Article Figures & Data
Figures
- Figure 1.
Characterizations of defect and sheep DRG neurons. A, Photograph of OD created in sheep joint. B, Photograph showing intact (top) lumbar region of a sheep spine and after transverse section (white dotted dissection line, bottom) to expose DRG (black circles). C, Acutely dissociated sheep DRG neurons in culture. D, Histogram showing area of each sheep DRG neuron imaged from whole DRG section, and the criteria used in this article for assigning neurons into small, medium, and large category. E, Histogram of neuronal diameters on which whole-cell patch clamp was performed.
- Figure 2.
OD neurons are more excitable than ctrl neurons. A, Percentage of neurons showing spontaneous activity in ctrl and OD condition (top) and a representative OD neuron with spontaneous activity (bottom). B, Percentage of neurons firing multiple APs on current injection (top) and a representative trace of multiple firing in response to 400 pA injected current (bottom). C, Schematic representation of AP properties (left) and distribution of HPD (right) and (D) RMP in ctrl and defect conditions. E, Representative traces of currents evoked from a neuron at different voltages and (F) plots of the inward and outwards currents in ctrl (black) and defect (red) conditions; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test. See Extended Data Figure 2-1 for breakdown of data according to weeks post-OD.
- Figure 3.
OD neurons have increased TRPV1 function than ctrl neurons. A, Patch-clamp traces showing a representative DRG neuron from the ctrl side firing AP spikes above baseline and same as baseline in response to capsaicin and αβ me-ATP, respectively. B, Bar graph showing percentage of neurons with above baseline firing activity in response to capsaicin C, Bar graph showing percentage of neurons with above baseline firing activity in response to αβ me-ATP. Numbers on the bars represent number of neurons in each condition firing above baseline. D, top, Representative Ca2+ trace from a neuron responding to capsaicin and KCl (positive control). Bottom, Magnitude of Ca2+ influx in response to capsaicin (Ctrl, n = 35, defect, n = 48). E, Percentage of neurons responding to capsaicin in each condition. F, top, Representative Ca2+ trace from a neuron responding to αβ me-ATP and KCl (positive control). Bottom, Magnitude of Ca2+ influx in response to αβ me-ATP (ctrl, n = 24, defect, n = 23). G, Percentage of neurons responding to αβ me-ATP in each condition. *p < 0.05, unpaired t-test.
- Figure 4.
OD and ctrl DRGs showed similar proportion of P2X3 and CGRP positive neurons. Ai, Representative brightfield (top) and anti-P2X3-antibody stained (bottom) image of a whole-sheep DRG in cross-section along with the percentage of neurons positive for P2X3 (Aii). Bi, Representative brightfield (top) and anti-CGRP-antibody stained (bottom) image of a whole-sheep DRG in cross-section along with the percentage of neurons positive for CGRP (Bii). Histograms of cross-sectional areas of neurons stained positive by anti-CGRP (C) and anti-P2X3 (D) antibodies. Numbers on the bars represent neurons positive for the respective antibody staining; *p < 0.05, **p < 0.01, χ2 test.
Tables
- Table 1
Scores of ODs in sheep by International Cartilage Repair Society (ICRS) macroscopic scoring system
Sheep # ICRS macroscopic score
(appearance of joint postmortem)1 3 2 2 3 2 4 2 5 3 6 2 Ctrl (n = 15) OD (n = 16) Mean SD Mean SD Diameter (μm) 39.5 8.309 39.5 7.611 Capacitance (pF) 71.0 41.24 80.5 47.24 RMP (mV) −48.3 13.39 −46.5 9.633 Threshold (pA) 116.7 155.5 100.0 225.8 HPD (ms) 3.1* 0.8886 4.5 2.151 AHP amplitude (mV) 14.0 7.548 14.0 9.126 Amplitude (mV) 101.6 18.17 103.5 11.17 *p < 0.05, unpaired t test.
Antibody Description Supplier Catalog Anti-TRPV1, mouse monoclonal Primary Proteintech 66983-1-Ig Anti-TRPV1, rabbit polyclonal Primary Abcam Ab3487 Anti-Trpv1, rabbit polyclonal Primary Abcam Ab31895 Anti-TRPV1, guinea pig polyclonal Primary Alomone AGP-118
Extended Data Figure 2-1
Extended data supporting Figure 2. Data points from main figures separated according to weeks post-OD. A, AP properties of sheep DRG neurons. Percentage of sheep DRG neurons which evoked AP upon application of capsaicin (B) and αβ m-ATP (C). D, Percentage and magnitude of Ca2+ influx of sheep DRG neurons in response to capsaicin and αβ m-ATP. E, percentage of P2X3 and CGRP positive sheep DRG neurons as assessed using immunohistochemistry. In all the panels, black = control, red = OD, circle = data from sheep two weeks post-OD, square = data from sheep four weeks post-OD and, triangle = data from sheep six weeks post-OD; *p < 0.05, **p < 0.01, unpaired t test. Download Figure 2-1, TIF file.
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