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. 2019 Nov 6;177(1):65–76. doi: 10.1111/bph.14846

Evidence for central antispastic effect of botulinum toxin type A

Ivica Matak 1,✉
PMCID: PMC6976784  PMID: 31444910

Abstract

Background and Purpose

Botulinum toxin type A (BoNT/A) injections into hyperactive muscles provide effective treatment for spasticity and dystonias, presumably due to its local effects on extrafusal and intrafusal motor fibres. A recent discovery of toxin's retrograde axonal transport to CNS might suggest additional action sites. However, in comparison to cholinergic peripheral terminals, functional consequences of BoNT/A direct central action on abnormally increased muscle tone are presently unknown. To address this question, the central effects of BoNT/A were assessed in experimental local spastic paralysis.

Experimental Approach

Local spastic paralysis was induced by injection of tetanus toxin (1.5 ng) into rat gastrocnemius. Subsequently, BoNT/A (5 U·kg−1) was applied i.m. into the spastic muscle or intraneurally (i.n.) into the sciatic nerve to mimic the action of axonally transported toxin. Functional role of BoNT/A transcytosis in spinal cord was evaluated by lumbar i.t. application of BoNT/A‐neutralizing antitoxin. BoNT/A effects were studied by behavioural motor assessment and cleaved synaptosomal‐associated protein 25 (SNAP‐25) immunohistochemistry.

Key Results

Tetanus toxin evoked muscular spasm (sustained rigid hind paw extension and resistance to passive ankle flexion). Subsequent injections of BoNT/A, i.m. or i.n, reduced tetanus toxin‐evoked spastic paralysis. Beneficial effects of i.n. BoNT/A and occurrence of cleaved SNAP‐25 in ventral horn were prevented by i.t. antitoxin.

Conclusions and Implications

Axonally transported BoNT/A relieves muscle hypertonia induced by tetanus toxin, following the trans‐synaptic movement of BoNT/A in the CNS. These results suggest that such direct, centrally mediated reduction of abnormal muscle tone might contribute to the effectiveness of BoNT/A in spasticity and hyperkinetic movement disorders.

What is already known

  • Supposedly, botulinum toxin A (BoNT/A) relieves spasticity and hyperkinesias due to its peripheral muscular actions.

  • In animals, BoNT/A enzymatic activity was detected in central motor regions after peripheral injections of BoNT/A.

What this study adds

  • In rats, BoNT/A reduced local spastic paralysis by direct action in the CNS.

  • These effects of BoNT/A were dependent on toxin's retrograde axonal transport and transcytosis within the ventral horn.

What is the clinical significance

  • BoNT/A efficacy in spasticity and movement disorders might involve central reduction of abnormal muscle tone.

Abbreviations

BoNT/A

botulinum toxin type A

DAS

digit abduction score

i.n.

intraneural

SNAP‐25

synaptosomal‐associated protein 25

TeNT

tetanus toxin

1. INTRODUCTION

Botulinum toxin (BoNT) serotypes A–G, potent neurotoxins from Clostridium botulinum, inhibit synaptic neurotransmitter release by proteolytic cleavage of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor proteins involved in Ca2+‐dependent neuroexocytosis (Pirazzini, Rossetto, Eleopra, & Montecucco, 2017). Serotype A (BoNT/A) is applied i.m. in low doses for long‐lasting reduction of abnormal skeletal muscle tone and/or hyperkinesia in hyperkinetic movement disorders and spasticity. It is approved for blepharospasm, hemifacial spasm, oromandibular and cervical dystonias, and upper and lower limb spasticity (Jankovic, 2017; Safarpour & Jabbari, 2018). Currently, it is believed that its beneficial effects in motor disorders are mediated entirely by local muscular neuroparalysis of both extrafusal and intrafusal cholinergic nerve endings, with indirect normalization of spinal reflexes and central processing of movement (Kaňovský & Rosales, 2011). However, based on immunodetection of BoNT/A‐truncated synaptosomal‐associated protein of 25 kDa (SNAP‐25), rodent studies demonstrated that enzymically active BoNT/A is axonally transported from injected muscle to the motor nuclei of brainstem and spinal cord (Antonucci, Rossi, Gianfranceschi, Rossetto, & Caleo, 2008; Caleo et al., 2018; Koizumi et al., 2014; Matak, Riederer, & Lacković, 2012; Restani et al., 2012). While the central action of BoNT/A is causally involved in its antinociceptive activity (Bach‐Rojecky & Lacković, 2009; Matak, Bach‐Rojecky, Filipović, & Lacković, 2011), the functional role of direct central activity of the toxin on normal motor function or abnormal muscle tone has not been investigated up to now.

Muscle hypertonia and hyperkinesia in dystonia and spasticity involves impaired balance between excitatory and inhibitory inputs to α‐motor neurons, and the pharmacological treatment aimed at controlling the excitatory drive or increasing the inhibitory transmission at the spinal cord level reduces their symptoms (Hallett, 2011; Kita & Goodkin, 2000). Tetanus toxin (TeNT), a homologue of the BoNTs, exerts spastic paralysis by blocking the synaptic inhibitory input to α‐motor neurons (Matthews, Fishman, & Wittenberg, 2014; Restani et al., 2012). Quantification of local spastic paralysis induced by low doses of TeNT, given i.m., might be used to study the effects of antispastic drugs (Kutschenko et al., 2012), although this possibility has not been examined so far. Here, we have used local spastic paralysis of the gastrocnemius muscle, induced by TeNT, in conscious rats to examine the possible central effects of BoNT/A on abnormal muscle tone.

2. METHODS

2.1. Animals

All animal care and experimental procedures were conducted in accordance with the European Union Directive 2010/63/EU and approved by the institutional review board (University of Zagreb School of Medicine) and Croatian Ministry of Agriculture ethical committees (permit no. EP 24‐2/2015). Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology (Curtis et al., 2018). Male Wistar Han rats (University of Zagreb School of Medicine, Croatia), 3–4 months old, 300–400 g weight, kept on 12‐hr/12‐hr light and dark cycle, three animals per cage with free access to food and water, were used in all experiments.

2.2. Pharmacological treatment

For i.m. injections, rats were anaesthetized with https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4233/https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=523 (70/7 mg·kg−1 i.p.). By employing 0‐ to 50‐μl luer tip Hamilton syringe coupled to 0.45 mm × 16 mm disposable needles, rats were percutaneously injected with 20 μl of neurotoxin divided into two injection sites (10 μl each site) into lateral and medial bellies of the right gastrocnemius. The doses of TeNT (1.5 ng) or BoNT/A (5 U·kg−1) were chosen based on previously used non‐systemic doses (Cui, Khanijou, Rubino, & Aoki, 2004; Matak et al., 2012; Matthews et al., 2014) and preliminary experiments.

For intraneural (i.n.) injection of BoNT/A or saline, a lateral skin incision was made on the right thigh and the sciatic nerve was blunt dissected through the muscles of anaesthetized animals. BoNT/A (5 U·kg−1, 2 μl) was slowly injected into the nerve trunk with a 0‐ to 10‐μl Hamilton syringe needle (Cat. No. #701, Hamilton, Bonadouz, Switzerland), as previously described (Bach‐Rojecky & Lacković, 2009; Matak et al., 2012).

