The Expressions Of VEGF In The Spinal Cords Of Rats With Neuropathic Pain And The Facilitating Effects On Noxious Stimuli

Objectives1. Intrathecal injection of exogenous VEGF and Flk-1 receptor antagonist were observed with chronic sciatic nerve compression model (chronic constriction injury of sciatic nerve, CCI) in rats exposed to thermal stimulation and mechanical pressure stimuli so as to explore the relationship between the sense of pain and VEGF and Flk -1 receptor.2. Immunohistochemical detection of the expressions of VEGF protein in CCI rat L4-5 spinal cord segment were conducted at different periods of time to understand the dynamic changes of the spinal cord and to study the response of VEGF to peripheral nerve injury.3. VEGF has proven to have neuroprotective effect, and the impact on neuropathic pain can be observed through this experiment to provide scientific basis for the neuropathic pain mechanism and the treatment of the disease.Methods1. Building of CCI rat modelThe CCI rat model was produced according to the method described earlier by Bennett- Xie. The rats were anesthetized with intra-peritoneal sodium pentobarbital (50mg/kg) and the abdomen was fixed. A small incision at midpiece of the left thigh was made, which was followed by retraction of the biceps femoris. The left sciatic nerve was isolated under a surgical microscope for about 8-10mm, and the nerves were tightly ligated four times with 4-0 chromicized catgut suture near the trigeminal proximal end for an interval of 1.0-1.5mm between every two nodes. The ligation intensity was controlled so as not to affect the blood circulation of peripheral nerves. The sham control rats were surgically prepared as described above except for the nerve ligation. All the surgical procedure was completed in a bioclean environment. The rats were each injected with 80,000 units of penicillin for 3 consecutive days to prevent infection. The rats were put into separate plastic rearing boxes paved with soft wood chips at the bottom.2. Behavioral testingThe hot-plate (RB-200 Intelligent Hot-plate Device, Science and Technology Co., Ltd. Chengdu UNITA) was used with the temperature maintained at 52℃to test the HWL to thermal stimuli of hindpaws. YT-100 Electronic Instrument (Science and Technology Co., Ltd. Chengdu too UNITA) was used to test the response of hindpaws to mechanical noxious stimuli. On 7^th day after surgery, hot plate and electronic measuring instruments were used to measure the HWLs and to evaluate whether the chronic nerve injury model was successful. Behavioral test methods were adopted to determine the success of the model. The HWLs were determined three times before the application of medicine in experiments, and the average was used as the contrast values (pre-administration values). After the administration of the medicine, the values of HWLs were tested 5, 10, 15, 20, 30, 45 and 60 minutes later respectively. The values of HWLs at different time after the administration of medicine were converted into percentage by (value after administration—pre-administration value) / pre-administration value×100%.3. Immunohistochemistry and image analysis were used to detect the expressions of VEGF protein in the L4-5 spinal cord of sham-operated rats and to detect the expressions of VEGF protein in L4-5 spinal cord in CCI rats on 1^st, 3^rd, 5(th), 7^th, 14^th and 18^th day so as to explore the impact of VEGF on noxious stimulation.Results1. HWLs compared between normal and CCI ratsThere were no significant differences between the basal HWLs to thermal stimuli (4.52±0.19; 4.54±0.18) or mechanical stimuli (5.12±0.39; 5.16±0.40) between left and right hindpaws in the non-operated rats. No significant different between the basal HWLs to thermal stimuli or mechanical stimuli in the left and right hindpaws were found. The HWL to thermal stimuli of left and right hindpaws in CCI rats was (2.86±0.12; 3.63±0.16). The HWL to mechanical stimuli of left and right hindpaws in CCI rats was (2.65±0.83; 3.78±0.24). There were significant differences between the left and right hindpaws in the normal and CCI groups in terms of the HWLs to thermal and mechanical stimuli (P<0.01) .The results indicated that CCI rats were more sensitive to injury stimulus than normal rats.2. Thirty-five neuropathic pain rats were divided into four groups received intrathecal injection of: (1) 10μl of saline (0.9% NaCl) as the control (n=9), (2) 0.1 nmol VEGF(n=8), (3) 0.2 nmol VEGF (n=9), or (4) 0.4 nmol VEGF (n=9).2. 