Hypothesis / aims of study
Recent studies on animals have demonstrated urothelial barrier remodeling following spinal cord injury (SCI): regenerative processes (increased cell proliferation, reduced cell differentiation, leading to reduced density of umbrella cells), as well as increased urothelial permeability. PUFAs derivatives seems to affect cell proliferation and differentiation in urothelium, but their role on urothelial barrier functions and morphological remodeling, in particular following SCI, remains currently unknown.
Therefore, the objectives of our study were 1) to characterize the morphological remodeling of urothelial barrier using for the first time Full Field Optical Coherence Tomography (FF-OCT), 2) to assess functional barrier remodeling (permeability), 3) as well as PUFAs derivatives expression in bladder samples in a mice model of chronic SCI.
Study design, materials and methods
Spinal cord injury was induced in male mice by a spinal cord transection between T3-T4 vertebrae (SCI group, n=30). Control mice were obtained following a sham surgery (without spinal cord transection) (control group, n=15). At four weeks, mice were sacrificed, and bladder were removed for 1) morphological study using FF-OCT; 2) permeability studies in Ussing chambers : paracellular and transcellular permeability were assessed by measuring the flux of sulfonic acid (SA, 400Da) and horseradish peroxidase (HRP, 44kDa) respectively, across the epithelial barrier from apical to serosal side; 3) transcriptomic analysis (qPCR) to characterize PUFAs derivatives mRNA expression.
Results
No change was observed in paracellular permeability.
However, a reduced transcellular permeability was observed in SCI as compared to sham mice (p=0.007) (Fig 1A). This was associated with a significant reduction in mRNA expression of ZO-1, CLND1 and CLND3, known to be involved in tight-junctions regulation (p<0.001, p<0.001, and p=0.018 respectively).
Furthermore, major morphological remodeling of the urothelium was observed in SCI group as compared to sham mice, using FFOCT: the umbrella cells were significantly smaller (diameter decreased by 2 fold; p=0.009; with no significant change in cell density) and the density of small superficial cells was significantly higher (increased by 7.5 fold, p=0.004)(Fig 1B, C and D).
These morphological changes were associated with a significant reduction in mRNA expression of differentiation markers of umbrella cells (CK20, UPK2)(Fig 1 E).
Finally, we showed a significant reduction in mRNA expression of key enzymes involved in polyunsaturated fatty acid (PUFA) metabolism (Cox-1, Cox-2, PTGES and PTGDS) in SCI mices as compared to sham (Fig 1F).
Interpretation of results
Our results showed functional and morphological remodeling of urothelium after SCI in a mice model.
Morphological remodeling may suggest a regenerative process with a higher density of small superficial cells and a smaller size of the umbrella cells, which appeared immature (loss of CK20 and UPK2 expression). In addition, we observed a significant reduction in mRNA expression of key enzymes involved in polyunsaturated fatty acid (PUFA) metabolism (Cox-1, Cox-2, PTGES and PTGDS) in the bladder of SCI as compared to sham. These morphological changes were associated with a reduced transcellular permeability.
Altogether our data suggest that SCI induced a functional remodeling of the urothelium reflecting an increased pro-regenerative epithelial response. In addition, this response is associated to a reduced expression of pro-inflammatory lipids mediators expression. This later could also contribute to and/or be associated with a reduced pro-resolutive PUFAs derivatives profile that could contribute to the maintenance of a pro-regenerative phenotype of the urothelium.