Spinal Cord Injury Causes Reduction of Galanin and Gastrin Releasing Peptid e mRNA Expression in the Spinal Ejaculation Generator of Male Rats

Abstract

Spinal cord injury (SCI) in men is commonly associated with sexual dysfunction, including anejaculation, and chronic mid-thoracic contusion injury in male rats also impairs ejaculatory reflexes. Ejaculation is controlled by a spinal ejaculation generator consisting of a population of lumbar spinothalamic (LSt) neurons that control ejaculation through release of four neuropeptides including galanin and gastrin releasing peptide (GRP) onto lumbar and sacral autonomic and motor nuclei. It was recently demonstrated that spinal contusion injury in male rats caused reduction of GRP-immunoreactivity, but not galanin-immunoreactivity in LSt cells, indicative of reduced GRP peptide levels, but inconclusive results for galanin. The current study further tests the hypothesis that contusion injury causes a disruption of GRP and galanin mRNA in LSt cells. Male rats received mid-thoracic contusion injury and galanin and GRP mRNA were visualized 8 weeks later in the lumbar spinal cord using fluorescent in situ hybridization. Spinal cord injury significantly reduced GRP and galanin mRNA in LSt cells. Galanin expression was higher in LSt cells compared to GRP. However, expression of the two transcripts were positively correlated in LSt cells in both sham and SCI animals, suggesting that expression for the two neuropeptides may be co-regulated. Immunofluorescent visualization of galanin and GRP peptides demonstrated a significant reduction in GRP-immunoreactivity, but not galanin in LSt cells, confirming the previous observations. In conclusion, SCI reduced GRP and galanin expression in LSt cells with an apparent greater impact on GRP peptide levels. GRP and galanin are both essential for triggering ejaculation and thus such reduction may contribute to ejaculatory dysfunction following SCI in rats.

Keywords: anejaculation; contusion spinal injury; lumbar spinal cord; sexual dysfunction; urogenital.

Figure 1

Fluorescent in situ hybridization for galanin and GRP mRNA. Representative confocal images (1 μm optical section) showing fluorescent signal for galanin (A) mRNA and GRP (B) mRNA in a single LSt cell, co-labeled with DAPI (C). White ellipse shown in C is representative of the region of interest used for optical density analysis. Boxed area in (C) is shown enlarged in (D) demonstrating that galanin and GRP mRNA particles are distinct. (E) Positive control probe showing labeling for POLR2A (Red) and UBC (Green) mRNA. (F) Negative control probe showing lack of labeling. Scale bars represent 10 μm (A–C), 2 μm (D), and 25 μm (E, F).

Figure 2

Galanin and GRP mRNA in LSt cells is reduced in SCI males. Representative confocal images (1 μm optical section) in a Sham (A–C, G–I) and SCI (D–F, J–L) animal showing SCI-induced reduction of fluorescent signal for galanin (A, D, G, J; Red) and GRP (B, E, H, K; Green) mRNA, in LSt cells in both L3 (A–F) and L4 (G–L) spinal levels. Merged images with DAPI are shown in (C, F, I, L). Scale bars represent 10 μm.

Figure 3

SCI reduced galanin and GRP mRNA in LSt cells. Quantitative analysis of fluorescent signal for Galanin (A) and GRP (B) mRNA in LSt cells in Sham (white bars) and SCI (black bars) groups. Data are expressed as mean ± SEM pixels above background for LSt cells in combined L3 and L4 spinal levels, and separately for L3 or L4 spinal levels. *Indicates statistically significant difference from Sham.

Figure 4

Association between Galanin and GRP mRNA in LSt cells. Scatterplots demonstrating the positive associations between fluorescent signal for galanin and GRP mRNA within LSt cells in combined L3 and L4 spinal levels (A), and separately for L3 (B) or L4 (C) spinal levels. Each circle represents the fluorescent pixels above background for galanin and GRP within one cell. Cells of sham animals are shown in white open circles, cells of SCI animals in filled blue circles. Lines show the linear relationship based on Pearson Product-Movement Correlation analyses for Sham (black line) and SCI (gray line) groups.

Figure 5

Galanin and GRP mRNA in LSt cells and dorsal horn neurons. Representative confocal images (1 μm optical section) showing fluorescent signal for galanin mRNA (red; A, D, G) or GRP (green; B, E, H) mRNA co-labeled with DAPI in blue in dorsal horn cells (A–C, D–F), while galanin and GRP are co-expressed in a LSt cell (G–I). Merged channels are shown in (C, F, I). Scale bars represent 5 μm.

Figure 6

Galanin and GRP immunofluorescence in LSt cells. Representative confocal images (1 μm optical section) showing immunofluorescent labeling of galanin (red; A, D, G, J) and GRP (green; B, E, H, K) in LSt cells of sham (A–C, G–I) and SCI (D–F, J–L) males in L3 (A–F) and L4 (G–L) spinal levels. Merged channels are shown in (C, F, I, L). Scale bar represent 25 μm.

Figure 7

SCI reduced GRP-immunofluorescence in LSt cells. Quantitative analysis of numbers of cells immunofluorescently labeled for galanin or GRP. Analyses were based on cells in combined L3 and L4 spinal levels (A), or separately for either L3 (B) or L4 (C) spinal levels in Sham (white bars) and SCI (black bars) groups. Data are expressed as mean ± SEM numbers of cells per section for the total numbers of galanin-ir cells (Total galanin; independent of GRP-ir) and GRP-ir cells (Total GRP; independent of galanin-ir). Analysis of the co-localization of galanin and GRP-immunoreactivity within LSt cells is shown as numbers of dual-labeled (Dual), or galanin-only labeled cells (Galanin only). GRP-only labeled cells weren't detected and hence not included on the graphs. *Indicates statistically significant difference from Sham. Data show that SCI did not reduce total numbers of galanin-ir LSt cells but reduced GRP-ir in LSt cells, resulting in fewer dual-labeled cells and more galanin-only LSt cells. This effect was most notable in L4 spinal levels.