In this study, we first conducted gene expression microarray experiments to examine transcriptional profiles in the ipsilateral dorsal root ganglia (DRG) of adult rats in a 4 day CFA model of inflammatory pain and generated a rat CFA model gene signature. Our second aim was to identify compounds that were negatively connected to this CFA gene signature using the Connectivity Map approach. Our third objective was to test the hypothesis that compounds identified in this way could reduce CFA-induced pain in this 4 day model. Reports on transcriptional profiling in inflammatory pain models are scarce. Yang et. al. examined gene expression changes in the inflamed tissues and the corresponding DRGs in a carrageenan model using a mini-array containing 100 cytokines, chemokines and related receptors . Parkitna et. al. conducted transcriptional profiling of 4000 genes in the lumbar section of rat spinal cord of a 3-day and a 14-day CFA model . The authors found a dramatic shift in the regulation of secretory vesicle trafficking in the spinal cord in the CFA model. The authors did not profile the L4-L5 DRG. However, they selected 4 transcripts that were changed in the spinal cord and tested them in the L4-5 DRG with qPCR. Consistent with our results, none of the 4 transcripts were significantly changed in the CFA DRG comparing to the naïve rats. To the best of our knowledge, there are no published DRG whole genome microarray studies following 4 days CFA inflammation in rats. We used the CFA model since commonly used NSAIDs such as naproxen that are used to treat OA pain are effective in this model and it is thus frequently used pre-clinically to identify new therapies for OA pain [11, 12].
By clustering differentially expressed genes by function or related pathways, we identified a number of genes that are implicated in the modulation of the immune system. Surprisingly, we found that the expression of these genes is decreased in the CFA-treated rats, suggesting suppression of the immune system at day 4 post-CFA treatment in this model system. These include genes in the eicosanoid signaling pathway, including phospholipase A2 group IVA (PLA2g4a), prostaglandin-endoperoxide synthase 1 (Ptgs1), and rat arachidonate 12/15-lipoxygenase. In addition, several genes such as BCL6, BTK and CNR2 that may modulate release of eicosanoids [20–22] are also reduced in the ipsilateral DRGs four days after CFA treatment. We speculate that these genes are early players that initiate inflammation and pain, and their signals are carried forth by other pathways that maintain chronic pain/inflammation with time.
Another group of genes down-regulated by CFA treatment encode proteins that regulate T cell function. These include IL21 receptor subtype II, IL2 receptor beta, RHOH (Ras homolog gene family member H), cannabinoid receptor 2, IKZF1, CASP8, and CD52 [23–31]. It is also noteworthy that IL22RA2, a soluble receptor for IL22 that antagonizes IL22 which is primarily produced by activated T cells , CD7 which promotes T cell apoptosis , Lgals2 (galectin-2), a proapoptotic effector of activated T-cells , and IL2 receptor gamma  are all increased in the CFA group compared to the naïve group. This may represent a mechanism of communication by the DRG that modulates T cell activity during inflammation or pain processing. In this regard, T cells have been recently suggested to play a role in neuropathic pain [36, 37]. In a model where DRG inflammation was induced by administration of epidural zymosan in incomplete Freund's Adjuvant , an increase of CCL2 (MCP-1) among other cytokines was observed, while IL-2 and IL-12 were decreased on day 3 post inflammation. Similarly, in a peripheral inflammation model induced by hind paw injection of carrageenan, both the Scya2 (the CCL2 mRNA) and the gene product MCP-1 (CCL2) were up-regulated following nociceptive stimuli . In that study, Scya2 mRNA was increased about 2-fold in the ipsilateral versus contralateral rat DRG and lasted up to 72 hour post carrageenan. Scya2 mRNA was localized to a subpopulation of vanilloid receptor 1 (TRPV1) containing neurons. Stimulation by a TRPV1 agonist, resiniferatoxin increased expression of Scya2 mRNA. These results are very similar to our findings where we found increased expression of the CCL2 gene in the DRG following CFA injection. CCL2 has also been reported to be up-regulated in the DRG in models of neuropathic pain ( and  for review). Thus, CCL2 seems to be a common signal elicited in different preclinical pain models.
Other genes that are induced in the DRG of CFA treated rats, include some that encode proteins either known to play a role in pain, promote neuronal growth/differentiation, or prevent neuronal death. One that stands out is neuromedin U (NMU) which has been shown to have an emerging physiological role in nociception upon binding to the NMU receptor 2 . Mice deficient in NMUR2 displayed reduced thermal nociceptive responses in the hot plate test, decreased thermal hyperalgesia following capsaicin injection and reduced the late phase response in the formalin test . In other studies, NMU inhibited inflammation-mediated memory impairment and neuronal cell-death in rodents . Ketterer et. al. (2009) have shown recently that NMU may signal via the hepatocyte growth factor (HGF) c-Met pathway. In our study, HGF is also increased in the DRG following CFA treatment. HGF is a pleiotropic cytokine which partly functions to promote neuronal survival and growth . HGF cooperates with nerve growth factor (NGF) to enhance axonal outgrowth from cultured dorsal root ganglion (DRG) neurons. HGF also enhances the neurotrophic activities of NGF in vivo where Met receptor signaling is required for the survival of a proportion of DRG neurons .
