Journal: Bone Research

Article Title: Neuron-to-vessel signaling is a required feature of aberrant stem cell commitment after soft tissue trauma

doi: 10.1038/s41413-022-00216-x

Figure Lengend Snippet: Genetic and pharmacological inhibition of TrkA signaling impairs vascular ingrowth after soft tissue trauma. a Schematic representation of experiment. (Created in Biorender.com). TrkA F592A mice have a point mutation which renders them susceptible to temporally controllable inhibition of TrkA catalytic activity, via administration of the small molecule 1NMPP1. Either TrkA WT or TrkA F592A animals (6–8 weeks old, male and female mice) were subjected to HO induction and both administered 1NMPP1 throughout the study period. Analyses performed at 21 d post-injury. b – d Representative immunohistochemical staining for TUBB3 (Beta III Tubulin, appearing green) and quantification among TrkA WT or TrkA F592A animals, as assessed at the distal tenotomy site as visualized using sagittal cross-sections. e – g Representative immunostaining for CD31 (appearing red) and quantification among TrkA WT or TrkA F592A animals. h – j EMCN (Endomucin) and CD146 co-immunohistochemical staining (appearing red and white) and quantification among TrkA WT or TrkA F592A animals. k Schematic representation of experiment. C57BL/6 J mice (6–8 weeks old, male and female mice) were administered AR786 or vehicle control throughout the study period. HO induction was performed (burn/tenotomy), with analysis at day 21. l – n Representative immunohistochemical staining for TUBB3 and quantification among control or AR786 treated animals, as assessed at the distal tenotomy site. o – q Representative immunostaining for CD31 (appearing red) and quantification among control or AR786 treated animals. Dashed white lines indicate edges of Achilles tendon. Scale bars: 100 μm. n = 4–5 animals per group. Individual dots in scatterplots represent values from single animal measurements, while mean and one SD are indicated by crosshairs and whiskers. * P < 0.05; ** P < 0.01. A two-tailed student’s t test was used for all comparisons

Article Snippet: In order to achieve temporal inhibition of TrkA catalytic activity in TrkA F592A animals, the small molecule 1NMPP1 was used – , (Aurora Analytics, LLC, Baltimore, MD).

Techniques: Inhibition, Mutagenesis, Activity Assay, Immunohistochemical staining, Staining, Immunostaining, Control, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: NGF-TrkA signaling in sensory nerves is required for skeletal adaptation to mechanical loads in mice

doi: 10.1073/pnas.1701054114

Figure Lengend Snippet: Inhibition of TrkA signaling by 1NMPP1 decreases load-induced bone formation in phenotypically normal TrkAF592A mice. (A and B) TrkAwt and TrkAF592A mice (n = 4–5) were simultaneously injected with NGF (5 μg/g BW) and 1NMPP1 (17 μg/g BW). Mechanical (A) and thermal (B) sensitivity was quantified immediately before and 24 h after treatment. (C–E) Phenotypic analysis of TrkAwt and TrkAF592A mice (n = 6) at 16 wk of age included body weight (H), femur length (I), or ultimate bending energy (J) as measured by three point bending of the femur. (F–I) Quantification of open field testing of TrkAwt and TrkAF592A mice (n = 3) by open field distance (F), total distance (G), open field speed (H), and average speed (I). (J–N) TrkAwt and TrkAF592A mice (n = 6) were subjected to 3 d of axial compression of the forelimb, with calcein and alizarin red injected 3 and 8 d after loading, respectively. Undecalcified sections from TrkAwt (J) and TrkAF592A (K) mice were used to quantify relative periosteal (L) and endosteal (M) bone formation rates. (N) Hotplate sensitivity testing was performed 3 d after loading. *P < 0.05 by t test. Data are presented as mean ± SE.

Article Snippet: TrkA F592A mice, which harbor a phenylalanine-to-alanine point mutation in exon 12 of the mouse Ntrk1 gene (F592A) that renders the endogenous TrkA kinase sensitive to inhibition by the membrane-permeable small molecule 1NMPP1 ( 14 ), are commercially available (Jackson Laboratory; stock no. 022362).

Techniques: Inhibition, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: NGF-TrkA signaling in sensory nerves is required for skeletal adaptation to mechanical loads in mice

doi: 10.1073/pnas.1701054114

Figure Lengend Snippet: Inhibition of TrkA signaling impairs load-induced nerve sprouting on the ulnar periosteal surface. (A-C) Nerves were visualized at the ulnar middiaphysis in Thy1-YFP mice that did not receive 1NMPP1, with representative cross-sections from nonloaded (A), Day 3 (B), and Day 7 (C) loaded limbs. Arrows indicate nerves visible on the medial surface. The cortical bone of the ulna is indicated as b. (D–F) Nerves were also visualized on the periosteal surface of the intact ulna in Thy1-YFP mice that did not receive 1NMPP1 by using confocal microscopy, with representative maximum intensity projections (MIP) from nonloaded (D), Day 3 (E), and Day 7 (F) loaded limbs. The periosteal surface of the ulna is indicated as b. (G and H) Nerves were visualized in TrkAF592A mice that received 1NMPP1 (40 μM) to inhibit TrkA signaling following load, with representative images from cross-sections (G) and MIP (H) 7 d after loading.

Article Snippet: TrkA F592A mice, which harbor a phenylalanine-to-alanine point mutation in exon 12 of the mouse Ntrk1 gene (F592A) that renders the endogenous TrkA kinase sensitive to inhibition by the membrane-permeable small molecule 1NMPP1 ( 14 ), are commercially available (Jackson Laboratory; stock no. 022362).

Techniques: Inhibition, Confocal Microscopy