Neuroprotective effect ofshenfuinjection after intra-arterial thrombolysis

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Abstract

BACKGROUND:

Electroacupuncture at Ren channel point can treat stroke and generate some neurodevelopment-related growth factors, which are associated with the proliferation and differentiation of stem cells.

OBJECTIVE:

To observe the effect of electroacupuncture at Ren channel point combined with injection of basic fibroblast growth factors (bFGF) on the expression of 5-bromo-2-deoxyuridine (BrdU)-labeled and BrdU/Neuroepthelial stem cell protein (Nestin)-labeled positive cells in subventricular zone of cerebral ischemic rats.

DESIGN:

Completely randomized grouping design and controlled trial.

SETTING:

Department of Acupuncture and Moxibustion, Shenzhen Hospital, Guangzhou University of Traditional Chinese Medicine.

MATERIALS:

Eighty-four adult healthy male Wistar rats were involved and divided into 5 groups by a lot: blank group (n=6), sham-operation group (n=6), model group (n=26), injection of bFGF group (bFGF group, n =24) and electroacupuncture at Ren channel combined with injection of bFGF group (bFGF group, n=22). Three time points were set in each of the latter three groups for observation: 7, 14 and 28 days after ischemia respectively. Mouse Br-dU and Nestin monoclonal antibody (analytical pure, China), G6805 electro-acupuncture apparatus (Huayi Medical Instrument, Shanghai) and Olympus FV 500 laser confocal microscope (Olympus Company) were used.

METHODS:

This trial was carried out in the Laboratory of Anatomy, Medical College of Sun Yat-sen University from September 2004 to July 2005. ① Except for blank group and sham-operation group, the rats in the other groups were prepared into rat models of local cerebral ischemia by suture-occluded method. The rats were reperfused, and then they were graded in neurologic deficits based on neurological scoring of Longa. Rat model was successful when the neurological scoring ranged from 1 to 4 points and used for the trial. Blank group was untouched; Right common carotid artery was isolated and right external carotid artery was dissected without embolism in the sham-operation group. On the 2nd day after operation, the rats in the combination group were given acupuncture at Chengjiang, Qihai and Guanyuan point, then, electro-acupuncture apparatus, with sparse wave 30 Hz, dense wave 100 Hz, intensity 6 to 15 V, was used to stimulate these three points. Stimulation standard was based on the slight trill appeared in the corresponding position, and the stimulation lasted 20 minutes. After being acupunctured, the rats were intramuscularly injected with 4 000 U/d, and administration was immediately after reperfusion, after this, bFGF was intramuscularly injected following acupuncture, once a day. In the bFGF group, the rats were intramuscularly injected with bFGF on the 2nd day after operation. The dosage and time of injection were the same as those in the combination group. The rats in the model group and sham-operation group were mounted on the acupuncture bench for 20 minutes on the 2nd day after operation, and acupuncture was omitted. ② The expressions of BrdU-labeled positive cells (reflect cell proliferation) and BrdU/Nestin-labeled positive cells (reflect neural stem cells proliferation) in the lateral subventricular zone were determined by immunofluorescence. Positive cells were counted in five visual fields using 200-fold Olympus FV 500 laser confocal microscope system. ③ One-way analysis of variance was used for multiple comparison of the mean of samples.

MAIN OUTCOME MEASURES:

The effect of electroacupuncture combined with injection of bFGF on the number of BrdU and BrdU/Nestin-labeled positive cells in subventricular region of cerebral ischemic side.

RESULTS:

Altogether 8,6 and 4 rats in model group, combination group and bFGF group died respectively during modeling, finally 6 rats respectively in the normal group and sham-operation group and 18 rats respectively i.e., 6 rats at each time point, in the model group, combination group and bFGF group were involved in the result analysis. ① 7 days after cerebral ischemia, BrdU and BrdU/Nestin-labeled positive cells in the model group were ( 272.6±36.9) and (65.3±11.8)/5 visual fields, respectively, which were more than those of normal group [(47.2±6.1), (15.6±4.2)/5 visual fields, P < 0.01], but less than those in the bFGF group ( 365.5±42.7), (82.9±12.2) /5 visual fields] and in the combination group [(427.8±46.9), (125.6±18.5) /5 visual fields, P < 0.01]. The BrdU and BrdU/Nestin-labeled positive cells in the bFGF group were significantly less than those in the combination group (P < 0.05). ② 14 days after cerebral ischemia, the BrdU and BrdU/Nestin-labeled positive cells in the model group were significantly more than those in the blank group [(184.7±28.1), (46.5±9.6) /5 visual fields, P < 0.01], but less than those in the bFGF group [(259.4±32.8), (68.1±10.1) /5 visual fields] and combination group [(308.6±34.2),(87.2± 16.9) /5 visual fields, P < 0.01]. The BrdU and BrdU/Nestin-labeled positive cells in the bFGF group were significantly less than those in the combination group (P < 0.05). ③ 28 days after cerebral ischemia, the BrdU and BrdU/Nestin-labeled positive cells in the model group were close to those in the normal group [(51.3±8.4), (17.8±5.3)/5 visual fields, P> 0.05], but significantly less than those in the bFGF group [(128.1 ±23.6), (41.3±11.4) /5 visual fields]and in the combination group [(162.5±26.1), (62.3±15.8) /5 visual fields, P < 0.01]. The BrdU and BrdU/Nestin-labeled positive cells in the bFGF group were significantly less than those in the combination group (P< 0.05). ④ There were no significant differences in BrdU/Nestin-labeled and Nestin-labeled positive cells between sham-operation group and normal group(P > 0.05).

CONCLUSION:

Electroacupuncture at Ren channel point alone or combined with injection of bFGF can significantly promote neural stem cell proliferation of rats after local cerebral ischemia. Electroacupuncture at Ren channel point and injection of bFGF have synergistic effect.

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