Chronic Spinal Cord Injury Reduces Gastrin-Releasing Peptide in the Spinal Ejaculation Generator in Male Rats

Abstract

Spinal cord injury (SCI) causes sexual dysfunction, including anejaculation in men. Likewise, chronic mid-thoracic contusion injury impairs ejaculatory reflexes in male rats. Ejaculation is controlled by a spinal ejaculation generator (SEG) comprised of a population of lumbar spinothalamic (LSt) neurons. LSt neurons co-express four neuropeptides, including gastrin-releasing peptide (GRP) and galanin and control ejaculation via release of these peptides in lumbar and sacral autonomic and motor nuclei. Here, we tested the hypothesis that contusion injury causes a disruption of the neuropeptides that are expressed in LSt cell bodies and axon terminals, thereby causing ejaculatory dysfunction. Male Sprague Dawley rats received contusion or sham surgery at spinal levels T6-7. Five to six weeks later, animals were perfused and spinal cords were immunoprocessed for galanin and GRP. Results showed that numbers of cells immunoreactive for galanin were not altered by SCI, suggesting that LSt cells are not ablated by SCI. In contrast, GRP immunoreactivity was decreased in LSt cells following SCI, evidenced by fewer GRP and galanin/GRP dual labeled cells. However, SCI did not affect efferent connections of LSt, cells as axon terminals containing galanin or GRP in contact with autonomic cells were not reduced following SCI. Finally, no changes in testosterone plasma levels or androgen receptor expression were noted after SCI. In conclusion, chronic contusion injury decreased immunoreactivity for GRP in LSt cell soma, but did not affect LSt neurons per se or LSt connections within the SEG. Since GRP is essential for triggering ejaculation, such loss may contribute to ejaculatory dysfunction following SCI.

Introduction

Spinal cord injury (SCI) is a devastating clinical injury with extreme cost and burden on patients and their families. Following initial stabilization after spinal cord injury, quality of life issues become increasingly important to patients. The SCI population is predominantly male, the majority of SCI men are younger than age 45, and ejaculatory dysfunction is extremely common among men with SCI.^1–11 Approximately 90-85% of men with SCI either do not achieve ejaculation or require the aid of intense penile vibratory stimulation or electro-ejaculation.^4,5,12,13 Surveys of men with SCI have demonstrated that regaining sexual function is an important goal, even surpassing that for recovery of walking among paraplegic men.^1,2,14 Unfortunately, treatment strategies to overcome ejaculatory dysfunction in SCI men have been impaired due to poor understanding of the mechanisms by which chronic injury so radically influences the spinal ejaculation generator (SEG).^8,12,15–17

Ejaculation is a reflex controlled by the SEG, which consists of a population of lumbar spinal cord neurons that were originally named for their thalamic projections: lumbar spinal thalamic or LSt cells. LSt cells are essential for ejaculatory reflexes, as cell-specific lesions of LSt cells ablate ejaculatory behavior and reflexes.^18,19 Studies in which the SEG was isolated from supraspinal connections (i.e., using anesthetized and spinal transected rats) have shown that LSt cells coordinate sensory inputs during sexual activity with coordinated autonomic and motor outputs via their inter-spinal connections to lumbar and sacral motor and autonomic preganglionic cells.^18,19 The thalamic projections of LSt cells are thus not involved in the central control of ejaculatory reflexes and instead have been hypothesized to relay ejaculation-related information to the brain and contribute to reward associated with ejaculation.²⁰ Likewise, supraspinal inputs to LSt cells are not essential to trigger ejaculation, but rather influence the SEG.²⁰ LSt cells are located in lamina X and medial lamina VII of L3 and L4^18,21 and co-express four neuropeptides: galanin, gastrin-releasing peptide (GRP), enkephalin, and cholecystokinin (CCK).^{18,20,22–25}

A similar population of neurons has been described in the human⁷ and mouse²⁶ spinal cord in the same spinal levels, evidence that the spinal ejaculation generator and the neuropeptide expression within is conserved among mammalian species. Moreover, LSt cells mediate ejaculatory reflexes via release of these neuropeptides onto autonomic and motor target neurons in the lumbosacral spinal cord. Pharmacological studies have demonstrated that galanin, CCK, GRP, or enkephalin receptor activation is required for ejaculatory reflex triggered by stimulation of the dorsal penile nerve,^22,23,27,28 a major relay of sensory inputs during sexual activity.^19,29,30 Specifically, intrathecal antagonist treatment of any of the receptors for these LSt neuropeptides completely prevented ejaculatory reflexes induced by dorsal penile nerve stimulation.^22,23,27 Moreover, GRP or mu opioid receptor activation by intrathecal agonist infusions is sufficient to trigger ejaculation in the absence of sensory stimulation.^22,23 Finally, agonist infusions of receptors for any of the LSt neuropeptides facilitate ejaculatory reflexes upon subthreshold stimulations of the dorsal penile nerve that are unable to trigger ejaculatory reflexes under control conditions.^22,23,27

Our laboratory has demonstrated that contusion injury at mid thoracic levels severely disrupts ejaculation in male rats even weeks after SCI.²⁸ The contusion injury model uses a single brief displacement of the spinal cord, is widely used and validated in rats,^31–33 and has been demonstrated to closely replicate the key pathophysiological features of human injury by producing either prolonged or rapidly applied extradural compression.^34–36 This was a critical observation, as previous studies had shown effects of select spinal lesions or lateral hemisections on erectile and pudendal nerve reflexes,^37–39 but detailed analyses of ejaculatory reflexes following chronic SCI had not been previously described. Moreover, in this study, ejaculatory dysfunction was demonstrated after removal of remaining supraspinal influences.²⁸ Hence, ejaculatory dysfunction was not caused by a reduction or loss of supraspinal influences following spinal injury, but rather it is hypothesized that chronic contusion injury caused long-term disruptions within the spinal ejaculation generator itself. In this study, we tested the hypothesis that chronic SCI caused alterations in LSt cells and the neuropeptides expressed within the LSt cells and axon terminals, thereby causing ejaculatory dysfunction.

The current set of studies test the hypothesis that SCI reduces the expression of neuropeptides galanin and GRP within LSt cells. GRP and galanin are exclusively expressed in LSt cells and are co-expressed in all LSt cells and their axon terminals.²³ GRP is a potent facilitator and infusions alone can trigger ejaculatory function,²³ while facilitative effects of galanin are dependent on sensory stimulation.²⁷ Moreover, galanin has been established as a reliable marker for LSt cells in rats,^{18,19,21–23,25,40,41} mice,²⁶ and human.⁷ Therefore, the current set of studies test the hypothesis that galanin and GRP peptide levels in LSt cells are reduced in male rats following contusion injury surgery, using immunofluorescence visualization. In addition, GRP and galanin immunoreactive axon terminals in putative contact with autonomic neurons in the upper lumbar and sacral levels were determined in order to examine potential loss of connections of LSt cells with these critical target areas.

Methods

Animals

Adult male Sprague Dawley rats (Charles River, Sherbrooke, Quebec, Canada, 225–250 g) were pair-housed in standard Plexiglas cages. Animals were housed on a 12-h light/dark cycle in a humidity and temperature-controlled room with lights off at 8 or 10 a.m. Food and water was available at all times. All studies were carried out in accordance with the National Institutes of Health guidelines involving vertebrate animals in research and were approved by the Institutional Animal Care and Use Committees at Western University (London, ON) and the University of Mississippi Medical Center (Jackson, MS).

Experiment 1: Effect of SCI on galanin and GRP immunoreactivity in LSt cell bodies and axon terminals

The main objective of the first study was to determine the effect of chronic contusion injury on immunofluorescence for galanin and GRP in LSt soma and axon terminals.

