Expression genetics identifies spinal mechanisms supporting formalin late phase behaviors

Abstract

Background: Formalin injection into rodent hind paws is one of the most commonly employed pain assays. The resulting nocifensive behaviors can be divided into two phases differing in timing, duration and underlying mechanisms. Spinal sensitization has long been felt to participate in the second phase of this response, although this sensitization is incompletely understood. By using correlative analysis between spinal gene expression and mouse strain-dependent intensity of late phase behavior, we hypothesized genes participating in variability of the response could be identified.

Results: Late phase formalin behavior scores among 10 inbred mouse strains were correlated with a spinal cord gene expression database constructed using expression arrays. Messenger RNA levels for several genes were highly correlated with the late phase behavioral responses. Most of these genes had already been implicated in mechanisms regulating pain and analgesia. One of the most strongly correlated genes, Mapk8 coding for c-Jun N-terminal kinase 1 (JNK1), was chosen for further analysis. Studies using additional strains of mice confirmed that spinal cord mRNA expression levels of Mapk8 followed the pattern predicted by strain-specific levels of formalin behavior. Interestingly, spinal cord JNK1 protein levels displayed an inverse relationship with mRNA measurements. Finally, intrathecal injections of the selective JNK inhibitor, SP600125, selectively reduced late phase licking behavior.

Conclusions: Wide differences in pain behaviors, including those resulting from the injection of formalin, can be observed in inbred strains of mice suggesting strong genetic influences. Correlating levels of gene expression in tissues established to be mechanistically implicated in the expression of specific behaviors can identify genes involved in the behaviors of interest. Comparing formalin late phase behavior levels with spinal cord gene expression yielded several plausible gene candidates, including the Mapk8 gene. Additional molecular and pharmacologic evidence confirmed a functional role for this gene in supporting formalin late phase responses.

Figure 1

Formalin-induced nocifensive behaviors. Video recordings of mice licking/biting after plantar hind paw injection of formalin were reviewed for 17 strains of inbred mice (n = 10-25/strain). Bars represent the mean ± S.E.M. percentage of samples featuring licking/biting in early (0-5 min; graph A) and late phase (10-60 min; graph B).

Figure 2

Haplotypic structure of the Mapk8 gene. Using SNP data for the 13 inbred mouse strains, for which it was available, the strains partitioned into 3 haplotypes. There were 86 SNPs available for this analysis. In this figure blue represents the common allele, yellow represents the minor allele, and white represents missing alleles.

Figure 3

Analysis of spinal cord Mapk8 expression in supplemental strains. In these experiments both real time qPCR and Western analysis were used to quantify mRNA and protein levels respectively. Graph A contains the results of qPCR experiments in which the relative levels of Mapk8 mRNA for high late phase responding strains were compared with low responding ones. In graph B, similar experiments were performed using spinal cord protein homogenates. For the protein experiments the spinal cord levels of JNK1 were normalized to the spinal cord levels in the intermediate-responding BALB/c mice arbitrarily set at a level of 1.0. For all experiments samples from n = 4 mice per strain were analyzed in triplicate. Bars represent mean ± S.E.M. Three strains within each group were compared to other strain group, *p < 0.05.

Figure 4

The effects of the JNK inhibitor, SP 600125, on formalin-induced nocifensive behaviors. For these experiments groups of mice (n = 8) first underwent intrathecal injection of SP 600125 or vehicle 3 h prior to formalin injection. The total duration of licking/biting time during early (0-5 min) and late phase (10 k-60 min) of the formalin response were measured and averaged for either the entire phase (graph A) or in 5-min intervals (graph B). Bars and symbols represent mean ± S.E.M. *p < 0.05, ***p < 0.001 compared to the vehicle group.