Sveučilište u Zagrebu Medicinski fakultet

Project team members

Ivica Matak, Research Associate, PhD (Department of Pharmacology, University of Zagreb School of Medicine, Zagreb, Croatia)

Petra Šoštarić, PhD candidate, dr. vet. med. (Department of Pharmacology, University of Zagreb School of Medicine, Zagreb, Croatia)

Višnja Drinovac Vlah, PhD, (Department of Pharmacology, University of Zagreb School of Pharmacy)

Ana Dugonjić Okroša. MPharm (Department of Pharmacology, University of Zagreb School of Pharmacy)

Collaborators and their roles:

Prof. Lidija Bach-Rojecky, PhD Department of Pharmacology, University of Zagreb School of Pharmacy and Biochemistry. Advices related to project coordination and implementation.

Prof. Zdravko Lacković, MD, PhD, the head and founder of Laboratory of Molecular Neuropharmacology (retired), University of Zagreb School of Medicine. Advices related to project coordination and implementation.

Dr. sc. Nikola Habek (MD) (Laboratory of Neurophysiology and “whole cell patch-clamp”, School of Medicine, University of Zagreb), advisor on introducing an experimental setup for electromyography, co-operation on electrophysiological experiments on cerebral cuts (not included in objectives and activities for implementation due to high risk for implementation).

Prof. dr. sc. Mario Cifrek and doc. dr.sc. Željka Lučev Vasić (Department of Electronic Systems and Information Processing, Faculty of Electrical Engineering and Computing, University of Zagreb), advices on introducing an experimental setup for electromyography, possible collaboration on joint setup construction and further collaboration.

External collaborators:

assist. prof. Ornella Rossetto, PhD; Marco Pirazzini, PhD, Research associate; Federico Fabris, M.Biotechnol / PhD candidate. Investigation of central effects of botulinum toxin serotypes other than type A botulinum toxin.

Botulinum toxin type A enzymatic activity (cleaved SNAP-25, green) in ventral horn following Its injection into the rat calf muscle.

Clostridial neurotoxins such as tetanus toxins (TeNT) and botulinum toxins serotypes A-G (BoNT/A-BoNT/G) are one of the most potent biological toxins, and causative agents of tetanus and botulism. Low therapeutic doses of BoNT/A injected into local muscles (in order of picograms to few nanograms) are used to treat hyperkinetic movement disorders and spasticity. Our preliminary studies indicate that the important part of its effect on abnormally increased muscle tone might occur in the central nervous system (CNS). Proposed project involves characterization of new preclinical models of hyperkinetic movement disorders, and characterization of the central effects of BoNT/A. The aim of present studies is to induce the imbalance of excitatory and inhibitory neurotransmission of motor regions with very low doses of TeNT and BoNT/A applied into the peripheral muscle, or into certain regions of the central nervous system. This w ill imitate hyperkinesia and hypertonia present in spasticity or dystonia. In animals w ith hyperkinesia, the effects of botulinum toxins will be characterized in extrafusal and intrafusal motor fibers, as well as central neurons of the ventral horn and brain stem. Central activity of BoNT/A will be prevented by employing intrathecally applied BoNT/A-neutralizing anti-toxin. Behavioral 2D and 3D video analysis of walking in normal and hyperkinetic animals, as well as motor tests of balance, fatigue, and the joint resistance to passive movement will be used to study the BoNT/A effects. The toxin effects will also be characterized by electromyography of spinal reflexes, and analysis of expression of neurotransmitter and proteins involved in motor neurotransmission. In the research fields of Neurology and Neuroscience, the expected scientific results are expected to advance the preclinical study of new therapeutic approaches to the treatment of movement disorders, the basic knowledge about the plasticity of the motor system, as well as the mechanisms of action of clostridial neurotoxins in the CNS.

The project is focusing on the research of effect of clostridial neurotoxins in motor nervous system, and attempts to develop new experimental models of hyperkinetic movement disorders such as dystonia and spasticity. These disorders are characterized by prolonged or intermittent unwanted muscular contractions that cause unwanted movements and abnormal position of the body and limbs. They are common movement disorders, with great influence on the quality of life. Up to now, the most important pharmacological treatment of dystonia and one of the most important therapeutic options for spasticity are botulinum toxin type A (BoNT/A) injections into hyperactive individual muscles or muscle groups. It is considered that one of the most important therapeutic effects of botulinum toxins is the blockage of cholinergic neuromuscular transmission leading to flaccid paralysis. However, the effects of botulinum toxin can hardly be explained solely by such action. In humans it was shown that botulinum toxin can act on activity or the excitability of motoneurons that innervate distant muscle groups. The time of onset or occurrence of the peak beneficial effect in the symptoms reduction does not accompany the time course of muscle flaccid paralysis. It has been suggested that, besides the neuromuscular junctions, BoNT/A could also act on the spinal reflex system involving gamma motor neurons and Ia muscular adherents that innervate the muscular spindle, and Ib muscular afferents innervating the Golgi’s tendon organ. However, the role of action on stretch reflex elements cannot explain all the effects of botulinum toxins, especially the effects at distant muscles or muscle groups (that cannot be explained by the toxin diffusion), and the duration of useful effect which often outlasts the duration of muscle weakness caused by primary flaccid paralysis.

