The safety of botulinum neurotoxin type A's intraarticular application in experimental animals.

In vivo studies of botulinum neurotoxin type A (BoNT-A) enabled characterization of its activity in the nociceptive sensory system separate from its preferred action in motor and autonomic nerve terminals. However, in the recent rodent studies of arthritic pain which employed high intra-articular (i.a.) doses (expressed as a total number of units (U) per animal or U/kg), possible systemic effects have not been conclusively excluded. Herein we assessed the effect of two pharmaceutical preparations, abobotulinumtoxinA (aboBoNT-A, 10, 20, and 40 U/kg corresponding to 0.05, 0.11, and 0.22 ng/kg neurotoxin) and onabotulinumtoxinA (onaBoNT-A, 10 and 20 U/kg corresponding to 0.09 and 0.18 ng/kg, respectively) injected into the rat knee, on safety-relevant readouts: digit abduction, motor performance and weight gain during 14 days post-treatment. The i. a. toxin produced dose-dependent impairment of the toe spreading reflex and rotarod performance, which was moderate and transient after 10 U/kg onaBoNT-A and ≤20 U/kg aboBoNT-A doses, and severe and long-lasting (examined up to 14 days) after ≥20 U/kg of onaBoNT-A and 40 U/kg aboBoNT-A. In addition, lower toxin doses prevented the normal weight gain compared to controls, while higher doses induced marked weight loss (≥20 U/kg of onaBoNT-A and 40 U/kg aboBoNT-A). Commonly employed BoNT-A formulations, depending on the doses, cause local relaxation of the surrounding muscles and systemic adverse effects in rats. Thus, to evade possible toxin unwanted local or systemic spread, careful dosing and motor testing should be mandatory in preclinical behavioral studies, irrespective of the sites and doses of toxin application.

Antinociceptive Actions of Botulinum Toxin A1 on Immunogenic Hypersensitivity in Temporomandibular Joint of Rats.

Botulinum neurotoxin type A1 (BoNT-A) reduces the peripheral peptide and cytokine upregulation in rats with antigen-evoked persistent immunogenic hypersensitivity (PIH) of the temporomandibular joint (TMJ). Herein, we examined the effects of two preparations of BoNT-A, abobotulinumtoxinA (aboBoNT-A; Dysport) and onabotulinumtoxinA (onaBoNT-A; Botox), on spontaneous and evoked nociceptive behaviors, as well as on central neuronal and astroglial activation. The antigen-evoked PIH was induced in rats via repeated systemic and unilateral intra-articular (i.a.) injections of methylated bovine serum albumin (mBSA). Rats were subsequently injected with unilateral i.a. aboBoNT-A (14 U/kg), onaBoNT-A (7 U/kg), or the vehicle (saline). After i.a. treatments, spontaneous and mechanically evoked nocifensive behaviors were assessed before and after the low-dose i.a. formalin (0.5%) challenge. The central effects of BoNT-A were assessed by an immunohistochemical analysis of cleaved synaptosomal-associated protein 25 (cSNAP-25) presence, c-Fos, GFAP, and CGRP expression in the trigeminal nucleus caudalis (TNC). Both BoNT-A preparations similarly reduced the formalin-induced spontaneous pain-related behaviors and mechanical allodynia of the hypernociceptive rats. Likewise, their effects were associated with the central occurrence of cSNAP-25 and reduction of c-Fos and GFAP upregulation in the TNC. BoNT-A antinociceptive activity on the PIH is associated with the toxin axonal transport to trigeminal sensory areas and reduction of neuronal and glial activation in central nociceptive regions.

Botulinum Toxin and Pain.

