Rafael Fortuna

Changes in contractile properties of muscles receiving repeat injections of botulinum toxin (Botox)

Botulinum toxin type A (BTX-A) is a frequently used therapeutic tool to denervate muscles in the treatment of neuromuscular disorders. Although considered safe by the US Food and Drug Administration, BTX-A can produce adverse effects in target and non-target muscles. With an increased use of BTX-A for neuromuscular disorders, the effects of repeat injections of BTX-A on strength, muscle mass and structure need to be known. Therefore, the purpose of this study was to investigate the changes in strength, muscle mass and contractile material in New Zealand White (NZW) rabbits. Twenty NZW rabbits were divided into 4 groups: control and 1, 3 and 6 months of unilateral, repeat injections of BTX-A into the quadriceps femoris. Outcome measures included knee extensor torque, muscle mass and the percentage of contractile material in the quadriceps muscles of the target and non-injected contralateral hindlimbs. Strength in the injected muscles was reduced by 88%, 89% and 95% in the 1, 3 and 6 months BTX-A injected hindlimbs compared to controls. Muscle mass was reduced by 50%, 42% and 31% for the vastus lateralis (VL), rectus femoris (RF) and vastus medialis (VM), respectively, at 1 month, by 68%, 51% and 50% at 3 months and by 76%, 44% and 13% at 6 months. The percentage of contractile material was reduced for the 3 and 6 months animals to 80-64%, respectively, and was replaced primarily by fat. Similar, but less pronounced results were also observed for the quadriceps muscles of the contralateral hindlimbs, suggesting that repeat BTX-A injections cause muscle atrophy and loss of contractile tissue in target muscles and also in non-target muscles that are far removed from the injection site.

Long-term repetitive mechanical loading of the knee joint by in vivo muscle stimulation accelerates cartilage degeneration and increases chondrocyte death in a rabbit model

BACKGROUND Excessive chronic loading is thought to be one factor responsible for the onset of osteoarthritis. For example, studies using treadmill running have shown an increased risk for osteoarthritis, thereby suggesting that muscle-induced joint loading may play a role in osteoarthritis onset and progression. However, in these studies, muscle-induced loading was not carefully quantified. Here, we present a model of controlled muscular loading which allows for the accurate quantification of joint loading. The aim of this study was to evaluate the effects of long-term, cyclic, isometric and dynamic, muscle-induced joint loading of physiologic magnitude but excessive intensity on cartilage integrity and cell viability in the rabbit knee. METHODS 24 rabbits were divided into an (i) eccentric, (ii) concentric, or (iii) isometric knee extensor contraction group (50 min of cyclic, submaximal stimulation 3 times/week for four weeks=19,500 cycles) controlled by the stimulation of a femoral nerve cuff electrode on the right hind limb. The contralateral knee was used as a non-loaded control. The knee articular cartilages were analysed by confocal microscopy for chondrocyte death, and histologically for Mankin Score, cartilage thickness and cell density. FINDINGS All loaded knees had significantly increased cell death rates and Mankin Scores compared to the non-loaded joints. Cartilage thicknesses did not systematically differ between loaded and control joints. INTERPRETATION Chondrocyte death and Mankin Scores were significantly increased in the loaded joints, thereby linking muscular exercise of physiologic magnitude but excessive intensity to cartilage degeneration and cell death in the rabbit knee.

In vivo Sarcomere Lengths and Sarcomere Elongations Are Not Uniform across an Intact Muscle

Sarcomere lengths have been a crucial outcome measure for understanding and explaining basic muscle properties and muscle function. Sarcomere lengths for a given muscle are typically measured at a single spot, often in the mid-belly of the muscle, and at a given muscle length. It is then assumed implicitly that the sarcomere length measured at this single spot represents the sarcomere lengths at other locations within the muscle, and force-length, force-velocity, and power-velocity properties of muscles are often implied based on these single sarcomere length measurements. Although, intuitively appealing, this assumption is yet to be supported by systematic evidence. The objective of this study was to measure sarcomere lengths at defined locations along and across an intact muscle, at different muscle lengths. Using second harmonic generation (SHG) imaging technique, sarcomere patterns in passive mouse tibialis anterior (TA) were imaged in a non-contact manner at five selected locations (“proximal,” “distal,” “middle,” “medial,” and “lateral” TA sites) and at three different lengths encompassing the anatomical range of motion of the TA. We showed that sarcomere lengths varied substantially within small regions of the muscle and also for different sites across the entire TA. Also, sarcomere elongations with muscle lengthening were non-uniform across the muscle, with the highest sarcomere stretches occurring near the myotendinous junction. We conclude that muscle mechanics derived from sarcomere length measured from a small region of a muscle may not well-represent the sarcomere length and associated functional properties of the entire muscle.

