Neuromuscular Recovery after Medial Collateral Ligament Disruption ...

Neuromuscular Recovery after Medial Collateral Ligament Disruption and Eccentric Rehabilitation Program JtROME LAURIN, ERICK DOUSSET, SERGE MESURE, and PATRICK DECHERCHI UMR CNRS 6233, Institut des Sciences du Mouvemnent: Etienne-JulesMarey, Equipe Plasticitý des Syst&mes Nerveux et Musculaire, Universit6 de la M&diterran6e (Aix-Marseille II) - Aix-Marseille UniversitW, Facult6 des Sciences du Sport, Pare Scientifique et Technologique de Luminy, Marseille, FRANCE

ABSTRACT LAURIN, J., E. DOUSSET, S. MESURE, and P. DECHERCHI. Neuromuscular Recovery after Medial Collateral Ligament Disruption and Eccentric Rehabilitation Program. Med. Sci. Sports Exerc., Vol. 43, No. 6, pp. 1032-1041, 2011. Purpose: Medial collateral ligament (MCL) rupture of the knee joint frequently occurs during sport activities. However, the optimal rehabilitation strategy after such lesion is unknown. The aim of this study was to assess the effects of progressive eccentric rehabilitation program on neuromuscular deficits induced by MCL transection. Methods: Rats were randomized as follows: (i) control group (C, n = 10) without any surgery; (ii) lesion groups in which neuromuscular measurements were made I (LI, n = 10) and 3 wk (L3, n = 9) after MCL transection by a 15- to 20-min surgery (this group was designed to determine changes induced by the MCL transection); and (iii) eccentric group (ECC, n = 7) in which rats performed a progressive 2-wk eccentric rehabilitation program beginning 1 wk after MCL transection surgery. Dynamic functional assessments were performed at weeks I and 3 after the MCL transection by measuring the maximal and minimal knee angles during the stance phase of the gait cycle. Neuromuscular measurements included 1) modulation of H-reflex in response to a 10-mM KCI injection, 2) analysis of the twitch relaxation properties of the quadriceps muscle, and 3) recording of metabosensitive and mechanosensitive afferents activity in response to chemical injections and to tendon patellar vibrations, respectively. Results: Our results indicated that H-reflex modulation induced by metabosensitive afferents was disturbed by MCL transection without any recovery despite rehabilitation program. Responses of both metabosensitive and mechanosensitive muscle afferents, as well as the muscle relaxation properties, were fully recovered after the eccentric rehabilitation program. Conclusions: Our results directly indicated an influence of progressive eccentric program on muscle afferents response after MCL section but apparently not for spinal reflex modulation. Key Words: H-REFLEX, GROUP 111AND IV AFFERENTS, MECHANOSENSITIVE AFFERENTS, TWITCH PROPERTIES, FUNCTIONAL REHABILITATION

ture damages was similar using rehabilitation alone compared with surgical procedure (19,35). However, recent studies (3,6) indicated that a rehabilitation program after isolated MCL rupture seem to be more appropriate. Indeed, surgical complication risks are avoided, and because MCL is highly vascularized, the cicatrization process seems to be effective (contrary to anterior cruciate ligament (ACL)). Consequently, isolated MCL lesions, including complete rupture, can be treated nonoperatively with early functional rehabilitation (30,36). However, long-term muscle impairments and functional deficits could persist despite the use of a functional rehabilitation program (18). Thus, a better understanding of neuromuscular dysfunction induced by MCL transection is required to assess the effectiveness of a treatment strategy. MCL lesions are accompanied by joint instability, which is associated with knee joint damages and neuromuscular deficits (14). It is largely admitted that sensorimotor disorders are related to changes or loss in sensory feedback from ligaments and/or other joint structures (14,2 1). Moreover, we have previously demonstrated that neuromuscular deficits induced by MCL transection could partially arise not only from alterations in metabosensitive (groups III and TV)

knee joint are frequently observed during sport edial collateral ligament (MCL)is the injuries the activities. Because this ligament primeofstatic stabilizer of the medial side of the knee, determining the optimal treatment strategy (operative treatment vs rehabilitation) after MCL lesion seems to be crucial. Operative treatment is recommended when MCL rupture is associated with other joint structure injuries (19,35). Other studies have shown that recovery from MCL rupture without joint struc-