This chapter is focused on analgesic mechanism of action of botulinum toxin type A (BoNT-A) including the action beyond peripheral nerve endings. With the exception of the meninges and possibly urinary bladder, the presence of BoNT-A activity in the periphery, cleaving SNAP25 as a target molecule, up to now was not convincingly shown. In contrast many reports demonstrated BoNT-A activity and the presence of cleaved SNAP25 in the brain and spinal cord. In a model of mirror pain BoNT-A analgesic effect can be achieved even without participation of peripheral nerve ending. Thus generalized hypothesis central or peripheral mechanism of action belongs to history, and there is a need to confirm or dispute the results with meninges, urinary bladder, and possibly with other, especially visceral organs.There are two general options for the central actions of BoNT-A: 1. The activity ends by silencing primary sensory neuron thereby stopping the pain information further in the CNS. 2. Or thereafter, indirectly or transsynaptically, BoNT-A triggers smaller or larger neural loops, forming memory of pain in the CNS that could explain the bilateral effects after unilateral peripheral administration, similar effect in mirror image allodynia and the like Intensive research has shown that peripherally administered BoNT-A reaches the CNS by axonal transport. There is increasing evidence that BoNT-A is preventing pain in a growing range of disorders. In the absence of unexpected findings, or an increase in the uncontrolled use of illicit preparations by uneducated persons, BoNT-A is emerging as a new long-lasting and relatively safe analgesic.

Vladimir Sertic: forgotten pioneer of virology and bacteriophage therapy.

Vladimir Sertic was a pioneer of bacteriophage research in the period between the two world wars. He was born and educated in Croatia, where he made his initial discoveries, and joined Félix d'Herelle's Laboratoire du Bactériophage in Paris in 1928. Original documents and a box with hundreds of sealed bacteriophages samples were kept in Sertic's Zagreb home for decades. Following Vladimir's death, his sister passed this archival material to Professor Zdravko Lackovic in 1989. Some years later, these artefacts were opened and studied. Additionally, we conducted a literature search using the term 'Vladimir Sertic' in the databases PubMed and Google Scholar. After a detailed examination of these data, we established a chronology of his work and compiled a list of his scientific publications. A complete bibliography, with the exception of those publications already cited here, is provided as an appendix. Sertic's key contributions included the exploration of the properties of phage lysins, the devising of a uniform bacteriophage classification system and, in collaboration with his protégé, Nikolai Boulgakov, the isolation of numerous bacteriophage strains, including the famous φX174. Finally it was Sertic's pioneering work in Zagreb that offered confirmation that phages are live agents.

Association of Intranasal and Neurogenic Dural Inflammation in Experimental Acute Rhinosinusitis.

BACKGROUND: Nasal cavity and sinus disorders, such as allergic rhinitis, rhinosinusitis, or certain anatomical defects, are often associated with transient or ongoing headaches. On the other hand, migraine headache patients often exhibit pain referral over the area of nasal sinuses and typical nasal autonomic symptoms involving congestion and rhinorrhea. Mechanism for convergence of nasal or sinus disorders and headaches is unknown. Herein, we examined the association of sino-nasal inflammatory pain with common preclinical indicators of trigeminovascular system activation such as dural neurogenic inflammation (DNI) and neuronal activation in brainstem nociceptive nuclei. METHODS: Nasal and paranasal cavity inflammation and pain was induced by formalin (2.5%/10 µl) or capsaicin (0.1%/10 µl) instillation at the border of maxillary sinus and nasal cavity in rats. Quantification of inflammation of nasal mucosa and DNI was performed by spectrophotometric measurement of Evans blue - plasma protein complex extravasation. Pain behavior was quantified by rat grimace scale (RGS). Nociceptive neuronal activation in caudal part of spinal trigeminal nucleus (TNC) was assessed by c-Fos protein immunohistochemistry. RESULTS: Capsaicin and formalin administered into rat nasal cavity increased plasma protein extravasation in the nasal mucosa and dura mater. Intensity of plasma protein extravasation in nasal mucosa correlated with extravasation in dura. Similarly, facial pain intensity correlated with nociceptive neuronal c-Fos activation in the TNC. CONCLUSION: Present data show that inflammatory stimuli in deep nasal and paranasal structures provoke distant intracranial changes related to trigeminovascular system activation. We hypothesize that this phenomenon could explain overlapping symptoms and comorbidity of nasal/paranasal inflammatory disorders with migraine.

New analgesic: Focus on botulinum toxin.

