Plasticity of TRPV1-Expressing Sensory Neurons Mediating Autonomic Dysreflexia Following Spinal Cord Injury

Abstract

Spinal cord injury (SCI) triggers profound changes in visceral and somatic targets of sensory neurons below the level of injury. Despite this, little is known about the influence of injury to the spinal cord on sensory ganglia. One of the defining characteristics of sensory neurons is the size of their cell body: for example, nociceptors are smaller in size than mechanoreceptors or proprioceptors. In these experiments, we first used a comprehensive immunohistochemical approach to characterize the size distribution of sensory neurons after high- and low-thoracic SCI. Male Wistar rats (300 g) received a spinal cord transection (T3 or T10) or sham-injury. At 30 days post-injury, dorsal root ganglia (DRGs) and spinal cords were harvested and analyzed immunohistochemically. In a wide survey of primary afferents, only those expressing the capsaicin receptor (TRPV1) exhibited somal hypertrophy after T3 SCI. Hypertrophy only occurred caudal to SCI and was pronounced in ganglia far distal to SCI (i.e., in L4-S1 DRGs). Injury-induced hypertrophy was accompanied by a small expansion of central territory in the lumbar spinal dorsal horn and by evidence of TRPV1 upregulation. Importantly, hypertrophy of TRPV1-positive neurons was modest after T10 SCI. Given the specific effects of T3 SCI on TRPV1-positive afferents, we hypothesized that these afferents contribute to autonomic dysreflexia (AD). Rats with T3 SCI received vehicle or capsaicin via intrathecal injection at 2 or 28 days post-SCI; at 30 days, AD was assessed by recording intra-arterial blood pressure during colo-rectal distension (CRD). In both groups of capsaicin-treated animals, the severity of AD was dramatically reduced. While AD is multi-factorial in origin, TRPV1-positive afferents are clearly involved in AD elicited by CRD. These findings implicate TRPV1-positive afferents in the initiation of AD and suggest that TRPV1 may be a therapeutic target for amelioration or prevention of AD after high SCI.

Keywords: capsaicin; colo-rectal distension; dorsal horn; dorsal root; dorsal root ganglion; high blood pressure; hypertension; hypertrophy.

Figure 1

High-thoracic (T3) spinal cord injury provoked hypertrophy of neurons in the L4/L5 dorsal root ganglion. (A) βIII-tubulin immunohistochemistry illustrating neuronal profiles in the L5 DRG. (B) Size-frequency distribution of pooled L4/L5 DRG neurons, reconstructed from profile distributions using recursive translation. Ganglia were harvested 3 months after sham-injury (gray) or complete T3 SCI (black). There was a small but significant rightward shift in size-frequency distribution in animals with T3 SCI (P < 0.05, K–S goodness-of-fit test). Scale bar = 70 μm.

Figure 2

High-thoracic (T3) spinal cord injury-induced selective hypertrophy of sensory neurons expressing the capsaicin receptor (TRPV1) and the artemin receptor (GFRα3) in the L4/L5 DRG. (A) Substance P (SP) – positive DRG neurons and their size-frequency distributions. (B) IB4-binding DRG neurons. (C) P2X3-positive DRG neurons. (D) TRPV1-positive DRG neurons. (E) GFRα3-expressing DRG neurons, known to express TRPV1. The overall proportions of immunopositive neurons [insets (A–E)] did not change for any subpopulation. Ganglia were harvested 3 months after sham-injury (gray) or complete T3 SCI (black). Scale bar = 50 μm. Asterisks indicate P < 0.05, K–S goodness-of-fit test.

Figure 3

High-thoracic (T3) spinal cord injury had no effect on medium-to-large sized neurons in the L4/L5 DRG expressing heavy neurofilament (NF200). (A) NF200-positive neurons did not undergo SCI-induced hypertrophy, nor did the proportion of neurons expressing NF200 change. (B) Hypertrophy of TRPV1-expressing DRG neurons was not accompanied by increased co-localization of TRPV1 and NF200. Ganglia were harvested 3 months after sham-injury (gray) or complete T3 SCI (black). Arrow: DRG neuron immunopositive for both TRPV1 and NF200. Scale bar = 70 μm.