Spinal contusion injury surgeries

Adult male rats were randomly divided into SCI (n = 9) and sham (n = 11) groups. Rats were deeply anesthetized with ketamine/xylazine (83 mg/kg and 13 mg/kg respectively, 1 mL/kg intraperitoneal injection), and backs of the rats were shaved and cleaned with betadine and ethyl alcohol (70%). A midline incision was made on the area overlying T6-7 and muscle and connective tissue surrounding the spinal column was dissected away using blunt dissection. All animals (SCI and sham) received a complete laminectomy of the sixth or seventh thoracic vertebra (T6-T7) and spinal columns were stabilized using forceps attached to the IH-400 base platform for contusion injuries. SCI (but not sham) animals received a contusion injury at the T6-T7 spinal levels of the spinal cord, using the IH-400 impactor (Precision Systems and Instrumentation, LLC, Lexington, KY). The impact rod was positioned 1.5 inches above the cord with the tip approximately 5 mm above the cord, and a force of 200 Kdyn was applied to the dura of the exposed spinal cord with a dwell time of 0 sec. Actual force delivered ranged from 199 to 214 Kdyn (mean: 205.56 ± 1.89 Kdyn). Spinal cord displacement values ranged from 582 to 1763 μm (mean: 1319.86 ± 180.32 μm). Displacement from two animals did not register with the contusion apparatus and was recorded as zero; these values were not included in the average calculations for displacement. Velocity of the impact rod ranged from 117 to 153 mm/sec (mean: 126.56 ± 3.52 mm/sec). Sham animals received all treatments except the application of the contusion injury. Following surgery, the layers of muscle and skin were sutured separately, with absorbable sutures (Ethicon, Mokena, IL; Cat# J415) and wound clips (9 mm; World Precision Instruments, Sarasota, FL; Cat# 500345) respectively.

To alleviate pain and prevent bacterial infection, all animals (SCI and sham) received subcutaneous injections of the analgesic carprofen (5 mg/mL/kg), and intramuscular injections of the antibiotic Baytril (5 mg/mL/kg; Bayer Pharmaceuticals, Leverkusen, Germany; Cat# RXBAYTRIL), immediately prior to surgery and for 3 days thereafter. Bladder dysfunction occurred in all SCI animals and bladders were manually emptied by light compression twice per day until bladder function returned. Sham animals were handled, but bladders were not expressed.

Locomotor testing

All SCI animals had noticeable paralysis of the hind limbs and locomotor activity was measured during the dark phase using an Open Field Apparatus (Med Associates Inc., St. Albans City, VT) surrounded by two 16 × 16 photobeam arrays for detection of vertical and horizontal movement. Locomotor activity was measured during a 15-min period and presented as ambulatory distance in the horizontal plane (horizontal distance in cm) and time spent rearing (vertical time in seconds). Testing sessions were conducted at 3 and 4 weeks after contusion or sham injury for all animals. Both measures of locomotor activity were analyzed using a two-way repeated analysis of variance (factors: SCI and time following surgery) and Holm-Sidak post hoc tests using 95% confidence levels. Sham and SCI animals differed statistically in both ambulatory distance and vertical time during both tests (Table 1). Locomotor data was missing from one sham animal and was not included in the statistical comparison. Analysis of LSt axon connections was conducted on a subset of the animals (Sham: n = 5; SCI: n = 5). Contusion data for these animals were : Force ranged from 201 to 214 Kdyn (mean: 205.8 ± 2.97 Kdyn); displacement values ranged from 1693 to 1763 μm (mean: 1698.67 ± 35.62 μm; displacement from two animals did not register with the contusion apparatus and was recorded as zero, and these values were not included in the average calculation); and velocity of the impact rod ranged from 122 to 124 mm/sec (mean: 123.2 ± 0.49 mm/sec).

Table 1.

Locomotor Activity in Sham and SCI Male Rats

			Horizontal distance (cm)	Vertical time (sec)
Exp 1: all animals
	Week 3	Sham (n = 9)	1,575.4 ± 89.9	150.2 ± 16.9
		SCI (n = 10)	1,006.5 ± 241.4	65.7 ± 30.4*
	Week 4	Sham (n = 9)	1,557.9 ± 163.1	124.2 ± 17.7
		SCI (n = 10)	1,056.1 ± 245.4	51.1 ± 9.2*
Exp 1: animals included in LSt axon terminal analysis
	Week 3	Sham (n = 5)	1623.1 ± 149.9	149.9 ± 18.3
		SCI (n = 5)	660.3 ± 157.6*	15.3 ± 6.3*
	Week 4	Sham (n = 5)	1823.9 ± 328.9	146.1 ± 32.3
		SCI (n = 5)	892.4 ± 197.8*	50.0 ± 14.1*
Exp 2: all animals
	Week 3	Sham (n = 13)	1532.8 ± 143.3	65.4 ± 6.68
		SCI (n = 22)	1053.1 ± 69.1*	40.5 ± 5.1*
	Week 4	Sham (n = 13)	1823.1 ± 121.7†	89.39 ± 10.1†
		SCI (n = 22)	1084.4 ± 69.4*	45.5 ± 5.6*
Exp 2: animals included in AR-Galanin analysis
	Week 3	Sham (n = 5)	1282.6 ± 155.5	85.0 ± 10.7
		SCI (n = 11)	953.9 ± 63.1	33.3 ± 4.8*
	Week 4	Sham (n = 5)	1552.1 ± 181.1	87.5 ± 14.2
		SCI (n = 11)	975.3 ± 96.6*	43.7 ± 8.0*

^*Significant difference from Sham controls.

^†Significant difference from Week 3.

Locomotor activity in Sham and SCI male rats shown as locomotor movement in the horizontal plane (horizontal distance in cm) and time spent rearing (vertical time in seconds), for each study and analysis at 3 and 4 weeks post-surgery (Week 3–4). Statistical analysis using two-way repeated analysis of variance revealed a main effect of SCI (Exp 1: all animals [vertical time: F(1,17) = 12.5; p = 0.003], Exp 1: animals included in LSt axon terminal analysis [horizontal distance: F(1,7) = 13.4; p = 0.008; vertical time: F(1,7) = 26.2; p = 0.001], Exp 2: all animals [horizontal distance: F(1,33) = 23.4, p < 0.001; vertical time: F(1,33) = 20.1, p < 0.001], Exp 2: animals included in AR-Galanin analysis [horizontal distance: F(1,13) = 13.1, p = 0.003; vertical time: F(1,13) = 29.1, p < 0.001]. Pairwise comparisons using Holms Sidak tests revealed significant reduction in locomotor activity in SCI animals compared with sham at 3 weeks (Exp 1: all animals [vertical time, p = 0.006], Exp 1: animals included in LSt axon terminal analysis [horizontal distance: p = 0.008, vertical time, p < 0.001]; Exp 2: all animals [horizontal distance: p < 0.001, vertical time: p = 0.011], Exp 2: animals included in AR-Galanin analysis [vertical time, p < 0.001]) and 4 weeks (Exp 1: all animals [vertical time, p = 0.016], Exp 1: animals included in LSt axon terminal analysis [horizontal distance: p = 0.01, vertical time, p = 0.004]; Exp 2: Aall animals [horizontal distance: p < 0.001, vertical time, p < 0.001], Exp 2: animals included in AR-Galanin analysis [horizontal distance: p = 0.002, vertical time, p = 0.002]). Main effect for Weeks was observed in Exp 2 [all animals horizontal distance: F(1,33) = 10.1, p = 0.003; vertical time, F(1,33) = 6.5, p = 0.016], and pairwise comparison showed that in Exp 2, Sham animals demonstrated greater locomotor activity in Week 4 compared with Week 3 (all animals (horizontal distance, p < 0.001; vertical time, p = 0.012). Data are expressed as mean ± standard error of the mean.

Exp, experiment; SCI, spinal cord injury; LSt, lumbar spinothalamic.