In 2010, Kissin concluded pessimistically that of the 59 new drugs introduced in the fifty-year period between 1960 and 2009 and still in use, only seven had new molecular targets. Of these, only one, sumatriptan, was effective enough to lead to the introduction of multiple drugs targeting the same target molecules (triptans) (Kissin, 2010). Morphine and acetylsalicylic acid (aspirin), introduced for the treatment of pain more than a century ago, continue to dominate biomedical publications despite their limited effectiveness in many areas (e.g., neuropathic pain) and serious adverse effects. Today, are we really closer to ideal analgesics that would work hard enough, long enough, and did not have unwanted side effects? The purpose of the present article is to analyze where we are now. Several drugs, like long-acting opioids or botulinum toxins open some hope. Advantage of botulinum toxin A is unique duration of action (months). New discoveries showed that after peripheral application botulinum toxin by axonal transport reaches the CNS. Major analgesic mechanism of action seems to be of central origin. Will botulinum toxin in the CNS bring new indications and or/adverse effects? Much more basic and clinical research should be in front of us. Although relatively safe as a drug, botulinum toxin is not without adverse effect. Policy makers, clinicians and all those applying botulinum toxin should be aware of that. Unfortunately the life without the pain is still not possible.

Transynaptic Action of Botulinum Neurotoxin Type A at Central Cholinergic Boutons.

Botulinum neurotoxin Type A (BoNT/A) is an effective treatment for several movement disorders, including spasticity and dystonia. BoNT/A acts by cleaving synaptosomal-associated protein of 25 kDa (SNAP-25) at the neuromuscular junction, thus blocking synaptic transmission and weakening overactive muscles. However, not all the therapeutic benefits of the neurotoxin are explained by peripheral neuroparalysis, suggesting an action of BoNT/A on central circuits. Currently, the specific targets of BoNT/A central activity remain unclear. Here, we show that catalytically active BoNT/A is transported to the facial nucleus (FN) after injection into the nasolabial musculature of rats and mice. BoNT/A-mediated cleavage of SNAP-25 in the FN is prevented by intracerebroventricular delivery of antitoxin antibodies, demonstrating that BoNT/A physically leaves the motoneurons to enter second-order neurons. Analysis of intoxicated terminals within the FN shows that BoNT/A is transcytosed preferentially into cholinergic synapses. The cholinergic boutons containing cleaved SNAP-25 are associated with a larger size, suggesting impaired neuroexocytosis. Together, the present findings indicate a previously unrecognized source of reduced motoneuron drive after BoNT/A via blockade of central, excitatory cholinergic inputs. These data highlight the ability of BoNT/A to selectively target and modulate specific central circuits, with consequent impact on its therapeutic effectiveness in movement disorders.SIGNIFICANCE STATEMENT Botulinum neurotoxins are among the most potent toxins known. Despite this, their specific and reversible action prompted their use in clinical practice to treat several neuromuscular pathologies (dystonia, spasticity, muscle spasms) characterized by hyperexcitability of peripheral nerve terminals or even in nonpathological applications (i.e., cosmetic use). Substantial experimental and clinical evidence indicates that not all botulinum neurotoxin Type A (BoNT/A) effects can be explained solely by the local action (i.e., silencing of the neuromuscular junction). In particular, there are cases in which the clinical benefit exceeds the duration of peripheral neurotransmission blockade. In this study, we demonstrate that BoNT/A is transported to facial motoneurons, released, and internalized preferentially into cholinergic terminals impinging onto the motoneurons. Our data demonstrate a direct central action of BoNT/A.

"Bunanje": XX Century Abuse of Atropa Belladonna Halucinogenic Berries in Continental Croatia.

