A novel anterograde neuronal tracing technique to selectively label spinal afferent nerve endings that encode noxious and innocuous stimuli in visceral organs.

This article is under embargo for 12 months from the date of publication in accordance with publisher copyright policy. This is the accepted version of the following article: Kyloh, M. and Spencer, N. J. (2014), A novel anterograde neuronal tracing technique to selectively label spinal afferent nerve endings that encode noxious and innocuous stimuli in visceral organs. Neurogastroenterology & Motility, 26: 440–444.], which has been published in final form at [http://dx.doi.org/10.1111/nmo.12266]. In addition, authors may also transmit, print and share copies with colleagues, provided that there is no systematic distribution of the submitted version, e.g. posting on a listserve, network or automated delivery

The district goes global : export vs FDI

Digitised version produced by the EUI Library and made available online in 2020

Characterization of primary afferent spinal innervation of mouse uterus

This Document is Protected by copyright and was first published by Frontiers. All rights reserved. it is reproduced with permission. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The primary afferent innervation of the uterus is incompletely understood. The aim of this study was to identify the location and characteristics of primary afferent neurons that innervate the uterine horn of mice and correlate the different morphological types of putative primary afferent nerve endings, immunoreactive to the sensory marker, calcitonin gene related peptide (CGRP). Using retrograde tracing, injection of 5–10 μL of 1,1′-didodecyl-3,3,3,3′-tetramethylindocarbocyanine perchlorate (DiI) into discrete single sites in each uterine horn revealed a biomodal distribution of sensory neurons in dorsal root ganglia (DRG) with peak labeling occurring between T13-L3 and a second smaller peak between L6-S1. The mean cross sectional area of labeled cells was 463 μm2 ± s.e.m. A significantly greater proportion of labeled neurons consisted of small cell bodies (<300 μm2) in the sacral spinal cord (S2) compared with peak labeling at the lumbar (L2) region. In both sections and whole mount preparations, immunohistochemical staining for CGRP revealed substantial innervation of the uterus by CGRP-positive nerve fibers located primarily at the border between the circular and longitudinal muscle layers (N = 4). The nerve endings were classified into three distinct types: “single,” “branching,” or “complex,” that often aligned preferentially in either the circular or longitudinal axis of the smooth muscles. Complex endings were often associated with mesenteric vessels. We have identified that the cell bodies of primary afferent neurons innervating the mouse uterus lie primarily in DRG at L2 and S1 spinal levels. Also, the greatest density of CGRP immunoreactivity lies within the myometrium, with at least three different morphological types of nerve endings identified. These findings will facilitate further investigations into the mechanisms underlying sensory transduction in mouse uterus

The presence of 5-HT in myenteric varicosities is not due to uptake of 5-HT released from the mucosa during dissection: use of a novel method for quantifying 5-HT immunoreactivity in myenteric ganglia

Author version made available according to Publisher copyright policy. This is the accepted version of the following article: 
Keating, D. J., Peiris, H., Kyloh, M., Brookes, S. J. H. and Spencer, N. J. (2013), The presence of 5-HT in myenteric varicosities is not due to uptake of 5-HT released from the mucosa during dissection: use of a novel method for quantifying 5-HT immunoreactivity in myenteric ganglia. Neurogastroenterology & Motility, 25: 849–853, 

which has been published in final form at 
http://dx.doi.org/10.1111/nmo.12189. 

In addition, authors may also transmit, print and share copies with colleagues, provided that there is no systematic distribution of the submitted version, e.g. posting on a listserve, network or automated delivery

Identification of the Visceral Pain Pathway Activated by Noxious Colorectal Distension in Mice

