Petr Dubový

Wallerian degeneration and peripheral nerve conditions for both axonal regeneration and neuropathic pain induction

Wallerian degeneration is a cascade of stereotypical events in reaction to injury of nerve fibres. These events consist of cellular and molecular alterations, including macrophage invasion, activation of Schwann cells, as well as neurotrophin and cytokine upregulation. This review focuses on cellular and molecular changes distal to various types of peripheral nerve injury which simultaneously contribute to axonal regeneration and neuropathic pain induction. In addition to the stereotypical events of Wallerian degeneration, various types of nerve damage provide different conditions for both axonal regeneration and neuropathic pain induction. Wallerian degeneration of injured peripheral nerve is associated with an inflammatory response including rapid upregulation of the immune signal molecules like cytokines, chemokines and transcription factors with both beneficial and detrimental effects on nerve regeneration or neuropathic pain induction. A better understanding of the molecular interactions between the immune system and peripheral nerve injury would open the possibility for targeting these inflammatory mediators in therapeutic interventions. Understanding the pleiotropic effects of cytokines/chemokines, however, requires investigating their highly specific pathways and precise points of action.

Bilateral changes of TNF-α and IL-10 protein in the lumbar and cervical dorsal root ganglia following a unilateral chronic constriction injury of the sciatic nerve

BackgroundThere is a growing body of evidence that unilateral nerve injury induces bilateral response, the mechanism of which is not exactly known. Because cytokines act as crucial signaling molecules for response of peripheral nerves to injury, they may be induced to mediate the reaction in remote structures.MethodsWe studied levels of tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10) proteins using ELISA in the ipsilateral and contralateral lumbar (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) from naïve rats, rats operated on to create unilateral chronic constriction injury (CCI) of the sciatic nerve, and sham-operated rats. Withdrawal thresholds for mechanical allodynia and thermal hyperalgesia were measured in the ipsilateral and contralateral hind and forepaws.ResultsThe ipsilateral hind paws of all rats operated upon for CCI displayed decreased withdrawal thresholds for mechanical allodynia and thermal hyperalgesia, while no significant behavioral changes were found in the contralateral hind paws and both forepaws. Significantly lower baseline levels of TNF-α and IL-10 protein were measured by ELISA in the lumbar than cervical DRG of naïve rats. Bilateral elevation of TNF-α was induced in both the lumbar and cervical DRG by unilateral CCI of the sciatic nerve for 7 and 14 days, while the level of IL-10 protein was increased bilaterally in the lumbar DRG 1 and 3 days after operation. IL-10 levels declined bilaterally even below baseline level in both cervical and lumbar DRG 7 days from CCI and normalized after 14 days. In contrast to no significant changes in TNF-α, level of IL-10 protein was significantly increased in the ipsilateral lumbar DRG after 3 days and bilaterally in the lumbar DRG after 14 days from sham operation.ConclusionsThe results of our experiments show a bilateral elevation of TNF-α and IL-10 not only in the homonymous DRG but also in the heteronymous DRG unassociated with the injured nerve. This suggests that bilaterally increased levels of TNF-α and IL-10 in DRG following unilateral CCI are linked with general neuroinflammatory reaction of the nervous system to injury rather than only to development and maintenance of neuropathic pain.

Intra- and Extraneuronal Changes of Immunofluorescence Staining for TNF- and TNFR1 in the Dorsal Root Ganglia of Rat Peripheral Neuropathic Pain Models

