Abnormal Reinnervation of Denervated Areas Following Nerve Injury Facilitates Neuropathic Pain.

An injury to peripheral nerves leads to skin denervation, which often is followed by increased pain sensitivity of the denervated areas and the development of neuropathic pain. Changes in innervation patterns during the reinnervation process of the denervated skin could contribute to the development of neuropathic pain. Here, we examined the changes in the innervation pattern during reinnervation and correlated them with the symptoms of neuropathic pain. Using a multispectral labeling technique-PainBow, which we developed, we characterized dorsal root ganglion (DRG) neurons innervating distinct areas of the rats' paw. We then used spared nerve injury, causing partial denervation of the paw, and examined the changes in innervation patterns of the denervated areas during the development of allodynia and hyperalgesia. We found that, differently from normal conditions, during the development of neuropathic pain, these areas were mainly innervated by large, non-nociceptive neurons. Moreover, we found that the development of neuropathic pain is correlated with an overall decrease in the number of DRG neurons innervating these areas. Importantly, treatment with ouabain facilitated reinnervation and alleviated neuropathic pain. Our results suggest that local changes in peripheral innervation following denervation contribute to neuropathic pain development. The reversal of these changes decreases neuropathic pain.

Combined shRNA over CRISPR/cas9 as a methodology to detect off-target effects and a potential compensatory mechanism.

Inhibition of genes is a powerful approach to study their function. While RNA interference is a widely used method to achieve this goal, mounting evidence indicates that such an approach is prone to off-target effects. An alternative approach to gene function inhibition is genetic mutation, such as the CRISPR/cas9 method. A recent report, however, demonstrated that genetic mutation and inhibition of gene expression do not always give corresponding results. This can be explained by off-target effects, but it was recently shown, at least in one case, that these differences are the result of a compensatory mechanism induced only by genetic mutation. We present here a combination of RNA inhibition and CRISPR/cas9 methods to identify possible off targets as well as potential compensatory effects. This approach is demonstrated by testing a possible role for Sema4B in glioma biology, in which our results implicate Sema4B as having a critical function. In stark contrast, by using shRNA over CRISPR/cas9 combined methodology, we clearly demonstrate that the Sema4B targeted shRNA effects on cell proliferation is the result of off-target effects. Nevertheless, it also revealed that certain splice variants of Sema4B are important for the ability of glioma cells to grow as individual clones.