The Central Mechanism In Onabotulinumtoxin A Treatment Of Trigeminal Neuralgia In Lab Rat Model

Trigeminal neuralgia is acknowledged as one of the most painful and common neuropathic disorders. It is characterized by paroxysmal attacks of one-sided intense electric shock-like or stabbing pains in areas supplied by the trigeminal nerve. The preferred treatment for trigeminal neuralgia is oral administration. If this fails,surgery could be an option. However, disappointing results have been seen in many patients who have undergone a variety of treatments. Onabotulinumtoxin A, an exotoxin produced by the Gram-positive, anaerobic and spore-forming bacterium Clostridium botulinum, has been widely used in the treatment of dystonia and in cosmetology. In recent years, an increasing number of researches have been done on its application in pain management. In 2012, we developed an improved technology roadmap of treating trigeminal neuralgia with Onabotulinumtoxin A and proved the long-term and effective pain relief with minor adverse reactions of Onabotulinumtoxin A in trigeminal neuralgia treatment with random, double-blind and placebo-controlled trials for the first time. Subsequent clinical studies have come to similar conclusion. However, the mechanism remains unclear. For this, a trigeminal neuropathic pain model is established by chronic constriction injury of the infraorbital nerve in rats to study Onabotulinumtoxin A’s therapeutic effects, treatment area and mechanism. This study is to lay a theoretical groundwork for Onabotulinumtoxin A treatment of trigeminal neuralgia and other clinical conditions associated with pain.Part I The pain behavior changes and histological changes of trigeminal neuralgia modelObjective Establish the trigeminal neuralgia model by chronic constriction injury of the infraorbital nerve in rats. Simulation of the classic trigeminal neuralgia. Observe pain behavior changes and histological changes after surgery.Methods Establish the trigeminal neuralgia model by chronic constriction injury of the infraorbital nerve in experimental group. The control group only exposed but not ligated infraorbital nerve. Observe behavior changes of the experimental and control groups before and every two days after the surgery using Von Frey filaments. Test histological changes at different time before and after the surgery with HE and MBP immunohistochemical staining.Results The experimental group enters into a refractory period 4 days after the surgery with a significant boost in the threshold of pain. On the 6th day, the threshold peaks at24.6±8.6g, which is a statistically significant result compared with the level before the surgery(P<0.05). Then the threshold drops to 1.6±0.3g(P<0.05) on the 14 th day,which flattens out until at least the 34 th day.There are no statistically significant differences between the pain thresholds before and after the surgery(P>0.05) in the control group.The pains thresholds of the experimental and control groups before the surgery are 13.5±3.6g and 12.6±5.5g between which no statistical significance is observed(P>0.05). The pain thresholds of the two groups on the 10 th day after the surgery are5.5±3.2g and 13.6±4.8g whose difference is of statistical significance, which remains as such until at least the 34 th day after the surgery.The HE and MBP immunohistochemical staining show there is significant demyelination in the experimental group and there is no significant change in control group.Conclusion Trigeminal neuropathic generated by the chronic constriction injury of the infraorbital nerve consistent with the clinical features and histological changes of classic trigeminal neuralgia. This is a simple and feasible model and it can be adopted for investigating the pathogenesis of trigeminal neuralgia and developing new therapies.Part II The antinociceptive activity of Onabotulinumtoxin A in trigeminal neuralgia modelObjective Treatment of trigeminal neuralgia model with Onabotulinumtoxin A. Observe the pain behavior changes after treatment.Methods On the 14 th day after the surgery, Onabotulinumtoxin A(3U/Kg and 10U/Kg) is injected to the whisker pad in the operated side of the experimental groups. The same volume of normal saline is injected in the control group. Observe pain behavior changes at different time before and after the surgery.Results Four days after the administration of Onabotulinumtoxin A(18 days after the surgery), significant boosts in pain thresholds are observed in both 3U and 10 U groups, which is statistically different from that of the control group(P<0.05). The antinociceptive effect lasts until at least 20 days after the Onabotulinumtoxin A administration(34 days after the surgery). The antinociceptive effect is