Another gene which is up-regulated in the DRG is the steroid-5-alpha-reductase (SRD5A2) which is a key enzyme in the conversion of several Δ4-3keto steroids, such as testosterone, progesterone, aldosterone and corticosterone, into their respective reductase derivatives. Morphine has been shown to increase SRD5A2 level and SRD5A2 inhibitor finasteride potentiates the antinociceptive effect of morphine, prevents the development of morphine tolerance in rats, suggesting that SRD5A2 plays a role in pain perception . SLC30A3 which is also increased in the current study is responsible for transport of zinc into synaptic vesicles. It may have a role in neuropathic pain .
We also observed a number of genes whose expression in DRG is modulated by CFA, though their roles in nociception have not been established, they may have indirect linkages to pain plasticity. Two genes involved in regulating circadian rhythm are modulated in the DRG of CFA treated rats versus the naïve rats. Pain perception is influenced by the circadian rhythm in humans and in animals . Expression of PER2 is increased, whereas expression of NPAS2 is decreased. Per/Cry form a heterodimer that interacts with the NPAS2/BMAL1 heterodimer to inhibit the transcription of Per and Cry . SFRS1 interacting protein 1 (PSIP1, or Lens epithelium-derived growth factor (LEDGF)) is up-regulated in response to stress and enhances the survival of neurons in the retina and optic nerve . We speculate that the increased expression of PSIP1 we observed may support neuronal growth in the DRG. ZHX2, a transcriptional regulator of neural progenitor cells, is also up-regulated in the DRG of CFA treated rats. Blocking ZHX2 function causes neuronal differentiation, whereas overexpression of ZHX2 or its ephrin-B1 intracellular domain disrupts the normal differentiation of cortical neural progenitor cells . ENC1 (or NRP/B), also a regulator of neuronal differentiation is increased in the DRG of CFA treated rats. Overexpression of NRP/B significantly induced neurite outgrowth in PC12 cells, whereas inhibition of NRP/B by antibodies or siRNA inhibited neurite outgrowth and suppressed the NGF-induced outgrowth of neurites [50, 51]. In summary, we have found a number of genes which regulate neuronal growth in the CFA model, suggesting a neuronal growth component to this peripheral inflammation model.
Using the Connectivity Map approach, we identified 5 compounds in the Broad database that were negatively connected with the CFA signature. These compounds belong to different chemical classes and their structural properties are different. They also bind to different targets. Fenoprofen, an NSAID, was among them. Another compound we identified with this approach was pyrvinium, a non-competitive androgen receptor inhibitor . Intrathecal administration of testosterone, an androgen derived from DHEA, has been shown to cause analgesia in neuropathic rats . Difenidol, also identified by Connectivity Mapping is used for treatment of vertigo. The precise molecular target of this drug is not known, although recently it was found to be a ligand for muscarinic receptors: M1, M3 and M4 , and may thus play a role in processing of pain stimuli .
Phenoxybenzamine, a non-selective α-blocker  was also identified as having negative connectivity with the CFA pain signature. Previous studies have reported efficacy of phenoxybenzamine in patients with various pain conditions. In one study, 40 patients with causalgia were treated successfully for their pain with phenoxybenzamine . In a case report, 3 out of 4 patients got relief from complex regional pain syndrome type I with phenoxybenzamine . In the current study, we examined effects of phenoxybenzamine in the 4 Day CFA model to determine whether the Connectivity Map approach could identify compounds that had analgesic potential in this model. Our data confirm the antinociceptive properties of phenoxybenzamine; this effect may be due to a direct blockade of adrenergic receptors that may have become supersensitive to catecholamines or perhaps due to possible sympathetic sprouting, as a result of increased expression of nerve growth regulating genes. Sympathetic sprouting in the DRG has been shown to occur in a study that examined gene expression profiles at 3 days post zymosan in incomplete Freund's adjuvant . Furthermore, antinociception has been reported for phenoxybenzamine in the rat hot plate test , the mouse tail flick test  and mouse writhing tests . Although we do not yet understand the basis for the diminished efficacy of phenoxybenzamine at the higher dose tested here (20 mg/kg), it is possibly due to excessive antagonism of the adrenergic receptors in addition to other mechanisms, such as its agonist-antagonistic properties . Regardless, our data suggest that the CFA pain model may have broader utility for testing compounds that modulate pain involving the sympathetic nervous system.