Address for correspondence: Erick Dousset, Ph.D., UMR CNRS 6233, Institui des Sciences du Mouvement: Etienne-Jules Marey, Equipe Plasticit6 des Syst6mes Nerveux et Musculaire, Universit6 de la M6diterran6e (AixMarseille I) - Aix-Marseille Universit6, Facult6 des Sciences du Sport, Parc Scientifique et Technologique de Luminy, CC910 - 163 Avenue de Luminy, F-13288 Marseille cedex 09, France; E-mail: erick.dousset@ univmed.fr. Submitted for publication March 2010. Accepted for publication October 2010. 0195-9131/11/4306-1032/0 MEDICINE & SCIENCE IN SPORTS & EXERCISE, Copyright© 2011 by the American College of Sports Medicine DOI: 10.1249/MSS.0b013e3182042956

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and niechanosensitive (mainly groups 1 and 11) quadriceps afferents response but also from impairments in quadriceps relaxation properties (23) lasting 5 wk. Mechanosensitive afferent fibers (group 1) originating from muscle spindle and Golgi tendons organs participated in the detection of muscle length changes and in force development sensitivity, respectively. They are well known to be involved in ot-motoneurons' discharge changes. In addition, the thinly myelinated group III and the unmyclinated group IV fibers (sensitive to mechanical and chemical muscular changes) are likely involved in the control of motor unit firing rates. Indeed, proprioceptive feedback from spindle Ia afferents could be reduced by groups lI[ and IV afferent fibers activity at the spinal level (34). These slowly conducting afferent fibers could also act on the motor cortical output to regulate the a--motoneurons' discharge (26). Furthermore, chemically induced discharges in groups I.1 and IV muscle afferents can reflexly increase the fusimotor discharge (17). Groups III and IV afferent fibers are selectively activated by different agents released during physical exercise and during inflarnmatoty process. More precisely, their activity is modified by inflammatory agents such as bradykinin, arachidonic acid, thromboxan A 2, prostaglandin, as well as metabolites substances released during muscle activity such as lactic acid (LA) and potassium (38,39). It has also been reported that an isolated ligament rupture may be associated with the alteration of some motor reflex properties by using the Hoffinann (H) reflex (20,29,31,37). However, other authors failed to detect H-reflex modulation during the following months after ACL rupture (13). Because the effects of ligament rupture on the spinal motor reflex mechanisms remained controversial, a first step to detect direct evidence about the influence of ligament injuries on spinal mechanisms could be the exploration of the mechanisms involved in H-reflex modulation. Research studies have also shown that isolated ligament rupture could induce an eccentric strength deficit compared to the uninjured side (25). However, few studies have reconmmended the use of functional rehabilitation program based on eccentric (i.e., lengthening) contractions to optimize the restoration of muscle function after a ligament rupture (9,32). It has been shown that the use of progressive, high-intensity eccentric exercises could be effective at improving muscle strength in both young and old patients with a ligament rupture (32). For example, Gerber et al. (9,11) have shown that the increase in quadriceps hypertrophy was twofold greater after the progressive eccentric rehabilitation without any apparent detriment on the ligament healing, and this effect persisted 1 yr after the lesion. Furthermore, another evidence indicated that an eccentric program may exert some benefit for restoring neuromuscular deficits after ligament injury. Strength gains and underlying neural adaptations were found to be greater and faster after an eccentric training compared with a concentric one (16). Although eccentric exercise seems to be interesting for rehabilitation, few evidence was found to

