Raquel Oliveira

Intrathecal administration of botulinum toxin type A improves urinary bladder function and reduces pain in rats with cystitis

Botulinum toxin A (Onabot/A) has been shown to have an antinociceptive effect. This might be due to an impairment of sensory nerves not only in the peripheral but also in the central nervous system. In this work, we analysed both systems by studying the effect of intrathecal (i.t.) administration of botulinum toxin A in an animal model of bladder pain and hyperactivity induced by cyclophosphamide (CYP).

Bisphosphonates induce the osteogenic gene expression in co-cultured human endothelial and mesenchymal stem cells.

Bisphosphonates (BPs) are known to affect bone homeostasis and also to have anti‐angiogenic properties. Because of the intimate relationship between angiogenesis and osteogenesis, this study analysed the effects of Alendronate (AL) and Zoledronate (ZL) in the expression of endothelial and osteogenic genes on interacting endothelial and mesenchymal stem cells, an issue that was not previously addressed. Alendronate and ZL, 10−12–10−6 M, were evaluated in a direct co‐culture system of human dermal microvascular endothelial cells (HDMEC) and human bone marrow mesenchymal stem cells (HMSC), over a period of 14 days. Experiments with the respective monocultures were run in parallel. Alendronate and ZL caused an initial dose‐dependent stimulation in the cell proliferation in the monocultures and co‐cultures, and did not interfere with their cellular organization. In HDMEC monocultures, the expression of the endothelial genes CD31, VE‐cadherin and VEGFR2 was down‐regulated by AL and ZL. In HMSC monocultures, the BPs inhibited VEGF expression, but up‐regulated the expression of the osteogenic genes alkaline phosphatase (ALP), bone morphogenic protein‐2 (BMP‐2) and osteocalcin (OC) and, to a greater extent, osteoprotegerin (OPG), a negative regulator of the osteoclastic differentiation, and increased ALP activity. In co‐cultured HDMEC/HMSC, AL and ZL decreased the expression of endothelial genes but elicited an earlier and sustained overexpression of ALP, BMP‐2, OC and OPG, compared with the monocultured cells; they also induced ALP activity. This study showed for the first time that AL and ZL greatly induced the osteogenic gene expression on interacting endothelial and mesenchymal stem cells.

Impairment of sensory afferents by intrathecal administration of botulinum toxin A improves neurogenic detrusor overactivity in chronic spinal cord injured rats

Spinal cord injury (SCI) often leads to neurogenic detrusor overactivity (NDO) due to sprouting of sensory afferents on the lumbosacral spinal cord. NDO is characterized by high frequency of voiding contractions and increased intravesical pressure that may lead to urinary incontinence. The latter has been described as one of the consequences of SCI that mostly decreases quality of life. Bladder wall injections of botulinum toxin A (Onabot/A) are an effective option to manage NDO. The toxin strongly impairs parasympathetic and sensory fibres coursing the bladder wall. However the robust parasympathetic inhibition may inhibit voiding contractions and cause urinary retention in patients that retain voluntary voiding. Here, we hypothesised that by restricting the toxin activity to sensory fibres we can improve NDO without impairing voiding contractions. In the present work, we assessed the effect of Onabot/A on sensory neurons in chronic (4weeks) SCI rats by injecting the toxin intrathecally (IT), at lumbosacral spinal cord level. This route of administration was shown before to have an effect on bladder pain and contractility in an animal model of bladder inflammation. We found that IT Onabot/A led to a significant reduction in the frequency of expulsive contractions and a normalization of bladder basal pressure while maintaining voiding contractions of normal amplitude. Cleavage of SNAP-25 protein occurred mainly at the dorsal horn regions where most of the bladder afferents end. Cleaved SNAP-25 was not detected in motor or preganglionic parasympathetic neurons. A significant decrease in CGRP expression, a peptide exclusively present in sensory fibres in the spinal cord, occurred at the L5/L6 segments and associated dorsal root ganglia (DRG) after Onabot/A injection in SCI animals. Onabot/A strongly increased the expression of ATF3, a marker of neuronal stress, in L5/L6 DRG neurons. Taken together, our results suggest that IT Onabot/A has a predominant effect on bladder sensory fibres, and that such effect is enough to control NDO following chronic SCI. The mechanism of action of Onabot/A includes not only the cleavage of SNAP-25 in sensory terminals but also impairment of basic cellular machinery in the cell body of sensory neurons.

Expression of cleaved SNAP-25 after bladder wall injection of onabotulinumtoxinA or abobotulinumtoxinA: A comparative study in the mice.

To study the expression of cleaved synaptosomal associated protein of 25u2009kDa (cSNAP‐25) in the bladder wall injected with onabotulinumtoxinA (onabotA) or abobotulinumtoxinA (abobotA) and compare the relative potency of these two brands.

Evidence for an urethro-vesical crosstalk mediated by serotonin

To study the contribution of urethral serotonin for the urethro‐vesical crosstalk

MP42-06 EXPRESSION AND FUNCTION OF SEROTONIN PARANEURONAL CELLS IN THE URETHRAL EPITHELIUM OF HUMAN AND RODENTS

persists following the resolution of colitis, and that linaclotide, an FDA approved guanylate cyclase-C (GC-C) agonist, is able to attenuate these changes [3]. We hypothesise that these CCH-induced changes are the result of altered sensitivity of afferents both within the colon and bladder wall and within the dorsal root ganglion (DRG), and that oral linaclotide administration may act to reduce this hypersensitivity. METHODS: We investigated healthy C57BL/6J mice and mice with CCH, 28 days after intra-colonic TNBS administration. CCH mice were randomly assigned to either chronic linaclotide (3mg/kg/day) or placebo (water) administration, consisting of a once daily oral gavage for 2 weeks prior to experimentation. Ex-vivo electrophysiological recordings determined bladder afferent and contractile sensitivity to abMe-ATP (30mM), carbachol (1mM), and capsaicin (10mM) as well as whole cell patch clamp of retrogradely traced bladder DRG neurons in all four groups. RESULTS: Bladder DRG from mice with CCH display hyperexcitability, with a significant reduction in rheobase compared to controls (p 0.01). CCH mice also display significantly enhanced afferent responses to abMe-ATP (p 0.001), carbachol (p 0.001), and capsaicin (p 0.001). CCH mice treated with linaclotide display attenuated DRG hyperexcitability and normalised afferent responses to agonists (p 0.01) compared to placebo (p 0.01). CONCLUSIONS: Mice with CCH also display increased bladder afferent excitability within both the DRG and bladder wall, indicating cross-organ sensitisation. Chronic oral administration of linaclotide, a locally acting GC-C agonist that inhibits colonic nociceptors, reverses these colitis-induced changes in bladder afferent sensitivity. Common sensory pathways may allow agents that reduce abdominal pain to improve urological symptoms. 1. Lamb, K., et al. AJPGI, 2006. 2. Ustinova et al, Neurourology and Urodynamics, 2010. 3. Grundy, L., et al., MP28-06. The Journal of Urology, 2016.