Previous work in a rabbit model of post-traumatic joint contractures shows that the mast cell stabilizer ketotifen decreases contracture severity. We show here that ketotifen decreases collagen gel contraction mediated by rabbit joint capsule fibroblasts when mast cells are present. Ketotifen was shown to decrease contracture severity and associated joint capsule fibrosis in an animal model of post-traumatic joint contractures. Ketotifen prevents the release of profibrotic growth factors from mast cells (MC). An Summary
Introduction
Recent work has shown that joint contracture severity can be decreased with the mast cell stabilizer ketotifen in association with decreased numbers of myofibroblasts and mast cells in the joint capsule of a rabbit model of post-traumatic contractures. Neuropeptides such as Substance P (SP) can induce mast cells to release growth factors. Using a gel contraction assay, we test the hypothesis that joint capsule cell-mediated contraction of a collagen gel can be enhanced with SP, but the effect is magnified in the presence of mast cells. Anterior elbow joint capsules were obtained at the time of surgical release from 2 men (age 34 and 54) and 1 woman (age 40) with chronic (> 1 year) post-traumatic joint contractures. The human mast cell line HMC-1 (Mayo Clinic, Rochester), SP and the NK1 receptor antagonist RP67580 (Sigma, Oakville, ON) were used. NK1 is the SP receptor. Neutralized Collagen solution composed with 58% Vitrogen 100 purified collagen mixed with HMC-1 cells only (7.5 105), human capsule cells (2.5 105), or human capsule cells (2.5 105) and 7.5 105 mast cells (1:3) were cast into 24- well tissue culture plates. In some experiments, SP (1 × 10−5 M) +/− RP67580 (0.5 mM) were added. The gels were maintained with 0.5 ml DMEM composed with 2% BSA and incubated at 37C for 12 h for gelation to occur. The gels were then detached from the wall and the bottom of culture plate wells, and photographed at regular intervals up to 72 hours. Gel contraction studies were carried out on passage 4 and done in triplicate for each patient. The average value of each patients triplicate was combined to give a mean contraction at each time point. Statistical analysis involved an ANOVA with posthoc Bonferroni correction. P < 0.001 was significant.Purpose
Method
The hypothesis is that mast cell numbers and neuropeptide containing nerve fibres are increased in the elbow joint anterior capsule of patients with post-traumatic contractures when compared to normal capsules. Capsules were obtained from two patients with chronic contractures following radial head fractures and two organ donor elbows free of contractures. Four sections from each capsule were double-labelled with specific antibodies to the mast cell marker chymase and the neuropeptide calcitonin gene-related peptide (CGRP). Species specific secondary fluorescent antibodies were used to detect the marker antibodies and cells were identified with a fluorescent nuclear marker (DAPI). Images were captured using a microscope (200x magnification) and five randomly selected areas were sampled for each section obtained from all joint capsules. Chymase positive cell numbers and numbers of nerve fibers (minimum length fifty micrometres) were gathered. The number of chymase positive mast cells was 6x greater in the contracture capsules when compared to normal capsules. In the contracture capsule, chymase positive mast cells represented 39% of total cells while in control capsules they represented 7% of total cells. Total cell numbers were similar in the capsules of both groups. The number of CGRP positive nerve fibres was increased 3x in the contracture capsule when compared to normal capsule. Mast cell numbers and neuropeptide positive fibre numbers are increased in the elbow joint anterior capsule of patients with post-traumatic contractures when compared to normal tissues. Neuropeptides such as CGRP can induce mast cell degranulation. Mast cells release profibrotic molecules such as transforming growth factor beta1 (TGF-b1), a myofibroblast upregulator. It has been described that TGF-b1 and myofibroblast numbers are elevated in human elbow joint capsules in post-traumatic contractures. While these trends are encouraging, more subjects are needed to determine whether the mast cell and neuropeptide nerve fibre findings can be generalised to larger numbers. If future work supports a myofibroblast - mast cell - neuropeptide - fibrosis axis in the joint capsule in post-traumatic contractures, then methods to modulate this axis, such as mast cell stabilisers, may be evaluated in animal models.
