The CatWalk gait analysis in assessment of both dynamic and static gait changes after adult rat sciatic nerve resection.

Functional repair of neurotmesis has been proven most challenging in regenerative medicine. Progress in this field has shown that functional repair not only requires axon regeneration, but also selectivity in target reinnervation. Although selectivity in target reinnervation still involves relatively unexplored avenues, evidence-based medicine, in the end, requires behavioral proof of repair. Therefore, there is a need for tests assessing behavioral deficits after neurotmesis. To date, behavioral tests for detecting both dynamic and static parameters are limited. The CatWalk gait analysis has been shown to detect a multitude of speed-controlled dynamic and static gait deficits after experimental spinal cord injury. Therefore, we here evaluated its use in detecting both dynamic and static gait deficits after neurotmesis. After rat sciatic nerve resection CatWalk testing was performed for 8 weeks. A large amount of dynamic and static gait parameters were detected to be immediately and severely affected in the ipsilateral paw, sometimes reaching levels of only 15% of those of the unaffected paw. We conclude that the CatWalk objectively detects dynamic and static gait impairments after sciatic nerve resection and future experiments are now required to prove which of these parameters are of particular interest to detect functional repair

Inverse relation between intensity of GFAP expression in the substantia gelatinosa and degree of chronic mechanical allodynia.

Glial cells are known to have a large impact on neuropathic pain conditions. Within the spinal cord, microglia rapidly respond to peripheral nerve injury, resulting in central sensitization and ultimately in the onset of enhanced pain behaviour. Astroglia respond with a short delay and are thought to contribute to the early maintenance of neuropathic pain. Nevertheless, it is unknown whether the roles of these glial cell types can be influenced by the chronicity of the neuropathology. Here, the persistent responses of astroglia and microglia to peripheral nerve injury within central pain networks in the upper dorsal horn laminae were studied. At 12 weeks after complete sciatic nerve injury, upregulation of glial fibrillary acidic protein (GFAP), but not complement receptor-3, could be detected in laminae II and III. Moreover, it was found that neuropathic animals with a higher degree of mechanical allodynia had a lower intensity of GFAP expression in lamina II (substantia gelatinosa). From these data we conclude that the role of astroglial responses in mechanical allodynia after peripheral nerve injury may be less straightforward as previously thought. Although astroglia are known to play a pro-nociceptive role in early neuropathic pain states, this role may shift to anti-nociception in more chronic pain states

Synaptic plasticity in the substantia gelatinosa in a model of chronic neuropathic pain.

Chronic neuropathic pain (CNP) is common after peripheral nerve injuries (PNI), but is rather refractory to available anti-pain medication. Advances in neuropathic pain research have identified cellular and molecular cues triggering the onset of neuropathic pain, but the mechanisms responsible for maintenance of chronic pain states are largely unknown. Structural changes such as sprouting of injured A-fibres into the substantia gelatinosa of the dorsal horn in the spinal cord have been proposed to relate to neuropathic pain in partial PNI models. Structural changes in central pain networks may also underlie the more persistent CNP following complete sectioning of a peripheral nerve, because this type of injury results in continuous and spontaneous afferent input to the spinal cord, which can trigger central sensitization. In the present study, the left sciatic nerve was completely sectioned and a 1-cm segment was removed to maintain a chronic pathology, whereas the right sciatic nerve was left intact. Mechanical allodynia was measured up to 84 days after injury, after which synaptic changes were studied in the lumbar substantia gelatinosa. The numbers of larger sized synaptophysin-immunoreactive presynaptic boutons were found to be increased in the substantia gelatinosa ipsilateral to the nerve injury. From these data we conclude that structural synaptic changes within the substantia gelatinosa are present months after complete nerve injury and that this plasticity may be involved in maintaining neuropathic pain states

Cellular and molecular insights into neuropathy-induced pain hypersensitivity for mechanism-based treatment approaches.

Neuropathic pain is currently being treated by a range of therapeutic interventions that above all act to lower neuronal activity in the somatosensory system (e.g. using local anesthetics, calcium channel blockers, and opioids). The present review highlights novel and often still largely experimental treatment approaches based on insights into pathological mechanisms, which impact on the spinal nociceptive network, thereby opening the 'gate' to higher brain centers involved in the perception of pain. Cellular and molecular mechanisms such as ectopia, sensitization of nociceptors, phenotypic switching, structural plasticity, disinhibition, and neuroinflammation are discussed in relation to their involvement in pain hypersensitivity following either peripheral neuropathies or spinal cord injury. A mechanism-based treatment approach may prove to be successful in effective treatment of neuropathic pain, but requires more detailed insights into the persistence of cellular and molecular pain mechanisms which renders neuropathic pain unremitting. Subsequently, identification of the therapeutic window-of-opportunities for each specific intervention in the particular peripheral and/or central neuropathy is essential for successful clinical trials. Most of the cellular and molecular pain mechanisms described in the present review suggest pharmacological interference for neuropathic pain management. However, also more invasive treatment approaches belong to current and/or future options such as neuromodulatory interventions (including spinal cord stimulation) and cell or gene therapies, respectively

Neuropathy-induced spinal GAP-43 expression is not a main player in the onset of mechanical pain hypersensitivity.