BACKGROUND: Atropa belladonna (Engl. deadly nightshade, Cro. velebilje, bunika) is a plant containing pharmacologically active, potentially toxic alkaloids: atropine, hyocyamine and scopolamine. The risk of poisoning in children is important because of possible confusion of black/dark blue belladonna fruit berries with other edible berries. There are many reports in literature of accidental intoxication but no report on traditional intentional usage to achieve hallucinogenic effects. SUBJECTS AND METHODS: Here we report purposeful ingestion of Atropa belladonna berries for hallucinatory effects among adolescents in Bjelovar region in north part of Croatia. This has been happening among children/adolescents while they were grazing animals. We visited a dozen villages in the region and spoke to the oldest mostly to the elderly residents. RESULTS: The existence of such abuse of Atropa belladonna berries in the first part of XX century was confirmed by eight narrators from five distinct places in the region. Interestingly this type of behavior had a specific name "bunanje", unknown in Croatian language, but clearly associated with local plant name bun or bunika. According to informants consumers of berries would develop delirium or hallucinations associated behavior, incoherent and meaningless speech. However nobody remembers any severe case of poisoning. At the regional hospital in Bjelovar in the Pediatric department, there is no record of poisoning with Atropa belladonna. To our knowledge this is the first report of intentional consumption of belladonna berries to achieve the hallucinogenic effect. CONCLUSIONS: The fact that the custom was observed in five distinct spots and it had its specific name "bunanje" suggest that those are not isolated random events but the type of practices; seasonal abuse of hallucinogenic berries of Atropa belladonna, among rural adolescents in the first part of XX century.

Involvement of substance P in the antinociceptive effect of botulinum toxin type A: Evidence from knockout mice.

The antinociceptive action of botulinum toxin type A (BoNT/A) has been demonstrated in behavioral animal studies and clinical settings. It was shown that this effect is associated with toxin activity in CNS, however, the mechanism is not fully understood. Substance P (SP) is one of the dominant neurotransmitters in primary afferent neurons transmitting pain and itch. Thus, here we examined association of SP-mediated transmission and BoNT/A antinociceptive action by employing gene knockouts. Antinociceptive activity of intraplantarly (i.pl.) injected BoNT/A was examined in mice lacking the gene encoding for SP/neurokinin A (tac1-/-) or SP-preferred receptor neurokinin 1 (tac1r-/-), compared to control C57Bl/6J wild type animals. BoNT/A action was assessed in inflammatory pain induced by formalin and CFA, and neuropathic pain induced by partial sciatic nerve ligation. BoNT/A activity in CNS was examined by c-Fos and BoNT/A-cleaved SNAP-25 immunohistochemistry. In wild type mice, acute (formalin-evoked) and chronic pain (neuropathic and inflammatory) was reduced by peripherally injected BoNT/A. In tac1-/- and tac1r-/- knockout mice, BoNT/A exerted no analgesic effect. In control animals BoNT/A reduced the formalin-evoked c-Fos expression in lumbar dorsal horn, while in knockout mice the c-Fos expression was not reduced. After peripheral toxin injection, cleaved SNAP-25 occurred in lumbar dorsal horn in all animal genotypes. BoNT/A antinociceptive activity is absent in animals lacking the SP and neurokinin 1 receptor encoding genes, in spite of presence of toxin's enzymatic activity in central sensory regions. Thus, we conclude that the integrity of SP-ergic system is necessary for the antinociceptive activity of BoNT/A.

Decompression endoscopic surgery for frontal secondary headache attributed to supraorbital and supratrochlear nerve entrapment: a comprehensive review.

In the last decade, a new surgical treatment modality was developed for frontal secondary headache, based on the assumption that the trigger of this pain entity is the entrapment of peripheral sensory nerves. The surgery entails a procedure, where an endoscopic approach is used to decompress the supraorbital and supratrochlear nerve branches, which are entrapped by the periosteum in the region of the corrugator supercilii muscle. Candidates for the surgery define their headache as moderate to severe persistent daily pressure or tension, localized in the frontal area, sometimes accompanied by symptoms of nausea and photophobia mimicking a primary headache-migraine. We created a step-by-step screening algorithm which is used to differentiate patients that have the highest chance for a successful surgical decompression. Up to now, published data regarding this type of surgery demonstrate long-lasting successful outcomes while adverse effects are minor. This article reviews and discusses from a surgeon's perspective decompression surgery for secondary headache attributed to supraorbital and supratrochlear nerve entrapment.

Activity of botulinum toxin type A in cranial dura: implications for treatment of migraine and other headaches.