stronger in the10 U group than in the 3U group but no statistical significance is observed(P>0.05).Conclusion Hypodermic Onabotulinumtoxin A injection to the whisker pad of model rats can effectively increase pain threshold and antinociception.Part III The site of action of OnabotulinumtoxinA in the treatment of trigeminal neuralgia in ratsObjective Study the site of action and the axonal transport of Onabotulinumtoxin A for the treatment of trigeminal neuralgia based on the results of the first two parts.Methods On the 13.5th day after the surgery(12 hours before the Onabotulinumtoxin A administration), inject colchicine, an axonal transport inhibitor, to the trigeminal ganglion. After 14 days, inject Onabotulinumtoxin A(10U/Kg) to the whisker pad in the operated side of the rats and monitor behavioral changes at different time.Compare with the control group and observe the role of axonal transport in Onabotulinumtoxin A treatment of trigeminal neuralgia.Detect c SNAP-25 as a marker of Onabotulinumtoxin A with Western blotting and immunofluorescence and observe the treatment area in the model.Results Inhibit the axonal transport with colchicine in the experimental group reduces the analgesic effect of Onabotulinumtoxin A. Compared with the control group, the pain threshold of the experiment group drops sharply with statistical significance from 4 days after the Onabotulinumtoxin A treatment(P<0.05).On the 7th day after the treatment, the level of c SNAP-25 in the subnucleus caudalis of spinal trigeminal nucleus has been detected to have a significant increase with Western blotting and immunofluorescence. After the colchicine infusion, the c SNAP-25 level undergoes a drastic decrease which is of statistical significance(P<0.05).Conclusion The inhibition of axonal transport results in a significant decrease of Onabotulinumtoxin A’s antinociceptive effects. Injected Onabotulinumtoxin A is transported to and then acts in the subnucleus caudalis of spinal trigeminal nucleus in trigeminal neuralgia models.Part IV The Mechanism of TRPs in Onabotulinumtoxin A for the treatment of trigeminal neuralgiaObjective To investigate the pain related TRPs(TRPA1, TRPV1, TRPV2 and TRPM8) in the mechanism of the antinociceptive activity of Onabotulinumtoxin A in treating the trigeminal neuralgia modelsMethods TRPA1, TRPV1, TRPV2 and TRPM8 are ion channels associated with pain in the TRPs family. To investigate the mechanism of the antinociceptive role of Onabotulinumtoxin A in treating the rat models, Western blotting analysis is adopted to detect the protein expression of TRPA1, TRPV1, TRPV2 and TRPM8 and their changes at different time. In the meantime, investigate impact of Onabotulinumtoxin A administration to the whisker pad in the operated side(3U/Kg and 10U/Kg) on the expression of these proteins and central sensitization mediated by TRPs ion channels.Results The Western blotting analysis shows significant increases in the expression of TRPA1 and TRPV1 in the subnucleus caudalis of spinal trigeminal nucleus on the14 th day after the chronic constriction injury of the infraorbital nerve(P<0.05), which remains considerably higher than the level in the control group on the 28 th day(P<0.05). The expression of TRPV2 increases sharply on the 7th day(P<0.05) and continues to increase on the 28 th day(P<0.05). TRPM8 level starts to rise on the 7th day, peaks on the 14 th day and remains statistically different from that of the control group(P<0.05).On the 14 th day after the Onabotulinumtoxin A administration, western blotting analysis shows the expression of TRPA1 and TRPV1 in subnucleus caudalis of spinal trigeminal nucleus drops sharply. A dose-response relationship between Onabotulinumtoxin A and the reduction of the expression of TRPA1 and TRPV1 has been observed for there are statistically significant differences between the levels of the two proteins in 3U and 10 U groups(P<0.05). On the 14 th day of the Onabotulinumtoxin A administration(10U/Kg), the expression of TRPV2 is drastically reduced whereas the level of TRPV2 in 3U is not statistically different from that of the control group(P>0.05) on the 14 th day after the surgery. Compared with the control group, Onabotulinumtoxin A does not affect the expression of TRPM8(3U/Kg and 10U/Kg)(P>0.05).Conclusion Sharp increases with dynamic changing processes have been detected in the expression of TRPA1, TRPV1, TRPV2 and TRPM8 in the subnucleus caudalis of spinal trigeminal nucleus in the trigeminal neuralgia models. Onabotulinumtoxin A can decrease central sensitization by dose-dependently reducing the expression of TRPA1, TRPV1 and TRPV2 in the subnucleus caudalis of spinal trigeminal nucleus and display antinociceptive effects on trigeminal neuralgia.