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validate the effect of eccentric rehabilitation on quadriceps neuromuscular finction after MCL injury. Thus, we hypothesized that an eccentric rehabilitation will change the neuromuscular mechanisms during the recovery period followed by MCL transection. This study was therefore designed to underline the effects of a progressive eccentric rehabilitation program on the recovery of H-reflex, metabosensitive and mechanosensitive afferents response, and contractile properties after MCL transection. To be complete, we also examined functional recovery assessing the effect of this program on the recovery of locomotion kinematics. MATERIALS AND METHODS Animals Thirty-six adult female Wistar rats, weighing 250-300 g (Charles River, Les Oncins, France), were housed in smoothbottomed plastic cages at 22°C with a 12-h light-dark cycle. Food (Purina rat chow) and water were available ad libitmn. Anesthesia and surgical procedures were performed according to the French law on animal care guidelines, and the Animal Care Committee of University Aix-Marseille [I and Centre National de la Recherche Scientifique approved our protocols. Furthermore, experiments have been carried out in accordance with the European Community's council directive of November 24, 1986 (86/609/EEC). No signs of screech, prostration, hyperactivity, anorexia, and no paw-eating behavior were observed through the experiment. Experimental Protocol Rats were randomized into four groups: (i) control group (C, n = 10) without any surgery; (ii) lesion groups (LI, n = 10 and L3, n = 9) in which neuromuscular measurements were made I and 3 wk after MCL transection, respectively; and (iii) eccentric group (ECC, n = 7) in which rats performed a progressive eccentric rehabilitation program froom weeks I to 3 after MCL transection. Thus, the effect of the rehabilitation program was determined: 1) in awake animals by measuring the maximal and minimal knee angles during the stance phase of the gait cycle on weeks I and 3; and 2) in anesthetized animals by measuring neuromuscular parameter 3-4 d after the last eccentric session. These measurements included (a) measurement of the H-reflex modulation in response to a 10-ranM KC1 injection known to activate metabosensitive afferent fibers (39,42), (b) analysis of the twitch relaxation properties of the quadriceps muscle, (c) recording of metabosensitive afferent activity in response to injection of various activating agents into the right femoral artery toward the contralateral muscle, and (d) recording of mechanosensitive fibers' response to tendon patellar vibrations. MCL Transection The methodological technique to induce MCL transection was already described in a previous study (23). Briefly, under anesthesia with chloral hydrate (Sigma-Aldrich Chimie

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SARL, Saint-Quentin Fallavier, France; 60 mg.kg- 1), the left knee MCL was isolated from the surrounding tissues with a I-cm medial skin and fascia incision without damaging the MCL. This ligament was then sectioned with small scissors without damaging the quadriceps muscle. Blood and nerve supplies were also kept intact. Finally, skin incision was sutured (Flexocrin 3-0; B. Braun Medical, Boulogne, France), and the animals were allowed to recover without hind limb immobilization in individual cages. The duration of total surgery was 15-20 min. In our previous study, we have already demonstrated that this surgical technique has no effect on our measured parameters, allowing us to conclude that the measured effects were due to the MCL injury itself and/or to the rehabilitation program (23). Eccentric Rehabilitation Program During eccentric sessions, animals were positioned in supine position. After a very light intramuscular injection (0.15 mL) of a mixture of 5 mL of ketamine (Virbac, Carros, France) and 2.5 rnL of largactil (Avensis, Paris, France), the body and the left hind limb were firmly held on a metallic support. Knee joint movements were imposed by a custommade ergometer as previously described (27). Muscle contractions were exclusively induced during each lengthening (eccentric) phases by a constant current stimulator (Multiprocess 16+, Physitech; Electronique M6dicale, Marseille, France) that delivered stimulation trains (frequency = 10 Hz, duration = 400 lis, contraction time = 2 s) through a pair of surface electrodes (electrodes ECG universal; Contr6le Graphique M6dical, Brie-Comte-Robert, France) fixed on the belly of the quadriceps muscle. In accordance to the recommendations of several studies (9), an early mobilization of the injured joint and a gradual increase in resistance exercise after ligament lesion were applied in the present study to be closer as possible to the protocol in use in physical therapy. Indeed, this rehabilitation program consisted of three eccentric sessions per week during a 2-wk period. This program began 1 wk after MCL trarsection. Each eccentric session was characterized by eight sets of 10 repetitions with 2 rmin of rest between each set. As in physical therapy, the stimulation intensity was determined from motor threshold (MT; i.e., stimulation intensity corresponding to the first observed quadriceps muscle response). Then, the intensity was increased by 0.1 x MT at each session from 1.5 x MT (during the first session) to 2 x MT (during the last session). Eccentric exercise was induced by imposing a 60' flexion of knee extensor muscles (from 1600 to 1000) at a speed of 20O.s ' during the first week of rehabilitation. Then, the range of motion was increased of 200 (from 180' to 1000) during the last three eccentric sessions. Each session lasted 20 rmin. Dynamic Functional Assessment of Hind Limb Recovery The functional recovery of left knee angle was evaluated in the locomotor glass lane, on weeks I and 3 after the MCL

