Stefano Cobianchi

Anti-allodynic efficacy of botulinum neurotoxin A in a model of neuropathic pain.

Neuropathic pain is typified by injuries to the peripheral and central nervous system and derives from such causes as cancer, diabetes, multiple sclerosis, post-herpetic neuralgia, physical trauma or surgery, and many others. Patients suffering neuropathic pain do not respond to conventional treatment with non-steroidal anti-inflammatory drugs and show a reduced sensitivity to opiates often associated with serious side effects. Recently, it has been demonstrated that botulinum neurotoxin serotype-A (BoNT/A) is able to induce analgesia in inflammatory pain conditions. The goal of this research was to test if BoNT/A was able to relieve also neuropathic pain symptoms. By using chronic constriction injury of the sciatic nerve, a mouse model of neuropathic pain, we observed that peripheral administration of BoNT/A strongly reduced the mechanical allodynia associated with this neuropathy. Remarkably, a single non-toxic dose of BoNT/A was sufficient to induce anti-allodynic effects, which lasted for at least 3 weeks. This result is particularly relevant since neuropathic pain is poorly treated by current drug therapies. This communication enlarges our knowledge on potentially new medical uses of BoNT/A in efforts to ameliorate human health conditions, with very important implications in the development of new pharmacotherapeutic approaches against neuropathic pain.

Differential effects of activity dependent treatments on axonal regeneration and neuropathic pain after peripheral nerve injury

Activity treatments are useful strategies to increase axonal regeneration and functional recovery after nerve lesions. They are thought to benefit neuropathy by enhancing neurotrophic factor expression. Nevertheless the effects on sensory function are still unclear. Since neurotrophic factors also play a fundamental role in peripheral and central sensitization, we studied the effects of acute electrical stimulation and early treadmill exercise on nerve regeneration and on neuropathic pain, and the relation with the expression of neurotrophins. After sciatic nerve section and suture repair, rats were subjected to electrical stimulation (ES) for 4h after injury, forced treadmill running (TR) for 5 days, or both treatments combined. Sciatic nerve section induced hyperalgesia in the medial area of the plantar skin in the injured paw. TR and ES differently but positively reduced adjacent neuropathic pain before and after sciatic reinnervation. ES enhanced motor and sensory reinnervation, and combination with TR induced strong agonistic effects in relieving pain. The differential effects of these activity treatments were related to changes in neurotrophic factor mRNA levels in sensory and motor neurons. ES speeded up expression of BDNF and GDNF in DRG, and of BDNF and NT3 in the ventral horn. TR reduced the levels of pro-nociceptive factors such as BDNF, NGF and GDNF in DRG. Combination of ES and TR induced intermediate levels suggesting an optimal balancing of treatment effects.

Botulinum neurotoxins and formalin-induced pain: Central vs. peripheral effects in mice

Neurotoxins affecting neuroexocytosis can represent an innovative pharmacological approach to the investigation of neural mechanisms of pain. Our interest has been focused on the use of botulinum neurotoxins (BoNTs), whose peripheral effects are extensively documented, while the effects on the central nervous system are much less clear. We have investigated both peripheral (sc into the hindpaw) and central (icv) effects of two BoNTs isoforms, BoNT/A and BoNT/B, on inflammatory pain. BoNT/A (sc: 0.937-15; icv: 0.937-3.75 pgtox/mouse) and BoNT/B (sc: 3.75, 7.5; icv: 1.875, 3.75 pgtox/mouse) were injected in CD1 mice and tested in the formalin test 3 days later. Licking response, as index of pain, and behavioral parameters, such as general activity and grooming, were recorded for 40 min during the test. BoNT/A partially affects the licking response in the second phase of formalin test in a similar magnitude of attenuation whether peripherally or centrally administered. BoNT/A does not significantly affect licking behavior during the first phase of the test. Peripheral administration of BoNT/B attenuates the licking response during the first phase not modifying the second phase, while the icv administration has hyperalgesic effect on the interphase of the formalin test. General activity and grooming behavior are not affected either by peripheral or by central administration of BoNTs. Our results show for the first time a central effect of BoNTs that differently modulate inflammatory pain depending both on serotype and on route of administration. Such data suggest BoNTs as a useful tool in the studies aimed at the comprehension of the mechanisms of inflammatory pain.

