Long-Term Effects of Botulinum Toxin Complex Type A Injection on Mechano- and Metabo-Sensitive Afferent Fibers Originating from Gastrocnemius Muscle

RESEARCH ARTICLE Long-Term Effects of Botulinum Toxin Complex Type A Injection on Mechano- and Metabo-Sensitive Afferent Fibers Originating from Gastrocnemius Muscle Guillaume Caron, Tanguy Marqueste, Patrick Decherchi* Aix-Marseille Université (AMU) and Centre National de la Recherche Scientifique (CNRS), UMR 7287, Institut des Sciences du Mouvement: Etienne-Jules MAREY (ISM-EJM), Equipe, Plasticité des Systèmes Nerveux et Musculaire, Parc Scientifique et Technologique de Luminy, Faculté des Sciences du Sport de Marseille, CC910 - 163 Avenue de Luminy, F-13288, Marseille, cedex 09, France * Abstract OPEN ACCESS Citation: Caron G, Marqueste T, Decherchi P (2015) Long-Term Effects of Botulinum Toxin Complex Type A Injection on Mechano- and Metabo-Sensitive Afferent Fibers Originating from Gastrocnemius Muscle. PLoS ONE 10(10): e0140439. doi:10.1371/ journal.pone.0140439 Editor: Michel R. Popoff, Institute Pasteur, FRANCE Received: April 20, 2015 Accepted: September 25, 2015 Published: October 20, 2015 Copyright: © 2015 Caron et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The aim of the present study was to investigate long term effects of motor denervation by botulinum toxin complex type A (BoNT/A) from Clostridium Botulinum, on the afferent fibers originating from the gastrocnemius muscle of rats. Animals were divided in 2 experimental groups: 1) untreated animals acting as control and 2) treated animals in which the toxin was injected in the left muscle, the latter being itself divided into 3 subgroups according to their locomotor recovery with the help of a test based on footprint measurements of walking rats: i) no recovery (B0), ii) 50% recovery (B50) and iii) full recovery (B100). Then, muscle properties, metabosensitive afferent fiber responses to potassium chloride (KCl) and lactic acid injections and Electrically-Induced Fatigue (EIF), and mechanosensitive responses to tendon vibrations were measured. At the end of the experiment, rats were killed and the toxin injected muscles were weighted. After toxin injection, we observed a complete paralysis associated to a loss of force to muscle stimulation and a significant muscle atrophy, and a return to baseline when the animals recover. The response to fatigue was only decreased in the B0 group. The responses to KCl injections were only altered in the B100 groups while responses to lactic acid were altered in the 3 injected groups. Finally, our results indicated that neurotoxin altered the biphasic pattern of response of the mechanosensitive fiber to tendon vibrations in the B0 and B50 groups. These results indicated that neurotoxin injection induces muscle afferent activity alterations that persist and even worsen when the muscle has recovered his motor activity. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by funding from Aix-Marseille Université (AMU) and the Centre National de la Recherche Scientifique (CNRS). Introduction Competing Interests: The authors have declared that no competing interests exist. Botulinum toxin complex type A (BoNT/A) is currently used to treat numerous medical conditions such as dystonia, neuromuscular disorders or pain. Its effects start between 2 and 5 days PLOS ONE | DOI:10.1371/journal.pone.0140439 October 20, 2015 1 / 20 Afferent Discharges and Botulinum Toxin Type A after injections and are maintained between 3 and 6 months [1]. BoNT/A exerts its action by preventing the exocytosis of acetylcholine vesicles at the neuromuscular junction eliciting flaccid paralysis [2,3]. Furthermore, BoNT/A induces central alterations [4–6] such as inhibition of glutamate [7], substance P [8,9], calcitonin gene related peptide (CGRP) [9,10] and to a lesser extend gamma-aminobutyric acid (GABA) release [11]. Those central changes could be due to indirect action of the toxin (decrease of the postsynaptic element activation following the decrease in presynaptic element activation) or to the toxin retrograde transport to the spinal cord (SC) and transcytosis [12–14]. BoNT/A also prevents acetylcholine releases by γ-motor endings in intrafusal muscle fibers [15,16]. The lack of γ-motor endings discharge in the injected muscle induce an intrafusal muscle fiber relaxation and then a decrease of afferent (Ia and II) inputs originating from spindles. At the spinal level, these changes reduce the direct excitation of agonist motoneurons and the indirect inhibition of antagonist motoneurons leading to a larger relaxation of agonist muscle (BoNT/A treated)[17]. Thus, the BoNT/A alters the central adjustments by mechanosensitive (Ia and II from muscle spindle and Ib from Golgi tendon organ)[18] and metabosensitive (III and IV) muscle afferent fibers [19]. Ia afferents detect muscle length and velocity while II afferents are mainly sensitive to instantaneous changes in muscle length [18,20]. Ib afferents are sensitive to forces variations [21–23]. Muscle afferent fibers from groups III and IV detect change in muscle metabolism [24,25] and in intramuscular pressure [26]. They are selectively stimulated during and after muscle fatigue [27] or by different agents such as bradykinin, capsaicin [28], lactic acid, H+ [25,29], arachidonic acid, prostaglandin [25], thromboxane A2 [30] and potassium chloride [31,32]. In a recent paper, we show that BoNT/A induces alterations in mechano- and metabosensitive afferent fibers when the muscle is to its paralysis apogee [19]. However, data are missing when the toxin is degraded and during muscle recovery. The main purpose of the present study was to measure, over recovery time, the effects of gastrocnemius BoNT/A injection on afferent fibers involved in the sensorimotor loop. The muscle afferent discharges from groups III and IV were recorded after direct electrical muscle stimulation inducing fatigue and intra-arterial injections of potassium chloride or lactic acid while the mechanosentive afferents discharges were recorded after tendon vibrations. The muscle properties (muscle weight, tetanus threshold, twitch amplitude and Fatigue Index) were also measured. Materials and Methods 1. Animals Twenty seven adult male Sprague Dawley rats, weighting 300–400g (Centre d’Elevage Roger Janvier1, Le Genest Saint Isle, France), were housed in smooth-bottomed plastic cages at 22°C with a 12-h light/dark cycle. Food (Safe1, Augy, France) and water were available ad libitum. An acclimation period of 1 week was allowed before the initiation of the experiment. Animals were randomized in 2 experimental groups: 1) untreated animals acting as control (Control, n = 6) and 2) treated animals in which the toxin was injected in the left muscle, the latter being itself divided into 3 subgroups ac (...truncated)