Loss of neurons from laminas I-III of the spinal dorsal horn is not required for development of tactile allodynia in the spared nerve injury model of neuropathic pain

Abstract

It has been proposed that death of inhibitory interneurons in the dorsal horn contributes to the neuropathic pain that follows partial nerve injury. In this study, we have used two approaches to test whether there is neuronal death in the dorsal horn in the spared nerve injury (SNI) model. We performed a stereological analysis of the packing density of neurons in laminas I-III 4 weeks after operation and found no reduction on the ipsilateral side compared with that seen on the contralateral side or in sham-operated or naive rats. In addition, we used two markers of apoptosis, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining and immunocytochemical detection of cleaved (activated) caspase-3. Neither of these methods demonstrated apoptotic neurons in the dorsal spinal cord 1 week after operation. Although TUNEL-positive cells were present throughout the gray and white matter at this stage, they were virtually all labeled with antibody against ionized calcium-binding adapter molecule 1, a marker for microglia. All animals that underwent SNI showed clear signs of tactile allodynia affecting the ipsilateral hindpaw. These results suggest that a significant loss of neurons from the dorsal horn is not necessary for the development of tactile allodynia in the SNI model.

Figure 1.

Graph showing 50% withdrawal thresholds to von Frey hairs in SNI (n = 12 ≤ 7 d; n = 6 ≥ 14 d) and sham-operated (sham; n = 6) rats. Results for ipsilateral (ipsi) and contralateral (contra) hindpaws are shown for each group, and each point represents the mean ± SEM. ^**Significant difference (p < 0.01) between the ipsilateral paw in SNI animals and contralateral paw in these animals and both paws in the sham-operated group (Kruskal-Wallis one-way ANOVA with Mann-Whitney post hoc test).

Figure 2.

Graph showing the mean values for the estimated numbers of neurons per 10 μm length of spinal cord for laminas I, II, and III in animals that had undergone SNI (n = 6) or sham operation (n = 6) 28 d previously. The individual values from each animal are also indicated (circles). The numbers of NeuN-immunoreactive cells in each lamina on the side ipsilateral (ipsi) to the SNI operation did not differ significantly from those on the contralateral (contra) side or from the sham-operated rats (one-way ANOVA).

Figure 3.

Cleaved caspase-3 immunoreactivity in the spinal cord of rats that had undergone SNI 7 d previously. a and b show staining for cleaved caspase-3 (green) in the medial part of the ipsilateral (a) and contralateral (b) dorsal horn. Numerous immunostained profiles are visible. c, d, Merging these images with NeuN staining (red) shows that the nuclei with strong or moderate immunoreactivity for cleaved caspase-3 are not NeuN positive. The rare instances of yellow in the merged images result from overlap of structures that are in different z-sections. e, f, Part of the ipsilateral dorsal horn from another rat that had undergone SNI. This section has been reacted sequentially with antibodies against cleaved caspase-3 (green) and GFAP (red). The nuclei with strong caspase immunoreactivity are surrounded by GFAP staining and can therefore be identified as astrocytes. Scale bars: (in a) a-d, 100 μm; (in e) e, f, 50 μm. Images are projected from 21 (a-d) or 15 (e, f) z-sections at 1 μm separation.

Figure 4.

Cleaved caspase-3 and TUNEL staining in the olfactory bulb of a naive 14-d-old rat. a, b, A single optical section shows two nuclei (arrows) that are TUNEL stained and immunoreactive for cleaved caspase-3. c, Both nuclei are stained with the NeuN antibody. d, Staining with DAPI shows that both cells have abnormal nuclear morphology, with peripherally clumped chromatin. Scale bar, 20 μm.

Figure 5.

TUNEL staining in the dorsal spinal cord 7 d after SNI and lack of coexistence with NeuN immunoreactivity. The drawing shows the positions of 86 TUNEL-positive nuclei that were present on the side ipsilateral to the SNI in five sections each from six rats, plotted onto an outline of the dorsal half of the spinal cord. Note that although the majority of TUNEL-positive nuclei are in the gray matter, there are also many throughout the white matter. The confocal images in the bottom part of the figure show a single optical section through a TUNEL-labeled nucleus in lamina I that is not NeuN immunoreactive. Other NeuN-positive and NeuN-negative nuclei are also visible. Scale bar for confocal images, 10 μm.

Figure 6.

The appearance of TUNEL staining, NeuN immunoreactivity, and DAPI in four consecutive optical sections (1-4) through the ipsilateral dorsal horn of a rat that underwent SNI 7 d previously. For each optical section, the top panel shows a merged image (NeuN, blue; TUNEL, green; DAPI, red), the middle panel shows TUNEL staining (green), and the bottom panel shows DAPI staining (red). A small TUNEL-positive nucleus with highly condensed chromatin is visible in the first three sections (arrowheads) and is closely apposed to a NeuN-immunoreactive cell with normal nuclear morphology (arrows), which can be seen in sections 2-4. Although the sequence of images scanned for DAPI clearly shows that these are two separate cells, the TUNEL labeling overlaps with NeuN immunoreactivity in the third section (shown with and without TUNEL labeling in insets) and could easily be mistaken for a TUNEL-positive NeuN-immunoreactive cell. z-spacing between sections, 1 μm; scale bar, 10 μm.

Figure 7.

TUNEL staining in a microglial cell in the superficial dorsal horn from a rat that had undergone SNI 7 d previously. The four panels show staining for TUNEL (green), DAPI (red), and Iba-1 (blue), together with a merged image. The nucleus of an Iba-1-immunoreactive microglial cell shows TUNEL staining (arrows). Several other microglia with TUNEL-negative nuclei are also visible, and some of these are marked with asterisks. Projected from three optical sections at 1 μm z-spacing. Scale bar, 10 μm.