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transection using an automatic analysis software (SimiMotion software; Simi, Unterschleissheim, Germany) associated with a numerical camcorder (MV 830i; Canon, Courbevoie, France). The latter was positioned perpendicular to the vertical plane of the chamber to get a sagittal view of the gait cycles. The glass walking track was 150 cm long, 9 cm wide, and 40 cm high. Heavy lighting was provided with two 500-W spotlights. Before recording, the lower back and the left hind limb of the rat were shaved. The hip joint (trochanter), the knee joint (condylus femoralis), the ankle joint (lateralis malleoIns), and the fifth metatarsal head were marked on the leg operated on with a black permanent marker. Rats were placed at one end of the glass lane, and a dark box was positioned at the other end to attract the rat. A stimulating noise and a strong light were applied to induce their locomotor activity. Such induced locomotion pattern was recorded with a 50-Hz acquisition frequency using SimiMotion software (Simi). On the basis of the video analysis, the system computed the different angle joints during the stance phase of the gait cycle. The start of the stance phase was considered to be the point where the foot touches the ground, and the end was considered to be the point where the foot leaves the ground to begin the swing phase. To ensure that the gait cycle was reliable, all rats performed the locomotor exercise in a daily familiarization session I wk before surgery. Maximal and minimal knee angles were averaged on three steps during the stance phase. The two-dimensional positions of the anatomical markers were tracked on each frame. Kinematic data were smoothed using a cubic smoothing spline procedure. The measured gait parameter was the knee joint angle defined as the intersection between the lines extended from the hip to the knee joint and the line from the knee joint to the ankle joint. On the basis of the position of the toe marker, the gait cycles were divided into a stance and a swing phase. The maximum and minfinum angles during the stance phase were computed within the three gait cycles and were kept for further analysis. In animals from the group C, maximum and minimum knee angles were recorded on weeks I and 3. Angles obtained from this group were compared with the angles obtained from the other groups (LI, L3, and ECC). Processing of the gait parameters was performed using MATLAB software (version 7.5.0 342 R2007b; MathWorks, Natick, MA). Surgery for Neuromuscular Measurements At I (LI group) and 3 wk (L3 and ECC groups) after the MCL transection, rats were anesthetized by an intramuscular injection of a solution containing a ternary mixture (5 mL of ketamine (Virbac), 2.5 mL of largactil (Avensis), 2 mL of domitor (Novartis, Mississauga, ON); 0.1 mL-100 g-1 body weight). Such mixture composed mainly by ketamine is well known to have no effect on H-reflex activity (15). Central temperature was maintained constant (around 380 C) with a homeothermic blanket (Harvard Apparatus, Holliston, MA) driven by a rectal thermal probe. Rats were tracheotomized, cannulated, and artificially ventilated (Harvard volumetric

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pump: rate of 40-60 rnin-1, tidal volume of 2-4 mL; Southlnatick, Holliston, MA). A catheter was inserted into

the right femoral artery and pushed up to the fork of the abdominal aorta to transport the chemnicals (i.e., KCI and LA) to the contralateral muscle (see below for recording nerve activity). This catheter was positioned to let the blood flow freely to the muscles of the left lower limb. To verify that the level of anesthesia remained constant throughout the operative procedure, the latter was assessed continually by monitoring the blood pressure and the HR with an electromanometer (model P23Db; Stathan, Hato Rey, Puerto Rico). Finally, the left femoral nerve was dissected fiee from surrounding tissues at a length of 2-2.5 cm. Animals were positioned in the supine position during all neuromuscular measurements. The knee and ankle were firmly held by clamps on a horizontal support to avoid disturbing movements and to maintain the 1000 knee joint angle during electrical nerve stimulations. H-reflex in Response to KCI Injection Single rectangular shocks were applied directly to the intact nerve using a pair of steel electrodes with a I-ms pulse generated by a constant current stimulator (Digitimer DS7 A; Welwin Garden City, Hertfordshire, UK). Stimulation electrodes were positioned on the surface of the nerve and located at 2-4 mm of the nerve insertion in the muscle. In accordance to a previous study (4), the stimulation frequency was fixed at 0.1 Hz to abolish the postactivation depression during the consecutive H-reflexes recording. H-reflex was recorded using bipolar needle electrodes (29-gauge; AD Instruments, MLA 1204 Needle Electrodes, 2-mm pin, Colorado Springs, CO) inserted in the belly of the vastus medialis muscle without any change in the location of the electrodes during the overall recordings. Furthermore, because muscle length is known to influence H-reflex amplitude, all potentials evoked were recorded for a given knee angle (i.e., 1000). The reflex signal was referred to a ground electrode implanted in an inert tissue, amplified, and filtered (2 k; 30 Hz to 10 kHz) with a differential amplifier (P2MP SARL, Marseille, France). The signal was recorded (Biopac MP 150 and AcqKnowledge software; Biopac System, Goleta, CA) and displayed on a coinputer using a data acquisition system (Biopac AcqKnowledge software; Biopac System) whose sample frequency was set at 2 kliz. Thus, M-wave and H-reflex were recorded in the vastus medialis muscle after femoral nerve stimulation. The stabilized maximal M-wave (M0,,) and H-reflex ( ) amplitudes were determined by incrementally increasing stimulation intensity (by 0.01-mA increments) from 0 mA until there was no further increase in wave amplitude. Stimulation intensity was fixed to obtain Hrn• and then fixed to obtain M,,,•. Then, two separate series of repetitive stimulations (interstimulus interval = 10 s) were performed in a randomized order and were separated by 15 miniof rest. One series was delivered at the H,a. intensity, and the second intensity. Associated twitches series was fixed at the M,4. were simultaneously recorded during the two series of stim-

