Segmental organization of spinal reflexes mediating autonomic dysreflexia after spinal cord injury

Abstract

Spinal cord injuries above mid-thoracic levels can lead to a potentially life-threatening hypertensive condition termed autonomic dysreflexia that is often triggered by distension of pelvic viscera (bladder or bowel). This syndrome is characterized by episodic hypertension due to sudden, massive discharge of sympathetic preganglionic neurons in the thoracolumbar spinal cord. This hypertension is usually accompanied by bradycardia, particularly if the injury is caudal to the 2nd to 4th thoracic spinal segments. The development of autonomic dysreflexia is correlated with aberrant sprouting of peptidergic afferent fibers into the spinal cord below the injury. In particular, sprouting of nerve growth factor-responsive afferent fibers has been shown to have a major influence on dysreflexia, perhaps by amplifying the activation of disinhibited sympathetic neurons. Using a model of noxious bowel distension after complete thoracic spinal transection at the 4th thoracic segment in rats, we selectively altered C-fiber sprouting, at specified spinal levels caudal to the injury, with microinjections of adenovirus encoding the growth-promoting nerve growth factor or the growth-inhibitory semaphorin 3A. This was followed by assessment of physiological responses to colorectal distension and subsequent histology. Additionally, anterograde tract tracers were injected into the lumbosacral region to compare the extent of labeled propriospinal rostral projections in uninjured cords to those in cords after complete 4th thoracic transection. In summary, overexpression of chemorepulsive semaphorin 3A impeded C-fiber sprouting in lumbosacral segments and mitigated hypertensive autonomic dysreflexia, whereas the opposite results were obtained with nerve growth factor overexpression. Furthermore, compared to naïve rats, there were significantly more labeled lumbosacral propriospinal projections rostrally after thoracic injury. Collectively, our findings suggest that distension of pelvic viscera increases the excitation of expanded afferent terminals in the disinhibited lumbosacral spinal cord. This, in turn, triggers excitation and sprouting of local propriospinal neurons to relay visceral sensory stimuli and amplify the activation of sympathetic preganglionic neurons in the thoracolumbar cord, to enhance transmission in the spinal viscero-sympathetic reflex pathway. These responses are manifested as autonomic dysreflexia.

Figure 1

Physiological responses to colorectal distension (CRD) two weeks after T4 spinal transection. Illustrative traces of pulsatile arterial pressure (PAP), mean arterial pressure (MAP) and heart rate (HR) before, during and after one minute CRD with rectal balloon catheter inflation (indicated by arrows) from injured rats with bilateral L6/S1 injections of (A) adenovirus expressing control green fluorescent protein (GFP Adts) or (B) nerve growth factor (NGF Adts). Note that both injured groups show autonomic dysreflexia with hypertension accompanied by bradycardia, but the severity of hypertension is almost two-fold greater and more prolonged with nerve growth factor over-expression.

Figure 2

Over-expression of nerve growth factor (NGF) in lumbosacral spinal levels augments calcitonin gene-related peptide (CGRP)⁺ afferent fiber sprouting 2 weeks post injury. Photomicrographs showing CGRP⁺ staining in S1 spinal segments injected with adenovirus expressing control green fluorescent protein (GFP Adts - left column) or NGF Adts (right column). Note the robust CGRP⁺ afferent fiber sprouting throughout the S1 injection site in response to NGF over-expression compared to GFP controls. Scale bars = 100 µm.

Figure 3

Injections of biotinylated dextran amine (BDA) tracer into L6/S1 spinal levels label more ascending projections after T4 transection. Longitudinal, horizontal sections at thoracolumbar spinal levels of (A) non-transected or (B) T4-transected rats approximately 5 mm rostral to 200 nl injections of BDA into the left L6/S1 dorsal commissure. After 2 weeks to allow tracer transport and verify physiologically that the injured rats had developed autonomic dysreflexia, histological processing revealed significantly more BDA-labeled projections (data not shown) within ipsilateral gray matter extending to rostral levels of the thoracic spinal cord. Scale bar = 0.5 mm

Figure 4

High magnification, dual immunofluorescent images at the mid-thoracic spinal leel two weeks after T4 transection demonstrate close proximity of (A) FluoroGold-labeled sympathetic preganglionic neurons (arrows) in the intermediolateral cell column and (B) biotinylated dextran amine (BDA)-labeled fibers originating from lumbosacral projection interneurons. Scale bar = 50 µm.

Figure 5

Schematic illustration depicting the etiology of autonomic dysreflexia evoked by pelvic visceral distension and other stimuli. Following complete spinal cord injury above the T6 level, sympathetic preganglionic neurons (blue) are released from descending medullo-spinal control (dashed arrow) and autonomic spinal reflexes are rendered hyperactive. Consequently, pelvic visceral sensory input (yellow) is relayed by propriospinal neurons (green) projecting from the dorsal gray commissure at the lumbosacral level to sympathetic preganglionic neurons and/or sympathetically-correlated interneurons located in the thoracolumbar cord (blue). Post-traumatic C-fiber sprouting into the lumbosacral cord (yellow) further amplifies the central signals (green) to elicit hypertension, ultimately causing profound peripheral vasoconstriction of splanchnic, muscle and cutaneous vascular beds. Subsequent stimulation of aortic depressor nerve (ADN) and carotid sinus nerve (CSN) baroreceptor afferents of the petrosal ganglion (red) is conveyed to the nucleus tractus solitarius that elicits bradycardia via activation of the nucleus ambiguus. Note that flushing is likely the result of inhibition of skin vasoconstrictor preganglionic neurons above the lesion by the baroreceptor reflex, and not due to the activation of sympathetic preganglionic neurons below the lesion by the dysreflexia-inducing stimulus.