背根神经节内P物质、降钙素基因相关肽免疫阳性神经元与阴茎包皮系带感觉信息的传递

目的：探讨背根神经节（DRG）内P物质（SP）、降钙素基因相关肽（CGRP）免疫阳性神经元与阴茎包皮系带感觉信息传递之问的关系。方法：通过荧光金（FG）逆行标记对大鼠阴茎包皮系带内神经末梢的来源作追踪定位，并结合SP、CGRP免疫荧光标记法，研究大鼠DRG内FG标记阳性神经元中SP、CGRP免疫阳性神经元的形态和分布。结果：FG逆行标记结果发现，大鼠阴茎包皮系带内的神经末梢起源于第6腰髓对应的背根神经节（L6-DRG）和第1骶髓对应的背根神经节（S1-DRG）的神经元。对这些神经元分别作SP、CGRP免疫荧光标记后发现，标记细胞大小不等，分别呈深红色和深绿色，沿神经束成行排列或散在分布。FG／SP、FG／CGRP双标记阳性细胞均为中小型，其数量分别占FG逆行标记阳性细胞总数的1／3和1／2，FG／SP／CGRP三标记阳性细胞占FG逆行标记阳性细胞总数的1／5。结论：大鼠L6-DRG和S1-DRG内的SP、CGRP免疫阳性神经元可能参与阴茎包皮系带感觉信息的传递。     

阴茎包皮及包皮系带内SP免疫阳性神经末梢的分布和来源

目的观察成人阴茎包皮和包皮系带内P物质(substanceP,SP)免疫阳性神经末梢的分布和来源。方法免疫组织化学法观察SP免疫阳性神经末梢的分布,荧光金(fluoro-gold,FG)逆行追踪和SP免疫荧光标记相结合法研究大鼠包皮系带内SP免疫阳性神经末梢的来源。结果成人阴茎包皮及包皮系带内均有密集的SP免疫阳性神经末梢存在,这些神经末梢主要位于表皮基底层,呈树枝状或念珠状分布,大多成束走行。阴茎系带处SP免疫阳性神经末梢的分布密度明显大于阴茎包皮处。FG逆标阳性细胞位于大鼠第六腰髓对应的背根神经节(L6-DRG)和第一骶髓对应的背根神经节(S1-DRG)。阳性细胞大中小不等,大多沿神经束成行排列或散在分布。SP免疫荧光标记细胞大多为中小型,呈深红色。FG/SP双标阳性细胞均为中小型,其数量占FG逆标阳性细胞总数的三分之一。结论SP参与了阴茎包皮及包皮系带感觉信息的传递。大鼠阴茎包皮系带内SP免疫阳性神经末梢源自于L6-DRG和S1-DRG。     

大鼠阴茎包皮和包皮系带内nNOS免疫阳性神经末梢的分布与起源

目的观察成年SD大鼠阴茎包皮和包皮系带内神经型一氧化氮合酶(nNOS)免疫阳性神经末梢的分布和起源。方法免疫组织化学法观察nNOS免疫阳性神经末梢的分布,荧光金(Fluoro-gold,FG)逆行追踪和nNOS免疫荧光标记相结合法研究大鼠包皮系带内nNOS免疫阳性神经末梢的起源。结果成年SD大鼠阴茎包皮和包皮系带内均有nNOS免疫阳性神经末梢存在,这些神经末梢主要位于表皮基底层和真皮乳头层,呈树枝状或念珠状分布。阴茎系带处nNOS免疫阳性神经末梢的图像光密度值(3.4±0.38)明显大于阴茎包皮处(2.2±0.45)。FG逆标阳性细胞位于大鼠第六腰髓对应的背根神经节(L6-DRG)(9.6±1.2)个/mm2和第一骶髓对应的背根神经节(S1-DRG)(1.2±0.2个/mm2)内。阳性细胞大中小不等,大多沿神经束成行排列或散在分布。nNOS免疫荧光标记细胞在L6-DRG和S1-DRG内分别为(24.2±2.6)个/mm2和(24.1±2.1)个/mm2,细胞大多呈中小型。FG/nNOS双标阳性细胞均为中小型,其数量接近FG逆标阳性细胞总数的一半。结论大鼠阴茎包皮系带内含有浓密的nNOS免疫阳性神经末梢,这些神经末梢源自于L6-DRG和S1-DRG。     