Do skeletal muscle properties recover following repeat onabotulinum toxin A injections

Onabotulinum toxin A (BTX-A) is a frequently used treatment modality to relax spastic muscles by preventing acetylcholine release at the motor nerve endings. Although considered safe, previous studies have shown that BTX-A injections cause muscle atrophy and deterioration in target and non-target muscles. Ideally, muscles should fully recover following BTX-A treatments, so that muscle strength and performance are not affected in the long-term. However, systematic, long-term data on the recovery of muscles exposed to BTX-A treatments are not available, thus practice guidelines on the frequency and duration of BTX-A injections, and associated recovery protocols, are based on clinical experience with little evidence-based information. Therefore, the purpose of this study was to investigate muscle recovery following a six months, monthly BTX-A injection (3.5 U/kg) protocol. Twenty seven skeletally mature NZW rabbits were divided into 5 groups: Control (n=5), zero month recovery - BTX-A+0M (n=5), one month recovery - BTX-A+1M (n=5), three months recovery - BTX-A+3M (n=5), and six months recovery - BTX-A+6M (n=7). Knee extensor strength, muscle mass and percent contractile material in injected and contralateral non-injected muscles was measured at each point of recovery. Strength and muscle mass were partially and completely recovered in injected and contralateral non-injected muscles for BTX-A+6M group animals, respectively. The percent of contractile material partially recovered in the injected, but did not recover in the contralateral non-injected muscles. We conclude from these results that neither target nor non-target muscles fully recover within six months of a BTX-A treatment protocol and that clinical studies on muscle recovery should be pursued.

Effects of Russian current and low-frequency pulsed current on discomfort level and current amplitude at 10% maximal knee extensor torque

Background: Low-frequency pulsed current (LFPC) and Russian current (RC), a type of kilohertz-frequency alternating current, have been frequently used for muscle strengthening in rehabilitation programs. Despite the popularity of these current types, it is unclear which is most effectively able to generate a similar submaximal torque with minimal discomfort and current amplitude. Objective: To compare discomfort and current amplitude between LFPC and RC to achieve a knee extensor torque of 10% of the maximal isometric voluntary contraction (MIVC). Methods: Twenty-two healthy subjects were subjected to three electrically elicited knee extensor submaximal contractions (10% MIVC) that were sustained for 10 seconds. The current amplitude required to achieve 10% MIVC and subjective discomfort were assessed directly by the electrical stimulator and with the Visual Analogue Pain Scale, respectively. A paired t-test was used to determine differences between the electrical currents (α = 0.05). Results: LFPC required significantly lower current amplitude (15%) and a lower discomfort level (50%) to achieve 10% of MIVC compared to RC. Conclusion: LFPC current seems to be more effective than RC with respect to discomfort level and current amplitude to produce 10% of MIVC.

The influence of cyclic concentric and eccentric submaximal muscle loading on cell viability in the rabbit knee joint

BACKGROUND Cartilage loading is associated with the onset and progression of osteoarthritis and cell death may play an important role in these processes. Although much is known about cell death in joint impact loading, there is no information on joints loaded by muscular contractions. The aim of this study was to evaluate the influence of muscle generated eccentric and concentric submaximal joint loading on chondrocyte viability. We hypothesised that eccentric muscle activation leads to increased cell death rates compared to concentric loading and to controls. METHODS 16 rabbits received either 50 min of uni-lateral, cyclic eccentric (n=8) or concentric (n=8) knee loading. Muscle activation for these dynamic conditions was equivalent to an activation level that produced 20% of maximum isometric force. Contralateral joints served as unloaded controls. Cell viability was assessed using confocal microscopy. FINDINGS Eccentric contractions produced greater knee loading than concentric contractions. Sub-maximal contractions caused a significant increase in cell death in the loaded knees compared to the unloaded controls, and eccentric loading caused significantly more cell death than concentric loading. INTERPRETATION Cyclic sub-maximal muscle loading of the knee caused increased chondrocyte death in rabbits. These findings suggest that low levels of joint loading for prolonged periods, as occurs in endurance exercise or physical labour, may cause chondrocyte death, thereby predisposing joints to degeneration.