Tissue collection

Five to six weeks after contusion or sham surgery, animals were transcardially perfused and brains and spinal cords removed. This time-point was selected based on our previous demonstration of severe disruption of ejaculatory reflexes at 4–6 weeks after contusion injury.²⁸ In these previous studies showing that SCI disrupted ejaculatory reflexes, all animals received an acute spinal transection 2 hours prior to measuring ejaculatory reflexes. Therefore, in order to examine expression of galanin and GRP under those same conditions, all animals in this experiment 1 also received an acute spinal transection under deep anesthesia using urethane (Sigma; Cat#U2500; 1.5 mg/kg). Two hours after transection, animals received an overdose of sodium pentobarbital (390 mg/mL/kg body weight; Fatal Plus; Vortech Pharmaceuticals, Dearborn, MI) and were perfused transcardially with 10 mL 0.9% sodium chloride solution (saline; Fisher; Cat# BP-358-212) followed by 500 mL of 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA; Cat# 19210) in in 0.1 M sodium phosphate buffer (PB). Spinal cords were removed and post-fixed for 1 h in the same fixative and next stored in 20% sucrose (in 0.1M PB containing 0.01% sodium azide (Fisher; Cat# S227)). Lumbosacral spinal cords (containing spinal levels L1-S1) were sectioned coronally into 12 parallel series of 35 μm sections using a sliding microtome (Micron; H400R; Germany) with a freezing stage (Physitemp, Clifton, New Jersey; BFS-5) and were stored in a cryopreservative solution containing 30% sucrose (Sigma; Cat# S7903), 30% ethylene glycol (Fisher; Cat#E178-1), and 0.01% sodium azide (Fisher; Cat# S227) in 0.1 M PB).

Immunofluorescence

General

Immunostaining was performed using free-floating sections at room temperature and with gentle agitation on a horizontal shaking platform. For each analysis, sections from all animals from all groups were immunoprocessed simultaneously. Sections were washed with phosphate-buffered saline (PBS: 0.1 M PB with 0.9% sodium chloride) between all incubations. Prior to incubation with primary antibodies, sections were treated with 1% H₂0₂ (Fisher; Cat# H323-500) for 10 min and antibody incubation solution (PBS containing 0.1% bovine serum albumin (Fisher; Cat# BP1605-100) and 0.4% Triton X-100 (Fisher; Cat# BP151-100). This antibody incubation solution was used for all primary and secondary antibody incubations. Following immunostaining, sections were mounted on SuperFrost Plus glass slides (Fisher; Cat# 22-037-246), cover-slipped with an aqueous mounting medium (Gelvatol) containing antifading agent 1,4-Diazabicyclo[2.2.2]octane (Sigma, Cat# D27802) and were stored at 4°C.

GRP/galanin/NeuN

One series of sections for each animal was incubated with a polyclonal rabbit antibody specifically recognizing GRP (Phoenix Pharmaceuticals, Burlingame, CA; RRID: AB_2722600, Cat# H-027-1; 1:20,000; 17 h), with biotinylated goat anti-rabbit (Vector Laboratories, Burlingame, CA; Cat# BA-1000; 1:500; 1 h), with ABC-elite (Vector; Cat# PK-6100; 1:1,000 in PBS; 1 h), with tyramine sample amplification (Perkin Elmer, Waltham, MA; Cat# SAT700B001EA; 1:250 in PBS with 1 uL of 3% H₂0₂/mL PBS; 10 min), and with Dylight 488-conjugated streptavidin (Invitrogen, Carlsbad, CA; Cat# 21832; 1:200; 30 min). Subsequently, sections were co-incubated with a polyclonal rabbit antibody specifically recognizing galanin (Peninsula Laboratories, San Carlos, CA; RRID: AB_518348, Cat# T4334; 1:3000) and a mouse monoclonal antibody against NeuN (Millipore Burlington, MA; RRID: AB_2298772, Cat# MAB377; 1:1,000; 17 h), with Dylight 550 donkey anti-rabbit (Invitrogen; Cat# SA5-10039; 1:100; 30 min) and subsequently with Dylight 650 goat anti-mouse (Invitrogen; Cat# SA5-10174; 1:100; 30 min).

ChAT/synaptophysin/galanin or GRP

One series of sections for each animal was incubated with a polyclonal rabbit antibody specifically against galanin (Peninsula; RRID: AB_518348, Cat# T4334; 1:3000; 17 h) or GRP (Phoenix; RRID: AB_2722600, Cat# H-027-13; 1:20,000; 17 h) and with Cy3 donkey anti rabbit (Jackson ImmunoResearch Laboratories Inc., West Grove, PA; Cat# 711165152; 1:200; 30 min). Tissue was subsequently incubated with a monoclonal mouse anti-ChAT (Millipore; RRID: AB_94647, Cat# MAB305; 1:800; 17 h), biotinylated donkey anti-mouse (Jackson; Cat# 715-065-151; 1:500; 1 h), ABC-elite (Vector; Cat# PK-6100; 1:1000 in PBS; 1 h), tyramine sample amplification (1:250; Perkin Elmer; Cat# SAT700B001EA; in PBS with 1 uL 3% H₂0₂/mL PBS), and Dylight 488-conjugated streptavidin (Invitrogen; Cat# 21832; 1:200; 30 min). Tissue was treated with a monoclonal mouse anti-synaptophysin (Sigma; RRID: AB_477523, Cat# S5768; 1:200; 17 h) and Cy5 donkey anti-mouse (Jackson; Cat# 715-175-151; 1:200; 30 min).

Antibody controls

Validation of the antibody against GRP was accomplished with pre-absorption with GRP peptide (Phoenix; RRID: AB_2722600, Cat# H-027-13; 10 μg/mL) which prevented all immunostaining. The galanin antibody has been validated previously.^{19,21–23,25,40,41} ChAT antibody (mouse anti-ChAT; Millipore; RRID: AB_94647, Cat# MAB305) was validated by conducting dual immunofluorescent staining with one of two other primary antibodies specifically recognizing ChAT: goat anti ChAT (Millipore; RRID: AB_11214092, Cat# AB144P; 1:100) or sheep anti ChAT (Invitrogen; Cat# OSC0042G, RRID: AB_10710007; 1:200). Complete co-labeling of ChAT-positive cell bodies and fibers in the lumbar spinal cord was observed with all three antibodies.

Analysis of galanin and GRP cell counts

The numbers of cells immunoreactive (-ir) for galanin, GRP, or both were counted using fluorescent microscope (Leica DM5000B) by two independent observers blinded to experimental treatments. For each animal, all sections were examined and numbers of immunoreactive (-ir) cells were counted per tissue section. NeuN staining was visualized when needed to confirm the location of galanin and GRP immunoreactivity within the cytoplasm. Total numbers of cells were calculated for each animal. There were no differences between groups in the numbers of analyzed sections containing galanin, GRP, or dual-labeled cells (Sham: 9.9 ± 1; SCI: 8.6 ± 0.8). However, in order to account for potential group differences due to individual differences in numbers of sections, the numbers of single and dual cells also were expressed as the number of cells per section (as in our previous papers).^18,21 Statistical comparisons between sham and SCI groups were conducted using Student's t-test with 95% confidence levels.