硬膜外腔植入髓核对大鼠脊髓背根神经节SP和CGRP表达的影响

目的 观察硬膜外腔植入异体髓核对大鼠L6-S1,脊髓背根神经节细胞SP和CGRP表达的影响,以期为椎间盘源性疼痛发病机制提供细胞生物学基础.方法 18只雄性SD大鼠(体重260-280g)随机分为三组:脂肪组、髓核组、假手术组,每组n=6.另外3只雄性SD大鼠用来提供异体脂肪和髓核.术后第30d取L4-L6脊髓背根神经节和脊髓背角,采用免疫组织化学染色方法 观察髓核对脊髓背角和背根神经节细胞肽类神经递质SP和CGRP表达的影响.结果 术后30d脂肪组大鼠与假手术组相比,脊髓背角和背根神经节细胞SP和CGRP表达没有显著性差异(P>0.05);髓核组与脂肪组和假手术组相比脊髓背角和背根神经节细胞SP和CGRP表达显著增加(P<0.05).结论 硬膜外腔植入异体髓核可引起脊髓背根神经节细胞SP和CGRP表达增加.     

大鼠盆神经节NOS和VIP阳性神经元对阴茎海绵体的支配

目的：研究大鼠盆神经节(MPG)中一氧化氮合酶(NOS)、血管活性肠肽(VIP)阳性神经元对阴茎海绵体的支配。方法：应用辣根过氧化物酶(HRP)逆行示踪技术结合免疫组织化学和酶组织化学双标技术,观察大鼠MPD中还原型辅酶Ⅱ(NADPFI,NOS标志物)阳性和VIP阳性神经元对阴茎海绵体的支配。结果：HRP阳性标记神经元主要分布于MPG内靠近阴茎神经的区域。HRP阳性标记神经元中72％(210／292)为VIP免疫阳性,83％(268／323)为NADPH组化反应阳性。结论：大鼠盆神经节中NOS、VIP阳性神经元对阴茎海绵体有直接支配,NO和VIP作为神经递质或调质参与了阴茎勃起。     

人工体神经-内脏神经反射弧恢复截瘫后直肠功能的神经追踪研究

目的 研究正常和建立人工体神经-内脏神经反射弧脊髓损伤大鼠肛门外括约肌-脊髓、直肠-盆神经节-脊髓之间的神经通路。方法 正常及建立人工体神经-内脏神经反射弧后截瘫的(以下简称模型组)雄性SD大鼠，用荧光金(FG)注射至肛门外括约肌(EAS)、直肠壁内、盆神经节(MPG)，行逆行神经追踪；模型组大鼠，采用麦芽凝集素结合的辣根过氧化物酶(WGA-HRP)作为顺行神经示踪剂，在大鼠脊髓左侧L4前角注入WGA-HRP。结果 正常组和模型组大鼠直肠壁注入FG后，MPG主体部可见大量标记神经元。正常组将FG注射入MPG后。FG标记神经元位于脊髓L6～S1骶髓副交感核(SPN)、后联合灰质核。正常鼠FG注射入肛门外括约肌后．标记神经元主要位于L5～S1脊髓的背内侧核(DM)区．背外侧核(DL)区可见少量标记神经元。模型组大鼠将FG注入盆神经节或肛门外括约肌后，左L4前角均可见FG标记神经元；左L4前角注入WGA-HRP后，肛门外括约肌和左侧MPG中均可见HRP阳性神经末梢。结论 正常大鼠盆神经节支配直肠的神经元主要分布于MPG主体部；脊髓内支配盆神经节的神经元主要位于脊髓L6～S1节段SPN；支配肛门外括约肌的运动神经元主要位于L5～S1脊髓DM区。建立人工反射弧后。大鼠L5～S1脊髓L4前根与L6前根吻合后所形成的“异类神经纤维”在MPG换元后，支配直肠壁和EAS。     