Figure 4

Capsaicin-sensitive dorsal root ganglion neurons increased in diameter caudal to, but not rostral to, high-thoracic spinal cord injury. (A–D) Size-frequency distributions of TRPV1-positive neurons from a rostral DRG (T1) and caudal (T5, L4/L5, L6/S1) DRGs, harvested 1 month after sham- (gray) or complete T3 SCI (black). The increase in size is the most pronounced in the most caudal ganglia. Proportions of TRPV1-positive neurons at each level are shown in the insets (A–D). Asterisks indicate P < 0.05, K–S goodness-of-fit test.

Figure 5

Capsaicin-sensitive neurons in the dorsal root ganglion exhibited increased TRPV1 signal intensity following high-thoracic spinal cord injury. (A) Size-intensity scatter plots of TRPV1-positive DRG neurons showing a marked upward scatter with T3 SCI. The increase in TRPV1 intensity was particularly pronounced in L6/S1 DRGs. There was a significant increase in signal intensity at 3 months after T3 SCI for both L4/L5 DRGs and L6/S1 DRGs (K–S goodness-of-fit tests on cumulative intensity-frequency distributions). (B) Representative images of TRPV1-expression in the L6/S1 DRG, from a sham-injured control (top) and an animal with T3 SCI (bottom). Scale bar = 50 μm.

Figure 6

Low-thoracic (T10) spinal cord injury elicited only modest changes in size of the most caudal capsaicin-sensitive dorsal root ganglion neurons. (A) No difference in size distributions of TRPV1-expressing DRG neurons in L4/L5 DRGs. (B) There was a small but significant rightward shift in the size-frequency distribution of L6/S1 DRG neurons (P < 0.05, K–S goodness-of-fit test), but this was much less dramatic than that which occurred after T3 SCI (see Figure 4). Ganglia were harvested 1 month after sham (gray) or complete T3 SCI (black).

Figure 7

Hypertrophy of capsaicin-sensitive afferents caudal to high-thoracic spinal cord injury was not accompanied by pronounced plasticity of their spinal projections. (A) One month after T3 complete SCI, there was a small but significant increase in TRPV1-positive axon density in the medial (med.) and lateral (lat.) parts of the most superficial laminae of the L4/L5 dorsal horn (boxed regions are shown enlarged). There was no evidence of TRPV1-positive axon extension into deeper laminae. (B) In the L6/S1 cord, there were no differences between sham-injured or T3 SCI animals in the dorsal horn, or in the dorsal gray commissure (DGC, boxed image enlarged to the right), the terminal field of the visceral medial collateral pathway. Asterisks: P < 0.05, Student’s t-test. Scale bars = 200 μm.

Figure 8

Intrathecal capsaicin attenuated colo-rectal distention (CRD) -induced autonomic dysreflexia in animals that survived for 1 month after complete T3 spinal cord injury. (A) A single intrathecal bolus of capsaicin (10 μl of 5 mg/ml in 50% DMSO) resulted in permanent degeneration of spinally projecting TRPV1-positive axons. Quantification shows TRPV1 axon density in the dorsal gray commissure (DGC). Asterisk indicates significant difference between vehicle-treated T3 SCI animals and both capsaicin-treated groups (P < 0.05, one-way ANOVA). Scale bar = 200 μm. (B) Beat-to-beat changes in blood pressure in response to colo-rectal distension (horizontal bars) from animals treated with intrathecal vehicle (Veh.), early capsaicin (48 h following T3 SCI, early Cap.), and late capsaicin (48 h prior to physiological recording, 28 days post-T3 SCI, late Cap.). (C) Quantitative cardiovascular responses to CRD in vehicle and capsaicin-treated rats, 30 days post-SCI. Capsaicin treatment, whether administered 48 h or 28 days after SCI, mitigated CRD-induced increases in systemic arterial pressure (SAP) and decreases in heart rate (HR). Resting SAP and HR were unaffected by intrathecal capsaicin. Asterisks: P < 0.05, one-way ANOVA.