REHABILITATION AFTER LIGAMENT TRANSECTION

ulation. The temporal profile of H,,/M,,a ratios was determined in the following experimental condition: six repetitive H-reflexes were elicited before the 10-mM KC1 (0.5 mL) injection, which corresponded to the reference ratio. Then, 15 consecutive H-reflexes were evoked immediately after the 10-i-M KCI (0.5 mL) injection. This KC1 injection process was also performed duri.ng the series of stimulation fixed at M,,,,, intensity. Each injection required 5-8 s to be completed. M- and H-waves were identified by measuring their latencies. The latter was defined as the time between the stimulus artifact to the apogee of the positive peak of first (M) and second (H) waves, respectively. Latencies were expressed in milliseconds. Each peak-to-peak amplitude of the H ax was normalized with the corresponding peak-topeak amplitude of the M,,,. in the train of stimulation to calculate the Hna,,IM.a ratio. After the KC1 injection, five pools of three consecutive reflex responses were averaged and compared with the reference reflex response (i.e., the first six averaged reflexes) to extract the peak of H-reflex variation. Moreover, Ma,, amplitude was also measured in the same manner as the Hi,na/Mmn,, ratio for several reasons: 1) to analyze a potential influence of groups III and IV metabosensitive afferents activation on M-wave and 2) to test whether the M-wave response was consistent across the experiment. Changes in the Hmax/Mniax ratio and Mmax amplitude are expressed in percentage of the corresponding reference values. Twitches associated with H-reflexes were analyzed in the same way. Finally, the H-reflex recovery was analyzed as follows: the three last averaged Hmax/M,m,.,, ratios (from the 13th to the 15th H-reflex response) were compared with the corresponding reference ratio (i.e., the first six H,,,a./M,,,, ratios). Twitch Measurement To measure quadriceps isometric force, a wire connected with a strain gauge (Microdynamometer S 60; Ugo Basile Narco Biosystem, Houston, TX) was fixed around and perpendicularly to the ankle. In these conditions, the knee angle was maintained in an isometric position at 100' during the femoral nerve stimulation. The contractile response of the quadriceps to femoral nerve stimulation was evoked with a neurostimulator (Digitimer DS7 A; Welwin Garden City, UK) that delivered single rectangular shocks (duration = 0.1 ins, frequency = 0.2 Hz). The current intensity used to evoke maximal twitch amplitude (from the beginning of the curve to the peak) was detemrained. Four electrical stimulations were perfonmed to be averaged. Contractions were recorded with Biopac MP] 50 system (sampled at 2000 Hz, filtered with low pass at 150 Hz) and analyzed with Biopac AcqKnowledge 3.9 software (Biopac System). Twitch measures were analyzed in terms of 1) maximal amplitude (A); 2) half relaxation time (HRT: time interval expressed in milliseconds between peak twitch tension and the half of the descending portion of the relaxation curve); and 3) the maximum relaxation rate (MNRR), defined as the slope of a tangent drawn to the steepest portion of the relaxation curve from the peak amplitude, was