家兔阴茎皮肤和阴茎背神经躯体感觉神经元的示踪定位

目的:明确家兔阴茎感觉神经躯体纤维来源及其节段性分布规律。方法:健康成年雄性新西兰家兔8只,随机均分为A、B两组,采用辣根过氧化物酶(HRP)逆行神经示踪技术,A组用微量注射器将50%HRP多点注射于阴茎皮下,B组注射于右侧阴茎背神经近侧断端内,留针时间30 m in,动物存活5 d后,4%多聚甲醛心脏灌注,取出腰骶段脊髓及相应背根神经节(DRG),对躯体传入和传出纤维来源的节段分布进行观察。结果:A组S2~S4节段脊神经节内出现大量HRP阳性标记感觉神经元,以S2和S4为多,呈明显节段性分布,各节段阳性神经元计数分别为:S2(215.0±10.2)、S3(242.2±8.3)、S4(109.7±8.4),组间差异具有统计学意义(P<0.01)。B组同侧S2-S4节段脊髓灰质前角腹外侧部出现部分阳性标记细胞,同侧S2~S4DRG内也出现了大量的HRP阳性细胞。结论:家兔阴茎皮肤感觉神经躯体传入和传出纤维投射来源与阴茎背神经一致,均源自骶髓前角及相应的脊神经节,并呈现明显的节段性分布。     

大鼠盆神经节胆碱乙酰转移酶和一氧化氮合酶阳性神经元对尿道外括约肌的支配

目的 观察大鼠盆神经节(MPG)中胆碱乙酰转移酶(ChAT)、一氧化氮合酶(NOS)阳性神经元对尿道外括约肌(EUS)的支配.方法 24只成年SD大鼠随机分为A组(n=12)和B组(n=12).应用辣根过氧化物酶(HRP)逆行示踪技术结合免疫组织化学和酶组织化学双标技术,观察A组大鼠MPG中ChAT阳性神经元和还原型辅酶Ⅱ(NADPH,NOS标志物)阳性神经元对EUS的支配.B组大鼠MPG内注射麦芽凝集素-辣根过氧化物酶(WGA-HRP)进行顺行神经追踪.结果 A组大鼠HRP标记神经元散在分布于MPG内.HRP标记神经元中43.3%(155/358)为ChAT免疫反应阳性,14.5%(49/339)为NADPH组化反应阳性.B组大鼠EUS内有HRP阳性神经末梢.结论 大鼠盆神经节中的神经元对EUS有直接支配,其神经递质有乙酰胆碱和一氧化氮.     

大鼠盆神经节胆碱乙酰转移酶和一氧化氮合酶阳性神经元对尿道外括约肌的支配

目的 观察大鼠盆神经节(MPG)中胆碱乙酰转移酶(ChAT)、一氧化氮合酶(NOS)阳性神经元对尿道外括约肌(EUS)的支配.方法 24只成年SD大鼠随机分为A组(n=12)和B组(n=12).应用辣根过氧化物酶(HRP)逆行示踪技术结合免疫组织化学和酶组织化学双标技术,观察A组大鼠MPG中ChAT阳性神经元和还原型辅酶Ⅱ(NADPH,NOS标志物)阳性神经元对EUS的支配.B组大鼠MPG内注射麦芽凝集素-辣根过氧化物酶(WGA-HRP)进行顺行神经追踪.结果 A组大鼠HRP标记神经元散在分布于MPG内.HRP标记神经元中43.3%(155/358)为ChAT免疫反应阳性,14.5%(49/339)为NADPH组化反应阳性.B组大鼠EUS内有HRP阳性神经末梢.结论 大鼠盆神经节中的神经元对EUS有直接支配,其神经递质有乙酰胆碱和一氧化氮.     

大鼠盆神经节胆碱乙酰转移酶和一氧化氮合酶阳性神经元对尿道外括约肌的支配

目的 观察大鼠盆神经节(MPG)中胆碱乙酰转移酶(ChAT)、一氧化氮合酶(NOS)阳性神经元对尿道外括约肌(EUS)的支配.方法 24只成年SD大鼠随机分为A组(n=12)和B组(n=12).应用辣根过氧化物酶(HRP)逆行示踪技术结合免疫组织化学和酶组织化学双标技术,观察A组大鼠MPG中ChAT阳性神经元和还原型辅酶Ⅱ(NADPH,NOS标志物)阳性神经元对EUS的支配.B组大鼠MPG内注射麦芽凝集素-辣根过氧化物酶(WGA-HRP)进行顺行神经追踪.结果 A组大鼠HRP标记神经元散在分布于MPG内.HRP标记神经元中43.3%(155/358)为ChAT免疫反应阳性,14.5%(49/339)为NADPH组化反应阳性.B组大鼠EUS内有HRP阳性神经末梢.结论 大鼠盆神经节中的神经元对EUS有直接支配,其神经递质有乙酰胆碱和一氧化氮.     

