Lidija Bach-Rojecky

Behavioral and immunohistochemical evidence for central antinociceptive activity of botulinum toxin A

Botulinum toxin A (BTX-A) is approved for treatment of different cholinergic hyperactivity disorders, and, recently, migraine headache. Although suggested to act only locally, novel observations demonstrated bilateral reduction of pain after unilateral toxin injection, and proposed retrograde axonal transport, presumably in sensory neurons. However, up to now, axonal transport of BTX-A from periphery to CNS was identified only in motoneurons, but with unknown significance. We assessed the effects of low doses of BTX-A injected into the rat whisker pad (3.5 U/kg) or into the sensory trigeminal ganglion (1 U/kg) on formalin-induced facial pain. Axonal transport was prevented by colchicine injection into the trigeminal ganglion (5 mM, 2 μl). To find the possible site of action of axonally transported BTX-A, we employed immunohistochemical labeling of BTX-A-truncated synaptosomal-associated protein 25 (SNAP-25) in medullary dorsal horn of trigeminal nucleus caudalis after toxin injection into the whisker pad. Both peripheral and intraganglionic BTX-A reduce phase II of formalin-induced pain. Antinociceptive effect of BTX-A was prevented completely by colchicine. BTX-A-truncated SNAP-25 in medullary dorsal horn (spinal trigeminal nucleus) was evident 3 days following the peripheral treatment, even with low dose applied (3.5 U/kg). Presented data provide the first evidence that axonal transport of BTX-A, obligatory for its antinociceptive effects, occurs via sensory neurons and is directed to sensory nociceptive nuclei in the CNS.

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.

Botulinum toxin type A in experimental neuropathic pain

Summary.A peripheral application of botulinum toxin type A (7 U/kg) has significantly reduced thermal and mechanical hypersensitivity in rats with the partial sciatic nerve transection as a classical model of surgical neuropathy.

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.

Lack of anti-inflammatory effect of botulinum toxin type A in experimental models of inflammation

Botulinum toxin type A (BTX‐A) has a long‐lasting antinociceptive activity and less clear effect on inflammation. It was proposed that these two effects share the same mechanism – the inhibition of neurotransmitter exocytosis from peripheral nerve endings. However, till now possible anti‐inflammatory action of BTX‐A did not evoke much attention. In the present paper, we investigate possible anti‐inflammatory action of the toxin in carrageenan and capsaicin models of inflammation in rats. BTX‐A (5 and 10 U/kg) was injected into the plantar surface of the rat right hind‐paw pad 5 days before the injection of the carrageenan (1%) or capsaicin (0.1%) at the same site. Carrageenan‐induced paw oedema and capsaicin‐induced protein extravasation were measured. Control, inflamed and BTX‐A pretreated inflamed paws were photographed and histopathological analysis (haematoxylin & eosin) was performed. Pretreatment with BTX‐A had no effect on the size of carrageenan‐induced paw oedema, measured as paw volume and weight or capsaicin‐induced plasma extravasations, measured by Evans blue as a marker of protein leakage. Neither macroscopic nor microscopic analysis showed a significant difference between BTX‐A pretreated and control inflamed tissue. Results show dissociation between the effect of BTX‐A on pain and inflammation thus questioning the validity of the suggested assumption about the common peripheral mechanism of action.

Involvement of μ-opioid receptors in antinociceptive action of botulinum toxin type A.

Botulinum toxin A (BTX-A) is approved for treatment of chronic migraine and has been investigated in various other painful conditions. Recent evidence demonstrated retrograde axonal transport and suggested the involvement of CNS in antinociceptive effect of BTX-A. However, the mechanism of BTX-A central antinociceptive action is unknown. In this study we investigated the potential role of opioid receptors in BTX-As antinociceptive activity. In formalin-induced inflammatory pain we assessed the effect of opioid antagonists on antinociceptive activity of BTX-A. Naltrexone was injected subcutaneously (0.02-2 mg/kg) or intrathecally (0.07 μg/10 μl-350 μg/10 μl), while selective μ-antagonist naloxonazine was administered intraperitoneally (5 mg/kg) prior to nociceptive testing. The influence of naltrexone (2 mg/kg s.c.) on BTX-A antinociceptive activity was examined additionally in an experimental neuropathy induced by partial sciatic nerve transection. To investigate the effects of naltrexone and BTX-A on neuronal activation in spinal cord, c-Fos expression was immunohistochemically examined in a model of formalin-induced pain. Antinociceptive effects of BTX-A in formalin and sciatic nerve transection-induced pain were prevented by non-selective opioid antagonist naltrexone. Similarly, BTX-A-induced pain reduction was abolished by low dose of intrathecal naltrexone and by selective μ-antagonist naloxonazine. BTX-A-induced decrease in dorsal horn c-Fos expression was prevented by naltrexone. Prevention of BTX-A effects on pain and c-Fos expression by opioid antagonists suggest that the central antinociceptive action of BTX-A might be associated with the activity of endogenous opioid system (involving μ-opioid receptor). These results provide first insights into the mechanism of BTX-As central antinociceptive activity.

Brain antioxidant capacity in rat models of betacytotoxic-induced experimental sporadic Alzheimer’s disease and diabetes mellitus

It is believed that oxidative stress plays a central role in the pathogenesis of metabolic diseases like diabetes mellitus (DM) and its complications (like peripheral neuropathy) as well as in neurodegenerative disorders like sporadic Alzheimers disease (sAD). Representative experimental models of these diseases are streptozotocin (STZ)-induced diabetic rats and STZ-intracerebroventricularly (STZ-icv) treated rats, in which antioxidant capacity against peroxyl (ORAC(-ROO)*) and hydroxyl (ORAC(-OH)*) free radical was measured in three different brain regions (hippocampus, cerebellum, and brain stem) by means of oxygen radical absorbance capacity (ORAC) assay. In the brain of both STZ-induced diabetic and STZ-icv treated rats decreased antioxidant capacity has been found demonstrating regionally specific distribution. In the diabetic rats these abnormalities were not associated with the development of peripheral diabetic neuropathy. Also, these abnormalities were not prevented by the icv pretreatment of glucose transport inhibitor 5-thio-D-glucose in the STZ-icv treated rats, suggesting different mechanism for STZ-induced central effects from those at the periphery. Similarities in the oxidative stress alterations in the brain of STZ-icv rats and humans with sAD could be useful in the search for new drugs in the treatment of sAD that have antioxidant activity.

Association of antinociceptive action of botulinum toxin type A with GABA-A receptor.

Abstract The mechanism of botulinum toxin type A (BTX-A) antinociceptive action in the central nervous system is little known. The potential interaction between BTX-A and GABAergic system has not been investigated previously. In the present study, we demonstrate prevention of BTX-A antinociceptive effect on formalin-induced inflammatory pain and partial sciatic nerve transection-induced mechanical allodynia by GABA-A antagonist bicuculline, thus suggesting association of the GABA-A receptors and antinociceptive action of BTX-A.

Lasting reduction of postsurgical hyperalgesia after single injection of botulinum toxin type A in rat

A single injection of low doses of botulinum toxin type A (3.5 U/kg) completely abolished secondary mechanical hyperalgesia throughout its duration in a model of post surgical pain after gastrocnemius incision in rat.

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.