Establishment Of The Rat IVRA Model And The Study For Intravenous Regional Anesthesia Effect Of Amitriptyline

Background Intravenous regional anesthesia (IVRA) is a local anaesthetictechnique introduced by Bier in 1908 and modified by Holmes for surgery ondistal extremities in human beings. IVRA is considered a simple, safe andreliable technique and is widely used in western countries. Experimentalstudies of IVRA were usually undertaken by using dog or rabbit models, butthese models are poor of simplicity and reproducibility, as well as beexpensive. Thus new animal model is expected. Amitriptyline is one ofclassic tricyclic antidepressants (TCAs) which have been used systemicallyfor therapy of chronic pain. Recently, some studies have shown thatamitriptyline blocks Na channels in a manner similar to classic localanesthetics. The purpose of this study was to establish a new IVRA animalmodel and use it to investigate the intravenous regional anesthesia effect,local toxicity and feasibility of amitriptyline as a local anesthetic.PART ONE Establishment of the rat IVRA modelObjectives To establish the new rat IVRA model and verify the diffusion ofthe drugs and the time influence of the tourniquet. Methods1) 16 healthy male adult SD rats weighing 190-240 g were randomly andequally divided into normal saline control groups and 0.5% lidocaineexperiment group. The rat's tail was divided into three parts equally: theproximal part, the middle part and the distal part. A 24 gauge needle wasinserted into vena caudalis at the distal part. The rat tail was exanguinated bywrapping it an Esmarch bandage, which was removed after a tourniquet wasapplied immediately distal to the proximal part to occlude arterial blood. A0.5ml solution was administered via the cannula closely following theapplication of the tourniquet. The end-point of the regional anesthesia wasdefined as the rat's lacking in a response to the tail clipping, and the sensoryrecovery after the tourniquet removal was defined as the tail-flicklatency(TFL) being less than 4 seconds. The following variables wererecorded: the tail clipping response and the TFL after drugs administration,the recovered time of the tail clipping response and the TFL after thetourniquet removal. The skin lesion at the second day.2) 3 healthy male adult SD rats were made into the IVRA model, and 0.5 mLof Methylene Blue was administered into the tail vein of each rat. The colorof the rats' tail was observed after injection of ink.3) 24 healthy male adult SD rats were randomly and equally divided into fourgroups which were different in tourniquet applying time. The tourniquetapplying time for the four groups were 10, 15, 30 and 60 min respectively.All animals were made into the rat IVRA model and given 0.5ml normalsaline. The following variables were recorded: the tail clipping response at 1minute after injection and 1 minute before the tourniquet removal. The TFL at 1,5,10,20,30,60 minute and the second day after the tourniquet removal.The skin lesion at 2, 5 day later.Results1). In experiment group, no responses to tail clipping were observed in theparts distal to tourniquet after drugs administration. After releasing thetourniquet, the response to tail clipping of the middle part and the distal partin lidocaine experiment group recovered at 17.5±11.2 and 26.4±19.6 min,the TFL of those recovered at 30.3±20.5 and 41.5±29.4 min, respectively.There were difference between the recovered time of the middle part and thedistal part(P＜0.05). In control groups, responses to tail clipping didn'tdisappear after injection.There were no significant differences of TFL atdifferent time point(P＞0.05). No skin lesion were found in two groups at thesecond day after the tourniquet removal.2) In all the three rats, the color distal to the tourniquet of the the tail becameblue after Methylene Blue administration.3) In all the groups, responses to tail clipping didn't disappear after injection.Only in the 60min-tourniquet group, the TFL exceeded 4 seconds at 1minuteafter the tourniquet removal and were significant longer than the baselineTFL(P＞0.05). There were no significant differences of TFL at different timepoint of the other three groups(P＞.05). No skin lesion were found in all thegroups at 2 day after the tourniquet removal.ConclusionsThe new rat IVRA model has essential characteristics similar to those ofIVRA in clinical practice and is economic, effective and technically easy toperform. PART TWO The relative IVRA potencies of Amitriptyline to Lidocaineand Bupivacaine in the rat IVRA modelObjective To compare the relative IVRA potencies of amitriptyline tolidocaine and bupivacaine by determining their respective median effectiveconcentrations(EC₅₀) in the rat IVRA model. Methods 90 healthy male SDrats weighing 190-240g were randomly and equally assigned intoamitriptyline group, lidocaine group and bupivacaine group. All animals inthe study were made into the rat IVRA model and 0.5ml of amitriptyline,lidocaine or bupivacaine of certain concentration was administered into thetail vein of every rat. The end-point of local anesthesia was defined as the ratlack of response to tail clipping. The EC₅₀ of amitriptyline, lidocaine andbupivacaine were measured by up-and-down method and the potency ratiofor the drugs was calculated. Results In the rat IVRA model, the EC₅₀ ofamitriptyline, lidocaine and bupivacaine were 0.11%, 0.13% and 0.06%,respectively. The potency of amitriptyline was 0.54 (95%CI, 0.38-0.71)relative to bupivacaine and was 1.18(95%CI, 1.52-0.84) relative to lidocaine.Conclusions The ratl IVRA mode can be used to compare the relativeanesthetic potencies of anesthetics. The anesthetic potency of amitriptyline is46% smaller than that of bupivacaine and 18% higher than that of lidocaine.PART THREE Comparison of regional anesthetic effect and local toxicitybetween Amitriptyline and Bupivacaine in the rat IVRA model Objectives The purpose of this study was to compare the durations ofregional anesthetic actions and local anesthetic-related impairment of sensoryand tissue by the equipotent amitriptyline and bupivacaine in the rat IVRAmodel.Methods 32 healthy male SD rats weighing 190-240 g were randomly andequally assigned into 4 groups, and the tail IVRA was performed with 0.5 mLof experimental agents. In Groupâ… , the rats were given 0.22% amitriptyline(2EC₅₀); in Groupâ…¡, 0.33% amitriptyline (3EC₅₀); in Groupâ…¢, 0.12%bupivacaine (2EC₅₀); and in Groupâ…£, 0.18% bupivacaine (3EC₅₀). Theend-point of the regional anesthesia was defined as the rat's lacking in aresponse to the tail clipping in the middle part. The sensory recovery after thetoumiquet removal was defined as the TFL of the distal part. being less than4 seconds. The following variables were recorded: The regional anesthesiaduration, the sensory recovery after the tourniquet removal. The tail clippingresponse, the TFL and the skin lesion in the rat tail at 2,5,9 days.Results After the release of the tourniquet, the anesthesia durations in Groupsâ… ,â…¡,â…¢andâ…£were 232.5±96.4, 380.0±127.9, 38.7±16.8, 51.3±17.7min,respectively. The sensory recovery times of Groupsâ… ,â…¡,â…¢andâ…£were327.5±162.3, 465.0±151.5, 51.3±16.9 and 69.3±14.1 min, respectively.The recovery times of anesthesia and sensory function were significantlylonger in Groupâ… ,â…¡than in Groupsâ…¢andâ…£(P＜0.001). The Groupâ…¡recovery times were significantly longer than Groupâ… (P＜0.01). Theanesthesia durations of amitriptyline were about six or seven times longerthan that of the equipotent concentration bupivacaine. Only in Groupâ…¡, sensory andl skin lesion of three rats were found at 2,5,9 days after thetoumiquet removal.Conclusions The regional anesthesia durations of amitriptyline were aboutsix or seven times longer than that of the equipotent concentrationbupivacaine. The regional intravenous amitriptyline may induce theconcentration-related neurotoxicity and histotoxicity. Thus, amitriptyline hasa narrower therapeutic window when compared with bupivacaine.