In patients with irritable bowel syndrome, visceral pain is evoked more readily following distension of the colorectum. However, the identity of extrinsic afferent nerve pathway that detects and transmits visceral pain from the colorectum to the spinal cord is unclear. In this study, we identified which extrinsic nerve pathway(s) underlies nociception from the colorectum to the spinal cord of rodents. Electromyogram recordings were made from the transverse oblique abdominal muscles in anesthetized wild type (C57BL/6) mice and acute noxious intraluminal distension stimuli (100–120 mmHg) were applied to the terminal 15 mm of colorectum to activate visceromotor responses (VMRs). Lesioning the lumbar colonic nerves in vivo had no detectable effect on the VMRs evoked by colorectal distension. Also, lesions applied to the right or left hypogastric nerves failed to reduce VMRs. However, lesions applied to both left and right branches of the rectal nerves abolished VMRs, regardless of whether the lumbar colonic or hypogastric nerves were severed. Electrical stimulation applied to either the lumbar colonic or hypogastric nerves in vivo, failed to elicit a VMR. In contrast, electrical stimulation (2–5 Hz, 0.4 ms, 60 V) applied to the rectum reliably elicited VMRs, which were abolished by selective lesioning of the rectal nerves. DiI retrograde labeling from the colorectum (injection sites 9–15 mm from the anus, measured in unstretched preparations) labeled sensory neurons primarily in dorsal root ganglia (DRG) of the lumbosacral region of the spinal cord (L6-S1). In contrast, injection of DiI into the mid to proximal colon (injection sites 30–75 mm from the anus, measured in unstretched preparations) labeled sensory neurons in DRG primarily of the lower thoracic level (T6-L2) of the spinal cord. The visceral pain pathway activated by acute noxious distension of the terminal 15 mm of mouse colorectum is transmitted predominantly, if not solely, through rectal/pelvic afferent nerve fibers to the spinal cord. The sensory neurons of this spinal afferent pathway lie primarily in the lumbosacral region of the spinal cord, between L6 and S1

Innervation Changes Induced by Inflammation in the Murine Vagina

© 2018 IBRO. Published by Elsevier Ltd. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (January 2018) in accordance with the publisher’s archiving policyVulvodynia is a prevalent chronic pain disorder associated with high medical costs and often ineffective treatments. The major pathological feature is proliferation of vaginal nerve fibers. This study aimed to develop a highly reproducible animal model to study neuroproliferation in the vagina and aid the identification of appropriately targeted treatments for conditions such as vulvodynia. Mild chronic inflammation was induced using microinjection of complete Freund’s adjuvant in the distal vagina of C57Bl/6 mice. Control mice received saline. Inflammation and innervation density were assessed at 7 and 28 days after a single administration or 14 days following repeated administration of complete Freund’s adjuvant or saline. Histochemistry and blinded-analysis of images were used to assess vaginal morphology (H & E) and abundance of macrophages (CD68-labeling), mast cells (toluidine blue staining, mast cell tryptase-immunoreactivity), blood vessels (αSMA-immunoreactivity) and nerve fibers immunoreactive for the pan-neuronal marker PGP9.5. Subpopulations of nerve fibers were identified using immunoreactivity for calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY). Single administration of complete Freund’s adjuvant resulted in vaginal swelling, macrophage infiltration, vascular proliferation and increased abundance of nerve fibers immunoreactive for CGRP, SP, VIP and/or PGP9.5 but not NPY, evident at seven days. Inflammation further increased following repeated administration of complete Freund’s adjuvant but nerve fiber proliferation did not. Nerve fiber proliferation continued to be evident at 28 days. The inter-individual differences within each treatment group were small, indicating that this model may be useful to study mechanisms underlying vaginal nerve fiber proliferation associated with inflammation

Anterograde labeling from lumbosacral DRG in vivo reveals spinal afferent nerve endings in the mucosa of three different mice.

