Nerve injury-induced tactile allodynia is present in the absence of FOS labeling in retrogradely labeled post-synaptic dorsal column neurons

Abstract

The dorsal column pathway consists of direct projections from primary afferents and of ascending fibers of the post-synaptic dorsal column (PSDC) cells. This pathway mediates touch but may also mediate allodynia after nerve injury. The role of PSDC neurons in nerve injury-induced mechanical allodynia is unknown. Repetitive gentle, tactile stimulus or noxious pinch was applied to the ipsilateral hindpaw of rats with spinal nerve ligation (SNL) or sham surgery that had previously received tetramethylrhodamine dextran in the ipsilateral n. gracilis. Both touch and noxious stimuli produced marked increases in FOS expression in other cells throughout all laminae of the ipsilateral dorsal horn after nerve injury. However, virtually none of the identified PSDC cells expressed FOS immunofluorescence in response to repetitive touch or pinch in either the nerve-injured or sham groups. In contrast, labeled PSDC cells expressed FOS in response to ureter ligation and labeled spinothalamic tract (STT) cells expressed FOS in response to noxious pinch. Identified PSDC neurons from either sham-operated or SNL rats did not express immunoreactivity to substance P, CGRP, NPY, PKCY, MOR, the NK1 and the NPY-Y1 receptor. Retrogradely labeled DRG cells of nerve injured rats were large diameter neurons, which expressed NPY, but no detectable CGRP or substance P. Spinal nerve injury sensitizes neurons in the spinal dorsal horn to repetitive light touch but PSDC neurons apparently do not participate in touch-evoked allodynia. Sensitization of these non-PSDC neurons may result in activation of projections integral to the spinal/supraspinal processing of enhanced pain states and of descending facilitation, thus priming the central nervous system to interpret tactile stimuli as being aversive.

Figure 1

Fluorescent images indicating labeling of the n. gracilis and the thalamus with tetramethylrhodamine dextran are shown. (A). The transverse section of the caudal medulla illustrates the site of injection of the retrograde tracer, identified by the red fluorescence within the n. gracilis. (B). Two pairs of injections of the rhodamine-dextran retrograde tracer were made 1 mm apart from each other into the right side of the thalamus. (C) Retrograde labeling from microinjection of the retrograde tracer into the n. gracilis is found in the labeled PSDC profiles in laminae III – IV of the dorsal horn ipsilateral to the injection of tracer in rats. (D). High power magnification of the retrogradely labeled PSDC profiles is shown.

Figure 2

Photomicrographs of the L₅ DRG obtained from sham-operated (left panels) and nerve-injured (right panels) rats are shown. The red immunofluorescence of the retrograde tracer is found in medium to large diameter cellular profiles of the DRG. Green immunofluorescence indicates labeling for substance P, CGRP and NPY in the DRG profiles. There is no evidence of colocalization of either substance P or of CGRP with tetramethylrhodamine dextran obtained from either the sham-operated or nerve-injured rats. Furthermore, the DRG obtained from sham-operated animals do not show labeling for NPY whereas it is present in the DRG of nerve-injured rats. Labeling for NPY is found in the medium to large diameter profiles of the DRG that also demonstrate the presence of the retrograde tracer.

Figure 3

Immunofluorescence for FOS expression in the n. gracilis and the L₅ spinal dorsal horn of sham-operated rats and of rats with SNL are shown in response to mechanical stimuli. (A). FOS expression was completely absent from the n. gracilis of sham-operated or nerve-injured rats after noxious pinch. This result is similar to observations made after light brush (not shown). (B). Both sham-operated and nerve-ligated rats demonstrate similar levels of evoked FOS expression throughout the dorsal horn of the spinal cord in response to noxious pinch. (C). FOS expression evoked by lightly stroking the hindpaw is increased throughout the dorsal horn of the nerve-injured rats relative to the sham-operated rats.

Figure 4

Double-labeling studies indicating retrograde tracer (red) and immunofluorescence for FOS (green) are shown for sham-operated and nerve-injured rats. FOS expression was evoked by light touch or by noxious pinch. There was very little to no labeling for FOS in any of the identified PSDC profiles in either the sham-operated or nerve-injured groups after either light or noxious stimuli.

Figure 5

Double-labeling studies indicating retrograde tracer (red) and immunofluorescence for FOS (green) are shown after ureter ligation (top panels) or noxious pinch (bottom panels). The upper panels also show PSDC profiles retrogradely labeled from the n. Gracilis and the lower panels show STT profiles retrogradely labeled from the thalamus. Low magnification images showing the dorsal horn are shown on the left and higher magnification images from the same section are shown on the right. Ligation of the ureter caused a prominent expression of FOS in the spinal dorsal horn, predominantly in the intermediate and deep laminae. Also visible are PSDC profiles that were retrogradely labeled by injection of tetramethylrhodamine dextran into the n. gracilis. The arrow indicates a retrogradely-labeled PSDC profiles that also expresses FOS in response to ureter ligation. Noxious mechanical pinch evoked FOS expression throughout the dorsal horn of the spinal cord. Retrograde labeling of STT neurons was accomplished by microinjection of tetramethylrhodamine dextran into the contralateral thalamus. The arrow indicates a retrogradely-labeled STT neuron that also expresses immunoreactivity for FOS.