In experiments examining the effect of the https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=242&familyId=26&familyType=GPCR receptor agonist R(+) https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1084, 3 mg·kg−1 dose was injected i.p. to conscious, briefly restrained animals. The rats were then returned to their home cages prior to behavioural measurement. The dose was chosen based on previously employed non‐sedative doses (Lorrai, Maccioni, Gessa, & Colombo, 2016).

In experiments examining the transcytosis of BoNT/A, the animals were anaesthetized with ketamine/xylazine and administered i.t. with 20 IU of BoNT/A antitoxin (National Institute for Biological Standards and Control, Potters Bar, UK) or equal volumes (20 μl) of normal horse serum (Gibco, ThermoFisher Scientific, Waltham, MA, USA). The injection was performed by employing luer tip Hamilton syringe coupled to a 26‐G sterile disposable needle, as described previously (Bach‐Rojecky & Lacković, 2009). A correct site of injection between the lumbar vertebrae was confirmed by the brief movement of rat tail and/or leg after the needle entered into the vertebral canal at the level of the cauda equina. The dose of BoNT/A antiserum was chosen based on our previous study (Caleo et al., 2018).

2.3. Behavioural motor tests

2.3.1. Resistance to passive ankle flexion

To assess the magnitude of TeNT‐evoked local rigidity, the rats were lifted by the experimenter's hand wrapped around the rat waist, and the ankle joint was flexed by pressing the hind paw interdigital pad area against a rectangular plastic platform (1.5 × 4 × 4 cm) mounted on the flat metal surface of zeroed digital scale. When a dorsiflexion with tibiotarsal angle of 90° was reached, the pressure exerted by experimenter was slowly relieved, and the resistance value in grams (g) was noted just before further elevation of the animal increased the tibiotarsal angle above 90°. After the rats were accustomed to handling for three separate training sessions, the resistance measured at 90° dorsiflexion was consistently low (around 10–20 g). Based on the preliminary testing of spastic animals, the cut‐off value was set to 150 g to prevent pain and discomfort. Two trials were made per measurement session and the mean value calculated.

2.3.2. Digit abduction score

To assess local spastic paralysis of the hind limb, the toe spreading reflex was measured upon lifting the animal from the ground by holding it around the waist. The reflex was quantified according to a semi‐quantitative scale, termed digit abduction score (DAS; Broide et al., 2013). The DAS scale is defined as 0 = separation of all toes; 1 = separation of four toes; 2 = separation of three toes; 3 = separation of two toes; and 4 = no toe separation.

2.3.3. Narrow beam walking

The animals were trained to walk across an elevated horizontal narrow beam (2.5 cm × 2.5 × 100 cm, elevated 50 cm above the ground), connecting a rectangular platform (10 × 10 cm) exposed to lamp light and a box‐like dark platform (25 × 25 cm) with narrow entrance (10 × 10 cm), as described by Carter, Morton, and Dunnett (2001). The beam was marked at 10‐cm distance from both platforms, and the transit time between the markings was measured (80‐cm distance). Two trials per measurement were made and the mean value calculated. Measurements were repeated if animal stumbled or stopped during the transit.

2.3.4. Rota‐rod

The animals were trained to maintain balance on a rota‐rod device with 8‐cm‐diameter rod rotating at 10 r.p.m. The cut‐off time (latency to fall) was set to 120 s. Two trials were made per measurement session and the mean value calculated.

2.4. Experimental protocol

Experimental design adhered to the BJP guidelines (Curtis et al., 2018). In all experiments, animals were assigned randomly into different experimental groups by using block randomization. The experimenter who performed the motor tests was blinded to the animal treatment. However, a particular visual appearance of hind limbs attributable to a certain treatment (e.g., hind paw extension in TeNT‐treated animals and limp appearance of the hind paw in BoNT/A i.m.‐treated animals) could not be masked during the animal manipulation.

At the end of all experiments, the animals were killed by deep ketamine/xylazine anaesthesia followed by transcardial perfusion with saline and buffered 4% paraformaldehyde fixative, or deep anaesthesia followed by decapitation.

2.4.1. Experiment 1: Effect of i.m. BoNT/A on TeNT‐induced calf muscle spasticity

The animals were injected into the gastrocnemius muscle with vehicle or TeNT (1.5 ng). Saline or BoNT/A (5 U·kg−1) was injected ipsilaterally into the gastrocnemius muscle on Day 7 after injection of vehicle/TeNT. Motor behaviour was measured on Days 0, 7 (prior to saline/BoNT/A), 8, 10, and 13 post‐TeNT. Number of animals per group = 6 in control groups (vehicle + saline, vehicle + BoNT/A) and seven in experimental spastic groups (TeNT + saline, TeNT + BoNT/A).

2.4.2. Experiment 2: Effect of i.n. BoNT/A injection into the sciatic nerve on TeNT‐induced spasticity

Vehicle or TeNT‐treated animals (1.5 ng i.m.) were injected i.n. with BoNT/A (5 U·kg−1 in 2 μl) 7 days post‐TeNT. Motor parameters were measured on Days 0 (prior to vehicle/TeNT), 1, 3, 7 (prior to saline/BoNT/A), 10, and 13 post‐TeNT. Number of animals per group = 7 in experimental control groups (vehicle + saline, vehicle + BoNT/A) and nine in experimental spastic groups (TeNT + saline, TeNT + BoNT/A).

2.4.3. Experiment 3: Effect of baclofen on TeNT‐induced spasticity

Vehicle or TeNT‐treated animals (1.5 ng i.m.) were injected with i.p. (R+) baclofen (3 mg·kg−1) on Day 7 post‐TeNT. Motor behaviour was measured prior to saline/baclofen and 1 hr post‐baclofen. Number of animals per group = 7 in experimental control groups (vehicle + saline, vehicle + baclofen) and nine in experimental spastic groups (TeNT + saline, TeNT + baclofen).

2.4.4. Experiment 4: Role of transcytosis in the effect of intrasciatic BoNT/A on TeNT‐induced spasticity

TeNT‐treated animals (1.5 ng i.m.) were injected i.n. with BoNT/A (5 U·kg−1/2 μl) 7 days post‐TeNT. The next day, 20–24 hr after BoNT/A, animals were injected i.t. with BoNT/A antiserum. Motor parameters were measured on Days 0 (prior to vehicle/TeNT), 1, 3, 7 (prior to saline/BoNT/A), 8 (prior to horse serum/antitoxin), 10, 12, and 14 post‐TeNT. Number of animals per group = 7 in experimental control groups (vehicle + saline, vehicle + baclofen) and nine in experimental spastic groups (TeNT + BoNT/A + horse serum, TeNT + BoNT/A + antitoxin).

2.4.5. Experiment 5: Examination of BoNT/A transcytosis in spinal cord after BoNT/A i.m. injection

In five animals, BoNT/A (5 U·kg−1) was injected into the left gastrocnemius, and 4 days after, they were injected with another 5 U·kg−1 injection into the right gastrocnemius. One day after second BoNT/A injection, animals were treated with BoNT/A antiserum, given i.t.. The animals were kept for a further 14 days, until they were killed by anaesthetic overdose followed by saline/fixative perfusion.

