| BackgroundItch has been defined as an unpleasant skin-specific sensation that provokes the desire to scratchreflex, thereby removing potentially harmful agents, such as a parasite, from the skin’s surface. However, itches also become chronic pathological conditions, such as chronic cholestasis. Unfortunately, effective treatment for chronic itch is lacking, and progress in the development of new therapies is hampered by insufficient (limited) knowledge of the neural circuits that underlie distinct pruritic sensation.It’s widely acknowledged that both pain and itch belong to nociceptive sensation, and many C-fibers respond both to algesic (pain-inducing) and pruritic (itch-inducing) compounds. The rostral ventromedial medulla (RVM) of the brainstem is a prominent component of the descending modulatory system for nociception at the spinal cord level, and neurons in the RVM have been implicated in the inhibition and facilitation of spinal nociceptive transmission. Recent studies have established that the RVM facilitation of nociceptive transmission in the spinal cord contributes to neuropathic pain. Thereby, it has been hypothesized that RVM facilitation of nociceptive transmission in the spinal cord might contribute to chronic pathological itch. Recent work has supported that the serotonin system in RVM was involved in descending facilitation in the spinal cord after tissue and nerve injury. Furthermore, spinal5-HT3receptor activation had been shown to contribute to the development of hyperalgesia in rats with spinal cord injury, and that blockade of spinal5-HT3receptors reduces hypersensitivity after L5-L6spinal nerve ligation. It is speculated that serotonergic signaling in RVM may modulate the activity of sympathetic outflow sensitive to pruritic signals.Objective1. To provide direct evidence that selective lesions of5-serotonergic neurons in the RVM by focal neurotoxin5,7-DHT treatment cause decreased pruritic behaviors induced by an intradermal microinjection of histamine(His), chloroquine(CQ), endothelin-1(ET-1) in the nape of the neck.2. To determine the role of the descending5-HT in mouse pruritic behaviors by selectively depleting functional phenotypes of5-HT in RVM neurons with regional RNA interference (RNAi) of tryptophan hydroxylase-2(TPH2), found in brain as the rate-limiting enzyme in neuronal serotonin biosynthesis and can be used as a marker of central5-HT-containing neurons, cause decreased pruritic behaviors induced by an intradermal microinjection of histamine(His), chloroquine(CQ), endothelin-1(ET-1) in the nape of the neck.Methods and Results1. Construction of mouse itch modelsMethods:Male C57BL/6J mice (8-10weeks old) were divided into naive group (N group, n=6), saline group (S group, n=6), histamine (His, n=6), chloroquine (CQ group, n=6) and endothelin-1group (ET-1group, n=6). The control group did not receive any treatment. After mice were received an intradermal microinjection of saline (100μl), histamine (500μg/100μl), chloroquine (200μg/100μl), endothelin-1(25ng/100μl) in the nape of the neck, pruritic behavior was quantified by recording the number of scratching bouts at5min intervals over the30min observation period.Results:The total scratching bouts in30min were5.83±1.67(N group),5.67±1.11(S group),91.83±24.17(His group),117.67±24.00(CQ group),187.83±38.50(ET-1group). The total scratching bouts were significantly increased in His group, CQ group, ET-1group as compared with that of in N group or S group (P<0.05), whereas there was no statistical difference between N group and S group, suggesting that intradermal microinjection of histamine, chloroquine and endothelin-1resulted in a dramatic increase in itch behavior.2. The change of pruritic behavior after selective lesions of5-serotonergic neurons in the RVM by focal neurotoxin5,7-DHT treatmentMethods:Four days after the injection of saline(0.5μl) or neurotoxin5,7-DHT(2μg/0.5μl) in the RVM area, male C57BL/6J mice (8-10weeks old) were divided into saline-histamine group (saline-His group, n=6), saline-chloroquine group (saline-CQ group, n=6) and saline-endothelin-1group (saline-ET-1group, n=6), DHT-histamine (DHT-His group, n=6), DHT-chloroquine (DHT-CQ group, n=6) and DHT-endothelin-1group (DHT-ET-1group, n=6). After mice were received an intradermal microinjection of saline(100μl), histamine(500μg/100μl), chloroquine(200μg/100μl), endothelin-1(25ng/100μl) in the nape of the neck, pruritic behavior was quantified by recording the number of scratching bouts at5min intervals during the30min observation period. After the mice were fully anesthetized and perfused, the brain and spinal cord were postfixed, transferred to30%sucrose, and cut into25-μm thick sections. The sections of the RVM area and spinal cord were processed for TPH or5-HT immunohistochemistry according to the standard protocols. Results:The total scratching bouts in30min were93.67±23.33(saline-His group),123.67±18.56(saline-CQ group),184.33±35.00(saline-ET-1group),12.83±4.89(DHT-His group),18.33±5.33(DHT-CQ group),60.33±10.33(DHT-ET-1group). The total scratching bouts were significantly decreased in DHT-His group, DHT-CQ group, DHT-ET-1group as compared with that of in saline-His group, saline-CQ group, saline-ET-1group, respectively (P<0.05). TPH positive neurons in RVM and5-HT immunoreactive expression in spinal cord were significantly decreased in DHT-His group, DHT-CQ group, DHT-ET-1group compared to saline-His group, saline-CQ group, saline-ET-1group, respectively (P<0.05). These results suggested that after mice were received selective lesions of5-HT containing neurons in the RVM by focal neurotoxin5,7-DHT treatment, pruritic behaviors induced by microinjection of histamine, chloroquine and endothelin-1in the RVM significantly decreased.3. Construction of mouse TPH2shRNA lentivirus and identification of interfering efficiency in vitroMethods:Contemplated by design, mouse TPH2gene shRNA oligonucleotide sequence was cloned into plasmid GV118. Human embryonic kidney293T cells and the plasmid we constructed,with the help of plasmid pHelper1.0and pHelper2.0,produce viral particles. And then Rattus norvegicus adrenal pheochromocytoma PC12cells was infected. Cell morphology and viral expression was observed under fluorescence microscopy to determine the PC12cells infected with lentivirus appropriate conditions and MOI. Real-time PCR and Western blot analysis were used to detect expression of TPH2in PC12cells.Results:Sequence analysis showed that the sequence of the plasmid LV-TPH2and LV-Con was corrected as designed.The titer of virus LV-TPH2-RNAi and LV-con-RNAi obtained form the293T cells were1×109TU/ml and2×109TU/ml. Infecting PC12cells to determine its MOI is100. Real-time PCR and Western blot detection with the normal control group decreased by about50%compared to the mRNA, protein is reduced by approximately60%, no significant change in the control virus group and normal group. These results suggest that this study successfully constructed a mouse TPH2shRNA lentiviral vector, and can effectively inhibit the TPH2expression in PC12cells.4. The change of pruritic behavior after selectively depleting functional phenotypes of5-HT in RVM neurons with regional RNAi of TPH2Methods:Seven days after the injection of control virus or siRNA-TPH-2virus in the RVM area, male C57BL/6J mice (8-10weeks old) were divided into control-histamine group (con-His group, n=6), control-chloroquine group (con-CQ group, n=6) and control-endothelin-1group (con-ET-1group, n=6), TPH-2-histamine (TPH-2-His group, n=6), TPH-2-chloroquine (TPH-2-CQ group, n=6) and TPH-2-endothelin-1group (TPH-2-ET-1group, n=6). After mice were received an intradermal microinjection of saline(100μl), histamine(500μg/100μl), chloroquine(200μg/100μl), endothelin-1(25ng/100μl) in the nape of the neck, pruritic behavior was quantified by recording the number of scratching bouts at5min intervals during the30min observation period. After the mice were fully anesthetized and perfused, the brain and spinal cord were postfixed, transferred to30%sucrose, and cut into25-μm thick sections. The sections of the RVM area and spinal cord were processed for TPH or5-HT immunohistochemistry according to the standard protocols.Results:The total scratching bouts in30min were104.17±11.56(con-His group),119.50±20.50(con-CQ group),169.22±334.83(con-ET-1group),31.00±11.33(TPH2-His group),55.17±10.22(TPH2-CQ group),48.00±5.33(TPH2-ET-1group). The total scratching bouts were significantly decreased in TPH2-His group, TPH2-CQ group, TPH2-ET-1group as compared with that of in con-His group, con-CQ group, con-ET-1group, respectively (P<0.05). TPH positive neurons in RVM and5-HT immunoreactive expression in spinal cord were significantly decreased in TPH2-His group, TPH2-CQ group, TPH2-ET-1group compared to con-His group, con-CQ group, con-ET-1group, respectively (P<0.05). These results suggested that after selectively depleting functional phenotypes of5-HT in RVM neurons with regional RNAi of TPH-2, pruritic behaviors induced by microinjection of histamine, chloroquine and endothelin-1significantly decreased.5. Statistical analysisThe quantitative data were expressed as mean and standard deviation, and analyzed with one-way ANOVA. Bonferroni’s Post Hoc test was used to compare the differences between every two groups. The qualitative data were analyzed with Kruskal-Wallis rank test. P<0.05was considered statistically significant.ConclusionOur data suggested that histamine, chloroquine and endothelin-1might signal through the5-HT pathway to induce pruritus sensation, while selective lesions of5-HT containing neurons in the RVM by focal neurotoxin5,7-DHT treatment, and selectively depleting functional phenotypes of5-HT in RVM neurons with regional RNAi of TPH-2, may antagonize the pruritus evoked by histamine, chloroquine and endothelin-1. Thus, this research was a novel exploration of mechanism for pruritic signal pathway. These results may provide a basis for the future development of antipruritic therapy. |
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