| Part ? pharmacokinetics and toxicology study of intradiscal injection of simvastatinObjective: To test the pharmacokinetics and toxicology of whole organs and tissues within 28 days after intradiscal injection of simvastatin in rabbits.To provide the information needed to support human clinical trials.Methods: Twelve male and twelve female rabbits were randomly divided into four groups: control group(0 mg/ml,control group),low dose group(0.1 mg/ml,low dose group),medium dose group(1 mg/ml,Medium dose group)and high dose group(10 mg/ml,High dose group,n=6).Simvastatin at different concentrations of 10 UL was injected into L3/4,L4/5 and L5/6 intervertebral discs in each group.Blood samples were collected in low-dose group and middle-dose group before operation,6 hours,24 hours,3 days,7 days,14 days and 28 days after operation,while in high-dose group,blood samples were collected before operation,0.5 hours,1 hour,3 hours,5 hours,8 hours,24 hours,3 days,7 days,14 days and 28 days after operation.The pharmacokinetics of blood samples were measured by LC-MS/MS.Cerebrospinal fluid was obtained before operation,3 h,5 h,8 h,24 h,3 d,7 d,14 d,28 d after surgery,and the drug concentration was measured.Blood routine,blood biochemistry and urine of all animals were analyzed and evaluated before the experiment and sacrifice.All animals were autopsied on the 28 th day.The heart,kidney,liver and spleen of each animal were observed and weighed.The intervertebral disc tissues were stained with hematoxylin and hematoxylin(H&E),and then qualitatively analyzed by optical microscopy.Results: 28 days after intradiscal injection of simvastatin,the weight of all rabbits increased steadily,and there was no significant difference among the four groups(p > 0.05);there was no significant difference in food residue among the four groups(p > 0.05);there was no significant difference in blood routine,blood biochemical and urine routine results among the four groups(p > 0.05);there was no significant difference in the weight of each organ among the four groups(p > 0.05).The serum concentration of simvastatin was lower than the minimum measurable concentration.There was no significant difference in the histological score of intervertebral disc between the control group,the low dose group and the Medium dose group(p > 0.05),but the histological score of intervertebral disc in the high dose group was significantly higher than that in the other three groups at 28 days(p > 0.05).Conclusion: 28 days after the injection of simvastatin into the intervertebral disc of rabbits,the gross observation of body weight,food consumption,blood routine,blood biochemistry,urine routine,cerebrospinal fluid examination and autopsy showed that three doses of simvastatin were injected into male and female animals respectively,showing no toxic effects.Microscopic histological evaluation of the intervertebral disc showed that the high dose group(10 mg/ml)had damage to the intervertebral disc tissue.Part ? Efficacy and dose effect of intradiscal injection of simvastatin sustained release gel in treatment of discogenic low back pain in rabbitsObjective: To verify the effectiveness of intradiscal injection of simvastatin sustained release gel in the treatment of discogenic low back pain in rabbits.Methods: X-ray-assisted disc puncture with 21 G was performed on 36 New Zealand rabbits to induce discogenic low back pain.Two weeks later,simvastatin was injected into the intervertebral disc.Thirty-six New Zealand rabbits were divided into two groups.In Group G1,L1/2 was the intervertebral disc degeneration segment(DDD group),L2/3 was the normal group(Control group),L3/4 was the medium dose treatment segment(1 mg/ml,DDDD + Medium group),L4/5 was the normal group,L5/6 was the intervertebral disc degeneration + hydrogel segment(DDD + GEL group).In G2 group,L1/2 was intervertebral disc degeneration segment,L2/3 was normal group,L3/4 was low dose treatment segment(0.3 mg/ml,DDD+Low group),L4/5 was normal group,L5/6 was high dose treatment segment(3 mg/ml,DDDD+High group).The weight and food residue of rabbits were recorded weekly.Blood routine,blood biochemistry and urine of all animals were analyzed and evaluated before the experiment and sacrifice.Rabbits were sacrificed 4,12 and 24 weeks after simvastatin injection.Before execution,the intervertebral height index(DHI)was evaluated by X-ray imaging,and the signal intensity of intervertebral disc was assessed by MRI.After execution,the intervertebral disc tissue was obtained and the water content of intervertebral disc was measured.Results: The X-ray results showed that the% DHI of DDD + High group was significantly higher than that of DDD group and DDD + GEL group at 12 and 24 weeks after injection(p < 0.05).The results of MRI showed that the scores of DDD + High group were significantly higher than those of DDD group and DDD + GEL group at 12 and 24 weeks after injection(p < 0.05).Histological score showed that the histological score of DDD + High group was significantly lower than that of DDD group(p < 0.05).The results of intervertebral disc water content showed that at 4,12 and 24 weeks after injection,the water content of DDD + High group was significantly higher than that of DDD group and DDD + GEL group(p < 0.05).Conclusion: The therapeutic efficacy and dose effect of simvastatin in rabbit model of discogenic low back pain were verified.Intra-disc injection of simvastatin is an effective strategy for the treatment of discogenic low back pain.Part ? The effect and possible mechanism of intradiscal injection of simvastatin in the treatment of discogenic low back pain in ratsObjective: To study the effect of intradiscal injection of simvastatin on discogenic low back pain in rats and its possible mechanism.Methods: A total of 30 adult female rats were used in this experiment.Twenty rats were randomly divided into sham operation group(Control group),intervertebral disc degeneration group(DDD group),intervertebral disc degeneration + hydrogel group(DDD + GEL group),intervertebral disc degeneration + simvastatin group(DDD + SIM group,n = 5).The mechanical pain threshold,cold sensation and standing status of drinking water in rats were measured before operation,1 day,1 week,2 weeks,3 weeks,4 weeks,5 weeks,6 weeks,7 weeks and 8 weeks after operation.The rats were killed at 8 weeks.The contents of NF-kappa B1,Rel A,GAP43,SP,CGRP,TRPM 8,IL-1? and TNF-? in the intervertebral disc,the corresponding contents of dorsal root ganglion(DRG)and plantar skin GAP43 and TRPM 8 were quantitatively detected by PCR.The corresponding intervertebral discs were stained with HE Alcin Blue Safronin O to detect their degeneration.Results: There was no significant difference in the mechanical pain threshold between the groups at each time point.From the first day to the 8th week after surgery,the cold-sensing response of the DDD group was significantly higher than that of the Control group(p < 0.05).At 7 and 8 weeks postoperatively,the cold-sensing response of the DDD+SIM group was significantly lower than that of the DDD+GEL group(p < 0.05).The average time of standing drinking water in the DDD group was significantly lower than that in the Control group from week 2 to week 8(p < 0.05).The average time of standing drinking water in the DDD+SIM group for 10 minutes was significantly higher than that in the DDD+GEL group at 6th,7th,and 8th week(p < 0.05).The levels of NF-?B1,Rel A,GAP43,SP,CGRP,TRPM8,IL-1? and TNF-? in the intervertebral disc of DDD+SIM group were significantly lower than those in DDD group(p < 0.05);GAP43 and TRPM8 in rat plantar skin.The content decreased significantly(p<0.05);the content of TRPM8 in DRG decreased significantly(p < 0.05).Conclusion: Simvastatin,on the one hand,inhibits the growth of nerve fibers in the intervertebral disc,and on the other hand,significantly reduces the amount of inflammatory factors in the intervertebral disc through the NF-KB pathway(inflammatory factors stimulate the new nerves in the intervertebral disc)to achieve pain relief..However,the specific mechanism still needs further research.This study provides new strategies and possible targets for the treatment of discogenic low back pain. |
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