Effects of activity-dependent strategies on regeneration and plasticity after peripheral nerve injuries

Peripheral nerve injuries result in loss of motor, sensory and autonomic functions of the denervated limb, but are also accompanied by positive symptoms, such as hyperreflexia, hyperalgesia and pain. Strategies to improve functional recovery after neural injuries have to address the enhancement of axonal regeneration and target reinnervation and also the modulation of the abnormal plasticity of neuronal circuits. By enhancing sensory inputs and/or motor outputs, activity-dependent therapies, like electrostimulation or exercise, have been shown to positively influence neuromuscular functional recovery and to modulate the plastic central changes after experimental nerve injuries. However, it is important to take into account that the type of treatment, the intensity and duration of the protocol, and the period during which it is applied after the injury are factors that determine beneficial or detrimental effects on functional recovery. The adequate maintenance of activity of neural circuits and denervated muscles results in increased trophic factor release to act on regenerating axons and on central plastic changes. Among the different neurotrophins, BDNF seems a key player in the beneficial effects of activity-dependent therapies after nerve injuries.

Short- but not long-lasting treadmill running reduces allodynia and improves functional recovery after peripheral nerve injury

We analyzed the effects of different treadmill running protocols on the functional recovery after chronic constriction injury (CCI) of the sciatic nerve in mice. We found that a treadmill protocol of short-lasting running (1 h/d for 5 days after CCI) reduced the neuropathy-induced mechanical allodynia and normalized the weight bearing and the sciatic static index of the injured hindpaw. At difference, a treadmill protocol of long-lasting running (1 h/d for more than 5 days after CCI) was unfavorable both for allodynia and for functional recovery. Behavioral results were correlated with immunofluorescence assays of microglia and astrocytes activation in L4/L5 lumbar spinal cord sections. We found a differential pattern of activation characterized by: (i) reduced microglia expression, after both short- and long-lasting treadmill running; (ii) reduced astrocytes expression after short-lasting treadmill running; and, (iii) persistence of astrocytes expression after long-lasting treadmill running. Finally, in sections of injured sciatic nerves, we analyzed the expression of Cdc2 and GAP-43 proteins that are both up-regulated during peripheral regenerative processes. Compared to mice subjected to long-lasting treadmill running, mice subjected to short-lasting treadmill running showed an acceleration of the regenerative processes at the injured sciatic nerve. Our data demonstrate that short-lasting treadmill running, by reducing the neuropathic pain symptoms and facilitating the regenerative processes of the injured nerve, have beneficial rehabilitative effects on the functional recovery after peripheral nerve injury.

Botulinum neurotoxin type A counteracts neuropathic pain and facilitates functional recovery after peripheral nerve injury in animal models.