<p>A, a single nerve axon ending is shown consisting of a bare unspecialized terminal with few varicosities. The arrows indicate the trajectory of this axon that is initially out of focus as it projects through the submucosa, then becomes in focus at the level of the mucosa. B, shows CGRP immunoreactivity of the region shown in panel A. The arrows indicate the same region of the axon that is in panel A. C, shows a superimposed image of panels B & A. The arrows indicate the anterogradely labeled axon is CGRP immunoreactive. D, shows a mucosal ending in a different animal consisting of a bifurcating axon, again with very few varicosities along its axon (see arrow). E, shows CGRP immunoreactivity of the region shown in panel D, see arrow. F, shows a superimposed image of panels E and D. This ending is CGRP positive, see arrow. G, shows another example from a different mouse of a varicose spinal afferent ending in the mucosa. The arrow shows a fine varicose ending. H, shows CGRP immunoreactivity of the region shown in G. The arrow indicates a lack of CGRP immunoreactivity of the ending shown by the arrow in panel G. I, shows a superimposed image of panels G and H. It can be seen that this mucosal ending is not CGRP positive (see arrows).</p

Anterograde labeling from lumbosacral DRG in vivo reveals two distinct types of spinal afferent nerve ending identified in the submucosa.

<p>A, shows a “branching-type” spinal afferent nerve ending in the submucosa. This ending ramifies extensively with many branching varicose axonal processes that align preferentially in the rostral-caudal axis, within the submucosa. The region indicated by the arrow in panel A is shown on expanded scale in panel B. The nerve endings indicated by the arrow in panel B are not CGRP positive (compare with arrow in panel C). Panel C shows CGRP immunoreactivity of the region shown in panel B. The arrow shows a lack of CGRP immunoreactivity of the ending indicated by the arrow in panel B. Panel D, shows a superimposed image of panels B & C. Panel E, shows a “complex-type” spinal afferent ending in the submucosa at the same level as the base of the Crypts of Lieberkhun. This complex type ending arises from a single spinal afferent axon and then ramifies into a complex structure with multiple varicose axons that branch in no apparent preferential orientation. The arrow in panel E indicates a varicose axonal ending that is not CGRP positive (compare with arrow at the same region in panel F). F, shows CGRP immunoreactivity of the same region shown in panel D. The arrow in panel F shows a lack of CGRP immunoreactivity of the ending in panel E. G, shows a superimposed image of E & F. It is clear that this complex-type spinal afferent ending is not CGRP immunoreactive - compare arrows in panels E, F & G.</p

Diagrammatic representation of the different types of spinal afferent endings identified in the mouse large intestine, following minute injections of dextran biotin into lumbosacral DRGs in vivo.

<p>A, shows a schematic of the nerve pathways utilized to transport dextran biotin to the distal colon. B, shows that 13 distinct types of nerve endings were identified. 1 shows varicose endings in longitudinal muscle which occurred extremely rarely. Numbers 2 & 3, refer two types of endings identified in myenteric ganglia. 2 refers to intraganglionic varicose endings (IGVEs) which are common, while 3 refers to rectal intraganglionic laminar endings (rIGLEs) which are rare, 4 refers to varicose endings in internodal strands. 5 refers to “branching-type” endings in the CM layer that run parallel to the CM fibres, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g006" target="_blank">Figure 6D</a>. 6 refers to “simple-type” afferent endings in the CM that consist of a single non-branching varicose axon, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g006" target="_blank">Figure 6A</a>. 7 refers to the “complex-type” endings in the CM that arise from a single axon and ramify extensively throughout the CM layer in no preferential orientation, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g005" target="_blank">Figure 5A</a>. 8, refers to “branching-type” spinal endings in the submucosa that ramify extensively in the rostral-caudal axis, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g009" target="_blank">figure 9A</a>. 9, refers to endings on blood vessels. 10, refers to rIGLEs in submucosal ganglia. 11, refers to “complex-type” endings at the level of the Crypts of Lieberkhun, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g009" target="_blank">Figure 9E</a>. 12, refers to simple type endings in the submucosa that consist of axon terminals that encircle the base of the Crypts with few or no varicosities, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112466#pone-0112466-g010" target="_blank">Figure 10A & 10D</a>. 13, refers to nerve endings that innervate into the mucosa. KEY: LM refers to longitudinal muscle, MG = myenteric ganglion, CM = circular muscle, SM = submucosa, SG = submucosal ganglion and CL = Crypts of Lieberkuhn.</p