2.5. Cleaved SNAP‐25 immunohistochemistry

Animals from experiments no. 4 and 5 were killed by anaesthetic overdose followed by tissue‐fixation perfusion. Lumbar spinal cords were removed and cryoprotected in sucrose (15% in fixative for 1 day and 30% in PBS for 2 days). Spinal cord coronal sections (35 μm) were cut on a freezing microtome and transferred to PBS‐filled wells for free floating immunohistochemical staining.

The antibody‐based procedures used here comply with the recommendations made by the British Journal of Pharmacology for reporting of Western blot or immunohistochemical studies (Alexander et al., 2018). Following inactivation of endogenous peroxidase and blocking in 10% normal goat serum, the sections were incubated with 1:2,000 rabbit polyclonal IgG antibody raised against the C‐terminal peptide of BoNT/A‐cleaved SNAP‐25 (provided by Prof. Ornella Rossetto, University of Padua, Italy) overnight at room temperature. The antibody binds specifically to BoNT/A‐cleaved SNAP‐25 and not the intact SNAP‐25 (Matak et al., 2011). The next day, slices were processed with Alexa Fluor 555 Tyramide Superboost Kit (cat no. B40923; Invitrogen, Carlsbad, CA, USA), which contains the HRP‐conjugated goat anti‐rabbit secondary antibody and Tyramide‐Alexa 555 substrate, according to the manufacturer's instructions. Controls with omitted primary antibody showed no specific staining, suggesting the lack of non‐specific binding of secondary antibody and/or Tyramide reagent. Sections were washed with PBS, mounted on glass slides with anti‐fading agent (Fluorogel, Electron Microscopy Sciences, Hatfield, PA, USA), and visualized with fluorescence microscope (Olympus BX51, Olympus, Tokyo, Japan) coupled to digital camera (Olympus DP70) and equipped with cellSens Dimension visualizing and quantification software (Olympus RRID:SCR_014551). Images shown in the figures were composed and processed for brightness and contrast using Adobe Photoshop Elements 9 (Adobe Systems, San Jose, CA, USA, RRID:SCR_014199). The average area of immunoreactivity (IR) to cleaved SNAP‐25 for a single animal was quantified based on three randomly chosen L4 slices. High magnification images (20×, obtained by employing 40× objective and 0.5× camera adapter) were used under constant low exposure time (18 ms) for lower background. The area covered by cleaved SNAP‐25 IR was quantified by employing constant manual threshold range (117–256) of red channel pixel intensity in all images. In a single slice, cleaved SNAP‐25 IR was quantified and summated in three non‐overlapping visual fields located in the lateral parts of ventral horn grey matter (3 × 0.14 mm2). Upon analysis, the images were coded for blinding to the animal treatment.

2.6. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). Results are presented as mean ± SEM and analysed by two‐way ANOVA for repeated measurements, followed by Bonferroni's post hoc test (P < .05 considered significant). The post hoc tests were performed only if F was significant (P < .05) and there was no variance inhomogeneity. Non‐parametric data (DAS) are presented as medians and analysed by Kruskal–Wallis non‐parametric one‐way ANOVA, followed by Dunn's multiple comparison post hoc test (P < .05 was considered significant). The cleaved SNAP‐25 IR data were analysed by non‐parametric Mann–Whitney t‐test (two group comparison) or Kruskal–Wallis non‐parametric one‐way ANOVA, followed by Dunn's multiple comparison post hoc test (P < .05 was considered significant). GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA, RRID:SCR_002798) was used for statistical analyses and graph drawing. The sample size and minimal number of animals per treatment group was predetermined based on preliminary experiments to predict the effect size for different measurements and according to a priori power analysis using free statistical software (G*power version 3.1., University of Düsseldorf, Germany, RRID:SCR_013726; Charan & Kantharia, 2013). The number of animals per group was calculated to be five or six based on estimated effect size of F = 0.4, α error probability = .05, power (1‐β) = 0.9, statistical test: ANOVA: repeated measures, within‐between interaction. The possibility of attrition due to various reasons was also taken into account, and a higher number of animals per group than required by the power analysis was used in all experiments. However, there was no loss of animals or data exclusion from analysis in experiments. Due to expected large differences in measured parameters between control and spastic treatments, a smaller number of rats was used in control non‐spastic groups to reduce the total number of animals used.

2.7. Materials

Lyophilized TeNT (Sigma Aldrich, St Louis, MO, USA) was reconstituted in 0.9% saline vehicle containing 2% BSA (Sigma Aldrich), stored in concentrated aliquots on −80°C, and further diluted with vehicle to obtain the necessary concentration for i.m. injections. Lyophilized BoNT/A (Botox®, Allergan, Irvine, CA, USA) was reconstituted in physiological saline. Baclofen (R(+) Baclofen; Sigma Aldrich) was dissolved in saline to obtain the required dose. Lyophilized polyclonal equine IgG‐based BoNT/A antitoxin (Botulinum type A antitoxin from National Institute for Biological Standards and Control, NIBSC code 14/174, Potters Bar, UK, validated by Li, Matoo, & Keller, 2012) was reconstituted in 0.9% saline to 1,000 IU·ml−1 concentration.

2.8. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Christopoulos et al., 2017; Alexander, Kelly et al., 2017).

3. RESULTS

3.1. Effect of i.m. and intrasciatic BoNT/A on TeNT‐evoked spastic paralysis

In conscious animals, TeNT injection into the gastrocnemius muscle induced unilateral rigid extension of the hind paw. This resulted in unilateral increased resistance to ankle dorsiflexion and impaired narrow beam and rota‐rod latencies. These deficits started to develop at Day 3 post‐TeNT injection.

Injection of BoNT/A (5 U·kg−1 i.m.) into the gastrocnemius of spastic animals gradually reduced the ankle flexion resistance from Day 1 to Day 3 post‐BoNT/A. Thereafter, the ankle flexion resistance remained low (similar to control non‐spastic group), until the end of experiment (Figure 1a). Toxin injection into the sciatic nerve reduced the resistance to passive ankle flexion, as well. The effect was evident at Day 3 and remained similarly low, thereafter, until the end of observation period (Figure 1b). The antispastic drug R(+) baclofen reduced the ankle flexion resistance 60 min after systemic i.p. treatment, as well (Figure 1c).

Figure 1.

