Despite the ample evidence of the beneficial effect of BoNTA on the bladder function in SCI patients, there is a limited understanding of the mechanisms by which BoNTA affects specific cell types in the bladder wall and regulates the expression of different genes, proteins and metabolites. To address the mechanism of the BoNTA activity reducing /preventing NDO, we carried out a longitudinal study of the molecular changes in the bladders of SCI patients, receiving BoNTA or placebo shortly after injury and at a 3 months’ time-point afterwards.

Bladder base biopsies aiming to include detrusor muscle were collected from nine SCI patients. The subjects were mean 41.4 (range 20-62) years old at time of injury, with motor and sensory complete lesions (AIS classification A) spinal cord injury at level C7-Th11. Inclusion and urodynamic evaluation were performed followed by the treatment with BoNTA or placebo. The participants were randomized, assessed urodynamically and received first treatment (BoNTA or placebo) within 3 months after injury (Visit 1- V1). The second urodynamic evaluation, treatment and second set of biopsy collection was three months later (Visit 2 – V2). Follow up with urodynamic evaluation, Quality of Life questionnaires and biopsy collection were done 12 months after first visit (Visit 3 – V3). Total RNA was isolated from one set of biopsies for the bulk RNA sequencing.  Single nuclei were isolated from post-fixed biopsies using 10x Genomics Chromium next GEM Single Cell Fixed RNA Sample Preparation Kit and snRNA sequencing carried out. Differentially expressed genes (DEGs) were identified by comparing each sample time point 0 (before drug intervention) with the subsequent time points.

Urodynamic studies showed that none of the patients in the BoNTA group (n = 5) developed NDO with contractions above 40 cm H2O during the follow up period, whereas two patients in the placebo group (n = 4) (PLA2 and PLA4) developed NDO. Direct comparison of the RNA sequencing analysis showed the effect of BoNTA on the signalling pathways playing a role in muscle contraction, cell division, and extracellular matrix organization, regulation of neurotransmitter release and synaptic organization. These genes were upregulated in the placebo patients and downregulated in the patients who received the BoNTA in agreement with UDI, which showed the development of NDO in placebo patients. We identified SV2A, SV2C and SYT2 as the key component of Neurotoxicity to Clostridium toxins pathways, up-regulated in PLA and significantly down-regulated in BoNTA group. snRNAseq identified 26 cell clusters present in the biopsies, with variable and time-dependent representation in the longitudinal samples. The immune cell clusters (macrophage/monocyte and neutrophils) were increased in the placebo group, concomitant with NDO development. We observed a decrease of urothelial markers, pointing at urothelial dysfunction in the placebo group.

Early BoNTA injections prevented activation of the smooth muscle-related and immune cell-related signalling pathways. None of the patients in the BoNTA group developed NDO with high amplitude contractions. snRNA sequencing demonstrated significant changes in the bladder wall composition between BoNTA and placebo groups. Immune cell infiltration into the bladder, leading to the permanent damage and organ remodelling, was effectively prevented by BoNTA. The patients receiving placebo showed a down-regulation of urothelial markers, indicative of the urothelial dysfunction, probably contributing to the NDO.

Early BoNTA injections into the bladders of SCI patients preserved organ architecture, effectively preventing immune cell infiltration and urothelial dysfunction leading to NDO.