Sodium Channel Blocking Agent Lidocaine In Vitro And In Vivo Biological Effects Of Intervention In The Articular Cartilage

With the development of social olden and the improvement of life quality, joint pain andosteoarthritis take more and more attention by people. In order to alleviate joint pain and theneed of the arthroscopic surgery, doctors often inject lidocaine into articular cavity.But as thetechnology becomes popularization, there are some reports about its side effects.The mostoutstanding is that the dissolution of articular cartilage after repeatedly lidocaine injection .Articular cartilage is a sort of transparent cartilage, covering on the surface of the joint.There isn＇t nerve or lymphatic vessels, the self-repairing ability is poor. Articular cartilagemainly constitutes with chondrocytes and matrix.The chondrocytes accounts for only 1%. Themain ingredients of the matrix is water,protein polysaccharides and collagen type II. Cartilagecells synthesize and secrete proteoglycans and type II collagen, maintain cartilage matrixmetabolism; The Matrix is around cartilage cells, making cartilage with elastic and tensionstrength. Cartilage cells and matrix depend on each other.The apoptosis or necrosis ofchondrocytes may affect the synthesis and secretion of the matrix; meanwhile the damage of thematrix will lead to the destruction of cartilage cells. In normal circumstances, matrix updateslowly,and the dynamic balance of matrix synthesis and secretion is affected by many kinds offactors, such as the regulation of the balance between TIMPs and MMPs.Cartilage cells are not excited cells. A variety of ion channels exist in their membrane.They are the foundation of various life activities : transporting materials,adjusting osmoticpressure,participating in electrical impulses formation,participating in signal transmission,which made cells adapted to environmental conditions. The activity of ion channels is affectedby many kinds of regulating factors such as agonists and blockers. For example,lidocaine canblock sodium ion channel,tetraethylammonium selectively block potassium ion channels.Lidocaine is a sort of sodium ion channel blockers, can selectively block voltage-gated ionchannel, resist Na⁺ flow through membrane which cause resting membrane potential rising.Under the circumstances, Ca²⁺ and H⁺ flow into cells increasely. Lower PH and osmoticpressure make cell＇s function damaged. Some reports that 0.1 mmol/L lidocaine can blocksodium ion channels , 2.5mmol/L lidocaine can induce cell apoptosis whice attribute to itscytotoxic effects. Its cytotoxic effects is dependent on time and concentration.In view of this, we hope to do an exhaustive research about the activity of cartilage cells,the synthesis and secretion of the matrix,the change of inflammatory factor and morphologicalchange under the intervention of clinical concentration＇lidocaine,so that we could get detaileddata which is benefit to clinic, avoiding iatrogenic chondrocytes damage. ObjectiveIn order to further reveal the influence of voltage-gated ion channel blockers- lidocaine onarticular cartilage, we design a series of experiments ,observing the effect of differentconcentrations and different interventing time of lidocaine on cartilage cells in vivo and in vitro.By means of measuring the articular cartilage cell＇s activityúsynthesis and secretion ofextracellular matrix＇súthe change of inflammatory factor and morphological change to exploreclinical safe concentration of lidocaine, in order to provide reference for the practical application.MethodsFifty-six New Zealand big white rabbits of two months old was divided into twoexperimental group randomly ,one for vitro and the another for vivo. In vitro group: some whiterabbits were air embolism executed. Cut down double knee cartilage with aseptic operation,separating articular cartilage cells. With different concentrations during different timelines ,weused lidocaine interve cartilage cells, detecting the apoptosis rate of chondrocytes with flowcytometry; testing the chondrocyte＇s ability of secreting collagen typeⅡúGAGúMMP-3 andTIMP-1 using ELISA;measuring the expression of collagen type mRNA andâ…¡aggrecan mRNAwith RT - PCR .Finally did statistical analysis. In vivo group: injected 5mmol/L lidocaine 0.5mlinto the rabbit＇s knees joints per week.Killed them respectively after 4 weeksú12 weeksú24weeks, observed the changes with immunohistochemical method and detected the expression ofcollagen typeâ…¡and GAG of joint fluid on the 1stú2ndú4th and 8th day with RT– PCR after oneinject.Resultsvitro group:1: apoptosis of cartilage induced by lidocaine has obvious time and concentrationdependence. The safe concentration is 2.5 mmol/L.2: 0. 2mmol/Lú2mmol/L lidocaine interve chondrocytes, 0.2 mmol/L group :early period(the third day) restrain GAG secretion, late-stage (the ninth day) GAG secretion increasessignificantly (p < 0.05). Comparing with control group ,the ability of secreting collagen type IIhas a decreasing trend of lidocaine group, without statistically difference(p > 0.05).2mmol/L group: reduces the secretion of GAG and collagen type II, but without statisticallydifference (p > 0.05).3:0. 2mmol/L lidocaine interve chondrocytes group: early period (the third day)restrainMMP - 3 secretion, late-stage (the ninth day) the secretion of MMP - 3 increases significantly (p< 0.05) comparing with control group, the secretion of TIMP - 1 increases slightly(p > 0.05) . 2mmol/L group: comparing with control group the content of MMP - 3 increases (p < 0.05).And the content of TIMP - 1 has no obvious changes(p > 0.05).4:Under 2mmol/L lidocaine interventing:collagen typeâ…¡mRNA is not statisticallydifferent comparing with control group; early (the third day) aggrecan mRNA expressionsdecreases, late-stage (the ninth day) aggrecan mRNA increases significantly (p < 0.05).vivo group:1:After lidocaine interventing 4 weeks, the general form, HE and immunohistochemicalstaining have no differences with autologous control of the rabbit knee cartilage.Their surface issmooth, their structure is intact, tide line is clear as usual. The quantity of cartilage cells does notchange.They line marshallingly.There is not disintegrating chondrocytes necrosis. After 12weeks and 24 weeks, cells becomes bigger. They are hyperchromatic with safranine O. Thoughboth groups are not statistically difference with control group, but the thickness of lidocainegroups becomes thickening .2: collagen typeâ…¡mRNA becomes higher at the first day after 5 mmol/L lidocaineinterventing,Then declines (p>0.05).At the first day aggrecan mRNA declines obviously,thengradually increases (p < 0.05).Conclusion1: The cell toxicity of lidocaine interventing cartilage cells is of concentration dependenceand time dependence.2: The injection of lidocaine with clinical concentration into joint lumen is safe, but if theperiod of treatment is too long, it will cause aggravating OA risk.