Figure 1

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Botulinum toxin type A (BoNT/A) and baclofen reduce the hind limb rigidity evoked by TeNT i.m. injection. TeNT (1.5 ng, i.m.)‐evoked resistance to ankle dorsiflexion (achieving 90° tibiotarsal angle) is reduced by BoNT/A i.m. injection into gastrocnemius (a) and intraneural injection (i.n.) into sciatic nerve (b). TeNT‐evoked increase in force required for ankle flexion is also reduced by intraperitoneal (i.p.) injection of spasmolytic drug R(+) baclofen (3 mg·kg−1) (c). BoNT/A (5 U·kg−1) significantly impairs the digit abduction score after i.m. injection (d), but not after i.n. injection (e). The DAS was measured 7 days after i.m. or i.n. BoNT/A and 14 days after TeNT i.m. injection. Veh; vehicle; sal, saline i.m. treatment; dotted horizontal line indicates time points of TeNT i.m., BoNT/A (i.m./i.n.), and baclofen (i.p.) treatments. (a–c) Mean ± SEM, * P < .05, significantly different from veh + sal, + P < .05, significantly different from TeNT + sal ; two‐way RM ANOVA followed by Bonferroni's post hoc test. (d,e) Data are represented as individual values and median (horizontal line) + P< .05, significantly different from veh + sal; one‐way non‐parametric ANOVA Kruskal–Wallis, followed by Dunn's post hoc test

In i.m. BoNT/A‐treated animals, unilateral flaccid paralysis was evident as impaired toe spreading reflex, assessed by DAS (Figure 1d). In contrast to that, BoNT/A i.n. did not significantly impair the DAS (Figure 1e).

BoNT/A or R(+) baclofen exerted no observable benefits in beam walking or rota‐rod latencies (Figure 2a,c). Similarly, BoNT/A i.n. did not improve beam walking, although it did slightly improve the rota‐rod performance at days 3 and 5 post‐BoNT/A treatment (Figure 2b). In control, non‐spastic animals, neither i.m. nor i.n. BoNT/A or R(+) baclofen impaired rota‐rod or beam walking performances, excluding the presence of any systemic effects of BoNT/A and sedative effects of R(+) baclofen.

Figure 2.

Figure 2

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Effects of BoNT/A and baclofen on TeNT (1.5 ng i.m.)‐induced functional motor deficits; i.m. BoNT/A (5 U·kg−1, i.m.) and intraperitoneal baclofen do not affect TeNT‐evoked impairment in beam walking and rota‐rod latencies (a and c). Intraneural BoNT/A (5 U·kg−1, i.n.) does not improve TeNT‐evoked impairment in beam walking latency, but it slightly improves rota‐rod latency (b) Veh, vehicle i.m. treatment; sal, saline treatment; vertical lines indicate time point of TeNT, BoNT/A, or baclofen treatments; mean ± SEM, * P < .05, significantly different from veh + sal, + P < .05, significantly different from TeNT + sal; two‐way RM ANOVA followed by Bonferroni's post hoc test

3.2. The antispastic effect of i.n. BoNT/A is prevented by i.t. antitoxin treatment

BoNT/A‐mediated benefits were expressed as a gradual reduction of ankle flexion resistance, starting 2 days following its i.n. injection (Figure 3b), resulting in a markedly improved voluntary use of the hind limb during standing and walking (Video S1). The i.t. lumbar injection of BoNT/A‐specific antitoxin prevented the beneficial effects of i.n. BoNT/A on rigid hind paw extension, dorsiflexion resistance, and hind limb use during standing and walking (Figure 3a,b and Video S2).

Figure 3.

Figure 3

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Beneficial effect of axonally transported BoNT/A on TeNT‐induced muscle hypertonia is dependent on BoNT/A transcytosis. The BoNT/A‐specific antitoxin (20 IU) injected i.t. at the level of lumbar spinal canal prevents intraneural (i.n.) BoNT/A (5 U·kg−1)‐mediated reduction of the TeNT‐evoked right hind paw extension (a) and resistance to 90° ankle dorsiflexion (b). Upper panel above the photographs shows the time course of TeNT, BoNT/A, and antitoxin treatments. (a) The middle and lower panels show the photo of same animal taken from different angle (representative of 7–9 animals per group). Photographs of calm, hand‐held animals were taken on Day 14 post‐TeNT/7 post‐BoNT/A. (b) On the graph, vertical lines indicate time point of TeNT, BoNT/A, and antitoxin treatments. N = 7–9 animals/group; mean ± SEM, + P < .05, significantly different from TeNT + saline + horse serum. # P < .05, significantly different from TeNT + BoNT/A + horse serum; two‐way RM ANOVA followed by Bonferroni's post hoc test

3.3. Cleaved SNAP‐25 occurrence in ventral horn after low dose i.n. and i.m. BoNT/A is prevented by spinal i.t. antitoxin

After BoNT/A injection into the sciatic nerve and subsequent horse serum i.t. treatment, cleaved SNAP‐25 immunofluorescence was observed in the ipsilateral ventral horn. In animals treated with i.t. antitoxin 24 hr after BoNT/A, very few individual neurites were observed in the ventral horn, suggesting the prevention of transcytosis of enzymatically active BoNT/A by the antitoxin (Figure 4).

Figure 4.

Figure 4

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Occurence of BoNT/A enzymic activity in the spinal cord after toxin intraneural (i.n.) injection is dependent on BoNT/A transcytosis; (a) i.t. lumbar administration of BoNT/A‐antitoxin (20 IU) reduces the occurrence of BoNT/A cleaved SNAP‐25 (indicated by arrows, immunofluorescent Alexa 555‐tyramide signal amplification) in the ventral horn when injected 1 day after BoNT/A application into the sciatic nerve (5 U·kg−1, right lower panel). Upper panel above the microphotographs shows the time course of TeNT, BoNT/A and antitoxin treatment, and the tissue preparation by perfusion. Scale bar = 250 μm. (b) Quantitative analysis of pixel intensity‐thresholded area of cleaved SNAP‐25 immunoreactivity in three non‐overlapping high magnification visual fields (433 μm × 323 μm = 0.14 mm2) located in lateral L4 ventral horn, average of three slices per animal; means ± SEM; N = 6 animals per group. + P < .05, significantly different from TeNT + saline + horse serum; one‐way non‐parametric ANOVA followed by Dunn's post hoc test

After i.m. injections of BoNT/A at different times relative to the i.t. antitoxin treatment (5 vs. 1 day before the antitoxin), cleaved SNAP‐25 was visible only in the left ventral horn ipsilateral to earlier BoNT/A treatment. Very little or no cleaved SNAP‐25 was observed in the right ventral horn ipsilateral to the BoNT/A injected i.m. 1 day prior to antitoxin (Figure 5). The antitoxin injection into the i.t. space did not alter the pre‐established flaccid paralysis on either side treated with BoNT/A (median DAS in both legs = 4, data not shown).

Figure 5.