Structural plasticity within the spinal nociceptive network may be fundamental to the chronic nature of neuropathic pain. In the present study, the spatiotemporal expression of growth-associated protein-43 (GAP-43), a protein which has been traditionally implicated in nerve fiber growth and sprouting, was investigated in relation to mechanical pain hypersensitivity. An L5 spinal nerve transection model was validated by the presence of mechanical pain hypersensitivity and an increase in the early neuronal activation marker cFos within the superficial spinal dorsal horn upon innocuous hindpaw stimulation. Spinal GAP-43 was found to be upregulated in the superficial L5 dorsal horn from 5 up to 10 days after injury. GAP-43 was co-localized with calcitonin-gene related peptide (CGRP), but not vesicular glutamate transporter-1 (VGLUT-1), IB4, or protein kinase-γ (PKC-γ), suggesting the regulation of GAP-43 in peptidergic nociceptive afferents. These GAP-43/CGRP fibers may be indicative of sprouting peptidergic fibers. Fiber sprouting largely depends on growth factors, which are typically associated with neuro-inflammatory processes. The putative role of neuropathy-induced GAP-43 expression in the development of mechanical pain hypersensitivity was investigated using the immune modulator propentofylline. Propentofylline treatment strongly attenuated the development of mechanical pain hypersensitivity and glial responses to nerve injury as measured by microglial and astroglial markers, but did not affect neuropathy-induced levels of spinal GAP-43 or GAP-43 regulation in CGRP fibers. We conclude that nerve injury induces structural plasticity in fibers expressing CGRP, which is regarded as a main player in central sensitization. Our data do not, however, support a major role of these structural changes in the onset of mechanical pain hypersensitivity

Stimulation of neurite outgrowth on neonatal cerebral astrocytes is enhanced in the presence of BDNF.

An area of increasing interest in spinal cord injury (SCI) research is the development of multi-factorial strategies to promote repair. In this respect, a prominent role is played by cell transplantation, the reparative effect of which can be enhanced by additional use of neurotrophic factors. Immature astrocytes have shown their merit in stimulating axon regeneration upon transplantation into the injured spinal cord. Brain-derived neurotrophic factor (BDNF) influences a wide range of descending axon tracts in the injured spinal cord. In the present study, we hypothesized that the neurite outgrowth of neonatal cortical neurons on immature astrocytes is enhanced in the presence of BDNF. To test this hypothesis, neonatal cortical neurons were cultured on neonatal astrocytes for 2 days in absence or presence of BDNF. The length of the longest neurite and the number of primary neurites per neuron were taken as measures to study neurite outgrowth. We show that BDNF dose-dependently enhanced neurite outgrowth of neonatal cerebral cortical neurons grown on immature astrocytes. Compared to conditions without BDNF, the length of the longest neurite increased by 25.5 and 28.8% in presence of 10 and 100 pg/ml BDNF, respectively. BDNF did not alter the density of the immature astrocytes. We conclude that the presence of BDNF enhances the neurite outgrowth on immature astrocytes. A multi-factorial strategy based on transplantation of neonatal astrocytes in the presence of additional BDNF is recommended and may stimulate axon regrowth after experimental injury to the central nervous system

Mice lacking L1 have reduced CGRP fibre in-growth into spinal transection lesions.

Repair strategies for spinal cord injury often focus on promoting regeneration of injured axons and stimulating subsequent functional recovery. Although many of these strategies have proven their merits, less is known about potential unwanted side-effects, such as sprouting of nociceptive CGRP immunoreactive axons, which may bring about pain-related behavior. Sprouting of CGRP axons into lesion sites spontaneously occurs after spinal cord injury (SCI). Using L1-deficient mice we show a reduction of such CGRP growth response. This reduction was specific for CGRP axons since the overall neurofilament positive fibre in-growth into the spinal lesion site was not affected. Our results may have important implications on the development and assessment of repair strategies that should not only stimulate functional recovery, but also prevent the development of pain or autonomic dysreflexia