A growing interest was recently focused on the use of Botulinum neurotoxin serotype A (BoNT/A) for fighting pain. The aim of this study was to investigate the effects of BoNT/A on neuropathic pain. It was observed that BoNT/A is able to counteract neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve both in mice and in rats. This effect is already present after a single intraplantar (i.pl.) or intrathecal (i.t.) neurotoxin administration that significantly reduces the sciatic nerve ligation-induced mechanical allodynia in mice and rats and thermal hyperalgesia in rats. This effect was evident starting 24 h after the administration of BoNT/A and it was long-lasting, being present 81 or 25 days after i.pl. injection of the higher dose in mice (15 pg/paw) and rats (75 pg/paw), respectively, and 35 days after i.t. injection in rats (75 pg/rat). Moreover, BoNT/A-injected mice showed a quicker recovery of the walking pattern and weight bearing compared to control groups. The behavioral improvement was accompanied by structural modifications, as revealed by the expression of cell division cycle 2 (Cdc2) and growth associated protein 43 (GAP-43) regeneration associated proteins, investigated by immunofluorescence and Western blotting in the sciatic nerve, and by the immunofluorescence expression of S100β and glial fibrillary acidic protein (GFAP) Schwann cells proteins. In conclusion, the present research demonstrate long-lasting anti-allodynic and anti-hyperalgesic effects of BoNT/A in animal models of neuropathic pain together with an acceleration of regenerative processes in the injured nerve, as evidenced by both behavioral and immunohistochemistry/blotting analysis. These results may have important implications in the therapy of neuropathic pain.

Assessment of sensory thresholds and nociceptive fiber growth after sciatic nerve injury reveals the differential contribution of collateral reinnervation and nerve regeneration to neuropathic pain

Following traumatic peripheral nerve injury reinnervation of denervated targets may be achieved by regeneration of injured axons and by collateral sprouting of neighbor undamaged axons. Experimental models commonly use sciatic nerve injuries to assess nerve regeneration and neuropathic pain, but behavioral tests for evaluating sensory recovery often disregard the pattern of hindpaw innervation. This may lead to confounding attribution of recovery of sensory responses to improvement in sciatic nerve regeneration instead of collateral reinnervation by the undamaged saphenous nerve. We used a standardized methodology to assess the separate contribution of collateral and regenerative skin reinnervation on sensory responses. Section and suture of the sciatic nerve induced loss of sensibility in the lateral and central areas of the injured paw, but nociceptive responses rapidly recovered by expansion of the intact saphenous innervation territory. We used electronic Von Frey and Plantar test devices to measure mechanical and thermal withdrawal thresholds in specific sites of the injured paw: lateral site innervated by the sciatic nerve, medial site that remained innervated by the saphenous nerve, and central site originally innervated by the sciatic nerve but affected by saphenous sprouting. After sciatic section, signs of early hyperalgesia developed in medial and central paw areas due to saphenous sprouting and expansion. The regenerating sciatic nerve fibers reached the paw at 3-4weeks and a late mechanical hyperalgesia was observed at the lateral site. Immunohistochemical staining of sensory fibers innervating the medial and lateral areas revealed a different pattern of skin reinnervation. Hypersensitivity in the intact saphenous nerve area was paralleled by early fiber sprout growth in the subepidermal plexus, but not entering the epidermis. On the other side, late sciatic hyperalgesia was accompanied by gradual skin reinnervation after 4weeks. The standardization of algesimetry testing in sciatic nerve injury models, as proposed in this study, provides a suitable model for studying in parallel neuropathic pain and sensory nerve regeneration processes. Our results also indicate that collateral sprouting and axonal regeneration contribute differently in the initiation and maintenance of neuropathic pain.

Early increasing-intensity treadmill exercise reduces neuropathic pain by preventing nociceptor collateral sprouting and disruption of chloride cotransporters homeostasis after peripheral nerve injury.

Abstract Activity treatments, such as treadmill exercise, are used to improve functional recovery after nerve injury, parallel to an increase in neurotrophin levels. However, despite their role in neuronal survival and regeneration, neurotrophins may cause neuronal hyperexcitability that triggers neuropathic pain. In this work, we demonstrate that an early increasing-intensity treadmill exercise (iTR), performed during the first week (iTR1) or during the first 2 weeks (iTR2) after section and suture repair of the rat sciatic nerve, significantly reduced the hyperalgesia developing rapidly in the saphenous nerve territory and later in the sciatic nerve territory after regeneration. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression in sensory neurons and spinal cord was reduced in parallel. iTR prevented the extension of collateral sprouts of saphenous nociceptive calcitonin gene–related peptide fibers within the adjacent denervated skin and reduced NGF expression in the same skin and in the L3 dorsal root ganglia (DRG). Injury also induced Na+–K+–2Cl− cotransporter 1 (NKCC1) upregulation in DRG, and K+–Cl− cotransporter 2 (KCC2) downregulation in lumbar spinal cord dorsal horn. iTR normalized NKCC1 and boosted KCC2 expression, together with a significant reduction of microgliosis in L3-L5 dorsal horn, and a reduction of BDNF expression in microglia at 1 to 2 weeks postinjury. These data demonstrate that specific activity protocols, such as iTR, can modulate neurotrophins expression after peripheral nerve injury and prevent neuropathic pain by blocking early mechanisms of sensitization such as collateral sprouting and NKCC1/KCC2 disregulation.

