Effects Of Epigenetic Regulation On Hyperalgesia, Cognitive Impairment, And BDNF-mediated Synaptic Plasticity In A Rat Model Of Bone Cancer Pain

Part Ⅰ: Application of a Modified Combined Spinal-EpiduralTechnique in Intrathecal Catheterization in RatObjectives: Intrathecal catheterization and drug delivery in rats has always been a very important method for neuroscience and pain research. Although the technique has been continually improved since the first report, the experience gained over the years suggested that some defects remained unsolved.Methods: Based on the modification of the standard epidural needle and lumbar needle, we aimed to develop a simple and practical technique for intrathecal catheterization, which was similar to the “needle-through-needle technique” used in combined spinal-epidural(CSE) anesthesia. At first, the tip of epidural needle was positioned at intervertebral foramen through lumbar skin incision, then spinal needle was inserted into the epidural needle to drill in at the dura, and finally intrathecal tube was advanced into subarachnoid space through the guide of the side hole on the body of epidural needle. For comparison, rats received intrathecal catheterization via either laminectomy at L3-4 or our modified method. The operation time, success rate, and the incidence of postoperative complication were recorded. Thermal paw withdrawal latency, mechanical paw withdrawal threshold and motor function were measured on pre-operative day 1 and post-operative day 1-5, respectively.Results: Compared with previously reported methods, our modified method was more simple and convenient in operation and could bring about fewer trauma, greater position precision, firmer catheters immobility, and minimal mortality or morbidity. It did not interfere with behavioral tests and motor coordination.Conclusion: We suggest that the modified method of intrathecal catheterization is well suitable for long-term behavior and pharmacology research on spinal cord.Part Ⅱ: Effects of HDAC and HAT Inhibitors on BDNF-mediatedSynaptic Plasticity in a Rat Model of Bone Cancer PainObjectives: Bone cancer pain(BCP) has a debilitating effect on cancer patients and is usually difficult to manage clinically. In association with BCP, we previously demonstrated that brain-derived nerve neurotrophic factor(BDNF) released from microglia contributes to hyperalgesia development in the spinal cord. BDNF is an important epigenetic target gene, regulated by the balance between histone acetyltransferase(HAT) and histone deacetylase(HDAC) enzymes. The role of HAT and HDAC in regulating BDNF in BCP is poorly understood. Therefore, we aim to study the pharmacological effects of curcumin(CUR; HAT inhibitor) and suberoylanilide hydroxamic acid(SAHA; HDAC inhibitor) on pain behavior as well as BDNF and synapsin(SYN1) expression in the spinal dorsal horn.Methods: 40 female SD rats were randomly divided into 5 groups: sham controls groups, BCP+vehicle groups, BCP+SAHA 25μg group, BCP+SAHA 5μg group, BCP+SAHA 1μg group. Bone cancer pain was induced by inoculation of Walker256 cells into left tibia of rats. On postoperative 6~8 day, rats received an intrathecal injection of SAHA in corresponding doses once daily. Ipsilateral mechanical withdrawal threshold(MWT) was measured on preoperative 1 day and postoperative 1~18 day, respectively. Another 32 female SD rats were randomly divided into 4 groups: sham group(SHAM); BCP + vehicles group(VEH); BCP + SAHA group(SAHA); BCP +CUR group(CUR). On the same three successive days, intraperitoneally DMSO, SAHA, and CUR daily was administrated in VEH group, SAHA group, and CUR group rats, respectively. MWT was measured on the same days as above. Another 48 female SD rats were used with the same grouping and treatments. Immunohistochemistry, Western blots, and real-time PCR were used to detect the expression of BDNF, SYN1, ac H3K9, and ac H4K16 in spinal cordResults: Intrathecally administered SAHA blocked bone cancer-induced mechanical hyperalgesia in a dose-dependent manner. Double immunofluorescence demonstrated that BDNF was colocalized with neurons and microglia while, ac H3K9, ac H4K16, and SYN1 were colocalized in neurons only. Systemic application of SAHA reversed BCP induced mechanical sensitization, increased acetylation of H3K9 and H4K16, but reduced the expression of BDNF and SYN1 in spinal dorsal horn. In contrast, treatment with CUR had no effect on pain threshold.Conclusion: HDAC inhibition can aid in the partial control of BCP development through the down-regulation of BDNF/SYN1 gene transcription.Part Ⅲ: Effects of Epigenetic Regulation on Cognitive Impairmentand Hippocampal BDNF/SYN1 Expression in a Rat Model of BoneCancer PainObjective: Previous studies indicated that chronic pain stimulation has a significant effect on learning and memory function by downregulation of hippocampal BDNF expression. A rat animal model of bone cancer pain(BCP) was used to observe the effects of HAT or HDAC inhibitor on learning and memory function and hippocampal BDNF and SYN1 expression to provide experimental basis for the prevention and treatment of bone cancer induced cognitive impairment.Methods: 28 female SD rats were randomly divided into 7 groups: normal group, sham group, and CBP 3, 6, 18, 12 day groups. Bone cancer pain was induced by inoculation of Walker256 cells into left tibia of rats. Western blotting was used to detect hippocampal BDNF/SYN expression at each time points. Double immunofluorescence of of BDNF, SYN1, ac H3k9, and ac H4K16 with neural cell markers(GFAP, OX42, and Neu N) were employed on hippocampus on postoperative12 days. Another 32 female SD rats were randomly divided into 4 groups: sham group(SHAM); BCP+vehicles group(VEH); BCP+SAHA group(SAHA); BCP +CUR group(CUR). On the 4 successive days(postoperative 10~14 day), intraperitoneally DMSO, SAHA, and CUR daily was administrated in VEH group, SAHA group, and CUR group rats, respectively. Morris Water Maze trials were conducted to measure the spatial learning and memory. Western blots and Real-time PCR were used to detect the expression of BDNF/SYN1 in hippocampal tissue.Results: Hippocampal BDNF/SYN1 increased significantly on postoperative 3 days and then decreased rapidly in following days. Double immunofluorescence demonstrated that BDNF and ac H3K9 were colocalized with microglia. Compared with sham group, hidden platform latency and swimming distance average in VEH group was significantly prolonged in the post-inoculation 11~13 day(2~4 days in water maze test). This trend could be significantly reversed by 4 consecutive days of application of SAHA. In the reference memory task, the time spent in the target quadrant in SAHA group was significantly increased compared with VEH group. SAHA could significantly improve bone cancer pain induced down-regulation of the BDNF and SYN1 expression in hippocampus 14 days after inoculation. There was no significant difference between the CUR group and the VEH group.Conclusion: Bone cancer pain could induce cognitive impairment through the downregulation of hippocampal BDNF/SYN1 expression. SAHA could improve the ability of learning and memory by epegentically increasing BDNF/SYN1 expression.