1. Several lines of evidence suggest that cytokines and their receptors are initiators of changes in the activity of dorsal root ganglia (DRG) neurons, but their cellular distribution is still very limited or controversial. Therefore, the goal of present study was to investigate immunohistochemical distribution of TNF-α and TNF receptor-1 (TNFR1) proteins in the rat DRG following three types of nerve injury.2. The unilateral sciatic and spinal nerve ligation as well as the sciatic nerve transection were used to induce changes in the distribution of TNF-α and TNFR1 proteins. The TNF-α and TNFR1 immunofluorescence was assessed in the L4-L5 DRG affected by nerve injury for 1 and 2 weeks, and compared with the contralateral ones and those removed from naive or sham-operated rats. A part of the sections was incubated for simultaneous immunostaining for TNF-α and ED-1. The immunofluorescence brightness was measured by image analysis system (LUCIA-G v4.21) to quantify immunostaining for TNF-α and TNFR1 in the naive, ipsi- and contralateral DRG following nerve injury.3. The ipsilateral L4-L5 DRG and their contralateral counterparts of the rats operated for nerve injury displayed an increased immunofluorescence (IF) for TNF-α and TNFR1 when compared with DRG harvested from naive or sham-operated rats. The TNFα IF was increased bilaterally in the satellite glial cells (SGC) and contralaterally in the neuronal nuclei following sciatic and spinal nerve ligature. The neuronal bodies and their SGC exhibited bilaterally enhanced IF for TNF-α after sciatic nerve transection for 1 and 2 weeks. In addition, the affected DRG were invaded by ED-1 positive macrophages which displayed simultaneously TNFα IF. The ED-1 positive macrophages were frequently located near the neuronal bodies to occupy a position of the satellites.4. The sciatic and spinal nerve ligature resulted in an increased TNFR1 IF in the neuronal bodies of both ipsi- and contralateral DRG. The sciatic nerve ligature for 1 week induced a rise in TNFR1 IF in the contralateral DRG neurons and their SGC to a higher level than in the ipsilateral ones. In contrast, the sciatic nerve ligature for 2 weeks caused a similar increase of TNFR1 IF in the neurons and their SGC of both ipsi- and contralateral DRG. The spinal nerve ligature or sciatic nerve transection resulted in an increased TNFR1 IF located at the surface of the ipsilateral DRG neurons, but dispersed IF in the contralateral ones. In addition, the SGC of the contralateral in contrast to ipsilateral DRG displayed a higher TNFR1 IF.5. Our results suggest more sources of TNF-α protein in the ipsilateral and contralateral DRG following unilateral nerve injury including macrophages, SGC and primary sensory neurons. In addition, the SGC and macrophages, which became to be satellites, are well positioned to regulate activity of the DRG neurons by production of TNF-α molecules. Moreover, the different cellular distribution of TNFR1 in the ipsi- and contralateral DRG may reflect different pathways by which TNF-α effect on the primary sensory neurons can be mediated following nerve injury.

Satellite glial cells express IL-6 and corresponding signal-transducing receptors in the dorsal root ganglia of rat neuropathic pain model.

There is a growing body of evidence that cytokines contribute to both induction and maintenance of neuropathic pain derived from changes in dorsal root ganglia (DRG), including the activity of the primary sensory neurons and their satellite glial cells (SGC). We used immunofluorescence and in situ hybridization methods to provide evidence that chronic constriction injury (CCI) of the sciatic nerve induces synthesis of interleukin-6 (IL-6) in SGC, elevation of IL-6 receptor (IL-6R) and activation of signal transducer and activator of transcription 3 (STAT3) signalling. Unilateral CCI of the rat sciatic nerve induced mechanoallodynia and thermal hyperalgesia in ipsilateral hind paws, but contralateral paws exhibited only temporal changes of sensitivity. We demonstrated that IL-6 mRNA and protein, which were expressed at very low levels in naïve DRG, were bilaterally increased not only in L4-L5 DRG neurons but also in SGC activated by unilateral CCI. Besides IL-6, substantial increase of IL-6R and pSTAT3 expression occurred in SGC following CCI, however, IL-6R associated protein, gp130 levels did not change. The results may suggest that unilateral CCI of the sciatic nerve induces bilateral activation of SGC in L4-L5 DRG to transduce IL-6 signalling during neuroinflammation.

Spatio-temporal changes of SDF1 and its CXCR4 receptor in the dorsal root ganglia following unilateral sciatic nerve injury as a model of neuropathic pain

