Spinal cord injury (SCI) results in loss of voluntary voiding control, due to damages in neuronal circuits controlling micturition. After an initial period of bladder areflexia, post-SCI neuroplasticity mediates the formation of a new micturition reflex, characterized by neurogenic detrusor overactivity (NDO), frequently concurrent with detrusor-sphincter-dyssynergia (DSD) [1]. These are known to be accompanied by dramatic changes in bladder tissue organization and innervation [2], and therefore, NDO management strategies classically target this organ. However, accumulating evidence suggests that efficient voiding also depends on urethral function,  with several studies supporting the idea that the control of bladder function might at least originate and/or be modulated by the urethra [3]. Nevertheless, this organ was always been less studied in SCI-induced urinary dysfunction studies and consequences of SCI lesions in urethral integrity have never been assessed. Given its importance in normal micturition, it is likely that SCI could reflect alterations in the urethra and that these could contribute to SCI-induced urinary impairment. Thus, this study aimed to describe the main consequences of SCI in the urethral tissue organization and innervation.

Female Wistar rats were divided into 3 experimental groups: spinal intact animals, and SCI animals left to recover 1 and 4 weeks after a largely incomplete spinal section at T8/T9 level (n=5/group). Before euthanasia, animals underwent 1h-cystometry under urethane anesthesia to evaluate bladder function. Afterwards, urethral tissues were collected and processed for paraffine impregnation to perform histologic colorations. Additional groups of animals were used for cryostat frozen sections to evaluate by immunohistochemistry the expression of markers for smooth muscle integrity (smooth muscle actin) and peripheral nerve fibres (B-III tubulin; Calcitonin gene-related peptide- CGRP; Tyrosine Hydroxylase- TH; Vesicular Acetylcholine transporter- VAChT) combined with a neuronal sprouting marker (Growth Associated Protein 43 -GAP43). Data was collected in distinct zones of the proximal urethra: mucosa, internal (IUS), and external urethral sphincter (EUS), and quantified by Image J.

Bladder contractions were abolished 1 week after SCI. At 4 weeks, NDO was already established, as shown by the increased frequency and amplitude of bladder contractions (p<0.05 versus spinal intact animals). Hematoxylin-Eosin staining revealed marked disorganization of the histological layers, with signs of cellular desquamation in the epithelium. At 4 weeks post-lesion, urethral layers were again evident, presenting fewer signs of cellular loss and a higher number of flattened cells. The height of the epithelium and lamina propria was increased after SCI (p<0.05 versus spinal intact animals for both time-points). The IUS was particularly affected, as evidenced by the dramatic decrease of the smooth-muscle actin expression observed 4 weeks post-lesion (p<0.001 versus spinal intact), whereas no significant changes were seen in the EUS. Sirius Red staining did not evidence signs of urethral fibrosis.  
The consequences of SCI on general innervation were assessed by β-III tubulin. In the IUS, denervation occurred 4 weeks after the lesion, while in the EUS this was already noted at 1-week post-SCI (p<0.05 versus spinal intact animals for both time-points). Analysis of CGRP showed sensory denervation in the mucosa (p<0.001 versus spinal intact animals), as well as in the EUS (p<0.05 versus spinal intact animals) already at 1-week and still present at 4-weeks post-SCI. Sympathetic denervation was seen only in the IUS, as shown by the reduction of TH expression at both time-points (p<0.01 versus spinal intact animals). Parasympathetic innervation, measured by VAChT immunolabelling, was not affected. The analysis of GAP-43 co-localization with the above neuronal markers suggested no signs of SCI-associated neuronal sprouting.

The organization of the urothelium was strongly affected at spinal shock, but recovered 4 weeks post-SCI, as changes reported in the SCI bladder. The integrity of the IUS was strongly affected, as shown by marked atrophy of the smooth muscle fibres. This is in contrast with muscle hypertrophy occurring in the bladder after SCI. This may occur as the urethra does not suffer the same type of mechanical demand that a hypercontractile bladder does. This is consistent with the lack of urethral SCI-induced fibrosis in this area, again in contrast with the SCI bladder. The sympathetic denervation of the IUS can be accounted for IUS atrophy, as sympathetic fibres may secrete trophic factors promoting growth and survival of IUS smooth muscle cells. Sympathetic denervation can, therefore, contribute to IUS atrophy. In turn, IUS atrophy may reduce the levels of urethral neurotrophins, necessary to maintain sympathetic and sensory nerve fibres. Accordingly, sympathetic denervervation was accompanied by a reduction in CGRP levels and lack of axonal sprouting.

Management of SCI-induced urinary incontinence has always been focused in modulation of bladder activity. The present results provide the first evidence that SCI causes profound and time-dependent changes in urethral tissue organization. Altogether, these observations demonstrate that rearrangement of the urethra may be linked to SCI-induced urinary dysfunction, possibly contributing to urinary incontinence in SCI rats. Hence, these findings support the need to further investigate urethral pathological mechanisms after SCI and may open the possibility of using the modulation of urethral activity combined with already established bladder therapies, as a therapeutical option to treat SCI-induced urinary dysfunction in human patients.

de Groat, W.C. and N. Yoshimura, Mechanisms underlying the recovery of lower urinary tract function following spinal cord injury. Progress in brain research, 2006. 152: p. 59-84.
Cruz, C.D. and F. Cruz, Spinal cord injury and bladder dysfunction: new ideas about an old problem. The Scientific World Journal, 2011. 11.
Birder, L.A., et al., Urethral sensation: basic mechanisms and clinical expressions. International Journal of Urology, 2014. 21: p. 13-16.