Sprouting of afferent and efferent inputs to pelvic organs after spinal cord injury

Abstract

Neural plasticity occurs within the central and peripheral nervous systems after spinal cord injury (SCI). Although central alterations have extensively been studied, it is largely unknown whether afferent and efferent fibers in pelvic viscera undergo similar morphological changes. Using a rat spinal cord transection model, we conducted immunohistochemistry to investigate afferent and efferent innervations to the kidney, colon, and bladder. Approximately 3-4 weeks after injury, immunostaining demonstrated that tyrosine hydroxylase (TH)-labeled postganglionic sympathetic fibers and calcitonin gene-related peptide (CGRP)-immunoreactive sensory terminals sprout in the renal pelvis and colon. Morphologically, sprouted afferent or efferent projections showed a disorganized structure. In the bladder, however, denser CGRP-positive primary sensory fibers emerged in rats with SCI, whereas TH-positive sympathetic efferent fibers did not change. Numerous CGRP-positive afferents were observed in the muscle layer and the lamina propria of the bladder following SCI. TH-positive efferent inputs displayed hypertrophy with large diameters, but their innervation patterns were sustained. Collectively, afferent or efferent inputs sprout widely in the pelvic organs after SCI, which may be one of the morphological bases underlying functional adaptation or maladaptation.

Keywords: Calcitonin gene-related peptide; Distribution; Peripheral system; Sprout; Tyrosine hydroxylase.

Figure 1.

Sensory afferents sprout in the renal pelvis rather than blood vessels in the kidney after SCI. (A–H) Representative immunofluorescence photomicrographs show that primary calcitonin gene-related peptidergic (CGRP⁺) sensory fibers are mainly found in the renal artery and pelvis. The density of CGRP⁺ fibers does not significantly change in the renal artery (*p > 0.05, unpaired t-test) but is greater (*p < 0.05) in the renal pelvis following SCI. (I–O) Sympathetic efferent projections sprout in the renal pelvis after SCI. Postganglionic sympathetic fibers are identified by double immunostaining for tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH). In naïve rats, some double labeled fibers are found in the muscle layer. Three weeks after T10-transection (T10-Tx), dense labeled fibers emerge. Statistical analysis shows a higher density of TH-labeling (*p < 0.05) in the renal pelvis compared to naïve rats (n = 6 per group). Scale bars: A, I, L = 100 µm; F, K, N = 30 µm.

Figure 2.

Postganglionic sympathetic efferents do not sprout in the renal vasculature following SCI. Peripheral sympathetic projections, identified by immunostaining with tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) antibodies, are present around the renal vascular beds, such as the arterioles (A, B, G), vasa recta (C, D, H), and arteries (E, F, I). After T10-transection (T10-Tx), the density of TH⁺ fibers does not change in the vasculature (all *p > 0.05, Unpaired t-test, n = 6 per group). Scale bar: 100 µm.

Figure 3.

Primary sensory projections are adjacent to sympathetic nerves in the kidney. (A, D) Immunofluorescent staining shows the innervation pattern of tyrosine hydroxylase positive (TH⁺) efferents and calcitonin gene-related peptide positive (CGRP⁺) axons in the kidney. TH⁺ terminals intertwine with CGRP⁺ afferents in the renal pelvis (B, E) and artery (C, F) in both naïve and SCI rats. Scale bars: A, D = 200 µm; C, F = 50 µm.

Figure 4.

Colonic sensory afferents sprout after SCI. (A, B) Immunostaining for calcitonin gene-related peptide (CGRP) in the colon sections harvested from naïve and SCI rats. In both cases, CGRP⁺ sensory terminals distribute in the lamina propria (C, D), muscularis mucosae (E, F), and muscle layer (G, H). (I–K) Statistical analysis shows denser CGRP labeling (all *p < 0.05, unpaired t-test) in the structures 3 weeks after SCI versus the naïve (n = 6 per group). Scale bars: B = 100 µm; H = 30 µm.

Figure 5.

Immunohistochemical changes in postganglionic sympathetic efferents in the colon following SCI. (A–J) Colonic sympathetic fibers are double immunostained by tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH). (K, L) Quantitative analysis demonstrates that denser TH⁺ labeling is reveled in the muscularis mucosae and muscle layer of the colon (both *p < 0.05, unpaired t-test) in SCI rats than naïve controls (n = 6 per group). Scale bars: F = 100 µm; J = 30 µm.

Figure 6.

Primary sensory afferents and postganglionic sympathetic fibers are present closely in the colon. Immunofluorescent photomicrographs illustrate postganglionic sympathetic projections identified by tyrosine hydroxylase (TH) and sensory afferents stained with calcitonin gene-related peptidergic (CGRP) in the colon. In naïve rats, TH⁺ fibers are mainly adjacent to CGRP⁺ terminals in the muscularis mucosae and muscle layer. Though they show disorganized distribution patterns in T10-transected (T10-Tx) rats, these 2 fibers twine together. Scale bars: A, D = 100 µm; C, F = 30 µm.

Figure 7.

Structural changes in the bladder following SCI. (A–C) Bladder morphological hypertrophy displays as thick smooth muscle layer (ML, black arrows) in SCI rats. Statistical analysis indicates that the organ weight and the thickness of the bladder wall are greater (*p < 0.05, **p < 0.01, unpaired t-test) in SCI rats than the naïve control (n = 6 per group). (D–F) Immunolabeling shows that CGRP⁺ afferents sprout in the bladder, identified with higher (**p < 0.01) density of labeling in the lamina propria (LP) and muscle layer (ML) after T10-Tx. (G–I) Postganglionic sympathetic fibers are identified by double immunostaining for tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH). After T10-transection, very few TH⁺ fibers emerge in most regions of the muscle layer (ML) after SCI. Quantitative analysis indicates lower TH⁺ fiber density in rats with SCI rats versus naïve controls (**p < 0.01). Scale bars: B = 100 μm; H = 50 μm.