There is a growing evidence that chemokines and their receptors play a role in inducing and maintaining neuropathic pain. In the present study, unilateral chronic constriction injury (CCI) of rat sciatic nerve under aseptic conditions was used to investigate changes for stromal derived factor-1 (SDF1) and its CXCR4 receptor in lumbal (L4–L5) and cervical (C7–C8) dorsal root ganglia (DRG) from both sides of naïve, CCI-operated and sham-operated rats. All CCI-operated rats displayed mechanical allodynia and thermal hyperalgesia in hind paws ipsilateral to CCI, but forepaws exhibited only temporal changes of sensitivity not correlated with alterations in SDF1 and CXCR4 proteins. Naïve DRG displayed immunofluorescence for SDF1 (SDF1-IF) in the satellite glial cells (SGC) and CXCR4-IF in the neuronal bodies with highest intensity in small- and medium-sized neurons. Immunofluorescence staining and Western blot analysis confirmed that unilateral CCI induced bilateral alterations of SDF1 and CXCR4 proteins in both L4–L5 and C7–C8 DRG. Only lumbal DRG were invaded by ED-1+ macrophages exhibiting SDF1-IF while elevation of CXCR4-IF was found in DRG neurons and SGC but not in ED-1+ macrophages. No attenuation of mechanical allodynia, but reversed thermal hyperalgesia, in ipsi- and contralateral hind paws was found in CCI-operated rats after i.p. administration of CXCR4 antagonist (AMD3100). These results indicate that SDF1/CXCR4 changes are not limited to DRG associated with injured nerve but that they also spread to DRG non-associated with such nerve. Functional involvement of these alterations in DRG non-associated with injured nerve in neuropathic pain remains to be elucidated.

Experimental models of peripheral neuropathic pain based on traumatic nerve injuries – An anatomical perspective

Peripheral neuropathic pain (PNP) frequently occurs as a consequence of nerve injury and may differ depending upon the type of insult and the individual patient. Progress in our knowledge of PNP induction mechanisms depends upon the utilization of appropriate experimental models in rodents based on various types of peripheral nerve lesions. In this review, we draw attention to current knowledge on basic cellular and molecular events in various experimental models used to induce the PNP symptoms. Spontaneous ectopic activity of axotomized and non-axotomized primary sensory neurons, the bodies of which are located in the dorsal root ganglion (DRG), seems to be a key mechanism of PNP induction. The primary sensory neurons are directly affected by nerve injury or indirectly by activated satellite glial cells and adjoining immune cells that release a variety of molecules changing the microenvironment of the neurons. Recently, it has become clear that molecules produced during Wallerian degeneration play an important role not only in axon-promoting conditions distal to nerve injury but also in initiation of neuropathic pain. The molecules, transported by the blood, influence afferent neurons and their axons not only in DRG associated, but also those not directly associated with the injured nerve (i.e., in the contralateral DRG or at different spinal segments). Generally, all experimental PNP models based on a partial injury of peripheral nerve segments contain mechanisms initiated by signal molecules of Wallerian degeneration.

Increased invasion of ED-1 positive macrophages in both ipsi- and contralateral dorsal root ganglia following unilateral nerve injuries

There is an increasing evidence that unilateral nerve injury induces cellular and molecular changes in the associated DRG not only on the ipsilateral but also in the contralateral side. In this investigation, ED-1+ macrophages were quantified by image analysis in the naïve L5 DRG (nDRG) and compared with the ipsi- and contralateral ones 2 and 4 weeks after unilateral sciatic nerve ligature and ventral root transection (VRT). A few ED-1+ macrophages were found in nDRG but not closely associated with the neuronal bodies. In contrast, following nerve injuries ED-1+ macrophages and their processes were frequently located close neuronal bodies and became their satellite cells. Moreover, an increased number of ED-1+ cells was found in the ipsilateral DRG 2 weeks after unilateral sciatic nerve ligature or VRT, but no significant differences were measured between 2 and 4 weeks after both types of nerve lesion. Contralateral DRG displayed a significant enhanced number of ED-1+ cells no sooner than 4 weeks from sciatic nerve ligature. In contrast, VRT induced a significant increased invasion of the ED-1+ cells in the contralateral DRG as early as 2 weeks after operation. Our experiments indicate that a significantly higher number of ED-1+ macrophages remained in both ipsi- and contralateral DRG up to 4 weeks from nerve injury. Based on results from different models of nerve injury, we suggest that more than one mechanism operates to stimulate the invasion of ED-1+ macrophages into the DRG including retrograde transport of factors produced during Wallerian degeneration or their delivery by blood flow. Signaling for macrophage invasion into DRG contralateral to nerve injury may be mediated by lost motoneurons or by interneurones.

