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Abstract
Purpose
We investigated whether periurethral injections of musclederived stem cells (MDSC) and chitosan/hydroapatite after denervation of rat's pudendal nerve could increase the leak point pressure over a long time period in a rat model of urinary incontinence.
Materials and Methods
Muscle-derived stem cells isolated from the gastrocnemius muscle of normal female rats were purified to obtain a myogenic population by using the preplate technique. The N group was the normal female rats, the D Group was the pudendal nerve transected group and the M Group was the MDSC/chitosan/hydroapatite composite gel injected group after pudendal nerve transection. The MDSC/chitosan/hydroapatite composite gel was injected into the proximal periurethral area. At 2 and 4 weeks, visually identified leak point pressure measurement was done with using the vertical tilt/intravesical pressure clamp model of urinary incontinence. The rats were then sacrificed and the periurethral tissues harvested for histological examination.
Results
The leak point pressure was significantly lower in the D group at each time compared with the N group, and the leak point pressure in the N and M groups were significantly higher than those in the D group at both 2 and 4 weeks. The persistence of MDSC over the period of study was verified by histological examination.
Figures and Tables
Fig. 1
Vertical tilt/intravesical pressure clamp model of urinary incontinence. Vertical tilt/intravesical pressure clamp model of urinary incontinence. (A) Acute spinal cord transection at the T9-T10 level was performed with the rat in a supine position and a transvesical catheter with a fire-flared tip (PE-90) was inserted into the dome of the bladder for measuring bladder filling and the pressure. (B) The intravesical pressure varied in 1-3cmH2O steps from zero upward until visual identification of the leak point (leak point pressure; LPP).
Fig. 2
Leak point pressure (LPP) at 2 weeks. The LPP in the D group was significantly lower at 1 month than the LPP at 1 month in the N and M groups. The LPP in the M group was significantly higher than that in the D group at 2 weeks (N: normal group, D: denervated group, M: denervated and muscle stem cell/chitosan/hydroapatite injected group). *: p<0.01 compared to N group, †: p<0.01 compared to D group.
Fig. 3
Leak point pressure (LPP) at 4 weeks. The LPP in the D group was significantly lower at 4 weeks than the LPP at 4 weeks in the N and M groups. The LPP in the M group was significantly higher than that in the denervation group at 4 weeks (N: normal group, D: denervated group, M: denervated and muscle stem cell/chitosan/hydroapatite injected group). *: p<0.01 compared to N group, †: p<0.01 compared to D group.
Fig. 4
Histology of the normal urethral sphincter of female rat. (A) Hematoxylin/eosin staining at 4 weeks. In the normal female rat urethral sphincter, a layer of striated muscle fibers (black arrow) encircles the smooth muscle fibers (red arrow). (B) MyHC (red) immunostaining of the specimen at 4 weeks post-treatment. In the normal female rat urethral sphincter, the striated muscle (white arrow) shows positive MyHC staining. (C) α-SMA (green) immunostaining of the specimen at 4 weeks post-treatment. The smooth muscle (red arrow) shows positive α-SMA staining, and the striated muscle (white arrow) shows blanks (A, B, C: ×100).
Fig. 5
Histology of the denervated urethral sphincter of female rat. (A) Hematoxylin/eosin staining at 4 weeks. The denervated proximal urethral sphincter also showed atrophic and thin circular striated fibers (black arrow). The smooth muscle is well preserved (red arrow). (B) MyHC immunostaining of specimen at 4 weeks post-treatment. The denervated proximal urethral sphincter, which is striated muscle (red arrow), shows negative MyHC staining. (C) α-SMA (green) immunostaining of the specimen at 4 weeks post-treatment. The smooth muscle (red arrow) shows positive α-SMA staining (A, B, C: ×100).
Fig. 6
Histology of the MDSC/chitosan/hydroapatite injected urethral sphincter of female rat. (A) Hematoxylin/eosin staining at 4 weeks. The MDSC/chitosan/hydroapatite injection area (black arrow) is well preserved. (B) MyHC immunostaining of the specimen at 4 weeks post-treatment. The area where the MDSC was injected area is PKH26 positive (red) and the striated muscle (white arrow) shows positive MyHC staining. (C) α-SMA immunostaining of the specimen at 4 weeks post-treatment. Muscular bundles (white arrow) are observed to be positive for α-SMA staining (green) on the periphery of the polymer (A, B, C: ×100).
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