Characterization of Inhbb, Heatr5a, & Cyp2s1 Expression in Dorsal Root Ganglia by in Situ Hybridization

Abstract Body : OBJECTIVES: Multiple studies have shown that gene expression changes occur in sensory neurons after peripheral nerve injury (PNI), including the expression of injury-specific genes. Much less is known about expression changes in stable genetic markers that delineate specific classes of somatosensory neurons after PNI. In this study, we examined expression changes in genes recently identified as markers of proprioceptive neurons. We generated anti-sense riboprobes (labelled RNA segments that are used to detect mRNA) that specifically identified the expression patterns of late embryonic markers after PNI. We hypothesized that expression of these late markers would decrease due to nerves acting in a regenerative state and could create a better understanding of the intrinsic factors that affect proprioceptive nerve regeneration.
METHODS: This study characterized the expression of proprioceptive genetic markers that are observed in mice on embryologic day e17.5 and continue to be expressed post-natally, specifically Inhbb, Heatr5a, and Cyp2s1. We viewed the expression patterns in lumbar dorsal root ganglion (DRG) neurons from intact animals and after PNI. Segments of the coding sequences for the genes of interest were subcloned and used to generate digoxigenin (DIG) labeled riboprobes. Control experiments demonstrated the validity of the riboprobes for these genes on brain tissue from adult mice when compared to a known proprioceptive marker parvalbumin (Pvalb). By creating these anti-sense riboprobes, the difference in expression pattern in lumbar L4-L6 DRGs between adult wild type mice and mice who underwent sciatic nerve transection surgery could be compared. The expression pattern differences were observed by using in-situ hybridization techniques. The number of dark staining pixels (n) that corresponded to the riboprobe were quantified over the area of the total DRG in μm^2, for the left (affected side) and right (control) lumbar DRG using ImageJ and CellSens software.
RESULTS: The expression density of probes for Inhbb, Heatr5a, and Cyp2s1 ranged between 8-13.1 n/10^5μm^2, 2.2-4.4 n/10^5μm^2, and 3.6-10.7 n/10^5μm^2, respectively, after normalizing labeled pixels (n) over the area of DRG (10^5μm^2) for experimental animals. There was no significant change in expression patterns for Inhbb, Heatr5a, and Cyp2s1 between the injured and control sides. However, the expression pattern results contributed to the validity of the DIG-labeled riboprobes when compared to Pvalb, which was tested on the same injured and control tissue samples.
CONCLUSIONS: This study confirmed that Inhbb, Heatr5a, and Cyp2s1 DIG-labeled riboprobes can be used to identify expression patterns within the DRG. The creation of additional riboprobes including early embryonic markers and increasing the sample size of animals tested would be needed to further characterize the expression patterns that occur after PNI with proprioceptive markers. Understanding gene expression changes after PNI and during nerve regeneration, could eventually lead to novel therapies that promote nerve regeneration in proprioceptive sensory nerves.