Altered cell metabolism in tissues of the knee joint in a rabbit model of Botulinum toxin A‐induced quadriceps muscle weakness

Quadriceps muscle weakness is frequently associated with knee injuries in sports. The influence of quadriceps weakness on knee joint homeostasis remains undefined. We hypothesized that quadriceps weakness will lead to tissue‐specific alterations in the cell metabolism of tissues of the knee. Quadriceps weakness was induced with repetitive injections of Botulinum toxin A in six 1‐year‐old New Zealand White rabbits for 6 months. Five additional animals served as controls with injections of saline/dextrose. Muscle weakness was assessed by muscle wet mass, isometric knee extensor torque, and histological morphology analysis. Cell metabolism was assessed for patellar tendon, medial and lateral collateral ligament, and medial and lateral meniscus by measuring the total RNA levels and specific mRNA levels for collagen I, collagen III, MMP‐1, MMP‐3, MMP‐13, TGF‐β, biglycan, IL‐1, and bFGF by reverse transcription and polymerase chain reaction. While the total RNA levels did not change, tissue‐specific mRNA levels were lower for relevant anabolic and catabolic molecules, indicating potential changes in tissue mechanical set points. Quadriceps weakness may lead to adaptations in knee joint tissue cell metabolism by altering a subset of anabolic and catabolic mRNA levels corresponding to a new functional and metabolic set point for the knee that may contribute to the high injury rate of athletes with muscle weakness.

A clinically relevant BTX-A injection protocol leads to persistent weakness, contractile material loss, and an altered mRNA expression phenotype in rabbit quadriceps muscles

Botulinum toxin type-A (BTX-A) injections have become a common treatment modality for patients suffering from muscle spasticity. Despite its benefits, BTX-A treatments have been associated with adverse effects on target muscles. Currently, application of BTX-A is largely based on clinical experience, and research quantifying muscle structure following BTX-A treatment has not been performed systematically. The purpose of this study was to evaluate strength, muscle mass, and contractile material six months following a single or repeated (2 and 3) BTX-A injections into the quadriceps femoris of New Zealand white rabbits. Twenty three skeletally mature rabbits were divided into four groups: experimental group rabbits received 1, 2, or 3 injections at intervals of 3 months (1-BTX-A, 2-BTX-A, 3-BTX-A, respectively) while control group rabbits received volume-matched saline injections. Knee extensor strength, quadriceps muscle mass, and quadriceps contractile material of the experimental group rabbits were expressed as a percentage change relative to the control group rabbits. One-way ANOVA was used to determine group differences in outcome measures (α=0.05). Muscle strength and contractile material were significantly reduced in experimental compared to control group rabbits but did not differ between experimental groups. Muscle mass was the same in experimental BTX-A and control group rabbits. We concluded from these results that muscle strength and contractile material do not fully recover within six months of BTX-A treatment.

Residual force enhancement following shortening is speed-dependent

The steady-state isometric force following active muscle shortening or lengthening is smaller (force depression; FD) or greater (residual force enhancement; RFE) than a purely isometric contraction at the corresponding length. The mechanisms underlying these phenomena are not explained within the context of the cross-bridge theory and are rarely studied in concert. Previous studies have shown RFE to be speed-independent. In the present study, we investigated if RFE preceded by active shortening is time-dependent by electrically evoking RFE in the human adductor pollicis muscle. The results shown that a slow stretch following FD fully re-established RFE compared to higher speeds of stretch. The mechanism(s) responsible for the recovery of RFE following a preceding shortening contraction (FD) might be associated with the recovery of cross-bridge based force and/or the re-engagement of a passive structural element (titin). Voluntary interaction with one’s environment involves highly coordinated shortening and lengthening muscle contractions. Therefore comprehending these history-dependent muscle properties in the context of movement control is paramount in understanding the behavior of in vivo motor control.

The effects of electrical stimulation exercise on muscles injected with botulinum toxin type-A (botox)

Botulinum toxin type A (BTX-A) is a frequently used treatment modality for a variety of neuromuscular disorders. It acts by preventing acetylcholine release at the motor nerve endings, inducing muscle paralysis. Although considered safe, studies suggest that BTX-A injections create adverse effects on target and non-target muscles. We speculate that these adverse effects are reduced by direct electrical stimulation (ES) exercising of muscles. The aims were to determine the effects of ES exercise on strength, mass, and contractile material in BTX-A injected muscles, and to investigate if BTX-A injections affect non-target muscles. Seventeen New Zealand White (NZW) rabbits were divided into three groups: (1) Control group received saline injections; (2) BTX-A group received monthly BTX-A (3.5 U/kg) injections into the quadriceps for six months and (3) BTX-A+ES group received monthly BTX-A injections and ES exercise three times a week for six months. Outcome measures included knee extensor torque, muscle mass, and contractile material percentage area in injected and contralateral, non-injected quadriceps. Glycogen depletion and direct muscle stimulation were used to assess possible muscle inhibition in non-injected quadriceps. ES exercise partially prevented muscle weakness, atrophy, and contractile material loss in injected muscles, and mostly prevented muscle degeneration in contralateral, non-injected muscles. Non-injected muscles of BTX-A+ES group showed higher force with direct muscle compared to nerve stimulation, and retained glycogen following the depletion protocol, suggesting that BTX-A inhibited activation in non-target muscles. We conclude that ES exercise provides some protection from degeneration to target and non-target muscles during BTX-A treatments.