Effect of applying p75NTR saporin to a punctured intervertebral disc on calcitonin gene-related peptide expression in rat dorsal root ganglion neurons

Background

Recent studies have revealed that the low-affinity nerve growth factor receptor, p75 neurotrophin receptor (p75NTR), is important in inflammatory pain. Moreover, p75NTR immunoreactive sensory nerve and dorsal root ganglion (DRG) neurons have been found to innervate lumbar intervertebral discs. The purpose of the current study was to investigate the effect of p75NTR saporin, a toxin used to destroy p75NTR, on calcitonin gene-related peptide (CGRP), an inflammatory neuropeptide associated with pain, in DRG neurons innervating punctured intervertebral discs in rats.

Methods

The neurotracer fluorogold (FG) was applied to the surfaces of L5/6 discs to label their innervating DRG neurons (n = 30). Of 30 rats, 10 were in a nonpunctured disc sham surgery control group (nonpuncture group), and the other 20 were in experimental groups in which intervertebral discs were punctured with a 23-gauge needle. p75NTR saporin was applied to the discs of 10 rats (puncture + p75NTR saporin group) and the other 10 received the same volume of saline (puncture + saline group). At 14 days after surgery, DRGs from L1 to L6 were harvested, sectioned, and immunostained for CGRP, and the proportions of CGRP-immunoreactive DRG neurons was evaluated.

Results

Of the FG-labeled neurons innervating the L5/6 disc, the proportion of CGRP-immunoreactive neurons was 32% ± 6% (mean ± SE) in the nonpuncture group, 47.2% ± 8% in the puncture + saline group, and 34.6% ± 9% in the puncture + p75NTR saporin group. The proportion of CGRP-immunoreactive neurons was significantly greater in the puncture + saline group compared with the nonpuncture and puncture + p75NTR saporin groups (P < 0.01).

Conclusions

Half of the DRG neurons innervating the discs were positive for CGRP in the puncture + saline group. CGRP is important for mediating inflammatory and nerve-injured pain and may be important in discogenic pain. However, p75NTR saporin suppressed CGRP expression in DRG neurons. Therefore, p75NTR may be an important receptor for mediating discogenic pain via CGRP expression.     

Characteristics of sensory DRG neurons innervating the lumbar facet joints in rats

The rat L5/6 facet joint, from which low-back pain can originate, is multisegmentally innervated from the L1 to L5 dorsal root ganglions (DRGs). Sensory fibers from the L1 and L2 DRGs are reported to non-segmentally innervate the paravertebral sympathetic trunks, whilst those from the L3 to L5 DRGs segmentally innervate the L5/6 facet joint. In the current study, characteristics of sensory DRG neurons innervating the L5/6 facet joint were investigated in rats, using a retrograde neurotransport method, lectin affinity- and immuno-histochemistry. We used four markers: (1) calcitonin gene-related peptide (CGRP) as a marker of small peptide containing neurons, (2) the glycoprotein binding the isolectin from Griffonia simplicifolia (IB4) or (3 the purinergic P2X₃ receptor for small, non-peptide containing neurons, and (4) neurofilament 200 (NF200) for small and large myelinated fibers. IB4-binding and CGRP and P2X₃ receptor containing neurons are typically involved in pain sensation, whereas NF200 is associated with pain and proprioception. Neurons innervating the L5/6 facet joints, retrogradely-labeled with fluoro-gold (FG), were distributed throughout DRGs from L1 to L5. Of FG-labeled neurons, the ratios of NF200 immunoreactive (IR) neurons and CGRP-IR neurons were 37% and 35% respectively. The ratio of IB4-binding and P2X₃ receptor-IR neurons was 10%, significantly less than the ratio of CGRP-IR neurons to FG-labeled neurons. The ratios of IB4-binding and P2X₃ receptor-IR neurons were significantly higher, and that of CGRP-IR neurons was significantly less in L1 and L2 DRGs than those in L3, L4 or L5 DRGs. Under physiological conditions in rats, DRG neurons transmit several types of sensations, such as proprioception or nociception of the facet joint. Most neurons transmitting pain are CGRP-IR peptide-containing neurons. They may have a more significant role in pain sensation in the facets via peptidergic DRG neurons.Tetsuhiro Ishikawa, Masayuki Miyagi, and Seiji Ohtori contributed equally to this work.     

