| PartⅠ Manufacturing nonbacterial prostate inflammatory pain models in ratsObjective: To explore the content of substance P and β-endorphin in blood plasm and the status of activation of astrocytes in the corresponding spinal cord after chemical irritation of the prostate of rats,to establish a rat model of nonbacterial prostatitis pain,to provide experimental basis for the treatment of Non-bacterial prostatitis pain.Methods: Twenty four healthy male SD rats were randomly divided into two groups:6 as sham operated group and the other 18 as Non-bacterial prostatitis models induced by injecting CFA to prostate.The rats in the Non-bacterial prostatitis model group were again randomly divided into 3 subgroups(6 in each group): CFA injection3 d group,CFA injection 7 d group,and CFA injection 10 d group.The prostate tissue of rats was stained by HE stain and,the content of sp and β-EP in hematoplasma of rats was detected by ELISA method,the changes in the total OD value of GFAP in L6-S1 spinal cord astrocytes of rats were detected and analyzed by immumofluorescence method combined with IPP 6 image measurement software.Results: 1、HE stain: The inflammatory reaction of prostate in CFA injection 3 d group was slight and in CFA injection 7 d group was strong,in addition,the inflammatory reaction of prostate in CFA injection 10 d group was basically consistent with the 7d group;2、ELISA method:Compared with sham operated group,the content of SP were higher and the content of β-EP decreased in the model group,which had statistically significant;3、Immumofluorescence method: Compared with sham operated group,The expression of GFAP was higher in the model group,which had statistically significant;4、The ELISA test results of SP andβ-EP and the expression results of GFAP was basically consistent with the extent of inflammatory reaction of prostate in the time phase.Conclusions: Non-bacterial prostatitis induced by CFA can cause increased release of pain transmitter substance P and decreased release of β-endorphin in plasma,enhanced activation of astrocytes in spinal dorsal horn and increased expression of GFAP,and induce obvious inflammatory pain which was most intense and stable on day 7 after CFA stimulation.Part II Activation of astrocytes in the L6-S1 spinal cord in non-bacterial postatitis pin of ratObjective: The study aimed to research the expression of brain natriuretic peptide(BNP)and natriuretic peptide recep-tor A(NPR-A)in dorsal root ganglia(DRG)of the spinal cord in a rat model of nonbacterial prostatitis pain(NPP),to investigate the relationship between the signaling pathway which BNP/NPR-A mediated and inflammatory pain caused by nonbacterial prostatitis。Methods: One hundred specific pathogen-free(SPF)male Sprague–Dawley rats were randomly divided into a control group(N=50)and a study group(N=50).The control group underwent prostatic injection of 0.1 ml of normal saline on days 3,7,10,14,and 28.The study group,or rat model of NPP,underwent prostatic injection of 0.1ml of complete Freund’s adjuvant on days 3,7,10,14,and 28.At the end of the study,the rats were euthanized,and the prostate tissues and dorsal root ganglia(DRG)of the spinal cord were removed.Histology was performed on the prostate tissue from the rats in the study group and control group.Real-time fluorescence-based quantitative polymerase chain reaction(PCR)and Western blot were used to study the expression of BNP and NPR-A m RNA and protein in the DRG from the rats in the study group and control group.Results: 1.HE staining of prostate tissue,the 3d group had a mild inflammatory response(vascular congestion,mild expansion of blood vessels and surrounding stroma shows a few neutrophils and lymphocytes infiltrating),the 7d had a intense inflammatory response(vasodilation congestion,severe and burst blood vessels and can be seen in the stroma around a lot of neutrophils and lymphocytes infiltrating),The inflammatory response on day 10 was the same as on day 7;The inflammatory response on day 14 and day 28 was the same as on day 3;2.In the rat model of NNP,the expression of BNP and NPR-A were significantly increased in dorsal root ganglia(DRG)of the spinal cord compared with the controls.Conclusions: In a rat model of CNP,the increased expression of BNP and NPR-A in dorsal root ganglia(DRG)of the spinal cord may have a role in pain signaling pathways associated with chronic prostatitis.Part III Effects of BNP,PKG inhibitors and BKCca inhibitors on K ion currents in nociceptive neurons of spinal cord central ganglion in rats with nonbacterial prostatitis painObjective: BNP,BKca inhibitor and PKG inhibitor were injected intrathecally into rat models of non-bacterial prostatitis pain,and to detect the K+ current of spinal cord central ganglion pain neuron in the rat models of non-bacterial prostatitis pain influences.Methods: There are 50 healthy male rats of SPF grade,10 were randomly selected as the sham operation group,and the remaining 40 were injected with Freund’s complete adjuvant into the prostate to prepare a non-bacterial prostatitis pain model;on the 7th day of modeling,BNP,BKca inhibitor and PKG inhibitor were injected into the nerve myelin sheath of rats,and they were randomly divided into sham operation control group,prostatitis pain group,intrathecal injected BNP group,intrathecal injected The BKca inhibitor + BNP group,and intrathecal injected the PKG inhibitor + BNP group,with 10 rats in each group,10 rats in each group.Five days after the intrathecal injection of BNP,the central ganglia of the spinal cord were extracted,and the K+ current on the Bkca channel of C pain-sensing neurons was detected by patch clamp technique.Results: The K+ current on the Bkca channel of C pain sensor: 1.The K+ current of the prostatitis pain group was lower than that of the sham operation group;2.The K+ current of the intrathecal injection of BNP group was higher than that of the prostatitis pain group;The K+ current of the intrathecal injection of BKca inhibitor and BNP was lower than that in the intrathecal BNP group.The K+ current of the intrathecal injection of PKG inhibitor and BNP was lower than that in the intrathecal BNP group.Conclusion: To active the BNP/NPR-A/PKG/ BKca signaling pathway can reduce the excitability of C-nociceptive neurons in spinal cord central ganglion of rats with NP pain,restore the hypersensitivity state of nociceptive neurons,and play an role in inhibit pain.Part IV Effect of intrathecal injection of BNP on activation of glial cells in spinal dorsal horn of rats with nonbacterial prostatitis pain and its analgesic effectObjective: to investigate the expression of astrocyte glial fibrillary acidic protein(GFAP)and microglial complement receptor-3(CR3/ CD11b)in cornu dorsale medullae spinalis of rats with Nonbacterial prostatitis pain,to explore the therapeutic efficacy and action mechanism of intrathecal injection of BNP alleviating chronic neuropathic pain,to establish the experimental base of the use of intrathecal injection of BNP alleviating chronic neuropathic pain.Methods: 1.Thirty healthy male SPF SD rats were randomly divided into sham operation control group,non-bacterial prostatitis pain group(NPP model)and NNP+ intrathecal injection BNP group(BNP group),with 10 rats in each group,The NNP model was established by intraprostatic injection of CFA,and the spinal cord of L6-S1 segment was extracted 5 days after intrathecal injection of BNP;2.the expression of GFAP and CR3/ CD11 b in cornu dorsale medullae spinalis of rats with immumofluorescence method combined with graphic image analysis software.Results: The cumulative optical density values of GFAP and CR3/ CD11 b expression in the NPP model group were higher than those in the sham operation group,with statistical significance(P < 0.01);The expression of GFAP and CR3/ CD11 b in NNP+ intrathecal injection BNP group were lower than those in NNP group,the differences were statistically significant(P < 0.01).Conclusion: Intrathecal injection of BNP can down-regulate the expressions of GFAP and CR3/ CD11 b in L6-S1 spinal cord of NNP rat model,and can inhibit the chronic pain caused by NNP. |
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