Individuals experiencing stress urinary incontinence (SUI) may exhibit impaired motor control of the pelvic floor muscles (PFM). The proper functioning of the motor control loop relies on the proprioception and its  transmission. Moreover, the muscle spindle is an essential component of proprioceptive conduction, and its proper functioning depends on its structural and functional integrity, and the presence of neurotrophic factors. The binding of target-derived neurotrophic factor NT-3 secreted by intrafusal muscle fibers to proprioceptive neurons plays an essential role in maintaining the conduction function of the muscle spindle. However, whether the decreased expression of NT-3 leads to the abnormal muscular spindle and proprioceptive transmission of PFM remains unclear. In this study, we investigated the morphology and expression of NT-3 protein in the PFM spindle and L6-S1 dorsal root ganglion(DRG) of the group SUI and Sham rats. Our hypothesis is that SUI rats will exhibit a higher rate of morphological variation in the PFM spindle and L6-S1DRG, as well as a lower expression level of the NT-3 protein in L6-S1DRG, as compared to Sham rats. This is the first study to analyze the proprioception and conduction of PFM in SUI rat models.

All procedures were approved by the Animal Research Committee and carried out per the approved guidelines. Five female Sprague-Dawley rats (SPF grade, nine weeks old, and body weight between 230 to 250g) were randomly selected as the Sham group. The remaining five rats underwent secondary vaginal balloon dilatation to make the SUI rat models. The operation of the Sham group was the same as in the SUI group, except that the vaginal balloon did not swell with water. PFM spindle and L6-S1 DRG samples from all animals were isolated for histologic evaluations on the 7th day after modeling. While anesthetized with urethane after the urodynamics evaluation, which includes bladder leak point pressure (BLPP) and abdominal leak point pressure (ALPP). PFM and L6-S1 DRG were rapidly removed, fixed with 4% paraformaldehyde for at least 24 hours, and then embedded into paraffin blocks for further processing.

(1)	  BLPP and ALPP in the SUI group were significantly lower than those in the Sham group (P < 0.05) after seven days of modeling, which indicated that the SUI rat model was successfully made. (Table 1)
(2)	The morphological changes of the PFM spindle and L6-S1DRG in the SUI group were more than in the Sham group. (Figure 1 A, B, C, D)
(3)	NT-3 was expressed in large-diameter neurons of L6-S1DRG. After 7th days of modeling, the positive signal of NT-3 in L6-S1DRG of the Sham group was more substantial than that of the SUI group. (Figure 1 E, F)

As hypothesized, the shape of the PFM spindle of the Sham group was intact, and its structure was regular. In contrast, the form of the PFM spindle in the SUI group showed different degrees of morphological variation, which indicated that the shape and structure of the PFM spindle might be destroyed by SUI modeling. Meanwhile, the large-diameter neurons of L6-S1DRG in the Sham group were arranged and stained uniformly, and the nuclear morphology was regular. In the SUI group, the cytoplasm density of DRG large-diameter neurons decreased, the cells contained vacuoles, some nuclei were stained lightly, and the nucleolus morphology and structure were irregular. These results suggest that SUI modeling may destroy the morphological structure of large-diameter neurons in L6-S1DRG, affecting its conduction function. In addition, NT-3 was expressed in large-diameter neurons of L6-S1DRG. After 7th days of modeling, the positive signal of NT-3 in L6-S1DRG of the Sham group was more substantial than that of the SUI group. Previous studies have shown that the proprioception of the perineum in female patients with SUI is decreased. After pelvic floor proprioception training, the therapeutic effect of SUI is significantly improved compared with the control group[1]. In addition, research showed that NT-3 deficiency rats had severe ataxia related to muscle spindle and proprioceptive neurons of DRG or its axons[2]. These results suggest that the abnormality of proprioceptive conduction of PFM caused by NT-3 deficiency may be related to the occurrence of SUI in the rat model.

NT-3 deficiency may lead to the abnormal structure of large-diameter neurons in the L6-S1 DRG and PFM spindle in the SUI rat model, resulting in abnormal proprioception and conduction of levator ani muscle, which may be the pathophysiological mechanism of SUI.

Liu, Jiaojiao, et al.  "Therapeutic effect of proprioception training combined with pelvic floor electrical stimulation biofeedback on postpartum pelvic floor dysfunction." Journal of Central South University. Medical Science, 2022, 47(9): 1253-1259.
Wright, D E, et al. "Muscle-derived neurotrophin-3 reduces injury-induced proprioceptive degeneration in neonatal mice.  " Journal of neurobiology, 2002, 50(3): 198-208.

Continence 7S1 (2023) 101000
DOI: 10.1016/j.cont.2023.101000