Analysis of galanin and GRP axon terminals

Tissue sections processed for triple immunofluorescence for ChAT, synaptophysin and galanin or GRP, as well as sections immunolabeled for galanin, GRP and NeuN were imaged using a Nikon D-Eclipse C1 laser-scanning confocal system (Nikon Corp, Minota, Tokyo, Japan) attached to a Nikon Eclipse E800 microscope (Nikon Corp) and EZ-C1 Gold version 3.80 software (Nikon Corp). Fluorophores were detected by three lasers at wavelengths of 488, 543, and 633 nm. Wavelengths were filtered by two dichroic mirrors filtering at 530 and 625 nm. Confocal z-stacks of optical images (1 μm optical sections taken at 60 × magnification at 1-2X zoom) were obtained through ChAT-ir cell soma in the sacral parasympathetic nucleus (SPN), intermediolateral (IML) nucleus, the central autonomic nucleus (CAN), and NeuN-labeled cells receiving galanin or GRP innervation in the central gray (CG) commissure of L6. Images were opened in ImageJ (NIH; 1.51W) and 10 or more cells (ChAT- or NeuN-ir) per area (IML, CAN, SPN, or CG) were analyzed for numbers of axon terminals in close apposition with the soma.

For each cell, optical sections in which the nucleus of the cell was visible were analyzed. For each optical section, the numbers of galanin or GRP positive terminals co-labeling synaptophysin or labeled for synaptophysin alone in close apposition to the soma were counted. A bouton was considered an axon terminal if immunoreactive for synaptophysin and counted as in close apposition if immunoreactive pixels were immediately adjacent to the soma membrane of the analyzed neuron. The perimeter of ChAT or NeuN cells was calculated for each optical section using imageJ and the numbers of galanin, GRP, or synaptophysin-only appositions were expressed per 100 μm of analyzed membrane. Averages per cell were calculated over the optical sections and averages per animal were calculated over the 10 cells. Using the same methods, galanin and GRP single-or dual-labeled axon terminals in close apposition to NeuN cells in central gray were calculated in sections stained for galanin, GRP, and NeuN. For this analysis, synaptophysin was not included as an axon terminal marker, and instead NeuN was used to confirm if galanin and GRP were localized to axon terminals. Comparisons between sham and SCI groups were conducted using Student's t-test with a 95% confidence interval.

Analysis of galanin and GRP axon terminals based on cell sizes

During the analysis described above, it was observed that galanin and GRP (LSt) contacts appeared to preferentially innervate large preganglionic neurons in the IML. Pearson product-movement correlation was conducted to test for statistically significant correlations between cell perimeter and the density of LSt contacts based on all ChAT-ir cells in sham and SCI groups and separately for IML, CAN, and SPN. Next, LSt innervation of ChAT-ir cells was again analyzed based on smaller and larger cells using soma perimeter smaller or greater than IML 60 μm, CAN 50 μm, and SPN 45μm). These sizes were chosen based on the overall distribution of cell sizes within these areas. Comparisons between sham and SCI groups within each area and cell size category were conducted using Student's t-test with a 95% confidence interval.

Experiment 2: Effects of chronic spinal cord injury on testosterone levels and androgen receptor expression

LSt cells express androgen receptors^42,43 and expression of galanin and GRP is regulated by testosterone in adult male rats.^44,45 Therefore, the goal of this experiment was to determine if changes in galanin and GRP immunoreactivity levels were associated with decreased testosterone levels and responsiveness following contusion injury.

Experimental groups and contusion injury

Testes and blood were collected from sham and SCI animals (Sham = 13, SCI = 22 [testes weights] or 18 [blood collection failed for four SCI animals]) and spinal cord tissue was obtained from a subset of these animals (Sham = 5, SCI = 10). Contusion or sham surgeries were performed as described above but without acute transection prior to perfusion. Animals used for this study received an actual force delivered of 200 to 236 Kdyn (mean: 207 ± 1.63 Kdyn). Spinal cord displacement values ranged from 864 to 1559 μm (mean: 1054 ± 35.49 μm). Velocity of the impact rod ranged from 117 to 122 mm/sec (mean: 120 ± 0.29 mm/sec). Blood collection was not performed on three of the SCI animals but means for the contusion injury values did not differ with this omission (mean: 207 Kdyn: 1047 μm; 120 mm/sec). Similar were the values for the subset of SCI rats for spinal cord collections (mean: 205 Kdyn; 1052 μm; 120 mm/sec).

Perfusion and tissue collection

Locomotor activity analysis was performed as described above (Table 1). Five weeks after contusion or sham surgery, during the early part of the dark phase (1–4 h after dark onset), animals received an overdose of sodium pentobarbital (concentration), and testes were extracted and immediately weighed. Blood was drawn from the inferior vena cava using a heparinized syringe (10 mL), kept on ice in heparin-coated eppendorf tubes (Fisher, Cat# 05-407-13C), centrifuged for 10 min at 4°C; serum was pipetted away from the red blood cells and frozen at −20°C. A subset of the rats was perfused and spinal cords were sectioned as described for experiment 1.

Testosterone radioimmunoassay

Blood was analyzed for testosterone levels using a Testo-RIA-CT kit (DiaSource ImmunoAssay; S.A. Louvain-la-Neuve, Belgium; Cat# KIR1709, Lot: 172004). All samples were processed in technical duplicates (conducted by the Analytical and Assay Core, University of Mississippi Medical Center). Four standards supplied in the Testo-RIA-CT kit were processed in technical duplicates. Three negative controls and two positive controls consisting of two standards were included throughout the run-in duplicate and values were as predicted by the kit (control 1: 60 ± 20 ng/dL, control 2: 279 ± 72 ng/dL). CV% was <10% in all samples.

Immunohistochemistry androgen receptor and galanin

All procedures were performed as in experiment 1, except that chromogens were used for visualization and slides were cover-slipped with Di-N-Butyl Phthalate in Xylene (DPX; Electron Microcopy Sciences, Hatfield, PA; Cat# 13512). One series of sections of all animals was incubated with rabbit polyclonal antibody specific for androgen receptor (Santa Cruz, Dallas, TX; RRID: AB_1563391, Cat# sc-816; 1:750;17 h), biotinylated goat anti-rabbit (Vector; Cat# BA-1000; 1:500; 1 h) ABC-elite (Vector; Cat# PK-6100; 1:1000 in PBS; 1 h). Sections were next incubated were incubated in 0.1 M PB containing 0.02% diaminobenzidine (DAB; Sigma Cat# D5905-100TAB), 2% nickel sulfate (Sigma; Cat# N4882-250G) and 0.012% H₂O₂ (10 min). Subsequently, sections were incubated with rabbit anti-galanin (Peninsula; RRID: AB_518348, Cat# T4334; 1:60,000; 17 h), biotinylated goat anti-rabbit (Vector; Cat# BA-1000; 1:500; 1 h), ABC-elite (Vector; Cat# PK-6100; 1:1,000; 1 h), and finally in PB containing 0.02% DAB with 0.012% H₂O₂ (10 min).

Analyses

Testes weights

Right and left testicles were weighed and averaged for each animal.

Testosterone RIA

Testosterone levels in the technical duplicates were averaged for each animal.

Androgen receptor

Sections containing L3 and 4 spinal levels were examined using brightfield microscope (Leica, Wetzlar, Germany; DM5000B). Numbers of neurons immunoreactive for galanin were counted and evaluated for co-localization with androgen receptor immunoreactivity. The percentages of galanin neurons with androgen receptor immunoreactivity were calculated for each animal. All comparisons between sham and SCI groups were determined using Student's t-test with 95% confidence levels.

Photography

Images for figures were captured using identical camera settings for all groups and were selected to be representative of the data. Images were only occasionally corrected for brightness to allow optimal visualization using Adobe Creative Cloud Photoshop 2019 (Adobe, San Jose, CA).

Results

Effect of SCI on galanin and GRP immunofluorescence in LSt cells

In sham animals, the distribution of galanin and GRP-ir cells and fibers was identical to that reported previously,^18,21,23,25 suggesting that sham surgery and treatments did not affect immunofluorescence for galanin or GRP. Galanin and GRP were co-expressed in a vast majority of cells in L3 and 4 spinal levels and located lateral to the central canal in laminae X and VII (Fig. 1), and axons co-localizing galanin and GRP were observed in the intermediolateral (IML) nucleus, central autonomic nucleus (CAN), sacral parasympathetic nucleus (SPN), and dorsal central gray (CG) commissure at L5-6 (Fig. 2), consistent with previous reports.²³

FIG. 1.