-In our previous studies, by examining the BoNT/A enzymatic activity in the CNS, we found that low doses of botulinum toxin, depending on axonal transport, are transported via peripheral nerves to the ventral horn of the spinal cord, specifically to cholinergic neurons surrounding the motor neurons. Our recent study has shown that the administration of BoNT/A – specific antitoxin into the cranial cerebrospinal space reduces the toxin’s enzyme activity in the facial nucleus when the toxin was applied to facial whisker pad muscles. These observations have demonstrated that the enzyme activity of BoNT/A in the motor part of the CNS should take place in the second order synapses by the trans-synaptic transport process. However, the role of botulinum toxin activity in the CNS on normal or hyperactive muscular tone, and the therapeutic role of such effect in the treatment of movement disorders is currently unknown. Our findings indicate that botulinum toxin type A centrally transported directly from the injected nerve reduces muscle hypertonia induced by intramuscular tetanus toxin administration (Matak, 2020). These findings indicate that an important part of its effect on abnormally increased muscle tone may occur in the central nervous system. This finding suggests that botulinum toxin in the CS may act on the imbalance between excitation and inhibition of transmission at different levels of the CNS. This disbalance is also the main mechanism for the development of muscular tone and hyperactivity disorders in many movement disorders such as spasticity and dystonia.

– The main expected results of the project are to identify the main therapeutic sites of BoNT/A action after peripheral application in the muscle, to identify possible central actions on individual behavioral and electrophysiological parameters as well as muscle atrophy, in order to provide a more complete picture of the mode of action of BoNT/A and possible side effects after therapeutic application.

– Incorporating new animal models of movement disorders that better represent the basic etiopathogenesis of neurological disorders characterized by muscular hyperactivity. The research activities included in the project will lead to the design of new animal models of muscular hyperactivity based on the modeling of neuronal hyperactivity in the various motor regions of the CNS and their behavioral and biochemical characterization.

– Elucidating the mechanisms of existing effective medicines using new models, with the possibility of identifying new targets for drugs (receptors and synapses in the CNS that they might act upon). In animal models of muscular hyperactivity, the effect of anticholinergics (botulinum toxins, antimuscarinic drugs) will be investigated, whose mechanism of action is currently unclear.

Toxins 2021 virtual meeting, held on 16^th of January 2021 (Accepted abstract)

LONG-TERM EFFECTS OF BOTULINUM NEUROTOXIN TYPE A ON EXPERIMENTAL MUSCLE HYPERTONIA

Ivica Matak^*, Petra Šoštarić.

Laboratory of Molecular Neuropharmacology, Department of Pharmacology, University of Zagreb School of Medicine, Zagreb, Croatia

*Corresponding author: Laboratory of Molecular Neuropharmacology, Department of Pharmacology, University of Zagreb School of Medicine, Zagreb, Salata 11, 10000 Zagreb, Croatia.
E-mail address:  ivica.matak@mef.hr (I. Matak).

Introduction and Objectives: Long-term effects of botulinum neurotoxin type A (BoNT-A) are the basis of its beneficial effects on neurological disorders characterized by muscle hyperactivity, such as spasticity and dystonias. Clinical reports suggest that BoNT-A–mediated normalization of muscle hypertonia does not necessarily correlate with or outlast the duration of muscular flaccid paralysis. The present aim was to reassess these clinical observations in a preclinical model of muscle hypertonia by characterizing the duration of toxin anti-spastic activity in relation to the duration of its muscular effects.

Methods: In male Wistar rats, the muscle hypertonia was evoked by tetanus neurotoxin (TeNT) injection into the gastrocnemius (1.75-2 ng total dose). After the development of spastic hypertonia, BoNT-A was similarly injected into the ipsilateral gastrocnemius muscle 7 days post-TeNT (1, 2 and 5 U/kg//20 µL). The effects of TeNT and BoNT-A were further examined by measurements of resistance to passive ankle dorsiflexion, digit abduction score (DAS), and Basso-Beattie-Bresnahan (BBB) Locomotor Rating Scale. Muscle atrophy was quantified by measurement of the medio-lateral calf diameter. After complete recovery of BoNT-A–mediated peripheral flaccid paralysis, the animals were re-injected with TeNT (day 49 after BoNT-A injection) and assessed behaviorally for another 19 days.

Results: In the immediate period following the first TeNT injection, all BoNT-A doses employed exhibited a similar reduction of the muscle hypertonia. The TeNT-evoked muscle hypertonia ended by day 28 post-TeNT, while the remaining BoNT-A–mediated flaccid paralysis recovered in a dose-dependent manner by day 42 post–BoNT-A. After the second TeNT injection, the anti-spastic activity of BoNT-A was still present up to day 68; however, it was less prominent in rats injected with the lowest BoNT-A (1 U/kg) dose. The level of persisting muscle atrophy, which did not show signs of recovery by the end of the experiment, was similar at all doses employed.

Conclusions: In line with clinical reports, we found that the antispastic effect of BoNT-A in rats persists after the lower limb recovery from flaccid paralysis in a dose-dependent manner. Long-term actions of BoNT-A on muscle hypertonia might be mediated by a mechanism distinct from its local muscular paralytic action.

Funding: Croatian Science Foundation (project ID: UIP-2019-04-8277)

Keywords: Botulinum neurotoxin type A; Flaccid paralysis; Muscle atrophy;
Muscle hypertonia; Tetanus neurotoxin