Radical hysterectomy to treat early-stage cervical cancer can yield good outcome; however, survivors may experience de novo lower urinary tract dysfunctions [1], which are due to damage of the pelvic nerve innervating the bladder during parametrial dissection. This study was conducted to demonstrate the effects of human amniotic fluid stem cells (hAFSCs) transplantation on bladder dysfunction and molecular changes in rats with bilateral pelvic nerve injuries (PNI).

Sixty female adult rats were divided into sham group, bilateral PNI rats and bilateral PNI rats with injection of hAFSCs. The stem cells were obtained from freshly collected amniotic fluid by routine amniocentesis of healthy pregnant donors. Cells were cultured and incubated. Passage 4-6 hAFSCs were collected and prepared to a final concentration of 1 x 1000000 cells/0.3 mL phosphate buffered saline. Because PNI interferes with bladder emptying, urine was manually expelled twice daily by gently depressing the lower abdomen. Cystometries at 10 and 28 days after sham or PNI were examined. Density of neurofilaments within bladder nerves and expressions of bladder protein gene product 9.5, growth-associated protein 43, nerve growth factor and p75 were studied using immunohistochemistry and real-time RT-PCR.

Density of neurofilaments of bladder nerves are decreased at 10 and 28 days after PNI, but improve after hAFSCs transplantation. Bilateral PNI cause overflow urinary incontinence and increase number of non-voiding contraction. However, those cystometric parameters improve after hAFSCs transplantation. Immunoreactivities and mRNAs of protein gene product 9.5 and growth-associated protein 43 in PNI bladders decrease at 28 days after PNI. Transplantation of hAFSCs accelerate the recovery of protein gene product 9.5 to sham levels. Immunoreactivities and mRNAs of nerve growth factor and p75 significantly increase after PNI, but both decrease after hAFSCs transplantation.

PNI may accumulate connective tissues in bladder wall and results in adverse effect on detrusor function to empty bladder. Our results show that bilateral PNI can abolish bladder contraction with consequent bladder distention and result in overflow incontinence; however, hAFSCs transplantation may recover all the cystometric results nearly to normal levels in PNI rats. The present study shows that the density of neurofilament in the PNI bladders remain decreased at 10 days or even 28 days after PNI. In addition, protein gene product 9.5 mRNA and immunoreactivities do not reach the sham level at 28 days after PNI, which is the same as the recovery time of neurofilament in the PNI bladder.After hAFSCs transplantation, the density of neurofilament and expression of protein gene product 9.5 may return to the sham levels, indicating nerve regeneration is at least one of the mechanisms involved in the process of recovery following injury to pelvic nerve. We find that growth-associated protein is involved in the mechanism of pelvic nerve regeneration in the PNI bladder. Transplantation of hAFSCs accelerates the recovery of growth-associated protein 43 to the sham levels at 10 days after PNI [2]. In addition to protein gene product 9.5 and growth-associated protein 43, nerve growth factor and p75 are also associated with the processes of denervation and nerve regeneration [3]. Immunoreactivities of nerve growth factor and p75 are up-regulated in the PNI bladders. Transplantation of hAFSCs restores the expressions of nerve growth factor and p75 to the sham levels at 10 days after PNI. The hAFSCs are multipotent like embryonic stem cells, grow easily in culture and have the potential to differentiate into a variety of cell types. The present study shows that hAFSCs may facilitate pelvic nerves regeneration and improve bladder dysfunction in bilateral PNI rats. Furthermore, as a non-invasive stem cell source, hAFSCs is readily available from routine amniocenteses.

Protein gene product 9.5, growth-associated protein 43, nerve growth factor and p75 are involved in the mechanism of PNI bladder nerve regeneration. Transplantation of hAFCs can improve PNI bladder dysfunction.

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