BACKGROUND AND PURPOSE: Although botulinum toxin type A (BoNT/A) is approved for chronic migraine treatment, its mechanism of action is still unknown. Dural neurogenic inflammation (DNI) commonly used to investigate migraine pathophysiology can be evoked by trigeminal pain. Here, we investigated the reactivity of cranial dura to trigeminal pain and the mechanism of BoNT/A action on DNI. EXPERIMENTAL APPROACH: Because temporomandibular disorders are highly comorbid with migraine, we employed a rat model of inflammation induced by complete Freund's adjuvant, followed by treatment with BoNT/A injections or sumatriptan p.o. DNI was assessed by Evans blue-plasma protein extravasation, cell histology and RIA for CGRP. BoNT/A enzymatic activity in dura was assessed by immunohistochemistry for cleaved synaptosomal-associated protein 25 (SNAP-25). KEY RESULTS: BoNT/A and sumatriptan reduced the mechanical allodynia and DNI, evoked by complete Freund's adjuvant. BoNT/A prevented inflammatory cell infiltration and inhibited the increase of CGRP levels in dura. After peripheral application, BoNT/A-cleaved SNAP-25 colocalized with CGRP in intracranial dural nerve endings. Injection of the axonal transport blocker colchicine into the trigeminal ganglion prevented the formation of cleaved SNAP-25 in dura. CONCLUSIONS AND IMPLICATIONS: Pericranially injected BoNT/A was taken up by local sensory nerve endings, axonally transported to the trigeminal ganglion and transcytosed to dural afferents. Colocalization of cleaved SNAP-25 and the migraine mediator CGRP in dura suggests that BoNT/A may prevent DNI by suppressing transmission by CGRP. This might explain the effects of BoNT/A in temporomandibular joint inflammation and in migraine and some other headaches.

Botulinum neurotoxin type A: Actions beyond SNAP-25?

Botulinum neurotoxin type A (BoNT/A), the most potent toxin known in nature which causes botulism, is a commonly used therapeutic protein. It prevents synaptic vesicle neuroexocytosis by proteolytic cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25). It is widely believed that BoNT/A therapeutic or toxic actions are exclusively mediated by SNAP-25 cleavage. On the other hand, in vitro and in vivo findings suggest that several BoNT/A actions related to neuroexocytosis, cell cycle and apoptosis, neuritogenesis and gene expression are not necessarily mediated by this widely accepted mechanism of action. In present review we summarize the literature evidence which point to the existence of unknown BoNT/A molecular target(s) and modulation of unknown signaling pathways. The effects of BoNT/A apparently independent of SNAP-25 occur at similar doses/concentrations known to induce SNAP-25 cleavage and prevention of neurotransmitter release. Accordingly, these effects might be pharmacologically significant. Potentially the most interesting are observations of antimitotic and antitumor activity of BoNT/A. However, the exact mechanisms require further studies.

Antinociceptive effect of botulinum toxin type A on experimental abdominal pain.

Visceral pain, especially in the abdominal region, represents one of the most common types of pain. Its chronic form is usually very hard to treat by conventional analgesic agents and adjuvants. We investigated the antinociceptive effect of botulinum toxin type A (BTX-A) in male Wistar rats in two models of visceral pain: peritonitis induced by intraperitoneal injection of 1% acetic acid and colitis induced by intracolonic instillation of 0.1% capsaicin. Pain was measured as the number of abdominal writhes. Additionally, referred mechanical sensitivity in the ventral abdominal area was evaluated by von Frey test and the extent of spinal c-Fos expression was immunohistochemically examined. BTX-A significantly reduced the number of abdominal writhes in both models of visceral pain after intrathecal application in a dose of 2 U/kg. In the experimental colitis model, BTX-A (2 U/kg) reduced both referred mechanical allodynia and c-Fos expression in the dorsal horn of the spinal cord (S2/S3 segments). In contrast to intrathecal administration, BTX-A (2 U/kg) administered into the cisterna magna had no effect on pain suggesting that the primary site of its action is a spinal cord.

Botulinum toxin A, brain and pain.

