Published online May 26, 2009.
https://doi.org/10.4111/kju.2009.50.5.480
The Effect of Human Muscle-Derived Stem Cells (MDSC) and Glycine-Isoleucine-Lysine-Valine-Alanine-Valine (GIKVAV) on the Cryo-Injured Bladder of Nude Mouse
Abstract
Purpose
In neurogenic bladder, both smooth muscle contraction and nerve regeneration are very important for functional improvement. Glycine-isoleucine-lysine-valine-alanine-valine (GIKVAV) is a peptide that can induce nerve regeneration in vivo. In this study, we evaluated bladder function after injection of muscle-derived stem cells (MDSCs) and GIKVAV into the cryo-injured bladder of nude mice.
Materials and Methods
Human muscle samples were obtained from the rectus abdominis muscle of 12 patients who underwent laparotomy. The purpose and entire method of the study were explained to the patients, and all subjects who participated in this study provided written informed consent. The MDSCs were isolated by a modified preplate technique, and only CD34+ human MDSC were extracted by use of Mini-MACS kits. The nude mice were subdivided into 5 groups (n=40): normal group (N, n=8), saline injection group after cryo-injury (S, n=8), GIKVAV injection group after cryo-injury (G, n=8), human MDSC injection group after cryo-injury (M, n=8), and GIKVAV and human MDSC injection group after cryo-injury (GM, n=8). At 2 weeks after injection, we compared the contractility of a bladder muscle strip of each group by organ bath and polygraph by using electronic field stimulation (EFS). Nerve regeneration was evaluated by choline acetyl transferase (ChAT) immunostaining.
Results
The contractile powers of the N, S, G, M, and GM groups were 3.58±0.27, 1.54±0.25, 1.54±0.31, 2.49±0.36, and 2.44±0.34 mN/mg, respectively, by EFS. The contractility of the bladder muscle strip in the S and G groups was lower than that in the N group. The contractile powers of the M and GM groups were lower than those of the N group but greater than those of the S and G groups. In ChAT immunohistochemical staining, nerve regeneration was increased in the G and GM groups compared with the S and M groups.
Conclusions
Nerve regeneration was induced by GIKVAV injection regardless of human MDSC injection. There was no direct effect of GIKVAV on bladder muscle contractility.
Fig. 1
The organ bath study of bladder contractility by use of electrical field stimulation. The average contractile power of the bladder muscle strip in all experimental groups (S, G, M, GM) was significantly less than in the normal control group (N). The average contractile power of the M and GM groups was significantly higher than that of the S and G groups. N: normal, N group, S: saline, S group, G: glycine-isoleucine-lysine-valine-alanine-valine, G group, M: muscle-derived stem cells, M group, GM: GIKVAV+MDSC, GM group, a: p<0.01 compared with S and G groups.
Fig. 2
H&E and fluorescent imaging of nude mouse bladder. The black arrows show the smooth muscle thickness of each group. The bladder smooth muscle layers of the S and G groups were thinner than those of the N group (A-C, ×100). The bladder smooth muscle layers of the M and GM groups were thicker than those of the S and G groups (D, E, ×100). Newly developed muscle layers were found in the M and GM groups. In fluorescent microscopy, PKH-labeled (red colored) injected cells were observed in the newly developed smooth muscle layers in the M and GM groups (I, J, ×100) but not in the N, S, and G groups (F-H, ×100). N: normal, N group, S: saline, S group, G: glycine-isoleucine-lysine-valine-alanine-valine, G group, M: muscle-derived stem cells, M group, GM: GIKVAV+MDSC, GM group.
Fig. 3
Immunohistochemical staining of choline acetyl transferase in nude mouse bladder. The nuclei were colored with DAPI (blue color). Four weeks after cryo-injury, immunofluorescence analysis showed a decreased expression (green color) of choline acetyltransferase in the S and M groups compared with the N group (A, B, D, ×100). After injection of GIKAV, the expression of choline acetyl transferase (green color) was increased in the G and GM groups compared with the S and M groups (C, E, ×100). N: normal, N group, S: saline, S group, G: glycine-isoleucine-lysine-valine-alanine-valine, G group, M: muscle-derived stem cells, M group, GM; GIKVAV+MDSC, GM group.
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