皮层电刺激联合康复治疗对脑缺血大鼠躯体运动功能的影响及机制

doi:10.3877/cma.j.issn.2095-655X.2019.03.015

目的

探讨皮层电刺激联合康复治疗对脑缺血大鼠躯体运动功能的影响及可能机制。

方法

采用随机数字表法将60只成年雄性SD大鼠分为假手术组、脑缺血组、脑缺血＋单纯运动治疗(跑台训练)组、脑缺血＋运动(跑台训练)＋皮层电刺激组，每组15只。制备大脑中动脉栓塞模型，筛选成功模型大鼠为实验对象。在患侧肢体相对应的皮层脑区外埋置刺激电极，术后第3天开始皮层电刺激，治疗持续至第13天。第14天时进行Bederson评分、疲劳转棒实验，之后取材进行免疫组织化学染色或免疫蛋白印记(Western Blot)，检测缺血侧脑组织酪蛋白激酶Ⅱ α亚基(CK2α)和钙调素依赖蛋白激酶Ⅱ(CaMK Ⅱ)表达水平，研究皮层电刺激联合康复治疗可能的作用机制。

结果

皮层电刺激联合康复治疗组大鼠存活率改善，Bederson评分为(0.38±0.15)分、疲劳转棒停留时间为(155.37±42.44)s，较脑缺血组有明显好转(t＝7.58，8.95；均P<0.01)，且结果优于单纯运动治疗组(t＝3.80，5.58；均P<0.05)。免疫组织化学染色结果可见脑缺血组CK2α及CaMK Ⅱ阳性率明显降低，与假手术组相比差异有统计学意义(t＝15.99，12.39；均P<0.01)。皮层电刺激联合康复治疗组CK2α、CaMK Ⅱ阳性比例分别为(24.75±4.55)%、(33.48±3.23)%，较脑缺血组明显升高，差异有统计学意义(t＝6.05，7.99；均P<0.01)，且阳性比例高于单纯运动治疗组(16.40±2.59)%、(22.53±6.44)%(t＝4.52，4.62；均P<0.05)。Western Blot结果显示，脑缺血组CK2α及CaMK Ⅱ表达水平显著下降，分别为假手术组的(0.31±0.12)、(0.39±0.12)倍，皮层电刺激联合康复治疗组CK2α及CaMK Ⅱ表达量明显升高，分别升高至假手术组的(0.81±0.08)、(0.76±0.19)倍，与脑缺血组相比差异有统计学意义(t＝10.68，4.73；均P<0.05)。

结论

皮层电刺激联合康复治疗可显著促进脑缺血大鼠运动功能恢复，其机制可能与上调运动皮层区CK2α及CaMK Ⅱ表达有关。

关键词: 脑缺血, 皮层电刺激, 联合康复, 酪蛋白激酶Ⅱ, 钙调素依赖蛋白激酶Ⅱ

Objective

To investigate the effect of combined rehabilitation therapy with cortical electrical stimulation on somatic motor function in rats with cerebral ischemia, and to explore its possible mechanism.

Methods

Sixty adult male SD rats were randomly divided into sham surgery group, cerebral ischemia group, cerebral ischemia+ simple exercise therapy (treadmill training) group, cerebral ischemia+ exercise (treadmill training)+ cortical electrical stimulation group, with 15 rats in each group. The middle cerebral artery occlusion (MCAO) was performed and only the successful models were selected as experimental objects. Stimulation electrodes were implanted outside the corresponding cortical area of the affected limbs. Electrical stimulation began on the 3rd day after operation, and the treatment lasted until the 13th day. On the 14th day, Bederson score and fatigue rotating rod test were performed. Immunohistochemical staining or Western Blot was used to detect the expression of CK2α and CaMK Ⅱ in ischemic brain tissue, and to study the possible mechanism of combined rehabilitation therapy with cortical electrical stimulation.

Results

The survival rate of rats in combined rehabilitation therapy with cortical electrical stimulation was improved, and the Bederson score and fatigue rod rotation time was (0.38±0.15), (155.37±42.44)s respectively, better than those in cerebral ischemia group (t=7.58, 8.95; all P<0.01), and also better than those in exercise only group (t=3.80, 5.58; all P<0.05). Immunohistochemical staining showed that the positive ratios of CK2α and CaMK II in cerebral ischemia group were significantly lower than those in sham group (t=15.99, 12.39; all P<0.01). The positive ratios of CK2α and CaMK Ⅱ in cortical electrical stimulation combined rehabilitation group were (24.75±4.55)% and (33.48±3.23)% respectively, which were significantly higher than those in cerebral ischemia group (t=6.05, 7.99; all P<0.01), and even higher than those in exercise only group (16.40±2.59)%, (22.53±6.44)% (t=4.52, 4.62; all P<0.05). Western Blot results showed that the expression of CK2α and CaMK Ⅱ in cerebral ischemia group decreased significantly to (0.31±0.12), (0.39±0.12) fold of sham group respectively, and those in combined rehabilitation therapy with cortical electrical stimulation increased obviously, which were (0.81±0.08), (0.76±0.19) fold of sham group, with significant differences compared with cerebral ischemia group (t=10.68, 4.73; all P<0.05).

Conclusion

Combined rehabilitation therapy with cortical electrical stimulation can significantly promote motor function recovery in rats with cerebral ischemia, and which may be related to up-regulation of CK2α and CaMK Ⅱ expression in motor cortex.