Botulinum neurotoxin type A (BoNT/A) is one of the most potent toxins known and a potential biological threat. At the same time, it is among the most widely used therapeutic proteins used yearly by millions of people, especially for cosmetic purposes. Currently, its clinical use in certain types of pain is increasing, and its long-term duration of effects represents a special clinical value. Efficacy of BoNT/A in different types of pain has been found in numerous clinical trials and case reports, as well as in animal pain models. However, sites and mechanisms of BoNT/A actions involved in nociception are a matter of controversy. In analogy with well known neuroparalytic effects in peripheral cholinergic synapses, presently dominant opinion is that BoNT/A exerts pain reduction by inhibiting peripheral neurotransmitter/inflammatory mediator release from sensory nerves. On the other hand, growing number of behavioral and immunohistochemical studies demonstrated the requirement of axonal transport for BoNT/A's antinociceptive action. In addition, toxin's enzymatic activity in central sensory regions was clearly identified after its peripheral application. Apart from general pharmacology, this review summarizes the clinical and experimental evidence for BoNT/A antinociceptive activity and compares the data in favor of peripheral vs. central site and mechanism of action. Based on literature review and published results from our laboratory we propose that the hypothesis of peripheral site of BoNT/A action is not sufficient to explain the experimental data collected up to now.

Botulinum toxin type A selectivity for certain types of pain is associated with capsaicin-sensitive neurons.

Unlike most classical analgesics, botulinum toxin type A (BoNT/A) does not alter acute nociceptive thresholds, and shows selectivity primarily for allodynic and hyperalgesic responses in certain pain conditions. We hypothesized that this phenomenon might be explained by characterizing the sensory neurons targeted by BoNT/A in the central nervous system after its axonal transport. BoNT/A's central antinociceptive activity following its application into the rat whisker pad was examined in trigeminal nucleus caudalis (TNC) and higher-level nociceptive brain areas using BoNT/A-cleaved synaptosomal-associated protein 25 (SNAP-25) and c-Fos immunohistochemistry. Occurrence of cleaved SNAP-25 in TNC was examined after nonselective ganglion ablation with formalin or selective denervation of capsaicin-sensitive (vanilloid receptor-1 or TRPV1-expressing) neurons, and in relation to different cellular and neuronal markers. Regional c-Fos activation and effect of TRPV1-expressing afferent denervation on toxin's antinociceptive action were studied in formalin-induced orofacial pain. BoNT/A-cleaved SNAP-25 was observed in TNC, but not in higher-level nociceptive nuclei. Cleaved SNAP-25 in TNC disappeared after formalin-induced trigeminal ganglion ablation or capsaicin-induced sensory denervation. Occurrence of cleaved SNAP-25 in TNC and BoNT/A antinociceptive activity in formalin-induced orofacial pain were prevented by denervation with capsaicin. Cleaved SNAP-25 localization demonstrated toxin's presynaptic activity in TRPV1-expressing neurons. BoNT/A reduced the c-Fos activation in TNC, locus coeruleus, and periaqueductal gray. Present experiments suggest that BoNT/A alters the nociceptive transmission at the central synapse of primary afferents. Targeting of TRPV1-expressing neurons might be associated with observed selectivity of BoNT/A action only in certain types of pain.

Comparison of analgesic effects of single versus repeated injection of botulinum toxin in orofacial formalin test in rats.

Long-term effectiveness and repeated administration of botulinum toxin A are the basis for its use in both neuromuscular disorders and certain painful conditions. Botulinum toxin A has been recently approved for migraine treatment, and its off-label use extends to other craniofacial pain disorders. However, recently it was reported that, after repeated injection, botulinum toxin loses its antinociceptive efficacy in rats. In present study with a similar design, we compared the effects of single and repeated injections of botulinum toxin in formalin-induced orofacial pain. No statistically significant differences were found between single or repeatedly treated animal groups. Our results are in line with the clinical experience and suggest that botulinum toxin can be re-administered in orofacial pain treatment.

Botulinum toxin's axonal transport from periphery to the spinal cord.