Up-regulation of p55 TNF alpha-receptor in dorsal root ganglia neurons following lumbar facet joint injury in rats

The rat L5/6 facet joint is multisegmentally innervated from the L1 to L6 dorsal root ganglia (DRG). Tumor necrosis factor (TNF) is a known mediator of inflammation. It has been reported that satellite cells are activated, produce TNF and surround DRG neurons innervating L5/6 facet joints after facet injury. In the current study, changes in TNF receptor (p55) expression in DRG neurons innervating the L5/6 facet joint following facet joint injury were investigated in rats using a retrograde neurotransport method followed by immunohistochemistry. Twenty rats were used for this study. Two crystals of Fluorogold (FG; neurotracer) were applied into the L5/6 facet joint. Seven days after surgery, the dorsal portion of the capsule was cut in the injured group (injured group n = 10). No injury was performed in the non-injured group (n = 10). Fourteen days after the first application of FG, bilateral DRGs from T13 to L6 levels were resected and sectioned. They were subsequently processed for p55 immunohistochemistry. The number of FG labeled neurons and number of FG labeled p55-immunoreactive (IR) neurons were counted. FG labeled DRG neurons innervating the L5/6 facet joint were distributed from ipsilateral L1 to L6 levels. Of FG labeled neurons, the ratio of DRG neurons immunoreactive for p55 in the injured group (50%) was significantly higher than that in the non-injured group (13%). The ratio of p55-IR neurons of FG labeled DRG neurons was significantly higher in total L1 and L2 DRGs than that in total L3, 4, 5 and 6 DRGs in the injured group (L1 and 2 DRG, 67%; L3, 4, 5 and 6 DRG, 37%, percentages of the total number of p55-IR neurons at L1 and L2 level or L3–6 level/the total number of FG-labeled neurons at L1 and L2 level or L3–6 level). These data suggest that up-regulation of p55 in DRG neurons may be involved in the sensory transmission from facet joint injury. Regulation of p55 in DRG neurons innervating the facet joint was different between upper DRG innervated via the paravertebral sympathetic trunks and lower DRG innervated via other direct routes. Y. Sakuma and S. Ohtori contributed equally to this work.     

Inhibiting nerve growth factor or its receptors downregulates calcitonin gene‐related peptide expression in rat lumbar dorsal root ganglia innervating injured intervertebral discs

Nerve growth factor (NGF) and its dual structurally unrelated receptors, tropomyosin‐related kinase A (TrkA) or p75 neurotrophin receptor (p75^NTR), cause the pathogenesis of discogenic pain. To investigate the sensory innervation of injured rat lumbar intervertebral disc (IVD), we examined the expression of neuropeptides such as calcitonin gene‐related peptide (CGRP) at dorsal root ganglia (DRG) by inhibiting NGF or its dual receptors. Sprague–Dawley rats with multiply punctured L5–L6 IVD were used. Six experimental groups were prepared: naïve, sham control, and four agent‐treated groups with punctured IVD (vehicle, anti‐NGF antibody, anti‐TrkA antibody, and anti‐p75^NTR antibody). Retrograde neurotracer Fluoro‐Gold (FG) was applied together except for the naïve group. Their lumbar DRG were harvested and immunolabeled for CGRP. FG‐labeled DRG neurons were most prevalent at L1 and L2 DRG, and the proportion of FG‐labeled CGRP‐immunoreactive DRG neurons in the vehicle group was significantly elevated (p < 0.05) compared with the sham group, while those of antibody‐treated groups, especially in the anti‐p75^NTR group, significantly decreased compared with the vehicle group (p < 0.05). Direct intradiscal application of antibody to NGF or its receptors suppressed CGRP expression, and p75^NTR antagonism induced the most profound suppression. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1614–1620, 2010     

Selective loss of calcitonin gene-related Peptide-expressing primary sensory neurons of the a-cell phenotype in early experimental diabetes

