The association of adelmidrol with sodium hyaluronate displays beneficial properties against bladder changes following spinal cord injury in mice

Abstract

The disruption of coordinated control between the brain, spinal cord and peripheral nervous system caused by spinal cord injury (SCI) leads to several secondary pathological conditions, including lower urinary tract dysfunction. In fact, urinary tract dysfunction associated with SCI is urinary dysfunction could be a consequence of a lack of neuroregeneration of supraspinal pathways that control bladder function. The object of the current research was to explore the effects of adelmidrol + sodium hyaluronate, on bladder damage generated after SCI in mice. Spinal cord was exposed via laminectomy, and SCI was induced by extradural compression at T6 to T7 level, by an aneurysm clip with a closing force of 24 g. Mice were treated intravesically with adelmidrol + sodium hyaluronate daily for 48 h and 7 days after SCI. Adelmidrol + sodium hyaluronate reduced significantly mast cell degranulation and down-regulated the nuclear factor-κB pathway in the bladder after SCI both at 48 h and 7days. Moreover, adelmidrol + sodium hyaluronate reduced nerve growth factor expression, suggesting an association between neurotrophins and bladder pressure. At 7 days after SCI, the bladder was characterized by a marked bacterial infection and proteinuria; surprisingly, adelmidrol + sodium hyaluronate reduced significantly both parameters. These data show the protective roles of adelmidrol + sodium hyaluronate on bladder following SCI, highlighting a potential therapeutic target for the reduction of bladder changes.

Fig 1. Acute effect of adelmidrol + sodium hyaluronate on histological parameters after SCI.

Control mice showed a normal musculature and no signs of edema (a, a1), whereas at 48 h post-SCI, mice showed clear signs of sub-mucosal edema and epithelial ulceration (b, b1). Adelmidrol + sodium hyaluronate treatment restored bladder structure (c, c1). The histological score summarizes these data (d) ***p< 0.001 vs Sham; ^###p< 0.001 vs SCI.

Fig 2. Acute effect of adelmidrol + sodium hyaluronate on mucus-producing cells after SCI.

PAS staining identifies granular glycogen deposits in bladder tissue and was performed 48 h after SCI. Control mice showed no staining of glycogen granules (a, a1). Mice subjected to SCI presented an augmented staining of glycogen in the bladder (b, b1), while mice treated with adelmidrol + sodium hyaluronate showed decreased staining (c, c1). These data are also visible in the glycogen score (d) ***p< 0.001 vs Sham; ^#p< 0.05 vs SCI.

Fig 3. Acute effects of adelmidrol + sodium hyaluronate on MC and on levels of chymase and tryptase.

Toluidine blue staining was used to identify mast cell infiltration (encircled), characterized by dark lilac blue granules: (a) Sham group; (b) SCI group; (c) SCI+ 2% adelmidrol and 0.1% sodium hyaluronate group. (d) Mast cell number per unit area of tissue (mast cell density). Figures are illustrative of at least 3 distinct experiments. Values are means ± SD of 10 animals for each group. ***p<0.001 vs Sham, ^###p<0.01 vs SCI. Immunohistochemical analysis showed no staining for chymase and tryptase in the Sham group (e and i). Increased chymase and tryptase expression was observed in bladder collected 48 h after SCI (f and j), while low levels of these proteins were found in mice treated with adelmidrol + sodium hyaluronate (g and k). The data are also presented graphically as percentage of total tissue area (h and l). h) ***p< 0.001 vs Sham; ^##p< 0.01 vs SCI; l) ***p< 0.001 vs Sham; ^##p< 0.01 vs SCI.

Fig 4. Acute effects of adelmidrol + sodium hyaluronate on ZO-1 and NGF expression in bladder after SCI.

Immunofluorescence for ZO-1 expression (red) in sham mice (a-c), SCI group (d-f) and SCI mice treated with adelmidrol + sodium hyaluronate (g-i). Immunofluorescence for NGF expression (red) in sham group (j-l), SCI group (m-o) and SCI mice treated with adelmidrol + sodium hyaluronate (p-r). Data are demonstrative of at least three separate experiments. Images are representative of all animals in each group. The graphs next to the respective panel represent the positive ZO-1 and NGF cells. All images were digitalized at a resolution of 8 bits into an array of 2048 x 2048 pixels. Pictures were captured at 100x magnification.

Fig 5. Acute effects of adelmidrol + sodium hyaluronate on NF-κB pathway and iNOS expression after SCI.

Representative western blots showing the effects of adelmidrol + sodium hyaluronate on: (a, a1) NF-κB p65 nuclear translocation, (b, b1) IκB-α degradation and (c, c1) iNOS expression at 48 h post-SCI. Adelmidrol + sodium hyaluronate treatment reduced NF-κB p65 translocation (a, a1), IκB-α degradation (b, b1), and iNOS expression (c, c1). Data are illustrative of at minimum three independent experiments. a) **p < 0.01 vs Sham; ^##p < 0.01 vs SCI; b) ***p < 0.001 vs Sham; ^###p < 0.001 vs SCI; c) **p < 0.01 vs Sham; ^##p < 0.01 vs SCI.

Fig 6. Chronic effects of adelmidrol + sodium hyaluronate on histological alteration after SCI.

Control mice showed a normal musculature and no signs of edema (a, a1), whereas at 7 days post-SCI, mice showed evident signs of sub-mucosal edema and epithelial ulceration (b, b1). Adelmidrol + sodium hyaluronate treatment restored bladder structure (c, c1). These data are also visible in the histological score (d) ***p< 0.001 vs Sham; ^##p< 0.01 vs SCI.

Fig 7. Chronic effects of adelmidrol + sodium hyaluronate on glycogen after SCI.

PAS staining to identify the granular glycogen deposits in bladder tissue was performed at 7 days after SCI. Control mice showed no staining of glycogen granules (a, a1). Mice subjected to SCI presented an increase of positive staining of glycogen in the bladder (b, b1), while mice treated with adelmidrol + sodium hyaluronate showed a decreased of positive staining in the bladder (c, c1). These data are also presented graphically in the glycogen score (d) ***p< 0.001 vs Sham; ^#p< 0.05 vs SCI.

Fig 8. Chronic effects of adelmidrol + sodium hyaluronate on ZO-1 and NGF expression in bladder after SCI.

Immunofluorescence for ZO-1 expression (red) in sham mice (a-c), SCI mice (d-f) and SCI mice treated with adelmidrol + sodium hyaluronate (g-i). Immunofluorescence for NGF expression (red) in sham mice (j-l), SCI mice (m-o) and SCI mice treated with adelmidrol + sodium hyaluronate (p-r). Data are representative of at minimum three independent experiments. Images are representative of all animals in each group. The graphs next to the respective panel represent the positive ZO-1 and NGF cells. All images were digitalized at a resolution of 8 bits into an array of 2048 x 2048 pixels. Pictures were captured at 100x magnification.

Fig 9. Chronic effects of adelmidrol + sodium hyaluronate on protein accumulation and bacterial growth in urine and in bladder of SCI mice.

The Bradford assay showed an increase in protein in urine 7 days after SCI, adelmidrol + sodium hyaluronate treatment reduced significantly protein accumulation in urine (c). Mice subjected to SCI showed an increase of bacterial colonies in both urine and bladder, while treatment with adelmidrol + sodium hyaluronate, reduced significantly bacterial growth (a and b respectively). a) **p < 0.01 vs Sham; ^#p < 0.05 vs SCI; b) ***p < 0.001 vs Sham; ^###p < 0.001 vs SCI; c) ***p < 0.001 vs Sham; ^#p < 0.05 vs SCI.