Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain

Abstract

Phenotypic modification of dorsal root ganglion (DRG) neurons represents an important mechanism underlying neuropathic pain. However, the nerve injury-induced molecular changes are not fully identified. To determine the molecular alterations in a broader way, we have carried out cDNA array on the genes mainly made from the cDNA libraries of lumbar DRGs of normal rats and of rats 14 days after peripheral axotomy. Of the 7,523 examined genes and expressed sequence tags (ESTs), the expression of 122 genes and 51 expressed sequence tags is strongly changed. These genes encompass a large number of members of distinct families, including neuropeptides, receptors, ion channels, signal transduction molecules, synaptic vesicle proteins, and others. Of particular interest is the up-regulation of gamma-aminobutyric acid(A) receptor alpha5 subunit, peripheral benzodiazepine receptor, nicotinic acetylcholine receptor alpha7 subunit, P2Y1 purinoceptor, Na(+) channel beta2 subunit, and L-type Ca(2+) channel alpha2delta-1 subunit. Our findings therefore reveal dynamic and complex changes in molecular diversity among DRG neurons after axotomy. Sequences reported in this paper have been deposited in the GenBank database (accession numbers BG 662484-BG 673712)

Figure 1

A scatter plot of cDNA array identifies outlying genes whose expression differs between control DRGs and the DRGs 14 days after peripheral axotomy. The line indicates that the ratio of nVol of axotomized DRG:control DRG is 1:1.

Figure 2

The strongly regulated genes with the changes in cDNA array ratio over 2 folds are identified in the DRGs 2, 7, 14, and 28 days (d) after peripheral axotomy. The ranges of the cDNA array ratio of nVol of axotomized DRG vs. normal DRG are presented with colors indicated in the last panel.

Figure 3

The strongly regulated ESTs with the changes over 2 folds are identified in the DRGs after axotomy. The ranges of the cDNA array ratio of nVol of axotomized DRG vs. normal DRG are presented with colors indicated in the last panel.

Figure 4

Some strongly regulated genes and ESTs are confirmed with RT-PCR or Northern blot. (A) The results of the semiquantitative RT-PCR. Size of the internal control was 500 bp, and the size of the target genes is 250 bp. Ratio indicates the signal intensity of examined gene vs. that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at different time points. (B) The results of Northern blot, with Bottom showing the internal control. Ratio indicates the signal intensity of examined gene in DRGs 14 days after axotomy vs. that in control DRGs.

Figure 5

(A–F") In situ hybridization of receptors and ion channels in L5 DRG 14 days after unilateral axotomy. Arrows point to large NPs and arrowheads point to small NPs. P2Y1-R is normally expressed in some NPs (A) and up-regulated in many large and small NPs after axotomy (A′, A"). PBDZ-R is hardly seen contralaterally (B) but expressed ipsilaterally in many small NPs (B′, B"). GABA_A-R α5 is normally expressed in some NPs (C) and up-regulated in many large NPs and some small NPs (C′, C"). Nicotinic acetylcholine-R α7 subunit (nAch-R α7) is expressed in some large NPs normally (D) and up-regulated in both large and small NPs (D′, D"). L-Ca²⁺ Ch α2δ-1 is normally seen in some small NPs (E) and increased in many small NPs after axotomy (E′, E"). Weak labeling of Na⁺ Ch β2 is normally seen in some NPs (F) and up-regulated ipsilaterally in many small NPs (F′, F"). [Bars = 50 μm (in A–F and A′–F′) and 20 μm (in A"–F")]. (G) The time course shows a constant increase in the percentage of NPs expressing these genes (P < 0.05 for P2Y1-R, PBDZ-R, nAch-R α7, and Na⁺ Ch β2 after 2 days, and for GABA_A-R α5 after 7 days, compared with control), except L-Ca²⁺ Ch α2δ-1 showing a transient increase after 2 days (P < 0.05, compared with control) and decrease to the control level at 28 days (P > 0.05, compared with control).