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normalized to A (MRR/A: mean relaxation rate constant, per millisecond), as suggested by Esau et al. (7) who have shown that MRR values are linearly related to A. Femoral Nerve Recordings As previously described by Laurin et al. (23), the proximal portion of femoral nerve was cut to exclude efferent discharges during nerve recordings. To analyze the response of the metabosensitive afferent fibers, the femoral nerve was positioned on a pair of bipolar tungsten electrode and immersed in paraffin oil. The neural signals were referred to a ground electrode implanted in a nearby tissue, amplified (10-100 K) and filtered (30-10 kHz) with a differential amplifier (P2MP SARL). The afferent discharge was recorded (Biopac MPI_50 and AcqKnowledge software; Biopac System) and fed into pulse window discriminators (P2MP SARL) that simultaneously analyzed afferent populations. The output of these discriminators, providing noise-free tracings (discriminated units), was displayed on a computer using a data acquisition system (Biopac AcqKnowledge software; Biopac System) at 1-s intervals (Hz). The discriminated units were counted and recorded on separate tracings. Metabosensitive response to chemical injections. Before injecting the chemical stimuli, a baseline recording was achieved to ensure that the discharge rate (Fimp,,1es) was stable. Baseline discharges were also recorded between chemical injections. The impulse activity was recorded without chemical injection during 180 s, and the recording was considered available only if the fluctuation of baseline impulse activity ranged between 100% and 103%. Consequently, variation of firing rate was related only to the stimuli applied and not to the environmental conditions. Once these resting levels of activity were established and stable, varying amounts of KCI (10 and 20 mM) and LA (1 and 2 mM) were randomly injected into the artery. We selected those concentrations because, in a previous study (5), we have shown that the change in discharge rate was significantly related to the doses of KCI (1, 5, 10, and 20 mM), whereas the activation by LA (0.5, 1, 2, and 3 rmM) culminated at the lowest concentrations (bell-shaped curve). Thus, we used physiological concentrations (42) where afferents' discharge was maximal. It is worth noting that the injected chemical agents in this investigation did not elicit muscle contraction as indicated by the recorded muscle tension. Thus, no neuromuscular blocking agent was used during afferent fibers recording. In accordance to Rybicki et al, (39), who have shown that the KCI concentration remained elevated in the muscle during 5-10 min after injection, there was a 10-rain delay between each injection to let the afferent activity go back to its baseline activity and to ensure that the KCI concentrations returned to their basal level. The poststimulus discharge firing rate was compared with the baseline discharge rate, and variations (AFimpuiscs) were expressed

in percentage of the corresponding baseline discharge firing rate, that is, baseline discharges corresponded to 100%. Analysis of the recorded afferent firing rate was performed

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using a specific MATLAB program (version 7.5.0 342 R2007b; MathWorks). Indeed, the discharge firing rate was measured on 20-s periods all along the recording period. On each 20-s period, the afferents' firing rate was calculated on different time intervals lasting from I to 20 s and was averaged between them. This process excluded any influence of the different time intervals used to calculate the variation in afferent firing rate. Thus, the MATLAB programn calculated the temporal profile of discharge rate variation and extracted the peak discharge rate after each injection. Baseline afferent discharge was analyzed in an 80-s period preceding each injection. Afterward, the postinjection 20-s period of peak discharge rate was compared with the corresponding baseline firing rate. Mechanosensitive response to tendon vibrations. Muscle tendon vibrations in the range of 10-100 Hz are known to activate muscle mechanosensitive fibers without activating muscle metabosensitive fibers. Low-frequency mechanical vibrations seem to preferentially activate static spindle afferents and perhaps also Golgi tendons organs, whereas high-frequency vibrations are well-known stimuli for dynamic spindle afferents (1). Thus, a mechanosensitive afferent discharge differed during one frequency vibration versus another. After a 15-min recovery period after injections, rectangular mechanical shocks were delivered perpendicularly to the longitudinal muscle axis on patellar tendon by a commercially available vibrator (Ling Dynamic System; LDS Group, Hertfordshire, UK) driven by a frequency generator (GenTrad Function Generator GF763AF; ELC, Annecy, France). Vibrations were applied for 5-s periods. The vibration frequency was increased step-by-step from 10 to 100 Hz while the discharge of afferent fibers was recorded. The maximal mechanosensitive afferents' discharge rate elicited by tendon vibrations was considered as the reference discharge rate (100%). The discharge rate (Finipulses) induced by the other frequencies of vibrations was expressed in percentage of the corresponding reference discharge rate. At the end of the experiment, rats were killed by an intraarterial pentobarbital overdose. Statistical Analysis All values are expressed in means ± SEM. Data processing was performed using statistical software (Statistica; StatSoft, Tulsa, OK). For all measurements, we used a one-way ANOVA completed with a Newman-Keuls post hoc. We used this statistical test to make intergroup comparisons in each measured variable (maximum and minimum knee angles, Hmax,Mr,ax, M,a. amplitude, MRR/A, HRT, or F'impuises). Differences were considered significant when P < 0.05. RESULTS Functional Assessment of Hind Limb Recovery The results showed a significant main effect (F = 6.808, P = 0.0005). Indeed, the maximum knee angle was significantly decreased (100.9' ± 2.00; -6.2%, P < 0,001) 1 wk