To evaluate the possible role of neuropeptide immunoreactive primary sensory neurons on the development of nociceptive dysfunction in diabetes, the absolute numbers of immunoreactive substance P and calcitonin gene-related peptide (CGRP) dorsal root ganglion (DRG) cell bodies were estimated in diabetic and nondiabetic BALB/C (p75(+/+)) and p75 receptor knockout (p75(-/-)) mice with unilateral sciatic nerve crush. The total numbers of immunoreactive substance P A-cells, substance P B-cells, CGRP A-cells, and CGRP B-cells in L5DRG were estimated using semithick consecutive sections and the optical fractionator. After 4 weeks of streptozotocin-induced diabetes, the number of immunoreactive CGRP A-cells was reduced from 692 +/- 122 to 489 +/- 125 (P = 0.004) in p75(+/+) mice on the noncrushed side. In p75(-/-) mice, there was no such effect of diabetes on the immunoreactive CGRP A-cell number. In p75(+/+) and p75(-/-) mice, there was no effect of diabetes on the immunoreactive CGRP B-cell number, nor was there any effect of diabetes on the immunoreactive substance P B-cell number. Sciatic nerve crush was associated with a substantial loss of L5DRG B-cells in diabetic and nondiabetic p75(+/+) mice and with substantial loss of immunoreactive substance P cells in diabetic p75(+/+) mice. In diabetic and nondiabetic p75(-/-) mice, there was no crush effect on neuropeptide expression. It is concluded that experimental diabetes in the mouse is associated with loss of immunoreactive CGRP primary sensory neurons of the A-cell phenotype, that this loss could play a role for the touch-evoked nociception in the model, and that the neuronal immunoreactive CGRP abnormality possibly is mediated by activation of the p75 neurotrophin receptor.     

Calcitonin gene-related peptide immunoreactive neurons with dichotomizing axons projecting to the lumbar muscle and knee in rats

Dorsal root ganglion (DRG) neurons with dichotomizing axons have been reported in several species and are thought to be related to referred pain. However, these neurons, which have dichotomizing axons to the lumbar muscles and to the knee, have not been investigated. Clinically, pain from the lumbar muscles is sometimes referred to the lower extremities. Two kinds of neurotracers [1,1-dioctadecyl-3,3,3,3-tetramethyl-indocarbocyanine perchlorate (DiI) and fluoro-gold (FG)] were used in the present double-labelling study. DiI crystals were placed in the left lower back muscle, and FG was applied to the medial side of the knee. Bilateral DRGs from L1 through L6 were immunoreacted with calcitonin gene-related peptide (CGRP) antibodies and observed under a fluorescence microscope. DRG neurons double-labelled with DiI and FG were recognized only in the ipsilateral DRGs from levels L1 to L6. Approximately 1% of DRG neurons innervating the low back muscles had other axons to the medial side of the knee. In double-labelled neurons, the ratio of CGRP-immunoreactive DRG neurons was 60%. This finding provides a possible neuroanatomical explanation for referred knee pain from the lower back since CGRP is a marker of sensory neurons typically involved with pain perception. However, these neurons are rare, and mechanisms of referred pain may be explained by the convergence–projection hypothesis.     

Possible mechanism of referred pain in the perineum and pelvis associated with the prostate in rats

PURPOSE: Since persistent pain in the perineum and pelvic floor associated with chronic prostatitis /chronic pelvic pain syndrome has been hypothesized to be referred pain, it might also be explained by neural mechanisms. MATERIALS AND METHODS: Dual retrograde fluorescent labeling and immunohistochemistry were identified as methods with which to investigate the neurogenic aspect of this status. The dual distribution of dorsal root ganglia (DRG) cells was determined after double retrograde fluorescent staining of the prostate and pelvic floor, and the prostate and perineum somatic nerves. Calcitonin gene-related peptide (CGRP) and substance P (SP) in dual labeled cells were determined by immunohistochemistry, giving possible insight into the cause of pelvic pain. RESULTS: Fluorescent double labeled cells were found in the lumbar and sacral DRG, while double labeled cells were distributed predominantly in L6 to S1 and L1 to L2 segment DRG in groups 1 and 2, respectively. On immunohistochemistry some of them were confirmed to contain CGRP and SP. Thus, there are crossover pathways between the prostate and pelvic floor. CONCLUSIONS: The findings that we present confirm that the peripheral process of DRG cells dichotomizes to the prostate, sphincter and somatic parties simultaneously. Some of these cells contain CGRP and SP, which indicate that referred pain in the perineum and pelvic floor may be caused by an axon reflex in the peripheral process of DRG neurons.