-derived neurotrophic factor(BDNF) and nerve growth factor(NGF). The levels of neurotoxic C3 and neuroprotective S100A10 astrocytes were evaluated on days 7 and 14. Results Histological analysis in phase I indicated that LIFU intervention on day 1 exacerbated tissue damage compared to the sham group, whereas intervention on day 3 showed no significant morphological differences compared to the sham group. That established day 3 as the safe window for intervention. In the second phase of the study, the LIFU group exhibited significantly higher average BBB scores on days 14 and 28 compared to the sham stimulation group. On day 28, the LIFU group′s average inclined plane test angle was significantly greater than the other groups′ averages. And both stride length and width had improved significantly more in the LIFU group than in the sham group, on average. Electrophysiological analysis on day 28 revealed that the LIFU group had significantly shorter motor evoked potential latencies and significantly higher amplitudes than the sham group. Immunofluorescence analysis on day 28 showed that the proportion of NF-H-positive areas, along with the expression of BDNF and NGF proteins, were significantly higher in the LIFU group, while neuron apoptosis was significantly less. The expression of the neurotoxic marker C3 was significantly downregulated in the LIFU group on day 7, while the neuroprotective marker S100A10 was significantly upregulated on day 14 compared to the sham group. Conclusions LIFU enhances the local nutritional microenvironment by modulating astrocyte phenotype switching, thereby suppressing neuronal apoptosis, promoting axon regeneration, and ultimately improving the recovery of motor function after SCI in rats. |
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