Neonatal pain is a significant clinical issue but the mechanisms by which pain is produced early in life are poorly understood. Studies have suggested that neonates may transduce noxious stimuli distinctly from adults.  Our recent work has linked the transcription factor serum response factor downstream of local growth hormone (GH) signaling to incision-related hypersensitivity in neonates. However, it remains unclear if similar mechanisms contribute to inflammatory pain in neonates. Our pilot studies showed that signal transducer and activator of transcription 1 (STAT1) was uniquely upregulated in the dorsal root ganglia (DRGs) of neonates after cutaneous and muscle inflammation.  Therefore, we investigated if distinct transcription factors or GH may modulate nociceptive behaviors in neonatal pups following cutaneous or muscle inflammation.

Inflammation was induced by 1 % carrageenan (in 0.9%NaCl) injection into either the right hairy hindpaw skin or hindpaw muscle at postnatal day 7 in Swiss Webster mice and compared to GH/ carrageenan treated and sham injected controls. Behavioral examinations of spontaneous paw guarding, thermal hypersensitivity (cutaneous), and mechanical withdrawal thresholds using von Frey filaments (cutaneous), or muscle squeezing assays were then performed in our groups. Results were correlated to gene expression in the L2/3 (skin) or L3/4/5 (muscle) DRGs at day one post injection using real-time PCR.  Mast cells and macrophages, and cytokine related activity were confirmed by immunohistochemistry, and Elisa, respectively. Finally, calcium imaging was performed in mice expressing an endogenous calcium indicator in sensory neurons (PirtCre:GCaMP6) using our novel ex vivo neonatal hindpaw muscle, tibial nerve, DRG, spinal cord preparation.
Carrageenan injection significantly induced spontaneous paw guarding, and mechanical and/or thermal hypersensitivity after either skin or muscle inflammation. Results corresponded with significant edema in the hind paws of carrageenan injected groups compared to GH/ carrageenan treated and sham injected controls. We also detected increased calcium transients in neurons from carrageenan treated mice compared to GH/carrageenan and naïve groups.   Surprisingly, while there were no significant changes in a series of select transcription factors, STAT1 was differentially altered in the DRGs after both skin and muscle inflammation. In addition, co-injection of GH/ carrageenan into the hindpaw suppressed DRG STAT1 expression. GH prevented infiltration of specific mast cells and macrophages after muscle injury while nerve targeted STAT1 inhibition specifically reduced macrophage infiltration into the muscle. Furthermore, GH treatment and neuronal STAT1 inhibition altered neuroimmune signaling in neonatal DRGs after muscle inflammation. Each of these interventions blocked the release of the cytokine MCP1 in the muscle after injury. 

Data suggest that STAT1 upregulation may be downstream of GH signaling and contribute to neonatal nociception during skin and muscle inflammation. GH and STAT1 signaling in neurons also appear to regulate neuroimmune signaling and muscle inflammation. Our study suggests that the mechanism of action by which GH reduces pain involves the inhibition of neuronal STAT1 which in turn prevents the recruitment of macrophages into the muscle during injury. Results could uncover new ways to treat inflammatory pain in neonates.