We examined the vasoconstrictive actions of neuropeptide Y (NPY) in the intact medial collateral ligament (MCL) of normal and anterior cruciate ligament (ACL) -deficient rabbit knees. Blood flow to the surgically exposed MCL was measured using high-resolution laser speckle imaging (LSI) before and after topical administration of NPY and the α1-adrenoreceptor agonist phenylephrine. In control rabbit knees, dose-dependent vasopressor responses were significantly greater than those in ACL-deficient knees, where there was little or no vasoconstrictor response. We conclude that chronic ACL deficiency markedly changes the vascular physiology and pharmacology of the surrounding articular tissues. To determine the effect of chronic ACL-deficiency on the physiologic responses to the potent sympathetic vasoconstrictor NPY. Abrogation of the vasoconstrictor response to both NPY and phenylephrine indicates that chronic ACL deficiency induces major changes in the vascular physiology of associated articular tissues. This study is the first to examine the vasoregulatory role of NPY in the MCL of unstable knee joints using LSI. In control rabbits, topical administration of NPY produced dose-dependent vasopressor responses (maximal effect at 10−10mol NPY). In ACL-transected knees there was little or no response to NPY (Figure 1). BIBP 3226 (selective NPY-Y1 receptor antagonist) did not affect the constrictor response to NPY in normal tissue, indicating that a receptor other than Y-1 mediates the response. Many neuropeptides participate in the post-traumatic inflammatory response. The sympathetic-derived NPY helps regulate inflammatory responses, is a vasoconstrictor and stimulates angiogenesis. Rupture of the ACL induces inflammation, hyperaemia and angiogenesis in the MCL. These changes in vascular physiology induced us to study the effect of ACL-deficiency on the actions of NPY in the MCL. Unoperated control (n=6) and 6-week ACL-transected (n=5) adult rabbits were used. Under anaesthesia, the MCL was surgically exposed and tissue blood flow was measured in real time using LSI as various doses and combinations of NPY, phenylephrine, and BIBP 3226 were administered topically. Possible causes of the reduced vasoconstrictive response to both NPY and phenylephrine in the MCL after 6wk of ACL-deficiency include change in the distribution or functionality of their specific receptors or inactivation of the associated down stream signalling pathways.
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Our aim was to determine the effect of denervation on repair-associated mRNA levels in the MCL after partial tear. Cohorts of rabbits underwent partial MCL tear with or without concomitant femoral nerve transection. Ligaments were harvested, RNA extracted and RT-PCR was performed using rabbit-specific primers for repair-associated molecules at three days, two wks, six wks and sixteen wks post-injury. Angiogenesis genes MMP3, MMP13, matrix components Collagen I and III and growth factors TGF-ß and NGF mRNA levels were increased in the denervated group at two-weeks post-injury (p<
0.05). Denervation significantly alters mRNA levels during the early stages of rabbit MCL healing. To determine the effect of denervation on repair-associated mRNA levels in the injured medial collateral ligament (MCL). Previous experiments revealed that denervation impairs healing of the MCL. We hypothesized that denervation would decrease repair-associated mRNA levels in the injured MCL when compared with normally innervated injured MCL. Adult, skeletally mature female rabbits were assigned to one of four groups: unoperated control, femoral nerve transection alone (denervated controls), MCL partial tear and denervated MCL partial tear. At three days, two weeks, six weeks or sixteen weeks post-surgery, cohorts of six rabbits from each experimental group were killed. Control rabbits were assessed at two weeks. Ligaments were harvested, RNA extracted and RT-PCR was performed using rabbit-specific primers. In the denervated injury group, mRNA levels of angiogenesis genes MMP-3 and MMP-13, matrix components Collagen I and III and growth factors TGF-ß and NGF had all increased at two-weeks post-injury, in comparison to non-denervated (p<
0.05). We also found increased levels of MMP-3 and NGF mRNA in the denervated group at sixteen weeks post injury (p<
0.05). The mRNA levels of the housekeeping gene GAPDH were increased in the denervated group only at three days post injury (p<
0.05). Of note, TGF-ß mRNA levels were significantly decreased in the denervated group at three days post injury (p<
0.05). Contrary to our initial hypothesis, denervation increases mRNA levels for many important molecules during the early stages of MCL healing. Additional research will be required to explain how and why denervation impairs ligament healing. No previous study has shown that innervation regulates mRNA levels in healing ligament.
Joint instability was induced by posterior cruciate ligament (PCL) transection. This resulted in significant changes in medial collateral ligament (MCL) gene expression as early as three days after injury that persisted as long as 6 weeks. We noted substantial changes in expression of matrix-metalloproteinases (MMPs) −1, −3 and -13, with reciprocal effects on their specific inhibitors TIMP-1 and −3. Sustained changes in expression of these angiogenesis-associated matrix-degrading enzymes likely account for the observed degradation of the mechanical properties of secondary stabilizers in chronically unstable joints. To determine changes in gene-expression induced by traumatic instability. Instability activates aberrant expression of angiogenesis-associated matrix metalloproteinases. PCL transection induces a significant increase in the expression of MMP-3 and decrease in its specific inhibitor TIMP-3 with opposite effects on MMP-1 and TIMP-1 as early as three days after injury. Understanding the changes in gene expression induced by instability may lead to specific treatments that could prevent the “collateral damage” to secondary stabilizing structures. Under anaesthesia, four cohorts of six adult rabbits underwent surgical transection of the PCL. Three days, and two, six and sixteen weeks later, the MCL was harvested and the relative expression of TGF-β, MMP-1, -3, and −13, and their tissues inhibitors, and urokinase-type plasminogen activator (uPA) was measured using semi-quantitative RT-PCR. Previous work revealed increased in blood flow by two weeks and increased vascular volume by six weeks in the MCL of PCL-deficient joints. These changes are preceded by substantial changes in expression of mRNA for matrix degradation enzymes involved in the early stages of angiogenesis. This aberrant expression of matrix metalloproteinases likely accounts for the progressive degradation of the mechanical properties of secondary stabilizing structures seen in chronic instability.
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