1 Intrathecal injection of VEGF shortened HWL to stimuli of hindpawsThe HWL to mechanical stimuli of left and right hindpaws in the contrast group were F_left/left=0.57, F_right/right=0.56 respectively. In the experimental group receiving an injection of 0.1nmol VEGF, the HWL to mechanical stimuli of left and right hindpaws were F_left/left=0.52, P=0.47; F_right/right=0.30, P=0.57 respectively. There existed no significant difference between the two groups. When the rats were injected with 0.2 noml VEGF, the HWL to mechanical stimuli of left and right hindpaws were F_left/left= 15.40, F_right/right=15.01 respectively. When the rats were injected with 0.4 noml VEGF, the HWL to mechanical stimuli of left and right hindpaws were F_left/left=19.03, F_right/right=18.60. In contrast with the contrast group, intrathecal injection of 0.2 or 0.4 nmol VEGF can shorten HWL to mechanical stimuli in the experimental group (P<0.01). VEGF could promote CCI rats' sensitivity to mechanical stimuli.2. 2 Effect of intrathecal injection of VEGF on HWL to thermal stimuliIn the contrast group, the HWL to thermal stimuli of left and right hindpaws were F_left/left=0.61, F_right/right=0.63 respectively. When the rats were injected with 0.1nmol VEGF, the HWL to thermal stimuli of left and right hindpaws were F_left/left= 46.82 and F_right/right=41.22 respectively. When the rats were injected with 0.2nmol VEGF, the HWL to thermal stimuli of left and right hindpaws were F_left/left=98.43 and F_right/right=92.32 respectively. When the rats were injected with 0.4nmol VEGF, the HWL to thermal stimuli of left and right hindpaws were F_left/left= 125.22 and F_right/right = 118.56 respectively. There existed significant differences between the contrast group and the CCI group in terms of the HWL to thermal stimuli of left and right hindpaws when the CCI rats were injected with 0.1nmol,0.2nmol and 0.4nmol VEGF respectively (P<0.01) .3. Effects of intrathecal administration of SU1498 on VEGF-induced decreases in HWLs0.4 nmol of VEGF was injected into CCI rats. 10 minutes later, the intrathecal injection of 0.4 nmol SU1498 was conducted to convert the HWLs to hot-plate stimulation (F_left/left= 124.35 and F_right/right= 120.42 down to F_left/left=45. 68 and F_right/right=43.83) and mechanical stimuli (F_left/left=20.35 and F_right/right=19.87 reduced to F_left/left= 7.62 and F_right/right=6.85) with a statistically significant difference (P <0.01). The facilitation of VEGF to noxious stimulation may be blocked by SU1498. The results suggested that the VEGF facilitatory response to noxious stimulation may be mediated through VEGFR2.4. Changes of VEGF protein expressions in the injury of spinal cord tissues of ratsThere were very few VEGF protein expressions in vascular endothelial cells, inneurons, or in glial cells in the spinal cord in the control group. On 1^st day,3^rd day,5^th day,7^th day,14^th day and 28^th day, there were a large number of VEGF protein expressions in the nucleus and cytoplasm of neurons in L4-5 spinal cord. On 3^rd day, the peak of VEGF protein expression reached 20.80±6.06,with the control group being 0.60±0.89 .Therefore, significant differences existed between the two groups. On 14^th day, VEGF protein expressions only existed in the cytoplasm, with the number of positive neurons being 7.80±2.17 and 2.60±1.82, significantly lower than on 7^th day, but still significantly higher than in the control group. On 3^rd day, L4-5 spinal cord glial cells began to appear in the expression of VEGF, with the number of positive glial cells being 13.25±3.16,and on 14^th day, reaching a peak of 35.83±6.11. On 28^th day, the number of positive neurons reduced to 21.50±4.93. Compared with the control group, there were significant differences in VEGF-positive cells of CCI groups (P <0.01). These results suggested that VEGF protein had high expressions in the spinal cord neurons, glial cells and vascular endothelial cells in CCI rats at different stages, which may participate in the information transfer and regulation of injurious stimuli in the spinal cord of rats and its mechanism remains to be studied in depth.The experimental results demonstrated that exogenous VEGF may prone to injurious stimuli in the spinal cord of rats with chronic neuropathic pain, and Flk-1 receptor was involved in the facilitation reaction process. The experiment provided a new perspective on further comprehending the molecular mechanism of the neuropathic pain.Conclusion1. The HWLs to thermal or mechanical stimuli in CCI group are much lower than in the normal group.2. Intrathecal injection of VEGF can shorten the HWLs to thermal or mechanical stimuli when compared with the injection of normal saline , with the degree of shortening related to the content of VEGF.3. Intrathecal injection of SU1498 of VEGF/Flk-1 in rat can convert the shortening of HWLs to thermal or mechanical stimuli induced by the injection of VEGF.4. After CCI in rats, the VEGF protein expressions in L4-5 spinal cord blood vessels, nerve cells, glial cells are time-dependent.