Bilateral elevation of interleukin-6 protein and mRNA in both lumbar and cervical dorsal root ganglia following unilateral chronic compression injury of the sciatic nerve

BackgroundCurrent research implicates interleukin (IL)-6 as a key component of the nervous-system response to injury with various effects.MethodsWe used unilateral chronic constriction injury (CCI) of rat sciatic nerve as a model for neuropathic pain. Immunofluorescence, ELISA, western blotting and in situ hybridization were used to investigate bilateral changes in IL-6 protein and mRNA in both lumbar (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) following CCI. The operated (CCI) and sham-operated (sham) rats were assessed after 1, 3, 7, and 14 days. Withdrawal thresholds for mechanical hyperalgesia and latencies for thermal hyperalgesia were measured in both ipsilateral and contralateral hind and fore paws.ResultsThe ipsilateral hind paws of all CCI rats displayed a decreased threshold of mechanical hyperalgesia and withdrawal latency of thermal hyperalgesia, while the contralateral hind and fore paws of both sides exhibited no significant changes in mechanical or thermal sensitivity. No significant behavioral changes were found in the hind and fore paws on either side of the sham rats, except for thermal hypersensitivity, which was present bilaterally at 3 days. Unilateral CCI of the sciatic nerve induced a bilateral increase in IL-6 immunostaining in the neuronal bodies and satellite glial cells (SGC) surrounding neurons of both lumbar and cervical DRG, compared with those of naive control rats. This bilateral increase in IL-6 protein levels was confirmed by ELISA and western blotting. More intense staining for IL-6 mRNA was detected in lumbar and cervical DRG from both sides of rats following CCI. The DRG removed from sham rats displayed a similar pattern of staining for IL-6 protein and mRNA as found in naive DRG, but there was a higher staining intensity in SGC.ConclusionsBilateral elevation of IL-6 protein and mRNA is not limited to DRG homonymous to the injured nerve, but also extended to DRG that are heteronymous to the injured nerve. The results for IL-6 suggest that the neuroinflammatory reaction of DRG to nerve injury is propagated alongside the neuroaxis from the lumbar to the remote cervical segments. This is probably related to conditioning of cervical DRG neurons to injury.

Role of inflammation and cytokines in peripheral nerve regeneration.

This chapter provides a review of immune reactions involved in classic as well as alternative methods of peripheral nerve regeneration, and mainly with a view to understanding their beneficial effects. Axonal degeneration distal to nerve damage triggers a cascade of inflammatory events alongside injured nerve fibers known as Wallerian degeneration (WD). The early inflammatory reactions of WD comprise the complement system, arachidonic acid metabolites, and inflammatory mediators that are related to myelin fragmentation and activation of Schwann cells. Fine-tuned upregulation of the cytokine/chemokine network by Schwann cells activates resident and hematogenous macrophages to complete the clearance of axonal and myelin debris and stimulate regrowth of axonal sprouts. In addition to local effects, immune reactions of neuronal bodies and glial cells are also implicated in the survival and conditioning of neurons to regenerate severed nerves. Understanding of the cellular and molecular interactions between the immune system and peripheral nerve injury opens new possibilities for targeting inflammatory mediators to improve functional reinnervation.

Morphological evidence of collateral sprouting of intact afferent and motor axons of the rat ulnar nerve demonstrated by one type of tracer molecule

In our experimental paradigm we sutured the distal stump of a transected musculocutaneous nerve to an intact ulnar nerve of the rat in an end-to-side fashion. We demonstrated the formation of collateral sprouts from intact afferent and motor axons by application of one type of molecule conjugated by two different fluorophores (Fluoro-Ruby and Fluoro-Emerald). Fluoro-Ruby and Fluoro-Emerald were applied distal to end-to-side suture into fresh cut ends of the ulnar and musculocutaneous nerves, respectively. Formation of collateral sprouts was evidenced by findings of mixed (a yellow to orange color) fluorescence labeling of spinal motoneurons and dorsal root ganglia neurons. Colocalization of two different tracers retrogradely transported to the neurons was verified by the individual green and red fluorescence profiles analyzed by means of the computer-assisted image-analyzing system. Our results unequivocally demonstrate that a nerve stump attached to an intact nerve can induce collateral sprouting of both afferent and motor axons.