Retrograde tracing of spinal cord connections to the cervix with pregnancy in mice

Abstract

In contrast to the uterus, the cervix is well innervated during pregnancy and the density of nerve fibers increases before birth. To assess neural connections between the cervix and the spinal cord, the cervix of pregnant mice was injected with the trans-synaptic retrograde neural tract tracer pseudorabies virus (PRV). After 5 days, the virus was present in nerve cells and fibers in specific areas of the sensory, autonomic, and motor subdivisions of the thoracolumbar spinal cord. In nonpregnant controls, the virus was predominantly distributed in laminae I-III in the dorsal gray sensory areas with the heaviest label in the substantia gelatinosa compared with the autonomic or motor areas. Labeled cells and processes were sparse in other regions, except for a prominent cluster in the intermediolateral column (lamina VII). Photomicrographs of spinal cord sections were digitized, and the total area with the virus was estimated. Compared with nonpregnant controls, the area with PRV was significantly decreased in all the spinal cord subdivisions in pregnant mice except in the intermediolateral column. However, areas with the virus were equivalent in mice injected with PRV at 4 days or 1 day before birth. These findings suggest that the predominant innervation of the murine cervix is from the sensory regions of the thoracolumbar spinal cord, and that these connections diminish with pregnancy. The results raise the possibility that the remaining connections from sensory and autonomic subdivisions, particularly the intermediolateral column, of the thoracolumbar spinal cord may be important for increased density of nerve fibers in the cervix as pregnancy nears term.

Figure 1

Photomicrographs of pseudorabies virus-labeled cells and fibers in sections in the lower thoracic spinal cord (T11) from a mouse that was injected with pseudorabies virus into the cervix when nonpregnant (top) or on day 15 of pregnancy (bottom). Pseudorabies virus label was most prevalent in sensory (dorsal horn) and autonomic (region surrounding and lateral to the central canal) subdivisions, as well as in the intermediolateral cell column (IML). Insets highlight details of virus-labeled neuronal cell bodies and fibers. Scale bar=100 μm and, in insets, 50 μm.

Figure 2

(Left) Photomicrographs of thoracic segment 11 spinal cord sections from a mouse that was injected with pseudorabies virus into the cervix when nonpregnant (top) or on day 15 of pregnancy (bottom). Dark stain indicates pseudorabies virus-labeled cells and fibers. (Right) Digitized pixelated images of pseudorabies virus label generated with Image Pro plus software were superimposed upon a drawing of the spinal cord segment cytoarchitecture from Molander et al. (1984). Isolated thin fibers or small punctate structures that reflect fibers in cross section were below the limit of imaging threshold detection. Scale bar=100 μm.

Figure 3

Estimated area of cells and fibers that contained pseudorabies virus in sensory, autonomic, and motor subdivisions of the lower thoracic and upper lumbar segments of spinal cord in mice that were injected with pseudorabies virus into the cervix when nonpregnant (NP), or on day 15 of pregnancy (D15), or on day 18 of pregnancy (D18). Data for area of cervix with pseudorabies virus (PRV) label were the sum of the number of pixels times the area/pixel in the thoracolumbar spinal cord (mean±S.E.M., n=4/group; see details in Materials and Methods). ‘a’ indicates P<0.05 versus NP group.