Galanin and gastrin-releasing peptide (GRP) immunoreactivity in sham and spinal cord injury (SCI) animals in L3 and L4 spinal levels. Representative fluorescent microscope images demonstrating immunofluorescent labeling for galanin (red; A, D, G, J, M), GRP (green; B, E, H, K, N), and merged channels (C, F, I, L, O) in a sham (A-C and G-I) and chronic spinal contusion injury (SCI; D-F, J-L, M-O) animal showing lumbar spinothalamic cells in the L3 (A-I) and L4 (G-O) levels of the spinal cord. Images in J-O demonstrate the reduced GRP-immunofluorescent signal, with M-O showing cells at a higher magnification. cc, central canal. Scale bar indicates 50 (A-L) or 25 (M-O) μm. Color image is available online.

FIG. 2.

Lumbar spinothalamic (LSt) axon terminal distribution in spinal autonomic regions. Representative fluorescent microscope images showing immunofluorescent labeling of LSt axons for galanin (red; A, D, G, J), gastrin-releasing peptide (GRP; green; B, E, H, K), and merged channels (with NeuN immunoreactivity in blue; C, F, I, L) innervating the intermediolateral cell column (IML; A-C), central autonomic nucleus (CAN; D-F), sacral parasympathetic nucleus (SPN; G-I), and central gray area (Central Gray; J-K) in a representative sham animal. Scale bar indicates 50 μm. Color image is available online.

SCI had no effect on numbers of galanin-ir cells, but significantly reduced numbers of GRP-ir cells (p = 0.03; Fig. 3). Since galanin and GRP are co-expressed in LSt cells, SCI therefore resulted in a significant reduction of numbers of dual-labeled cells (p = 0.03; Fig. 3) and increased numbers of cells that were galanin-ir without co-labeling of GRP-ir (p < 0.001; Fig. 3). No neurons single labeled for GRP were noted in either sham or SCI groups. These effects of SCI were observed when data were expressed as cells per animal (Fig. 3A) or as cells per analyzed section per animal (Fig. 3B). Qualitatively, this reduction appeared to be particularly robust in L4 compared with L3 (Fig. 1). In sham males, 84.9% ± 3.9 of galanin cells co-labeled GRP, while this percentage was significantly reduced by SCI to 39.7% ± 4.6 (p < 0.001). GRP was co-labeled with galanin in 99.6% ± 0.4 in sham and 99.0% ± 1 in SCI animals. Thus, SCI selectively reduced GRP expression within galanin-positive LSt cells.

FIG. 3.

Spinal cord injury (SCI) reduces gastrin-releasing peptide (GRP) immunoreactivity in lumbar spinothalamic neurons. Quantitative analysis showing numbers of cells immunoreactive for galanin and GRP (Dual), or only for galanin (Galanin Only) or GRP (GRP Only) in the sham (Sham) or contusion spinal injury (SCI) groups. Data are shown as total numbers of cells that were counted (A) or as cells per analyzed section (B). Data are expressed as mean ± standard error of the mean. * indicates significant difference from Sham group.

Effect of SCI on LSt innervation of ChAT neurons in IML, CAN, and SPN

Robust innervation of galanin-ir and GRP-ir axons was observed in IML, CAN, and SPN with basket-like structures surrounding ChAT-ir pre-ganglionic neurons (Fig. 4). Previous studies demonstrated that these axon terminals originate from LSt cells.^18,46 However, SCI did not have a significant effect on numbers of galanin-ir or GRP-ir axon terminals in close apposition to ChAT-ir cells in IML (Fig. 5A, 5B), CAN, or SPN (Fig. 6). Group differences also were not detected in numbers of synaptophysin-only containing axon terminals in close contact to ChAT-ir neurons or in total numbers of contacts (Fig. 5A, 5B and Fig. 6). However, in the IML, it was observed that only a subset of ChAT-ir neurons received galanin-ir or GRP-ir axon inputs. Therefore, further correlation analysis based on all cells per group (rather than based on averages per animal) was conducted to reveal that there was a significant positive correlation between the size of the ChAT-ir cell in IML and numbers of galanin-ir axon inputs (Table 2), even though the numbers of inputs were expressed per 100 μm of soma membrane to eliminate effects of differences in soma size (Table 2, Fig. 5, and Fig. 6). Hence, larger ChAT-ir neurons in IML received denser LSt axon innervation (Fig. 5C, 5D and Fig. 4A).

FIG. 4.

Lumbar spinothalamic (LSt) axon terminals innervating autonomic preganglionic neurons. Representative confocal images (1-μm optical sections) in a sham animal showing galanin-immunoreactive (red) and synaptophysin-immunoreactive (blue) axon terminals in close proximity to ChAT-immunoreactive neurons (green) in intermediolateral cell column (IML; A), central autonomic nucleus (CAN; B), and sacral parasympathetic nucleus (SPN; C). An enlarged view at higher magnification (D) demonstrates axon terminals in close apposition to ChAT-ir soma membrane that are either immunoreactive for galanin and synaptophysin (magenta; red arrows), or synaptophysin only (blue; white arrows). Scare bars indicates 50 μm (A-C) or 5 μm (D). Schematic drawings (based on Paxinos Atlas80) of L1 (E), L4 (F), and L6 (G) demonstrate the anatomical locations of LSt cells and the areas of analysis of LSt axon terminal inputs to cells in IML, CAN, SPN, and central gray (CG). Color image is available online.

FIG. 5.

Spinal cord injury (SCI) did not affect lumbar spinothalamic axon terminal innervation of intermediolateral cell column (IML). Quantitative analysis of galanin- (A-G) or gastrin-releasing peptide (GRP)-immunoreactive (B-H) axon terminals in close apposition (Contacts) to ChAT-immunoreactive cells in IML in Sham (white bars and circles) and SCI (black bars and circles) groups. (A and B) Numbers of contacts are expressed per 100 μm of ChAT-ir soma membrane, for axon terminals co-labeled for synaptophysin (Syn) and galanin (A) or GRP (B), single labeled for Syn, and the sum of these synaptophysin and galanin (A) or GRP (B) and synaptophysin-only contacts (Total). C and D demonstrate a positive correlation of numbers of galanin- (C) or GRP-immunoreactive (D) contacts and the size of ChAT-ir cells using the Pearson Product-Movement Correlation (see Table 2 for statistical data). Line represents the slopes in Sham (black: Galanin - y = 0.7336x - 27.216, R² = 0.6215; GRP - y = 0.4116x - 16.013, R² = 0.787) and SCI (gray: Galanin - y = 0.8943x - 33.732, R² = 0.6949; GRP - y = 0.4533x - 17.593, R² = 0.6376) groups. E-H). Numbers of contacts immunoreactive for galanin (E) or GRP (F) on ChAT-ir cells with soma perimeters equal to or larger (E,F), or smaller than 60 μm (G,H). Data are expressed as ± standard error of the mean. * indicates significant difference from Sham group.

FIG. 6.

Spinal cord injury (SCI) did not affect lumbar spinothalamic axon terminal innervation of central autonomic nucleus (CAN) or sacral parasympathetic nucleus (SPN). Quantitative analysis of galanin- (A-C) or gastrin-releasing peptide (GRP)-immunoreactive (B-D) axon terminals in close apposition (Contacts) to ChAT-immunoreactive cells in Sham (white bars) and SCI (black bars) groups. Numbers of contacts are expressed per 100 μm of ChAT-ir soma membrane, for axon terminals co-labeled for synaptophysin and galanin (A, C) or GRP (B, D), single labeled for synaptophysin (Syn), and the sum of these dual labeled synaptophysin and galanin (A, C) or GRP (B, D) plus synaptophysin-only contacts (Total). Data are expressed as mean ± SEM.

