Transection of the pelvic or vagus nerve forestalls ripening of the cervix and delays birth in rats

Abstract

Innervation of the cervix is important for normal timing of birth because transection of the pelvic nerve forestalls birth and causes dystocia. To discover whether transection of the parasympathetic innervation of the cervix affects cervical ripening in the process of parturition was the objective of the present study. Rats on Day 16 of pregnancy had the pelvic nerve (PnX) or the vagus nerve (VnX) or both pathways (PnX+VnX) transected, sham-operated (Sham) or nonpregnant rats served as controls. Sections of fixed peripartum cervix were stained for collagen or processed by immunohistochemistry to identify macrophages and nerve fibers. All Sham controls delivered by the morning of Day 22 postbreeding, while births were delayed in more than 75% of neurectomized rats by more than 12 h. Dystocia was evident in more than 25% of the PnX and PnX+VnX rats. Moreover, on prepartum Day 21, serum progesterone was increased severalfold in neurectomized versus Sham rats. Assessments of cell nuclei counts indicated that the cervix of neurectomized rats and Sham controls had become equally hypertrophied compared to the unripe cervix in nonpregnant rats. Collagen content and structure were reduced in the cervix of all pregnant rats, whether neurectomized or Shams, versus that in nonpregnant rats. Stereological analysis of cervix sections found reduced numbers of resident macrophages in prepartum PnX and PnX+VnX rats on Day 21 postbreeding, as well as in VnX rats on Day 22 postbreeding compared to that in Sham controls. Finally, nerve transections blocked the prepartum increase in innervation that occurred in Sham rats on Day 21 postbreeding. These findings indicate that parasympathetic innervation of the cervix mediates local inflammatory processes, withdrawal of progesterone in circulation, and the normal timing of birth. Therefore, pelvic and vagal nerves regulate macrophage immigration and nerve fiber density but may not be involved in final remodeling of the extracellular matrix in the prepartum cervix. These findings support the contention that immigration of immune cells and enhanced innervation are involved in processes that remodel the cervix and time parturition.

FIG. 1.

Percent of rats that delivered at least one pup relative to the day postbreeding in Sham-operated (Sham, n = 13) or nerve transected groups (PnX, pelvic; VnX, vagus; PnX+VnX, pelvic and vagus). By 1700 h (indicated as 5pm in figure) of Day 22 postbreeding, 2 of 9 PnX rats, 3 of 6 VnX rats, and 3 of 5 PnX+VnX rats had delivered a pup. Symbols for groups on Days 22 and 23 are offset to visualize data.

FIG. 2.

Data are mean densities of cell nuclei (±SEM) (3 sections/rat) in the cervix of Sham-operated rats (Sham, n = 14; 4–6/group), PnX pregnant rats (n = 22; 4–11/group), VnX rats (n = 16; 4–8/group), or PnX+VnX rats (n = 12; 3–5/group) with respect to day (D) postbreeding. All prepartum [71] or postpartum (PP) groups were significantly reduced compared to the nonpregnant (NP) group (n = 3) (ANOVA, F = 19.30, df = 11). b, P < 0.05 versus Day 21 Sham (ANOVA, F = 110.4, df = 2). *, P < 0.05 versus Day 22 PP Sham (ANOVA, F = 5.004, df = 3).

FIG. 3.

Data shown are the mean ODs ± SEM of picrosirius red-stained sections (3 sections/rat; n = 3–10 rats/group) normalized to cell nuclei density to correct for hypertrophy of cervix with pregnancy. Photomicrographs of birefringent polarized light were converted to grayscale images. Regions of reduced collagen content and scattered fibrils were inversely related to OD as previously described [5, 40] (details of NIH Image J analyses are in Materials and Methods). Compared to OD of nonpregnant controls, OD was significantly increased (less collagen content and structure) in cervix of prepartum [71] and postpartum (PP) groups (P < 0.05; ANOVA, F = 5.984, df = 11). Rat numbers/group are the same as those specified in Figure 2 legend. b, P < 0.05 versus Day (D) 21 Sham (P < 0.05, t-test; t = 3.039, df = 6); c, P < 0.05 versus Day 22 PnX rats (delayed birth) (ANOVA, F = 4.094, df = 2); #, P < 0.05 versus ≥ Day 22.5 PP PnX rats (ANOVA, F = 4.411, df = 2).

PLATE I. Figures 4 and 5.

Fig 4. Left panels) Macrophages stained with ED-1 antibody (specific dark brown-stained cells indicated by open arrows) and cell nuclei counterstained with hematoxylin (indicated by circles of pale violet stain) in a section of cervix on Day 21 of pregnancy from corresponding Sham and neurectomized groups. e, endothelium. Bar = 50 μm. Right panels) Data are mean numbers of macrophages in the cervix (±SEM) (3 sections/rat; numbers of rats/group are specified in Fig. 2 legend) normalized to cell nuclei number/μm³ to account for variability among individuals or field of view and hypertrophy with pregnancy. Day (D) 21 and 22 groups are prepartum, while Day 22 Shams (white bar, top panel) and Day ≥22.5 groups (cross-hatched bar) are postpartum at term or with delayed birth, respectively. The density of macrophages in the nonpregnant Sham group was 0.048 ± 0.011 (n = 3, data not shown). n/a, not applicable, since all Sham controls delivered by Day 22 postbreeding. a, P < 0.05 versus nonpregnant Sham (ANOVA, F = 3.823, df = 11); b, P < 0.05 versus prepartum Day 21, same group (ANOVA; PnX, F = 3.6, df = 2; VnX, F = 5.6, df = 2); *, P < 0.05 versus postpartum Day 22 Shams (ANOVA, F = 2.7, df = 3); #, P < 0.05 versus Day 22 PnX rats with delayed birth (Student t-test, t = 2.6, df = 6). Fig 5. Left panels) Cervix from rats on Day (D) 21 of postbreeding. Nerve fibers are stained dark brown with peripherin antibody (arrows) and counterstained with hematoxylin (nuclei are pale violet). Open spaces are blood vessels (V), some of which contain erythrocytes. Group designations and number of rats/group are the same as in Figure 4. Bar = 25 μm. Right panels) Data are mean nerve fiber densities in cervix of Sham and neurectomized groups (±SEM) (3 sections/rat, n = 3–10 rats/group) normalized to cell nuclei number/μm³ × 10³ to adjust for variability among individuals and hypertrophy with pregnancy. The area of cervix with nerve fibers in the nonpregnant Sham group was 0.49 ± 0.03 (n = 3, data not shown). a, P < 0.05 versus nonpregnant Sham (Kruskal-Wallis); b, P < 0.05 versus prepartum Day 21 Sham group (ANOVA, F = 20.98, df = 2); c, P < 0.05 versus Day 22 VnX group with delayed birth (ANOVA, F = 5.412, df = 2); *, P < 0.05 versus prepartum Day 21 Sham (ANOVA, F = 3.306, df = 6).