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after MCL rupture (LI group) compared with the C group (107.50 ± 2.20). No difference was observed between the C group and the two other groups (L3 and ECC). Likewise, no difference was observed between the ECC group and the LI or L3 groups (Fig. 1A). As shown in Figure lB, minimum knee angle was also significantly decreased (73.40 + 1.70; -6.8%, P < 0.05) 1 wk after the ligament lesion (LI group) compared with the C group (78.8' ± 2.30). Figure 1B also exhibits that there was no difference between the C group and the two other groups (L3 and ECC), and no difference was also observed between the ECC group and the LI or L3 group.

Changes in

Hmax/Mmax

Ratio after Metabosensitive

Afferent Fibers Activation

A 0.60

0 Control o Li

H_ax/M_,x ratio

L3 U El ECC

0.50+ 0.40 0.30 0.20-0.10

0.00

B

The mean latencies of the M- and H-waves were measured at 1.4 ± 0.1 and 4.3 ± 0.2 ms for the C group, at 1.3 ±0.1 and 4.2 + 0.2 ms for the LI group, at 1.3 ± 0.1 and 4.1 + 0.2 ms for the L3 group, and at 1.3 ± 0.1 and 3.6 ± 0.2 ms for the ECC group, respectively. No difference was observed in Hila/M,,,ay ratio between groups (Fig. 2A) before chemical injection. However, the results concerning the H-reflex

05 -5'

-101+ -15 + -20 + -25 +

A

7

1

-30+III T Maximum Knee Angle (degree) 109

Control M oUL M L3

ElECC

107 105 103

-35-L H.../M... (% of the corresponding reference ratio)

FIGURE 2-Changes in Umax/Mrnx ratio after MCL rupture. A, Before the 10-mM KCI injection. B, After the 10-mM KCI injection. The peak variation of H,a_IM,x ratio is expressed in percentage of the

corresponding reference ratio. Asterisks indicate a significant difference in injured groups (Li, L3, and ECC) compared with C group (*P < 0.05, **P < 0.01).

101 99 97 95

B 88 - Maxinum Knee Angle (degree) 8684828078 76" 74"

response to this injection showed a significant main effect (F = 3.166, P = 0.03). After the 10-mM KCI injection, the decrease in peak variation of the H,,/Mmax ratio was significantly higher in the C group (-23.1% ± 4.9%) compared with those in the other groups during the activation of metabosensitive afferent fibers (LI = -3.6% ± 3.8%, P < 0.01; L3 = -5.0% ± 3.1%, P < 0.01; ECC G -7.8% ± 3.2%, P < 0.05) (Fig. 2B). In all groups, the H,,a,/Mm, ratio was fully recovered at the end of the recording period (recovery period, i.e., 3 wk after injury) compared with that during the initial period (corresponding to the reference ratio; data not shown). Changes in

Mmax

Amplitude

72 7068 66 FIGURE I-Time course of knee angle during the stance phase of the gait cycle during 3 wk. A, Maximal angle. Asterisks indicate a significantly lower angle in the injured ligament LI group compared with the C group (***P < 0.001). B, Minimal angle. The asterisk indicates significant differences between injured ligament LI group and C group (*P < 0.05).

REHABILITATION AFTER LIGAMENT TRANSECTION

Figure 3 shows that the KCI injection did not alter the M,,a amplitude in each group, suggesting that the H-reflex recording conditions were similar always. Muscular Relaxation Properties The results showed a significant main group effect (F = 4.690, P = 0.007). As shown in Figure 4, the MRR/A

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A

4.