Figure 5

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Occurrence of cleaved SNAP‐25 in spinal cord following BoNT/A i.m. injection depends on toxin transcytosis. (a) Lumbar i.t. administration of BoNT/A‐specific antitoxin (20 IU) prevents the occurrence of cleaved SNAP‐25 (bright immunofluorescence, Alexa 555‐tyramide signal amplification) in the right ventral horn ipsilateral to BoNT/A injected 1 day before antitoxin (5 U·kg−1, right gastrocnemius, ventral horn on the right side of the coronal section). In comparison to that, left ventral horn ipsilateral to BoNT/A (5 U·kg−1, left gastrocnemius) injected 5 days before antitoxin shows abundant cleaved SNAP‐25. The panel below the magnified ventral horn images indicate the time course of BoNT/A and antitoxin treatments on the respective side. Scale bar = 500 μm. (b) Quantitative analysis of pixel intensity‐thresholded area of cleaved SNAP‐25 immunoreactivity in three non‐overlapping high magnification (20×) visual fields (433 μm × 323 μm = 0.14 mm2) per single L4 slice, average of three slices per animal (N = 5 animals per group). BoNT/A + antitox Δ 5d, BoNT/A injected 5 days before antitoxin; BoNT/A + antitox Δ 1d, BoNT/A injected 1 day before antitoxin. + P < .05, significantly different from BoNT/A Δ 5d; two‐tailed Mann–Whitney U‐test

4. DISCUSSION

In the present study, local muscle hyperactivity was evoked by low‐dose TeNT i.m. injection into the rat gastrocnemius. TeNT, unlike BoNT/A and other BoNT serotypes, induces muscular spasm following its axonal transport and transcytosis into ventral horn inhibitory synapses which control the motor neuron activity (Brooks, Curtis, & Eccles, 1957; Matthews et al., 2014; Restani et al., 2012). Although the muscular hyperactivity in hyperkinetic movement disorders and spasticity is etiologically different, it is similarly associated with impaired inhibitory control of lower motor neurons (Hallett, 2011; Kita & Goodkin, 2000). Converging mechanisms of muscle hypertonia in dystonia, spasticity, and tetanus are in line with therapeutic efficacy of common drugs (Hassel, 2013; Kita & Goodkin, 2000) and supported by the efficacy of the GABAB receptor agonist baclofen in TeNT‐evoked spastic paralysis (Figure 1c). Thus, TeNT‐evoked local spastic paralysis could be employed to induce the motor neuron disinhibition and exaggerated excitatory drive, although TeNT‐induced local muscle spasm does not reflect the full complexity of clinical disorders. The most common form of naturally occurring spasticity caused by upper motor neuron lesion is the result of complex compensatory plastic changes at different levels, such as altered supraspinal inputs and/or consequent dysfunction of segmental spinal modulation (Segal, 2018). Thus, the use of TeNT‐evoked local spasm as a model of naturally occurring spasticity may be limited only to certain aspects, such as the disinhibition of local spinal interneurons.

In rats with established TeNT‐evoked focal spastic paralysis, BoNT/A was injected i.m. into the gastrocnemius and, to mimic the effect of axonally transported toxin, i.n. into the sciatic nerve. Intrasciatic injection was chosen based on previous studies, which demonstrated the occurrence of cleaved SNAP‐25 in the spinal cord without observable ipsilateral flaccidity of the hind limb (Matak et al., 2012), and centrally mediated antinociceptive effects (Bach‐Rojecky & Lacković, 2009), suggesting the retrograde axonal transport of BoNT/A from the nerve trunk. Quantification of toe spread reflex by DAS scale was used to evaluate the BoNT/A‐mediated local muscular action, as previously described (Broide et al., 2013). Both modes of BoNT/A application relieved the TeNT‐evoked spastic paralysis. In line with local neuromuscular effect on the neuromuscular junction, i.m. BoNT/A produced a rapid onset of spasm relief (within 24 hr), which was concurrent with prominent DAS impairment (Figure 1a,d). After i.n. BoNT/A, the relief of local spastic paralysis was observed after 48–72 hr, without significant DAS impairment (Figures 1b,e and 3b), suggesting a spasm‐relieving effect, not related to the peripheral flaccid paralysis.

Previously, we have shown that the occurrence of cleaved SNAP‐25 within the facial motor nucleus after toxin application into the whisker pad muscles is prevented by BoNT/A‐specific antitoxin applied into the lateral ventricles or cisterna magna. These experiments demonstrated BoNT/A trans‐synaptic migration into secondary synapses via the extracellular fluid (Caleo et al., 2018). In the present study, BoNT/A‐antitoxin injected i.t. into the lumbar spinal canal prevented the i.n. BoNT/A‐mediated relief of TeNT‐induced spastic paralysis (Figure 3a,b; Videos S1 and S2). The antitoxin also reduced the i.n. BoNT/A‐induced SNAP‐25 cleavage in the ventral horn (Figure 4). These observations indicate a modulation of motor function by retrogradely transported and transcytosed, enzymatically active BoNT/A. Similarly, i.t. antitoxin prevented the occurrence of cleaved SNAP‐25 in the ventral horn after BoNT/A injection into the gastrocnemius (Figure 5), while the DAS impairment resulting from BoNT/A muscular action was unaffected by the antitoxin. The present observations suggest that, after retrograde axonal transport and transcytosis of i.m.‐injected BoNT/A, peripheral and central effects co‐occur. However, central BoNT/A effects might not be easily distinguished since they are preceded by a prominent flaccid paralysis.

In line with the reduction of overt muscle tone observed here and selectivity of BoNT/A action in excitatory synapses (Grumelli, Corradini, Matteoli, & Verderio, 2010), it is likely that transcytosed BoNT/A primarily reduces the excitatory inputs onto motor neurons. Previous colocalization studies show that, after i.m. injection of the toxin, BoNT/A‐cleaved SNAP‐25 in motor nuclei of facial and hind limb muscles was mostly present in https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2480‐positive neurites surrounding the motor neurons and https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=193#1013)‐expressing synaptic contacts with their somas (Cai, Francis, Brin, & Broide, 2017; Caleo et al., 2018; Matak et al., 2012). Cholinergic synapses with direct excitatory input on motor neurons, known as C‐boutons or C‐terminals, reduce the after‐hyperpolarization of motor neurons via muscarinic https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=14 receptors (Miles, Hartley, Todd, & Brownstone, 2007). The BoNT/A effect on C‐boutons or other synapses in the ventral horn awaits further functional characterization.

The clinical significance of central BoNT/A effects is presently unknown. Peripheral effects could be more dominant or mask the central toxin effects. However, clinical benefits exerted by i.m. BoNT/A often do not follow the degree of peripheral weakness. Several clinical observations are difficult to explain only by local peripheral action of BoNT/A:

  • Distant effects in non‐injected muscles and limbs. A reduction of motor neuronal excitability was reported in the hand muscle of patients treated in the neck muscles for spasmodic torticollis (Wohlfarth, Schubert, Rothe, Elek, & Dengler, 2001) and in the lower facial muscles of patients treated in the orbicularis oris for hemifacial spasm (Ishikawa et al., 2010). In spastic patients, the observed reduction of recurrent inhibition of distant non‐injected muscles cannot be explained by the peripheral actions of BoNT/A on either local or distant neuromuscular junction or muscle spindles (Aymard, Giboin, Lackmy‐Vallée, & Marchand‐Pauvert, 2013; Marchand‐Pauvert et al., 2013). Distant, even contralateral motor benefits have been reported in patients treated for focal dystonias and stiff man syndrome, which were interpreted as evidence of systemic diffusion or a direct central effect of the toxin (Giladi, 1997; Girlanda, Quartarone, Sinicropi, Nicolosi, & Messina, 1996; Liguori, Cordivari, Lugaresi, & Montagna, 1997).

  • Reduction of spasm frequency in focal dystonias (such as blepharospasm and torticollis) and stiff person syndrome, which cannot be interpreted by toxin action on muscle relaxation alone (Giladi, 1997; Liguori et al., 1997; Valls‐Sole, Tolosa, & Ribera, 1991).

