Integrated omics analysis unveils a DNA damage response to neurogenic injury.

Spinal cord injury (SCI) evokes profound bladder dysfunction. Current treatments are limited by a lack of molecular data to inform novel therapeutic avenues. Previously, we showed systemic inosine treatment improved bladder function following SCI in rats. Here, we applied multi-omics analysis to explore molecular alterations in the bladder and their sensitivity to inosine following SCI. Canonical pathways regulated by SCI included those associated with protein synthesis, neuroplasticity, wound healing, and neurotransmitter degradation. Upstream regulator analysis identified MYC as a key regulator, whereas causal network analysis predicted multiple regulators of DNA damage response signaling following injury, including PARP-1. Staining for both DNA damage (γH2AX) and PARP activity (poly-ADP-ribose) markers in the bladder was increased following SCI, and attenuated in inosine-treated tissues. Proteomics analysis suggested that SCI induced changes in protein synthesis-, neuroplasticity-, and oxidative stress-associated pathways, a subset of which were shown in transcriptomics data to be inosine-sensitive. These findings provide novel insights into the molecular landscape of the bladder following SCI, and highlight a potential role for PARP inhibition to treat neurogenic bladder dysfunction.

Neuropilin 2 Is a Novel Regulator of Distal Colon Contractility.

Appropriate coordination of smooth muscle contraction and relaxation is essential for normal colonic motility. The impact of perturbed motility ranges from moderate, in conditions such as colitis, to potentially fatal in the case of pseudo-obstruction. The mechanisms underlying aberrant motility and the extent to which they can be targeted pharmacologically are incompletely understood. This study identified colonic smooth muscle as a major site of expression of neuropilin 2 (Nrp2) in mice and humans. Mice with inducible smooth muscle-specific knockout of Nrp2 had an increase in evoked contraction of colonic rings in response to carbachol at 1 and 4 weeks following initiation of deletion. KCl-induced contractions were also increased at 4 weeks. Colonic motility was similarly enhanced, as evidenced by faster bead expulsion in Nrp2-deleted mice versus Nrp2-intact controls. In length-tension analysis of the distal colon, passive tension was similar in Nrp2-deficient and Nrp2-intact mice, but at low strains, active stiffness was greater in Nrp2-deficient animals. Consistent with the findings in conditional Nrp2 mice, Nrp2-null mice showed increased contractility in response to carbachol and KCl. Evaluation of selected proteins implicated in smooth muscle contraction revealed no significant differences in the level of α-smooth muscle actin, myosin light chain, calponin, or RhoA. Together, these findings identify Nrp2 as a novel regulator of colonic contractility that may be targetable in conditions characterized by dysmotility.

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury.

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and subsequent bladder wall remodeling. To facilitate assessment of the cellular composition of the bladder wall in non-injured control and spinal cord injured mice, we developed an optimized dissociation protocol that supports high cell viability and enables the detection of discrete subpopulations by flow cytometry. Spinal cord injury is created by complete transection of the thoracic spinal cord. At the time of tissue harvest, the animal is perfused with phosphate-buffered saline under deep anesthesia and bladders are harvested into Tyrode's buffer. Tissues are minced prior to incubation in digestion buffer that has been optimized based on the collagen content of mouse bladder as determined by interrogation of publicly available gene expression databases. Following generation of a single cell suspension, material is analyzed by flow cytometry for assessment of cell viability, cell number and specific subpopulations. We demonstrate that the method yields cell populations with greater than 90% viability, and robust representation of cells of mesenchymal and epithelial origin. This method will enable accurate downstream analysis of discrete cell types in mouse bladder and potentially other organs.

Dorsal rhizotomy for treatment of bilateral intercostal neuralgia following augmentation mammaplasty: case report and review of the literature.

Intercostal neuralgia may develop following breast augmentation. The authors describe a woman who suffered 2 years of severe pain associated with cutaneous hypaesthesia in a T3-T5 distribution. Serial, placebo-controlled T3-T5 dorsal root nerve blocks provided temporary pain relief. The patient experienced immediate and lasting pain relief (34 months) following bilateral T3-T5 dorsal rhizotomies. This case provides anecdotal evidence that dorsal rhizotomy is a beneficial intervention for refractory intercostal neuralgia.

Activity-dependent slowing of conduction velocity in uninjured L4 C fibers increases after an L5 spinal nerve injury in the rat.

Growing evidence suggests that uninjured afferents may play an important role in neuropathic pain following nerve injury. The excitability of nociceptive neurons in the L4 spinal nerve appears to be enhanced following an injury to the adjacent L5 spinal nerve. In this study, we investigated whether the action-potential conduction properties of unlesioned, unmyelinated fibers are also altered. A teased-fiber technique was used to record from single C fibers from the L4 spinal nerve of the rat in vitro. Repeated electrical stimulation of the tibial nerve was used to investigate activity-dependent slowing of conduction velocity. Twin pulse stimulation at a 50 ms interpulse interval allowed investigation of supranormal conduction velocity. Blinded experiments were performed 8-10 days after sham surgery and after an L5 spinal nerve ligation (L5 SNL). Activity-dependent slowing revealed two populations of C fibers, a "nociceptor" population with a large degree of activity-dependent slowing and a "non-nociceptor" population with a smaller degree of activity-dependent slowing. Both populations showed enhanced activity-dependent slowing of conduction velocity and enhanced supranormal conduction velocities in lesioned animals compared to sham animals. Activity-dependent slowing was also enhanced after an L5 SNL in the mouse. These alterations in conduction velocity may reflect changes in expression of ion channels responsible for the membrane excitability. These data provide additional evidence that a nerve injury leads to persistent alterations in the properties of adjacent uninjured, unmyelinated fibers.

Erythropoietin protects sensory axons against paclitaxel-induced distal degeneration.

Paclitaxel causes a sensory polyneuropathy with characteristic features of distal axonal degeneration. Although the exact mechanisms underlying distal axonal degeneration are unknown, paclitaxel-induced axonal degeneration has been shown to be associated with an increase in detyrosinated tubulin. Here we show that recombinant human erythropoietin prevents axonal degeneration in sensory neurons in vitro and this effect is associated with downregulation of detyrosinated tubulin. Furthermore, in an animal model of paclitaxel-induced distal sensory polyneuropathy, recombinant human erythropoietin protects against distal axonal degeneration. These findings suggest that recombinant human erythropoietin may be useful as a therapy to prevent paclitaxel-induced sensory polyneuropathy in patients undergoing chemotherapy.