"114'T A

2

... .........

0

(% baseline activity) L3 E2ECC El

112t not

+

-2 108+

-4

_T_

I

106+

-6 4104 +

P ControlI

-8 M1iYo, (% of the corresponding reference amplitude)

-10

3 L3 IDECC

102 + IO -L10mM

FIGURE 3-Peak variation of Ml,as after MCL rupture. The peak variation of jimf., data was analyzed in the same way as the peak var-

20mM

B

iation of the I]m.,/Mo,o, ratio.

114-T

ratio results exhibited a significant decrease in LI (28.0 ± 1.5 is-1; - 19.4%, P < 0.05) and L3 (26.4 ± 1.3 ms- ; -23.8%, P < 0.05) when compared with the C group (34.7 ± 2.3 ms-1). There was no significant difference between the ECC and C groups. The results showed a significant main effect (F = 3.357, P = 0.029). Regarding FtRT, no significant difference between LI and C groups was reported (Fig. 4). Furthermore, when compared with the C group (15.0 + 1.0 ins), FtRT was increased in the L3 group (19.3 ± 0.9 ins; +29%, P < 0.05). When the ECC group was compared with the C group, no significant difference was shown.

AFi 0 ,,v•, (% baseline activity)

r÷

112+ 110+ I 1084+ 106+ 1044+

_I

1024+ t0ooI I mM

2 mM

FIGURE -5--Metabosensitive fiber responses to chemical injections. A,

KCI injections (10 and 20 mM). Asterisks indicate a significant attenuation in the LI group compared with the C group (*P < 0.05). The cross indicates a significantly higher response in the ECC group compared

Response of Metabosensitive Fibers to Chemical Injections For the 10-mM KCt injection, our results showed a significant main effect (F = 7.174, P = 0.0009) except for the 20-mM KCI injection. A significant main effect was also shown (F = 6.199, P = 0.002) for the l-rmM LA injection only. KCI injections. In Figure 5A, the variation of the metabosensitive afferents' discharge after the 10-maM KCI injection 4 0

T

U2HRT(nas)I Ia MRR/A Ratio

r 35 ./¢

30 25

-U1

20t 15

-

10

Control

LI

L3

ECC

FIGURE 4-Time course of absolute values of the MRR/A ratio and HRT. The asterisk indicates a significantly lower ratio in the Ll and L3 groups compared with the C group (*P < 0.05). The cross indicates a significantly higher value in the L3 group compared with the C group (+P < 0.05).

1038

Official Journal of the American College of Sports Medicine

with the LI group (+P < 0.05). B, LA injections (I and 2 nmM). The

asterisk indicates a significant attenuation in the LI group compared with the C group (*P < 0.05). The cross indicates a significantly higher response in the ECC group compared with the LI group (+P < 0.05).

was significantly decreased in the LI group (101.8% ± 0.7%) compared with the C group (108.6% ± L 1%, P < 0.01) and the ECC group (107.6% ± 1.5%, P < 0.05). No significant difference was observed between the LI and L3 groups. For the 20-mM KCI injection, there was no significant difference between groups. No differences were observed between the ECC group and the C group and between the ECC group and the L3 group. LA injections. As seen in Figure 5B, the decrease of metabosensitive afferents' discharge variation after the 1-mM LA injection was significant (P < 0.01) in the L1 group (102.8% ± 0.7%) compared with that in the C gr'oup (108.2% ± 1.4%). No difference was observed among the C, L3, and ECC groups. Furthermore, a significant (P < 0.05) response was found after the 1-mM LA injection for ECC group (110.4% ± 1.8%) compared with the Ll group, but no significant difference was observed between the Li and L3 groups. No significant difference was observed for the 2-rmM LA injection between groups. No differences were observed between the ECC group and the C group and between the ECC group and the L3 group.

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Response of Mechanosensitive Fibers to Patellar Tendon Vibrations The results showed a significant main group effect (F = 3.501, P < 0.001). Significant differences were observed in low-frequency vibrations when the C group was compared with the LI group at 10 Hz (P < 0.05) and at 40 Hz (P< 0.05) as shown in Figure 6. No significant difference was observed when we compared the C group with the L3 or the ECC group. Comparison between ECC and LI groups exhibited significant differences at 10 Hz (P