  • Clinical benefits not necessarily concurrent with the extent and duration of muscular paralysis. In patients treated with BoNT/A for dystonia and spasticity, the apparent muscle weakness was negligible or lasted for less time than the clinically observed beneficial effects (Bjornson et al., 2007; Eek & Himmelmann, 2016; Priori, Berardelli, Mercuri, & Manfredi, 1995). In writer's cramp patients, a greater BoNT/A‐induced weakness was observed after writing‐induced muscle activity in comparison to no muscle activity immediately after toxin injection (Chen et al., 1999). This observation suggested that the increased muscle activity leads to increased BoNT/A entry into the neuromuscular junctions. However, the time point of maximal muscle weakness did not correlate with the time point of greatest therapeutic benefit, and there was no significant difference in the observed therapeutic benefit in both patient groups (Chen et al., 1999).

Beyond the neuromuscular relaxation, BoNT/A may modify motor neuron activity by neuroparalytic action on (a) intrafusal nerve endings of muscle spindles and consequent modification of stretch reflexes (Giladi, 1997; Rosales & Dressler, 2010) and/or (b) cholinergic synapses between recurrent axonal collaterals and Renshaw interneurons involved in recurrent inhibition, provided by axonal transport (Marchand‐Pauvert et al., 2013; Mazzocchio & Caleo, 2015). However, these mechanisms cannot account for the effects in blepharospasm and hemifacial spasm, as mentioned above, because facial muscles and facial motor neurons lack their respective muscle spindles and recurrent axonal collaterals (Kitai, Tanaka, Tsukahara, & Yu, 1972; Whitehead, Keller‐Peck, Kucera, & Tourtellotte, 2005).

The present results suggest that modification of motor function following BoNT/A transcytosis within the CNS might contribute to the antispastic action of i.m. BoNT/A. Central effects might participate in the fine tuning of motor activity in movement disorders and augment the toxin's peripheral effects (Matak, Lacković, & Relja, 2016). In particular, central BoNT/A action might mediate prolonged duration of beneficial action after the resolution of peripheral weakness (Mazzocchio & Caleo, 2015) or reduced excitability of motor neurons innervating the distant, non‐injected muscles. In conclusion, the present results point to central actions of BoNT/A on pathologically increased muscle tone, involving a mechanism that is distinct from its well‐known action in peripheral cholinergic terminals.

AUTHOR CONTRIBUTIONS

I.M. presented the conception and design of the study, acquisition and analysis of the data, draft of the manuscript and figures, and approval of the final manuscript.

CONFLICT OF INTEREST

The author declares no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14207, https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14208, and https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14206, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Video S1. Intraneuronal (i.n.) sciatic injection of BoNT/A improves the hind‐limb use in TeNT‐evoked spasticity. The video shows representative external appearance and short walking sequence of a single rat with TeNT‐evoked spastic paralysis of the right gastrocnemius muscle prior to, and 7 days post‐treatment with i.n. BoNT/A (5 U kg−1)/6 days post i.t. control treatment with normal horse serum. Note the extended position and the inability to flex the hind paw, resulting in disuse of TeNT‐injected hind‐limb (day 0, seconds 0–11 of the video). Marked improvement of the local spastic paralysis restoring the ability to flex the hind limb, ground placement of the hind paw, and hind limb use during walking is visible 7 days after i.n. BoNT/A (seconds 12–21 of the video).

Click here for additional data file. (34MB, mp4)

Video S2. BoNT/A specific antitoxin prevents beneficial effects of i.n. BoNT/A on hind limb use in TeNT‐induced spastic paralysis. The video shows representative external appearance and short walking sequence of a single rat with TeNT‐evoked spastic paralysis prior to, and 7 days post‐treatment with i.n. BoNT/A (5 U kg−1)/6 days post intrathecal lumbar treatment with BoNT/A‐specific antitoxin (20 IU in 20 μL). Local spastic paralysis and disuse of the hind‐limb was similar at day 0 (seconds 0–4 of the video), and day 7 post BoNT/A/day 6 post antitoxin (seconds 5–17 of the video).

Click here for additional data file. (29.3MB, mp4)

ACKNOWLEDGEMENTS

Supported by European Social Fund and Croatian Ministry of Science, Education and Sport (Project no. HR.3.2.01‐0178).

Antibody to cleaved SNAP‐25 and the BoNT/A‐specific antitoxin were kindly provided, respectively, by Prof. Ornella Rossetto (University of Padua, Padua, Italy) and Dr Thea Sesardic (National Institute for Biological Standards and Control, Potters Bar, United Kingdom). Zdravko Lacković and Maja Relja (University of Zagreb School of Medicine, Zagreb, Croatia) and Matteo Caleo (CNR Institute of Neuroscience, Pisa, Italy) provided advices regarding the data representation and preparation of the manuscript. Božica Hržan (University of Zagreb School of Medicine, Zagreb, Croatia) gave excellent technical support regarding animal treatment and randomization.

Matak I. Evidence for central antispastic effect of botulinum toxin type A . Br J Pharmacol. 2020;177:65–76. 10.1111/bph.14846