Table 2.

Correlation Analyses of ChAT-ir Soma Size and Axon Terminal Inputs

Galanin			Correlation coefficient	p value	Number of cells
	IML	Sham	0.788	<0.001	75
		SCI	0.834	<0.001	86
	CAN	Sham	0.403	0.003	54
		SCI	0.414	<0.001	65
	SPN	Sham	0.459	<0.001	102
		SCI	0.339	<0.001	103

GRP			Correlation coefficient	p value	Number of cells
	IML	Sham	0.887	<0.001	62
		SCI	0.801	<0.001	64
	CAN	Sham	0.361	0.02	41
		SCI	0.48	<0.001	51
	SPN	Sham	0.216	0.128	51
		SCI	0.271	0.014	81

Pearson analyses to determine correlations between the mean perimeter size of ChAT-ir cell soma and the numbers of galanin-ir or GRP-ir axon terminals in close contact to these ChAT-ir cells in IML, CAN, and SPN. Putative axon terminal inputs were expressed as numbers per 100 μm of soma perimeter of ChAT-ir cells and based on all cells (Number of cells) per treatment group (Sham or SCI).

-ir, immunoreactive; IML, intermediolateral; SCI, spinal cord injury; CAN, central autonomic nucleus; SPN, sacral parasympathetic nucleus; GRP, gastrin-releasing peptide.

The possibility that SCI preferentially affected LSt inputs only to larger or smaller cells (soma perimeter above or below 60 μm) was therefore tested. Results showed that SCI had no effect on numbers of galanin-ir or GRP-ir axon inputs to either smaller or larger ChAT-ir neurons (Fig. 5E-H). In addition, SCI did not affect ChAT-ir neurons (Table 3 and Table 4): SCI did not alter the soma perimeter or numbers of large and small ChAT-ir cells, or the percentages of ChAT-ir cells that had a soma perimeter larger or smaller than 60 μm. Similar analyses were also performed in SPN and CAN, and similar positive correlations were noted with larger cells receiving greater numbers of galanin-ir and GRP-ir axon terminal inputs (Table 3 and Table 4). Further analyses of numbers of galanin-ir and GRP-ir axon terminals into smaller and larger cells in SPN and CAN revealed no effects of SCI (Table 3 and Table 4) and SCI did not affect ChAT-ir neurons in these areas (Table 3 and Table 4).

Table 3.

Galanin-ir LSt Innervation of Autonomic Nuclei

		Contacts per 100 μm
Galanin		Galanin	Syn	Total	ChAT-ir Soma Perimeter (μm)	# of ChAT-ir Cells	% of ChAT-ir Cells
IML >60 μm	Sham	33.3 ± 2.5	12.1 ± 1.2	45.4 ± 3.1	80.6 ± 3.7	7.2 ± 1.5	46 ± 5.6
	SCI	32.6 ± 5.6	17.2 ± 1.5	49.9 ± 4.7	74.1 ± 3.7	7.8 ± 1.9	45 ± 8.6
IML <60 μm	Sham	6.8 ± 1.2	31 ± 3.4	37.8 ± 2.9	48.6 ± 1.8	7.8 ± 0.6	54 ± 5.6
	SCI	5.4 ± 1.9	32.7 ± 2.5	38.1 ± 2.6	44.8 ± 1.6	9.4 ± 2.3	55 ± 8.6
CAN >50 μm	Sham	15.2 ± 1.5	14.5 ± 1.8	29.7 ± 2.5	63.1 ± 1.2	6.2 ± 1.2	60.6 ± 14.6
	SCI	14.5 ± 2.4	12.9 ± 1.3	27.4 ± 1.6	57.9 ± 1.4	5.6 ± 1.5	45.7 ± 11.4
CAN <50 μm	Sham	12.1 ± 1.9	19 ± 1.9	31.1 ± 2.9	42.1 ± 1.8	4.6 ± 1.8	39.4 ± 14.6
	SCI	9.3 ± 0.7	16.1 ± 1.8	25.4 ± 1.6	43.8 ± 1.3	7.4 ± 2.2	54.3 ± 11.4
SPN >45 μm	Sham	18.6 ± 0.6	12.4 ± 0.8	31 ± 1.3	52.2 ± 0.1	14.4 ± 2.1	69.5 ± 7.3
	SCI	17.8 ± 0.5	10.8 ± 1.2	28.7 ± 1	52.5 ± 0.5	11.8 ± 2	55.5 ± 4.7
SPN <45 μm	Sham	12.6 ± 1.6	15.9 ± 1.2	28.5 ± 1.3	40.8 ± 0.9	6 ± 1.1	30.5 ± 7.3
	SCI	13.6 ± 2.3	13.1 ± 1.7	26.6 ± 2.2	39.5 ± 0.4	8.8 ± 0.5	44.5 ± 4.7

Analysis of galanin-ir axon terminals in close apposition to ChAT-ir neurons in IML, CAN, and SPN. Numbers of galanin-ir axon terminals in close apposition to ChAT -ir neurons (Contacts) are expressed per 100 μm ChAT-ir cell soma perimeter and shown separately for larger and smaller ChAT -ir cells. All galanin-ir axon terminals co-labeled synaptophysin-ir. In addition, numbers of other synaptophysin-ir axon terminals are listed (Syn). Parameters of ChAT-ir neurons are also included: mean soma perimeter of large and small ChAT-ir cells, numbers of cells included in analyses (# of ChAT-ir Cells), and percentages of ChAT-ir cells that were smaller or larger than the mean threshold perimeter listed in the table. There were no statistical differences between groups. All data are expressed as mean ± standard error of the mean.

-ir, immunoreactive; LSt, lumbar spinothalamic; IML, intermediolateral; SCI, spinal cord injury; CAN, central autonomic nucleus; SPN, sacral parasympathetic nucleus.

Table 4.

GRP-ir LSt Innervation of Autonomic Nuclei

		Contacts per 100 μm
GRP		GRP	Syn	Total	ChAT-ir soma perimeter (μm)	# of ChAT-ir Cells	% of ChAT-ir Cells
IML >60 μm	Sham	19.2 ± 1.7	9.3 ± 1.3	28.5 ± 2.3	81.6 ± 2.3	3.6 ± 0.5	29.9 ± 4.3
	SCI	19.3 ± 2.3	10.3 ± 1.3	29.6 ± 2.7	77.3 ± 3.4	2.8 ± 0.7	20.7 ± 4.2
IML <60 μm	Sham	1 ± 0.2	17.9 ± 0.8	18.9 ± 0.7	42 ± 1.2	8.8 ± 1.5	70.1 ± 4.3
	SCI	1.9 ± 0.4	18.5 ± 1	20.4 ± 0.8	44.6 ± 0.9	10.2 ± 0.6	79.3 ± 4.2
CAN >50 μm	Sham	8.1 ± 3	12.8 ± 1.4	20.9 ± 1.9	62.8 ± 3	4.6 ± 1.8	49.6 ± 11
	SCI	4.5 ± 1.5	15.9 ± 0.8	20.5 ± 1.2	58.7 ± 1.4	4.8 ± 1.6	49.8 ± 9.2
CAN <50 μm	Sham	2.6 ± 1.4	15.4 ± 0.6	18 ± 0.9	40.5 ± 0.7	3.6 ± 0.5	50.4 ± 11
	SCI	2.7 ± 0.4	16.8 ± 1.1	19.4 ± 0.9	43.8 ± 0.9	4.4 ± 1.1	50.2 ± 9.2
SPN >45 μm	Sham	3.3 ± 1	20.3 ± 1.6	23.7 ± 1.1	48.4 ± 0.5	14.4 ± 2.1	69.5 ± 7.3
	SCI	10.3 ± 3.3	13.8 ± 2	24.2 ± 2.3	48.1 ± 1	11.8 ± 2	55.5 ± 4.7
SPN <45 μm	Sham	6.7 ± 1.4	18.6 ± 1.4	25.4 ± 2.5	40.2 ± 0.4	6 ± 1.1	30.5 ± 7.3
	SCI	9.6 ± 1.5	14.5 ± 1	24.1 ± 1.2	38.7 ± 0.6	8.8 ± 0.5	44.5 ± 4.7