Tubulization with chitosan guides for the repair of long gap peripheral nerve injury in the rat.

Biosynthetic guides can be an alternative to nerve grafts for reconstructing severely injured peripheral nerves. The aim of this study was to evaluate the regenerative capability of chitosan tubes to bridge critical nerve gaps (15 mm long) in the rat sciatic nerve compared with silicone (SIL) tubes and nerve autografts (AGs). A total of 28 Wistar Hannover rats were randomly distributed into four groups (n = 7 each), in which the nerve was repaired by SIL tube, chitosan guides of low (∼2%, DAI) and medium (∼5%, DAII) degree of acetylation, and AG. Electrophysiological and algesimetry tests were performed serially along 4 months follow‐up, and histomorphometric analysis was performed at the end of the study. Both groups with chitosan tubes showed similar degree of functional recovery, and similar number of myelinated nerve fibers at mid tube after 4 months of implantation. The results with chitosan tubes were significantly better compared to SIL tubes (P < 0.01), but lower than with AG (P < 0.01). In contrast to AG, in which all the rats had effective regeneration and target reinnervation, chitosan tubes from DAI and DAII achieved 43 and 57% success, respectively, whereas regeneration failed in all the animals repaired with SIL tubes. This study suggests that chitosan guides are promising conduits to construct artificial nerve grafts.

Prevention of NKCC1 phosphorylation avoids downregulation of KCC2 in central sensory pathways and reduces neuropathic pain after peripheral nerve injury

Summary Inhibition of NKCC1 activation after peripheral nerve injury prevents decrease of KCC2 in the central pain projecting areas and is associated with reduction of hyperalgesia. ABSTRACT Neuropathic pain after peripheral nerve injury is characterized by loss of inhibition in both peripheral and central pain pathways. In the adult nervous system, the Na+–K+–2Cl− (NKCC1) and neuron‐specific K+–Cl− (KCC2) cotransporters are involved in setting the strength and polarity of GABAergic/glycinergic transmission. After nerve injury, the balance between these cotransporters changes, leading to a decrease in the inhibitory tone. However, the role that NKCC1 and KCC2 play in pain‐processing brain areas is unknown. Our goal was to study the effects of peripheral nerve injury on NKCC1 and KCC2 expression in dorsal root ganglia (DRG), spinal cord, ventral posterolateral (VPL) nucleus of the thalamus, and primary somatosensory (S1) cortex. After sciatic nerve section and suture in adult rats, assessment of mechanical and thermal pain thresholds showed evidence of hyperalgesia during the following 2 months. We also found an increase in NKCC1 expression in the DRG and a downregulation of KCC2 in spinal cord after injury, accompanied by later decrease of KCC2 levels in higher projection areas (VPL and S1) from 2 weeks postinjury, correlating with neuropathic pain signs. Administration of bumetanide (30 mg/kg) during 2 weeks following sciatic nerve lesion prevented the previously observed changes in the spinothalamic tract projecting areas and the appearance of hyperalgesia. In conclusion, the present results indicate that changes in NKCC1 and KCC2 in DRG, spinal cord, and central pain areas may contribute to development of neuropathic pain.