REFERENCES

  1. Alexander, S. P. H. , Christopoulos, A. , Davenport, A. P. , Kelly, E. , Marrion, N. V. , Peters, J. A. , … CGTP Collaborators (2017). The Concise Guide To PHARMACOLOGY 2017/18: G protein‐coupled receptors. British Journal of Pharmacology, 174, S17–S129. 10.1111/bph.13878 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander, S. P. H. , Kelly, E. , Marrion, N. V. , Peters, J. A. , Faccenda, E. , Harding, S. D. , … CGTP Collaborators (2017). The Concise Guide To PHARMACOLOGY 2017/18: Transporters. British Journal of Pharmacology, 174, S360–S446. 10.1111/bph.13883 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alexander, S. P. H. , Roberts, R. E. , Broughton, B. R. S. , Sobey, C. G. , George, C. H. , Stanford, S. C. , … Ahluwalia, A. (2018). Goals and practicalities of immunoblotting and immunohistochemistry: A guide for submission to the British Journal of Pharmacology. British Journal of Pharmacology, 175, 407–411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Antonucci, F. , Rossi, C. , Gianfranceschi, L. , Rossetto, O. , & Caleo, M. (2008). Long distance retrograde effects of botulinum neurotoxin A. The Journal of Neuroscience, 28, 3689–3696. 10.1523/JNEUROSCI.0375-08.2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Aymard, C. , Giboin, L. S. , Lackmy‐Vallée, A. , & Marchand‐Pauvert, V. (2013). Spinal plasticity in stroke patients after botulinum neurotoxin A injection in ankle plantar flexors. Physiological Reports, 1, e00173 10.1002/phy2.173 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bach‐Rojecky, L. , & Lacković, Z. (2009). Central origin of the antinociceptive action of botulinum toxin type A. Pharmacology Biochemistry and Behavior, 94(2), 234–238. 10.1016/j.pbb.2009.08.012 [DOI] [PubMed] [Google Scholar]
  7. Bjornson, K. , Hays, R. , Graubert, C. , Price, R. , Won, F. , McLaughlin, J. F. , & Cohen, M. (2007). Botulinum toxin for spasticity in children with cerebral palsy: A comprehensive evaluation. Pediatrics, 120, 49–58. 10.1542/peds.2007-0016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Broide, R. S. , Rubino, J. , Nicholson, G. S. , Ardila, M. C. , Brown, M. S. , Aoki, K. R. , & Francis, J. (2013). The rat Digit Abduction Score (DAS) assay: A physiological model for assessing botulinum neurotoxin‐induced skeletal muscle paralysis. Toxicon, 71, 18–24. 10.1016/j.toxicon.2013.05.004 [DOI] [PubMed] [Google Scholar]
  9. Brooks, V. B. , Curtis, D. R. , & Eccles, J. C. (1957). The action of tetanus toxin on the inhibition of motor neurones. The Journal of Physiology, 135, 655–672. 10.1113/jphysiol.1957.sp005737 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cai, B. B. , Francis, J. , Brin, M. F. , & Broide, R. S. (2017). Botulinum neurotoxin type A‐cleaved SNAP25 is confined to primary motor neurons and localized on the plasma membrane following intramuscular toxin injection. Neuroscience, 352, 155–169. 10.1016/j.neuroscience.2017.03.049 [DOI] [PubMed] [Google Scholar]
  11. Caleo, M. , Spinelli, M. , Colosimo, F. , Matak, I. , Rossetto, O. , Lacković, Z. , & Restani, L. (2018). Transynaptic action of botulinum neurotoxin type A at central cholinergic boutons. The Journal of Neuroscience, 38, 10329–10337. 10.1523/JNEUROSCI.0294-18.2018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Carter, R. J. , Morton, J. , & Dunnett, S. B. (2001). Motor coordination and balance in rodents. Current Protocols in Neuroscience. Chapter 8:Unit 8.12 [DOI] [PubMed] [Google Scholar]
  13. Charan, J. , & Kantharia, N. (2013). How to calculate sample size in animal studies? Journal of Pharmacology and Pharmacotherapeutics, 4(4), 303 10.4103/0976-500x.119726 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chen, R. , Karp, B. I. , Goldstein, S. R. , Bara‐Jimenez, W. , Yaseen, Z. , & Hallett, M. (1999). Effect of muscle activity immediately after botulinum toxin injection for writer's cramp. Movement Disorders, 14, 307–312. 10.1002/1531-8257(199903)14:2<307::AID-MDS1016>3.0.CO;2-3 [DOI] [PubMed] [Google Scholar]
  15. Cui, M. , Khanijou, S. , Rubino, J. , & Aoki, K. R. (2004). Subcutaneous administration of botulinum toxin A reduces formalin‐induced pain. Pain, 107, 125–133. 10.1016/j.pain.2003.10.008 [DOI] [PubMed] [Google Scholar]
  16. Curtis, M. J. , Alexander, S. , Cirino, G. , Docherty, J. R. , George, C. H. , Giembycz, M. A. , … Ahluwalia, A. (2018). Experimental design and analysis and their reporting II: Updated and simplified guidance for authors and peer reviewers. British Journal of Pharmacology, 175, 987–993. 10.1111/bph.14153 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Eek, M. N. , & Himmelmann, K. (2016). No decrease in muscle strength after botulinum neurotoxin‐A injection in children with cerebral palsy. Frontiers in Human Neuroscience, 10, 506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Giladi, N. (1997). The mechanism of action of botulinum toxin type A in focal dystonia is most probably through its dual effect on efferent (motor) and afferent pathways at the injected site. Journal of the Neurological Sciences, 152, 132–135. 10.1016/S0022-510X(97)00151-2 [DOI] [PubMed] [Google Scholar]
  19. Girlanda, P. , Quartarone, A. , Sinicropi, S. , Nicolosi, C. , & Messina, C. (1996). Unilateral injection of botulinum toxin in blepharospasm: Single fiber electromyography and blink reflex study. Movement Disorders, 11, 27–31. 10.1002/mds.870110107 [DOI] [PubMed] [Google Scholar]
  20. Grumelli, C. , Corradini, I. , Matteoli, M. , & Verderio, C. (2010). Intrinsic calcium dynamics control botulinum toxin A susceptibility in distinct neuronal populations. Cell Calcium, 47, 419–424. 10.1016/j.ceca.2010.03.002 [DOI] [PubMed] [Google Scholar]
  21. Hallett, M. (2011). Neurophysiology of dystonia: The role of inhibition. Neurobiology of Disease, 42, 177–184. 10.1016/j.nbd.2010.08.025 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Harding, S. D. , Sharman, J. L. , Faccenda, E. , Southan, C. , Pawson, A. J. , Ireland, S. , … NC‐IUPHAR (2018). The IUPHAR/BPS guide to PHARMACOLOGY in 2018: Updates and expansion to encompass the new guide to IMMUNOPHARMACOLOGY. Nucl Acids Res, 46, D1091–D1106. 10.1093/nar/gkx1121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hassel, B. (2013). Tetanus: Pathophysiology, treatment, and the possibility of using botulinum toxin against tetanus‐induced rigidity and spasms. Toxins (Basel), 5, 73–83. 10.3390/toxins5010073 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ishikawa, M. , Takashima, K. , Kamochi, H. , Kusaka, G. , Shinoda, S. , & Watanabe, E. (2010). Treatment with botulinum toxin improves the hyperexcitability of the facial motoneuron in patients with hemifacial spasm. Neurological Research, 32(6), 656–660. 10.1179/174313209x431129 [DOI] [PubMed] [Google Scholar]
  25. Jankovic, J. (2017). Botulinum toxin: State of the art. Movement Disorders, 32, 1131–1138. 10.1002/mds.27072 [DOI] [PubMed] [Google Scholar]
  26. Kaňovský, P. , & Rosales, R. L. (2011). Debunking the pathophysiological puzzle of dystonia—With special reference to botulinum toxin therapy. Parkinsonism & Related Disorders, 17(Suppl 1), S11–S14. 10.1016/j.parkreldis.2011.06.018 [DOI] [PubMed] [Google Scholar]
