Targeted ablation of mesenteric projecting sympathetic neurons reduces the hemodynamic response to pain in conscious, spinal cord-transected rats

Abstract

Individuals with spinal cord injuries above thoracic level 6 (T(6)) experience episodic bouts of life-threatening hypertension as part of a condition termed autonomic dysreflexia. The paroxysmal hypertension can be caused by a painful stimulus below the level of the injury. Targeted ablation of mesenteric projecting sympathetic neurons may reduce the severity of autonomic dysreflexia by reducing sympathetic activity. Therefore, cholera toxin B subunit (CTB) conjugated to saporin (SAP; a ribosomal inactivating protein that binds to and inactivates ribosomes) was injected into the celiac ganglion to test the hypothesis that targeted ablation of mesenteric projecting sympathetic neurons reduces the pressor response to pain in conscious, spinal cord-transected rats. Nine Sprague-Dawley male rats underwent a spinal cord transection between thoracic vertebrae 4 and 5. Following recovery (5 wk), all rats were instrumented with a radio telemetry device for recording arterial pressure and bilateral catheters in the gluteus maximus muscles for the infusion of hypertonic saline (hNa(+)Cl(-)). Subsequently, the hemodynamic responses to intramuscular injection of hNa(+)Cl(-) (100 microl and 250 microl, in random order) were determined. Following the experiments in the no celiac ganglia injected condition (NGI), rats received injections of CTB-SAP (n = 5) or CTB (n = 3) into the celiac ganglia. CTB-SAP rats, compared with NGI and CTB rats, had reduced pressor responses to hNa(+)Cl(-). Furthermore, the number of stained neurons in the celiac ganglia and spinal cord (segments T(6)-T(12)), was reduced in CTB-SAP rats. Thus, CTB-SAP retrogradely transported from the celiac ganglia is effective at ablating mesenteric projecting sympathetic neurons and reducing the pressor response to pain in spinal cord-transected rats.

Fig. 1.

Analog recordings of arterial pressure before and during intramuscular injection of hypertonic saline in three spinal cord-transected rats with: 1) no celiac ganglia injection (NGI), 2) CTB injected into the celiac ganglia, and 3) CTB-SAP injected into the celiac ganglia. Dotted line indicates injection of hypertonic saline (250 μl). The pressor response to intramuscular injection of hypertonic saline was notably lower in the rat with celiac ganglion injection of CTB-SAP.

Fig. 2.

Change in mean arterial pressure in response to 100 μl and 250 μl of hNa⁺Cl⁻ injected into the gluteus maximus muscle in the NGI group, CTB celiac-injected group, and CTB-SAP celiac-injected group. The control data for the CTB and CTB-SAP rats are presented separately. Specifically, the 3 animals that received CTB were matched with their response before injection (NGI _CTB). The 5 animals that received CTB-SAP were matched with their response before injection (NGI _CTB-SAP). There was no difference in the arterial pressure response to 100 μl or 250 μl of hNa⁺Cl⁻ between the NGI and CTB groups. However, the arterial pressure response to both 100 μl and 250 μl of hNa⁺Cl⁻ were significantly lower in the CTB-SAP group documenting an attenuated autonomic dysreflexia. *P < 0.05 CTB-SAP vs. NGI and CTB.

Fig. 3.

Neuronal number (A), average soma area per neuron (B), and cresyl violet-stained celiac neurons (C) from NGI rats, as well as rats that had CTB or CTB-SAP injected into the celiac ganglia. Counts of celiac neurons in the CTB-SAP group showed a significant reduction in the number of neurons compared with the NGI and CTB groups. These results are consistent with C showing many celiac neurons in the NGI and CTB groups and few neurons in the CTB-SAP group. However, of the 2,711 neurons analyzed, the soma area (B) was not different between the three groups. Scale bar (bottom right) = 25 μm. *P < 0.05 CTB vs. CTB-SAP.

Fig. 4.

CTB-immunoreactive neurons of the T₆–T₇ spinal cord from rats that had CTB (A) or CTB-SAP (B) injected into the celiac ganglia. CTB-immunoreactive sympathetic preganglionic neurons (SPNs) were found on both sides of the column of the T₆ and T₇ spinal cord in CTB rats (A). However, virtually all of the CTB-immunoreactive SPNs were eliminated in the CTB-SAP group (B). Scale bar (bottom right) = 25 μm.

Fig. 5.

Cresyl violet-stained neurons of the dorsal root ganglia from rats that had CTB (A) or CTB-SAP (B) injected into the celiac ganglia. It is clear that CTB-SAP did not alter neurons within the dorsal root ganglia. Scale bar (bottom right) = 25 μm.