Key words: Brain ischemia, Cortical electrical stimulation, Combined rehabilitation, Casein Kinase Ⅱ, Calcium/calmodulin-dependent protein kinase-Ⅱ

表1 各组大鼠脑缺血后不同时间死亡数分布情况

组别	例数(只)	0～24 h(只)	5～48 h(只)	9～72 h(只)	4～7 d(只)	8～14 d(只)	总死亡数(只)	总死亡率(%)
假手术组	15	1	0	0	0	0	1	6.67
脑缺血组	15	1	1	2	0	1	5	33.33
脑缺血＋单纯运动治疗组	15	2	0	0	1	1	4	26.67
脑缺血＋运动＋皮层电刺激组	15	1	1	0	0	0	2	13.33

表1 各组大鼠脑缺血后不同时间死亡数分布情况

组别	例数(只)	0～24 h(只)	5～48 h(只)	9～72 h(只)	4～7 d(只)	8～14 d(只)	总死亡数(只)	总死亡率(%)
假手术组	15	1	0	0	0	0	1	6.67
脑缺血组	15	1	1	2	0	1	5	33.33
脑缺血＋单纯运动治疗组	15	2	0	0	1	1	4	26.67
脑缺血＋运动＋皮层电刺激组	15	1	1	0	0	0	2	13.33

表2 术后第14天各组大鼠脑缺血后Bederson神经功能评分比较(分，±s)

组别	例数	神经功能评分
假手术组	14	0.00±0.00
脑缺血组	10	1.70±1.25^a
脑缺血＋单纯运动治疗组	11	0.91±0.70
脑缺血＋运动＋皮层电刺激组	13	0.38±0.15^bc
F值	?	14.72
P值	?	<0.01

表2 术后第14天各组大鼠脑缺血后Bederson神经功能评分比较(分，±s)

组别	例数	神经功能评分
假手术组	14	0.00±0.00
脑缺血组	10	1.70±1.25^a
脑缺血＋单纯运动治疗组	11	0.91±0.70
脑缺血＋运动＋皮层电刺激组	13	0.38±0.15^bc
F值	?	14.72
P值	?	<0.01

表3 术后第14天各组大鼠脑缺血后疲劳转棒停留时间比较(s，±s)

组别	例数	疲劳转棒停留时间
假手术组	14	196.45±24.28
脑缺血组	10	76.48±14.49^a
脑缺血＋单纯运动治疗组	11	107.51±27.33
脑缺血＋运动＋皮层电刺激组	13	155.37±42.44^bc
F值	?	37.83
P值	?	0.00

表3 术后第14天各组大鼠脑缺血后疲劳转棒停留时间比较(s，±s)

组别	例数	疲劳转棒停留时间
假手术组	14	196.45±24.28
脑缺血组	10	76.48±14.49^a
脑缺血＋单纯运动治疗组	11	107.51±27.33
脑缺血＋运动＋皮层电刺激组	13	155.37±42.44^bc
F值	?	37.83
P值	?	0.00

表4 各组大鼠CK2α及CaMK Ⅱ阳性细胞率比较(%，±s)

组别	例数	CK2α	CaMK Ⅱ
假手术组	4	29.58±3.10	43.88±5.16
脑缺血组	4	13.59±4.19^a	14.53±3.36^a
脑缺血＋单纯运动治疗组	4	16.40±2.59	22.53±6.44
脑缺血＋运动＋皮层电刺激组	4	24.75±4.55^bc	33.48±3.23^bc
F值	?	16.01	29.21
P值	?	0.001	0.000

表4 各组大鼠CK2α及CaMK Ⅱ阳性细胞率比较(%，±s)

组别	例数	CK2α	CaMK Ⅱ
假手术组	4	29.58±3.10	43.88±5.16
脑缺血组	4	13.59±4.19^a	14.53±3.36^a
脑缺血＋单纯运动治疗组	4	16.40±2.59	22.53±6.44
脑缺血＋运动＋皮层电刺激组	4	24.75±4.55^bc	33.48±3.23^bc
F值	?	16.01	29.21
P值	?	0.001	0.000

图1 各组大鼠运动皮层CK2α阳性表达荧光染色图(IHC-Fr ×200)

图2 各组大鼠运动皮层CaMK Ⅱ阳性表达荧光染色图(IHC-Fr ×200)

表5 各组大鼠缺血侧运动皮层CK2α、CaMK Ⅱ表达水平比较(±s)

组别	例数	CK2α	CaMK Ⅱ
假手术组	3	1.00±0.00	1.00±0.00
脑缺血组	3	0.31±0.12^a	0.39±0.12^a
脑缺血＋单纯运动治疗组	3	0.53±0.08^b	0.63±0.16
脑缺血＋运动＋皮层电刺激组	3	0.81±0.08^bc	0.76±0.19^b
F值	?	41.40	10.64
P值	?	0.000	0.004

表5 各组大鼠缺血侧运动皮层CK2α、CaMK Ⅱ表达水平比较(±s)

组别	例数	CK2α	CaMK Ⅱ
假手术组	3	1.00±0.00	1.00±0.00
脑缺血组	3	0.31±0.12^a	0.39±0.12^a
脑缺血＋单纯运动治疗组	3	0.53±0.08^b	0.63±0.16
脑缺血＋运动＋皮层电刺激组	3	0.81±0.08^bc	0.76±0.19^b
F值	?	41.40	10.64
P值	?	0.000	0.004

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The effect and possible mechanism of combined rehabilitation therapy with cortical electrical stimulation on somatic motor function for cerebral ischemia rats

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The effect and possible mechanism of combined rehabilitation therapy with cortical electrical stimulation on somatic motor function for cerebral ischemia rats