  27. Kilkenny, C. , Browne, W. J. , Cuthill, I. C. , Emerson, M. , & Altman, D. G. (2010). Animal research: Reporting in vivo experiments: The ARRIVE guidelines. PLoS Biology, 8, e1000412 10.1371/journal.pbio.1000412 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kita, M. , & Goodkin, D. E. (2000). Drugs used to treat spasticity. Drugs, 59, 487–495. 10.2165/00003495-200059030-00006 [DOI] [PubMed] [Google Scholar]
  29. Kitai, S. T. , Tanaka, T. , Tsukahara, N. , & Yu, H. (1972). The facial nucleus of cat: Antidromic and synaptic activation and peripheral nerve representation. Experimental Brain Research, 16, 161–183. [DOI] [PubMed] [Google Scholar]
  30. Koizumi, H. , Goto, S. , Okita, S. , Morigaki, R. , Akaike, N. , Torii, Y. , … Kaji, R. (2014). Spinal central effects of peripherally applied botulinum neurotoxin A in comparison between its subtypes A1 and A2. Frontiers in Neurology, 5, 98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kutschenko, A. , Reinert, M. C. , Klinker, F. , Paulus, W. , Hesse, S. , & Liebetanz, D. (2012). Accurate quantification of tetanus neurotoxin‐induced focal spasticity in mice using complex running wheels. Journal of Neuroscience Methods, 205, 45–48. [DOI] [PubMed] [Google Scholar]
  32. Li, D. , Matoo, P. , & Keller, J. E. (2012). New equine antitoxins to botulinum serotypes A and B. Biologicals, 40, 240–246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Liguori, R. , Cordivari, C. , Lugaresi, E. , & Montagna, P. (1997). Botulinum toxin A improves muscle spasms and rigidity in stiff‐person syndrome. Movement Disorders, 12, 1060–1063. 10.1002/mds.870120636 [DOI] [PubMed] [Google Scholar]
  34. Lorrai, I. , Maccioni, P. , Gessa, G. L. , & Colombo, G. (2016). R(+)‐baclofen, but not S(−)‐baclofen, alters alcohol self‐administration in alcohol‐preferring rats. Frontiers in Psychiatry, 7, 68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Marchand‐Pauvert, V. , Aymard, C. , Giboin, L. S. , Dominici, F. , Rossi, A. , & Mazzocchio, R. (2013). Beyond muscular effects: Depression of spinal recurrent inhibition after botulinum neurotoxin A. The Journal of Physiology, 591, 1017–1029. 10.1113/jphysiol.2012.239178 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Matak, I. , Bach‐Rojecky, L. , Filipović, B. , & Lacković, Z. (2011). Behavioral and immunohistochemical evidence for central antinociceptive activity of botulinum toxin A. Neuroscience, 186, 201–207. 10.1016/j.neuroscience.2011.04.026 [DOI] [PubMed] [Google Scholar]
  37. Matak, I. , Lacković, Z. , & Relja, M. (2016). Botulinum toxin type A in motor nervous system: Unexplained observations and new challenges. Journal of Neural Transmission (Vienna), 123, 1415–1421. 10.1007/s00702-016-1611-9 [DOI] [PubMed] [Google Scholar]
  38. Matak, I. , Riederer, P. , & Lacković, Z. (2012). Botulinum toxin's axonal transport from periphery to the spinal cord. Neurochemistry International, 61, 236–239. 10.1016/j.neuint.2012.05.001 [DOI] [PubMed] [Google Scholar]
  39. Matthews, C. C. , Fishman, P. S. , & Wittenberg, G. F. (2014). Tetanus toxin reduces local and descending regulation of the H‐reflex. Muscle & Nerve, 49, 495–501. 10.1002/mus.23938 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mazzocchio, R. , & Caleo, M. (2015). More than at the neuromuscular synapse: Actions of botulinum neurotoxin A in the central nervous system. The Neuroscientist, 21, 44–61. 10.1177/1073858414524633 [DOI] [PubMed] [Google Scholar]
  41. Miles, G. B. , Hartley, R. , Todd, A. J. , & Brownstone, R. M. (2007). Spinal cholinergic interneurons regulate the excitability of motor neurons during locomotion. Proceedings of the National Academy of Sciences of the United States of America, 104, 2448–2453. 10.1073/pnas.0611134104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pirazzini, M. , Rossetto, O. , Eleopra, R. , & Montecucco, C. (2017). Botulinum neurotoxins: Biology, pharmacology, and toxicology. Pharmacological Reviews, 69, 200–235. 10.1124/pr.116.012658 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Priori, A. , Berardelli, A. , Mercuri, B. , & Manfredi, M. (1995). Physiological effects produced by botulinum toxin treatment of upper limb dystonia. Changes in reciprocal inhibition between forearm muscles. Brain, 118, 801–807. [DOI] [PubMed] [Google Scholar]
  44. Restani, L. , Giribaldi, F. , Manich, M. , Bercsenyi, K. , Menendez, G. , Rossetto, O. , … Schiavo, G. (2012). Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons. PLoS Pathogens, 8, e1003087 10.1371/journal.ppat.1003087 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rosales, R. L. , & Dressler, D. (2010). On muscle spindles, dystonia and botulinum toxin. European Journal of Neurology, 17(Suppl 1), 71–80. 10.1111/j.1468-1331.2010.03056.x [DOI] [PubMed] [Google Scholar]
  46. Safarpour, Y. , & Jabbari, B. (2018). Botulinum toxin treatment of movement disorders. Current Treatment Options in Neurology, 20, 4 10.1007/s11940-018-0488-3 [DOI] [PubMed] [Google Scholar]
  47. Segal, M. (2018). Muscle overactivity in the upper motor neuron syndrome: Pathophysiology. Physical Medicine and Rehabilitation Clinics of North America, 29, 427–436. 10.1016/j.pmr.2018.04.005 [DOI] [PubMed] [Google Scholar]
  48. Valls‐Sole, J. , Tolosa, E. S. , & Ribera, G. (1991). Neurophysiological observations on the effects of botulinum toxin treatment in patients with dystonic blepharospasm. Journal of Neurology, Neurosurgery, and Psychiatry, 54, 310–313. 10.1136/jnnp.54.4.310 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Whitehead, J. , Keller‐Peck, C. , Kucera, J. , & Tourtellotte, W. G. (2005). Glial cell‐line derived neurotrophic factor‐dependent fusimotor neuron survival during development. Mechanisms of Development, 122, 27–41. 10.1016/j.mod.2004.09.003 [DOI] [PubMed] [Google Scholar]
  50. Wohlfarth, K. , Schubert, M. , Rothe, B. , Elek, J. , & Dengler, R. (2001). Remote F‐wave changes after local botulinum toxin application. Clinical Neurophysiology, 112, 636–640. 10.1016/S1388-2457(01)00478-3 [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Video S1. Intraneuronal (i.n.) sciatic injection of BoNT/A improves the hind‐limb use in TeNT‐evoked spasticity. The video shows representative external appearance and short walking sequence of a single rat with TeNT‐evoked spastic paralysis of the right gastrocnemius muscle prior to, and 7 days post‐treatment with i.n. BoNT/A (5 U kg−1)/6 days post i.t. control treatment with normal horse serum. Note the extended position and the inability to flex the hind paw, resulting in disuse of TeNT‐injected hind‐limb (day 0, seconds 0–11 of the video). Marked improvement of the local spastic paralysis restoring the ability to flex the hind limb, ground placement of the hind paw, and hind limb use during walking is visible 7 days after i.n. BoNT/A (seconds 12–21 of the video).

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Video S2. BoNT/A specific antitoxin prevents beneficial effects of i.n. BoNT/A on hind limb use in TeNT‐induced spastic paralysis. The video shows representative external appearance and short walking sequence of a single rat with TeNT‐evoked spastic paralysis prior to, and 7 days post‐treatment with i.n. BoNT/A (5 U kg−1)/6 days post intrathecal lumbar treatment with BoNT/A‐specific antitoxin (20 IU in 20 μL). Local spastic paralysis and disuse of the hind‐limb was similar at day 0 (seconds 0–4 of the video), and day 7 post BoNT/A/day 6 post antitoxin (seconds 5–17 of the video).

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