Axonal transport of enzymatically active botulinum toxin A (BTX-A) from periphery to the CNS has been described in facial and trigeminal nerve, leading to cleavage of synaptosomal-associated protein 25 (SNAP-25) in central nuclei. Aim of present study was to examine the existence of axonal transport of peripherally applied BTX-A to spinal cord via sciatic nerve. We employed BTX-A-cleaved SNAP-25 immunohistochemistry of lumbar spinal cord after intramuscular and subcutaneous hind limb injections, and intraneural BTX-A sciatic nerve injections. Truncated SNAP-25 in ipsilateral spinal cord ventral horns and dorsal horns appeared after single peripheral BTX-A administrations, even at low intramuscular dose applied (5 U/kg). Cleaved SNAP-25 appearance in the spinal cord after BTX-A injection into the sciatic nerve was prevented by proximal intrasciatic injection of colchicine (5 mM, 2 µl). Cleaved SNAP-25 in ventral horn, using choline-acetyltransferase (ChAT) double labeling, was localized within cholinergic neurons. These results extend the recent findings on BTX-A retrograde axonal transport in facial and trigeminal nerve. Appearance of truncated SNAP-25 in spinal cord following low-dose peripheral BTX-A suggest that the axonal transport of BTX-A occurs commonly following peripheral application.

Central action of peripherally applied botulinum toxin type A on pain and dural protein extravasation in rat model of trigeminal neuropathy.

BACKGROUND: Infraorbital nerve constriction (IoNC) is an experimental model of trigeminal neuropathy. We investigated if IoNC is accompanied by dural extravasation and if botulinum toxin type A (BoNT/A) can reduce pain and dural extravasation in this model. METHODOLOGY/PRINCIPAL FINDINGS: Rats which developed mechanical allodynia 14 days after the IoNC were injected with BoNT/A (3.5 U/kg) into vibrissal pad. Allodynia was tested by von Frey filaments and dural extravasation was measured as colorimetric absorbance of Evans blue-plasma protein complexes. Presence of dural extravasation was also examined in orofacial formalin-induced pain. Unilateral IoNC, as well as formalin injection, produced bilateral dural extravasation. Single unilateral BoNT/A injection bilaterally reduced IoNC induced dural extravasation, as well as allodynia (lasting more than 2 weeks). Similarly, BoNT/A reduced formalin-induced pain and dural extravasation. Effects of BoNT/A on pain and dural extravasation in IoNC model were dependent on axonal transport through sensory neurons, as evidenced by colchicine injections (5 mM, 2 µl) into the trigeminal ganglion completely preventing BoNT/A effects. CONCLUSIONS/SIGNIFICANCE: Two different types of pain, IoNC and formalin, are accompanied by dural extravasation. The lasting effect of a unilateral injection of BoNT/A in experimental animals suggests that BoNT/A might have a long-term beneficial effect in craniofacial pain associated with dural neurogenic inflammation. Bilateral effects of BoNT/A and dependence on retrograde axonal transport suggest a central site of its action.

Botulinum toxin type A reduces pain supersensitivity in experimental diabetic neuropathy: bilateral effect after unilateral injection.

We investigated antinociceptive activity of botulinum toxin type A (BTX-A) in a model of diabetic neuropathic pain in rats. Male Wistar rats were made diabetic by a single intraperitoneal injection of streptozotocin (80mg/kg). Sensitivity to mechanical and thermal stimuli was measured with the paw-pressure and hot-plate test, respectively. The formalin test was used to measure sensitivity to chemical stimuli. Diabetic animals with pain thresholds lower for at least 25% compared to the non-diabetic group were considered neuropathic and were injected with BTX-A either subcutaneously (3, 5 and 7U/kg) or intrathecally (1U/kg). Mechanical and thermal sensitivity was measured at several time-points. After peripheral application, BTX-A (5 and 7U/kg) reduced mechanical and thermal hypersensitivity not only on ipsilateral, but on contralateral side, too. The antinociceptive effect started 5days following BTX-A injection and lasted at least 15days. Formalin-induced hypersensitivity in diabetic animals was abolished as well. When applied intrathecally, BTX-A (1U/kg) reduced diabetic hyperalgesia within 24h supporting the assumption of retrograde axonal transport of BTX-A from the peripheral site of injection to central nervous system. The results presented here demonstrate the long-lasting pain reduction after single BTX-A injection in the animals with diabetic neuropathy. The bilateral pain reduction after unilateral toxin application and the effectiveness of lower dose with the faster onset after the intrathecal injection suggest the involvement of the central nervous system in the antinociceptive action of BTX-A in painful diabetic neuropathy.