Analysis of GRP-ir axon terminals in close apposition to ChAT-ir neurons in IML, CAN, and SPN. Numbers of GRP-ir axon terminals in close apposition to ChAT -ir neurons (Contacts) are expressed per 100 μm ChAT-ir cell soma perimeter and shown separately for larger and smaller ChAT -ir cells. All GRP-ir axon terminals co-labeled synaptophysin-ir. In addition, numbers of other synaptophysin-ir axon terminals are listed (Syn). Parameters of ChAT-ir neurons are also included: mean soma perimeter of large and small ChAT-ir cells, numbers of cells included in analyses (# of ChAT-ir Cells), and percentages of ChAT-ir cells that were smaller or larger than the mean threshold perimeter listed in the table. There were no statistical differences between groups. All data are expressed as mean ± standard error of the mean.

-ir, immunoreactive; GRP, gastrin-releasing peptide; LSt, lumbar spinothalamic; Syn, IML, intermediolateral; SCI, spinal cord injury; CAN, central autonomic nucleus; SPN, sacral parasympathetic nucleus.

Effect of SCI on LSt innervation of neurons in central gray

A dense plexus of galanin-ir and GRP-ir axons was observed in the dorsal central gray (CG) at L5-6-S1 levels. Scattered ChAT-ir neurons also were observed in this general area, but these did not receive putative contacts from galanin-ir and/ GRP-ir axon terminals. Therefore, analysis was conducted of NeuN-ir cells within the CG receiving such putative contacts (Fig. 7A). Analysis revealed a slight, but significant reduction of axon terminals containing only galanin-ir and not GRP-ir onto CG neurons, but SCI did not affect dual-labeled or GRP-ir only axon terminals expressing only GRP-ir in close contact to NeuN cells (Fig. 8A).

FIG. 7.

Spinal cord injury (SCI) did not affect lumbar spinothalamic axon terminal innervation of CG neurons. Representative confocal image (1-μm optical sections) in a sham animal showing galanin- (A; red) and gastrin-releasing peptide (GRP)-immunoreactive (B; green) axon terminals in close proximity to a NeuN-immunoreactive neuron (blue) in Central Gray with merged channels shown in (C). White arrows indicate galanin and GRP dual labeled-axon terminals in close apposition to the soma; green arrow indicates a putative contact labeled for GRP only. Scale bar indicates 5 μm. (D) Quantitative analysis of axon terminals labeled for galanin-only (Galanin), GRP-only (GRP) or both galanin and GRP (Dual) in close apposition to NeuN-labeled soma of CG cells (Contacts) expressed per 100 μm of soma membrane. Data are expressed as mean ± standard error of the mean. * indicates significant difference from Sham group. Color image is available online.

FIG. 8.

Spinal cord injury (SCI) did not affect measures of testosterone release or action. Testes weights (A; in grams) and testosterone plasma concentrations (in ng/dL; B) in Sham (white bars) and SCI (black bars) groups. Data are expressed as mean ± standard error of the mean. Representative images from a sham (C, E, G) and SCI (D, F, H) animal showing chromogen labeling for androgen receptors (black nuclei) and galanin (brown) in lumbar spinothalamic cells (C, D), sacral nucleus of the bulbocavernosus (SNB; E-F), sacral parasympathetic nucleus (SPN), and central gray (CG; G-H). Arrows indicate general locations of SNB, SPN, and CG cells immunoreactive for androgen receptors. cc, central canal. Scale bars indicate 50 μm. Color image is available online.

Effects of SCI on testosterone levels and androgen receptor expression

There were no apparent effects of SCI on indicators of testosterone release or action. There were no differences between sham and SCI groups in testes weights or testosterone plasma concentrations (Fig. 8A, 8B). In addition, 100% of galanin-ir LSt cells co-localized AR-ir in both sham and SCI groups (Fig. 8C, 8D). Finally, robust AR-ir was noted in IML, CAN, CG, SPN and sacral nucleus of the bulbocavernosus (SNB) while qualitative assessment revealed no apparent differences between sham and SCI animals (Fig. 8E-H).

Discussion

The current findings support the hypothesis that chronic contusion injury significantly impacts the SEG, specifically leading to reduced levels of immunoreactivity for GRP within LSt cells, indicative of reduced neuropeptide levels for GRP. Since GRP is a potent facilitator of ejaculatory reflexes, such loss may contribute to ejaculatory dysfunction after SCI in male rats. In contrast, SCI did not significantly impact galanin-ir in LSt cells or the presence of LSt cells per se. Moreover, LSt axon terminals localizing galanin and GRP were apparently unaffected by SCI. In summary, following SCI, LSt cells and their connections that comprise the SEG were intact, but levels of GRP within the LSt soma were reduced. These findings lead to the hypothesis that GRP agonists may be a valuable option for intervention or treatment of ejaculatory dysfunction following SCI.

Chronic contusion injury has previously been demonstrated to result in ejaculatory dysfunction in male rats, namely the inability to trigger ejaculatory reflexes by stimulation of the dorsal penile nerve.²⁸ In these studies, dorsal penile nerve-stimulated ejaculatory reflex was analyzed at 4 or 6 weeks post injury under urethane anesthesia, using parameters of expulsion and emission phases of ejaculation.²⁸ In SCI males, dorsal penile nerve stimulation was unable to trigger ejaculation, using stimulation frequencies that reliably trigger reflexes in control animals nor using super-threshold frequencies.²⁸ Moreover, these studies were conducted in rats that received an acute spinal transection prior to testing in order to isolate the SEG from any remaining supraspinal influences. Hence, ejaculatory dysfunction was not caused by alterations in supraspinal influences on the SEG at the time of testing. Therefore, it was hypothesized that ejaculatory dysfunction following chronic SCI was caused by deficits within the SEG.

Moreover, chronic contusion injury impacted both parameters of expulsion and emission, which are mediated by separate populations of motor, sympathetic, and parasympathetic neurons that are located in different spinal levels in the lumbar and sacral spinal cord.²⁰ Therefore, anejaculation is unlikely to be caused by changes in the individual motor and premotor neurons, but rather driven by changes in LSt cells, as these form the pivotal part of the SEG that regulates the coordination of motor and autonomic responses via intraspinal projections.^{18,19,25,40,41,46,47} Such SCI-induced changes may include loss of LSt cells, their neuropeptides, or their connections with autonomic and motor cells.

The first main finding of the current set of studies that SCI reduced immunoreactivity for GRP within LSt cells supports this hypothesis. Immunoreactivity for galanin was unaffected, demonstrating that SCI did not result in a disruption or cell death of the LSt cells per se. Hence, the loss of immunoreactivity for GRP is indicative of a reduction of peptide levels for GRP within LSt cells. The mechanisms by which SCI at mid-thoracic levels caused a reduction of GRP in LSt cells at L3 and 4 spinal levels are unknown and will be subject for future investigations. However, it is less likely that these alterations were caused by acute inflammatory processes as those occurring at the site of injury,^48–51 but rather are due to long term changes in supraspinal influences to the SEG, and epigenetic modifications of GRP expression in LSt cells.^52–56

The finding that one of the neuropeptides expressed in LSt cells (i.e., GRP) is affected without an apparent effect on another (i.e., galanin) is surprising. One possible explanation includes epigenetic modification specifically of the GRP gene without affecting the galanin gene. Alternatively, we hypothesize that galanin peptide levels also may have been affected by SCI, but that the current study failed to detect such changes due to potentially high levels of galanin neuropeptide in LSt cells and the limited sensitivity of immunofluorescence as a quantitative assay for peptide detection. Likewise, GRP peptide levels may normally be lower compared with galanin and thus may more readily fall below threshold of immunofluorescent detection following SCI. Therefore, future studies will test these hypotheses using quantitative analyses of mRNA expression levels for galanin and GRP following SCI.

