Multiple sclerosis (MS) is the most prevalent neurological disorder in young people. Among the most incapacitating symptoms, urinary incontinence due to neurogenic detrusor overactivity is reported by the majority of patients. The transient receptor potential vanilloid 1 (TRPV1) is a receptor described to have an important role in bladder dysfunction, particularly if associated with NDO. TRPV1 desensitization with agonists, such as resiniferatoxin (RTX), has been shown to improve bladder function in various animal models. In the context of MS, a recent study showed that TRPV1 knockout mice were protected from disease progressions, presenting delayed disease onset, myelin preservation and reduced clinical scores [1]. Here, we investigated if MS-induced bladder dysfunction can be attenuated by TRPV1 desensitization with RTX in Experimental Auto-immune Encephalitis (EAE).

In this study, female Wistar rats were used and EAE was induced by a single injection in the flank of 100µl of an emulsion containing myelin basic protein (MBP) and Complete Freund’s Adjuvant (CFA) [2]. Cyclosporin A (Cyc) was subcutaneous injected 3 times a week until the end of the experimental time (6 weeks). Animals were monitored daily and the thermal and mechanical sensitivity were evaluated to follow the disease progression. Four weeks after disease induction, received 100µl of intrathecal RTX 0,1µg/kg or its vehicle (10% ethanol in sterile saline). In the following week, animals were again submitted to behavioural testing to determine cutaneous sensitivity. At the end of the experimental period, rats received subcutaneous urethane (1.2 g/Kg) and underwent 1-hour cystometry. At the end, the lumbosacral spinal cord, associated dorsal root ganglia (DRG) and the sciatic nerve were collected and processed for immunohistochemical analysis. the markers analysed included MBP, TRPV1, calcitonin gene-related peptide (CGRP), isolectin-IB4 (IB4) binding, Iba-1 and Glial fibrillary protein (GFAP).

EAE animals developed fluctuating heightened sensitivity to mechanical and hot stimuli during disease progression and presented increased frequency of bladder reflex contractions, accompanied by a decrease in the bladder amplitude of contractions. Intrathecal administration of RTX, but not vehicle, improved cutaneous sensitivity to mechanical stimulation and bladder function, with a reduction of the frequency of bladder contractions. 
Tissue analysis showed reduced spinal but not peripheral MBP expression, when compared with tissue obtained from non-manipulated animals.  Spinal CGRP was not altered by EAE but TRPV1 expression and IB4 binding were reduced. Labelling intensity of Iba-1 and GFAP marker was elevated in EAE rats in comparison with non-manipulated rats. While intrathecal vehicle did not change the spinal expression of those markers, a reduction of the labelling intensity of TRPV1, CGRP and IB4 was found following intrathecal RTX. Likewise, intrathecal RTX also lead to a decrease in the immunostaining intensity of Iba-1 and GFAP.
In the DRG of intact animals we observed a high intensity TRPV1 and CGRP labelling in small-to-medium sized cell bodies (20-180 µm2 area) In EAE animals, TRPV1 and CGRP labelling was also found in larger cell bodies (120-600 µm2). No differences in labeling intensity were found in IB4-binding. After intrathecal RTX, but not vehicle, we found a decrease in the number of cell bodies labelled with CGRP, TRPV1 and IB4.

Results show that EAE animals developed pain, reflected by increased mechanical and thermal sensitivity that fluctuated during the experimental period and is suggestive of a relapsing and remitting form of disease. EAE animals also developed bladder dysfunction, characterized by high frequency and low amplitude of bladder reflex contractions. This was accompanied by demyelination restricted to the spinal cord, glial activation, as well as a reduction in the expression of the peripheral markers, TRPV1 and IB4 binding. Intrathecal RTX injection, and consequent central TRPV1 desensitization [3] improved pain levels and bladder reflex activity. Altogether, results suggest EAE-induced pain and bladder dysfunction reflect changes in glial activation at the spinal cord level as well as impairment of the central processes of peripheral afferents conveying sensory input to the superficial laminae of the cord.

The present study described the emergence of changes in bladder function in an EAE animal model, indicatives of neurogenic detrusor overactivity (NDO). As intrathecal RTX improved bladder dysfunction, it is likely that TRPV1 is an important player in MS pathophysiology and may constitute an attractive therapeutic target to alleviate symptoms.

Paltser G, Liu XJ, Yantha J, et al. TRPV1 gates tissue access and sustains pathogenicity in autoimmune encephalitis. Mol Med. 2013: 19:149-59
Thibault K, Calvino B, Pezet S. Characterisation of sensory abnormalities observed in an animal model of multiple sclerosis: a behavioural and pharmacological study. Eur J Pain. 2011 Mar: 15:231 e1-16
Cruz CD, Charrua A, Vieira E, Valente J, Avelino A, Cruz F. Intrathecal delivery of resiniferatoxin (RTX) reduces detrusor overactivity and spinal expression of TRPV1 in spinal cord injured animals. Exp Neurol. 2008 Dec: 214:301-8