Fig 1.
Principle of fluorescent tracer injection and DiI signal after CCI.
(A) Fluoroemerald (FE, green) is applied proximal to the site of injury; it is taken up by damaged neurons and transported to the DRG. DiI (red) is injected into the hindpaw immediately after the surgical procedure. It permeates the axonal membrane and diffuses along the axon. Membrane disruption, however, impedes further diffusion towards the DRG. (B). One week after CCI, the sciatic nerve was excised and cryosected to examine native DiI intensity. Compared to the distal sciatic nerve (left panel), transections proximal to the site of injury (right panel) exhibit a clearly decreased DiI intensity. Nuclei were visualized with blue DAPI (n = 4, representative sample, scale bar = 40 μm).
Table 1.
Tracer combinations and their interpretation.
Fig 2.
Flow cytometric detection of damaged and intact neurons for qPCR.
DRGs L3-5 were harvested and cells isolated 7 days after CCI. The sorting strategy to identify neurons positive for Fluoroemerald (FE) and DiI is shown in (A). Initially, cells were gated for size and granularity, before excluding dead cells using Sytox Blue and haematopoetic cells using CD45-Ab Cy7. The remaining cells were sorted for DiI and FE. FACS plots of negative control (B left), contralateral (B middle) and ipsilateral (B right) DRG cells are displayed in the lower panel. DiI+/FE- cells are considered to be spared neurons, FE+ cells are damaged neurons. Both populations were obtained for further analysis (n = 3, representative example).
Fig 3.
Samples of damaged neurons (red) exhibit within-group similarity and considerable group difference to both adjacent (yellow) and contralateral (green) neurons. In contrast, these two groups cannot be discriminated from each other (hierarchical clustering with complete linkage for 200 probe sets with highest MAD. (Color key: row z-score -2 (dark blue) to +2 (white)).
Fig 4.
Correlation analysis between microarray and qPCR.
For genes tested by qPCR, differential expression in damaged vs. contralateral neurons was compared to microarray results. The scatter plot shows a fair correlation.
Fig 5.
(A-J) Expression levels in damaged and adjacent neurons relative to contralateral neurons are shown for ion channels and neuropeptides previously described in neuropathic pain (mean ± SEM, n = 2, paired t-test, *p < 0.05 compared to contralateral).
Fig 6.
CRH expression in DRG neurons after CCI.
(A) mRNA from DRG neurons 7 d after CCI was analyzed by qPCR, It shows a strong, significant upregulation in damaged neurons and, to a smaller degree, in adjacent neurons compared to contralateral neurons (mean ± SEM, n = 2, paired t-statistics *p < 0.05 compared to contralateral). (B) Immunohistochemistry for CRH in DRG neurons after CCI. Ipsilateral DRG were obtained 7 d after CCI or sham surgery and immunostained for CRH. Immunoreactivity for CRH was very low and nearly undetectable in sham controls (right panel). DRGs from neuropathic animals (left panel) show a robust immunoreactivity of CRH, detected in the cytoplasm of small, medium and large size cell bodies (scale bar: 60 αm, two representative samples, n = 3).
Fig 7.
(A-G) Expression levels in damaged and adjacent neurons relative to contralateral neurons are shown for genes not yet known in neuropathic pain (mean ± SEM, n = 2, paired t-test, *p < 0.05 compared to contralateral).
Table 2.
Genes upregulated in damaged DRG neurons compared to contralateral control.
Table 3.
Genes downregulated in damaged DRG neurons compared to contralateral control.
Table 4.
Genes upregulated in damaged DRG neurons compared to adjacent non-damaged neurons.
Table 5.
Genes downregulated in damaged DRG neurons compared to adjacent non-damaged neurons.