Findings from the current study also showed that SCI did not cause structural alterations or reductions of LSt axon connections as numbers of axon terminals defined by galanin and GRP-ir were largely unaltered. This is in agreement with a previous study in female rats showing lack of effects of a complete spinal transection at T7-9 on overall immunoreactivity for galanin fibers in L4 and L6 levels, while a decrease was noted in dorsal horn in L1.⁵⁷ However, it is possible that functional changes in LSt axons that affect neuropeptide release were not detected using the methods of analysis in this study, which was strictly based on presence of immunoreactivity for galanin and GRP. Hence, changes in storage, transport, and release of galanin and GRP in and from large dense core vesicles⁵⁸ were not examined. Nonetheless, the findings that the SEG appears intact morphologically is positive and promising for future development of intervention options, such as use of GRP agonists.

Galanin has been demonstrated in LSt cells in rat, mouse, and human and is a reliable marker of LSt cells.^7,18,25,26 Moreover, galanin and GRP are completely co-localized within LSt cells and their axons,²³ which was again demonstrated in the current study. However, galanin and GRP appear to have different functional roles for the control of ejaculatory reflexes. Pharmacological studies have shown GRP to be a potent facilitator of ejaculatory reflexes and intrathecal infusions of GRP can trigger ejaculation in the absence of sensory stimulation.²³ Thus, the reduction in GRP peptide level may greatly contribute to ejaculatory dysfunction following SCI. In contrast, facilitative effects of galanin infusions are dependent on concurrent stimulation of the dorsal penile nerve.²⁷ We have hypothesized that actions of galanin thus require co-release and actions of other LSt neuropeptides, such as GRP. One mechanism by which the LSt neuropeptides may co-activate downstream post-synaptic neurons are via heteroreceptor complexes.⁵⁹ Such heterodimers have been described for galanin with other receptors, including serotonin⁶⁰ and mu opioid receptors.⁶¹ Moreover, it is currently unclear exactly where galanin and GRP receptors are expressed within the SEG, except limited evidence for potential expression of GRP receptor in SPN cells,⁶² and effects of SCI on receptor expression or function have not been explored.

Findings from the current study demonstrated that chronic contusion injury did not affect numbers, size or overall somatic synaptic connectivity of ChAT-ir autonomic preganglionic neurons in IML, CAN, or SPN. Changes in morphology and synaptic input to sympathetic preganglionic neurons have been reported after complete midthoracic transection or hemisection and in thoracic levels below the lesion, showing reduced soma size and shrunken dendrites in the acute phase after injury^63–67 or increased soma size both rostral and caudal to the injury.⁶⁸ However, transection-induced changes in soma size of thoracic sympathetic preganglionic cells were transient in rats^66,68 and humans⁶⁵ and returned to baseline levels longer-term after injury. Thus, the findings of the current study showing a lack of change in soma size of sympathetic preganglionic cells at 5 weeks post contusion injury is consistent with those earlier findings.

Complete spinal transection also has been shown to reduce synaptic innervation of sympathetic preganglionic cells,^63,64,69 consistent with a complete loss of supraspinal inputs, while hemisections of the corticospinal tract caused increased synaptic inputs from the rostral ventral medulla to sympathetic preganglionic cells,⁷⁰ suggesting sprouting and regeneration.⁷¹ In the current study, no changes in overall synaptic inputs to sympathetic preganglionic cells were detected following chronic contusion injury and may thus suggest that this injury either spared the majority of supraspinal influences on these preganglionic cells or that sprouting of remaining axons had occurred. Fewer studies have investigated effects of SCI on sacral preganglionic cells in the SPN.

Historically, SPN cells have been considered parasympathetic, act on motor cells in pelvic ganglia via the pelvic nerve and stimulate secretions during the emission phase.²⁰ However recent evidence has shown that SPN cells share genetic signatures and dependencies with the thoracolumbar preganglionic cells, thus consistent with the characterization of sympatheticfunction.⁷² A previous study in female cats demonstrated that complete spinal transection reduced synaptic innervation of SPN cells.⁷³ In the current study, SCI did not affect SPN cells or their synaptic inputs and the discrepancy may be due to type of injury, time after injury, animal species, and sex. An interesting additional observation of the current study was that LSt cells appear to preferentially innervate larger ChAT-ir cells. It is currently unclear if different or overlapping subpopulations of preganglionic autonomic cells innervate different urogenital organs.

There is some evidence that in the female cat SPN, a dorsal portion of preganglionic cells participates in bowel control, while a lateral population is involved in bladder control.⁷⁴ The latter population has larger soma and receives greater synaptic innervation.⁷⁴ Ejaculatory function involves coordinated control of multiple urogenital structures, including bladder, prostate, and seminal vesicles, and thus innervation by LSt cells to the majority of preganglionic neurons in these spinal regions may indicate the level of coordination of multiple urogenital organs during ejaculatory reflex.⁷⁵ In addition to LSt axon projections to ChAT-ir preganglionic neurons, LSt cells also have dense projections to the dorsal central gray (CG) at L5-S1 levels. Again, SCI did not affect LSt axon projections to cells in this general area. Moreover, the exact functional role of the LSt connections to this area is not understood. CG contains heterogenous populations of interneurons, including cells that in turn project to SNB motor neurons that innervate the bulbocavernosus muscle and may thus be involved in the expulsion phase of ejaculation.⁷⁶

Finally, results from the current showed that contusion injury did not significantly impact levels of testosterone and androgen receptor localization within the SEG after contusion injury. Hence, reduced GRP immunoreactivity was not caused by a loss of testosterone or androgen sensitivity. Levels of testosterone in the current study were relatively low in both groups, consistent with the typical low levels of testosterone during the early part of the dark phase and potential effects of surgery-related stressors.⁷⁷ In SCI men, androgen deficiency as defined by low testosterone levels is noted in approximately 35% of patients and only after chronic SCI.⁷⁸ The exact contributing factors to this androgen deficiency in men are not fully understood and may include high body mass index and low physical activity.⁷⁸

While LSt cells and their connections at the level of the spinal cord are essential for ejaculation^18,19 it is important to note that this spinal control center is in turn under descending inhibitory (and potentially excitatory) influence of supraspinal sites.^79–81 Much remains unknown about the precise nature and origins of these supraspinal influences to the SEG, and may include possible inputs from the caudal raphe nuclei, nucleus paragigantocellularis,^82–85 paraventricular nucleus of the hypothalamus,⁸⁶ and the periaqueductal gray.⁸⁷ Investigation of the exact origins of supraspinal inputs to the SEG and the neurotransmitter content of such inputs will be of great value and will be needed to form the basis for examination of effects of contusion injury on such inputs.

In conclusion, the current findings demonstrate that contusion injury at mid-thoracic levels caused a reduction of GRP within LSt cells, without an apparent effect on LSt cell axon projections to autonomic nuclei within the spinal cord. LSt cells function as spinal interneurons to mediate ejaculatory reflexes.^18,19 Recent evidence has highlighted the plastic responses of spinal interneurons to spinal injury and the potential for therapeutic interventions targeting spinal interneurons.⁸⁸ Hence, the current results add to that proposal. Given that GRP is a strong facilitator of ejaculatory reflex, injury-induced reduction of GRP may contribute to ejaculatory dysfunction following SCI and form a putative target for treatment options.

Author Disclosure Statement

No competing financial interests exist.