Figure 4.

Inhibition of JAK1/2 kinases with ruxolitinib reduces NHO development after SCI in vivo. (A) Western-blots of whole muscle lysates collected at day 7 from SCI+CDTX mice that were treated with either vehicle control or ruxolitinib (60 mg/kg bi-daily) from day 0–7 post surgery. Western-blots of whole muscle lysates were probed with rabbit anti-pSTAT3 Y705 mAb, and rabbit anti-total STAT3 mAb, then band fluorescence was quantified and ratio of signal intensity of pSTAT3 versus total STAT3 calculated for each individual mouse. Each lane and each dot represents a separate mouse, n = 4–5/treatment group. Data represented as mean ± SD, ^*p = 0.03 by Mann-Whitney test. (B) Measurement of NHO volume by micro CT (μCT) in mice which received SCI+CDTX and treated with either vehicle control or ruxolitinib (60 mg/kg bi-daily) from day 0–7 post surgery. (i) NHO volumes were quantified in vivo by μCT at indicated time points post-surgery illustrating the reduction in NHO development. Each dot represents a separate mouse, n = 4–10 mice/treatment/time point. Data represented as mean ± SD, ^**p = 0.0076, ^*p = 0.031, and p = 0.015 respectively by Mann-Whitney test. (ii) Representative μCT images at 7 days post-surgery (C) Masson's trichrome staining 3 weeks post-surgery confirming the development of multiple NHO bone and collagen+ foci (crosshatches) within the muscle in vehicle treated mice, which are reduced after ruxolitinib treatment, and absent in control mice (SHAM+CDTX) (D) Immunohistochemistry staining of serial sections from SCI+CDTX mice 3 weeks post-surgery (top and middle panels). Mice were treated with vehicle or ruxolitinib (60 mg/kg bi-daily) from day 0–7 post surgery. Stains were performed with either rat anti-F4/80 mAb, rabbit anti-collagen I (CT1), or anti-osterix antibodies. Isotype control (rat IgG2b for F4/80; Rabbit IgG for CT1, and Osterix) are also shown to confirm specificity of staining. In vehicle treated mice CT1⁺ NHO foci are present within the damaged muscle (crosshatch), these foci are surrounded by F4/80⁺ macrophages and have Osterix⁺ cells lining the NHO foci surface (arrows). After ruxolitinib treatment there are still F4/80⁺ macrophages within the damaged muscle, however there are less CT1⁺ NHO foci with Osterix⁺ cells lining the surface. ^*symbols denote the same anatomical landmark in each image. NHO development is absent in SHAM+CDTX mice 3 weeks post-surgery, with no CT1, and Osterix expression (bottom panel). (E) Quantification of F4/80 expression via IHC confirmed that ruxolitinib treatment did not change F4/80⁺ macrophage expression within the hamstrings of vehicle vs. ruxolitinib treated mice, 7 days post-surgery. Each dot represents a separate mouse, n = 2–5/treatment group/sectional depth. Four different depths were analyzed for each sample with at least 50 μm between each depth. Data represented as mean ± SD. All images taken at 40X magnification, scale bar represents 50 μm.