Bilateral activation of glial cells and cellular distribution of the chemokine CCL2 and its receptor CCR2 in the trigeminal subnucleus caudalis of trigeminal neuropathic pain model.

Glial cells activated by peripheral nerve injury contribute to the induction and maintenance of neuropathic pain by releasing neuromodulating cytokines and chemokines. We investigated the activation of microglia and astrocytes as well as the cellular distribution of the chemokine CCL2 and its receptor CCR2 in the trigeminal subnucleus caudalis (TSC) ipsilateral and contralateral to infraorbital nerve ligature (IONL). The left infraorbital nerve was ligated under aseptic conditions, and sham controls were operated without nerve ligature. Tactile hypersensitivity was significantly increased bilaterally in vibrissal pads of both sham- and IONL-operated animals from day 1 to 7 and tended to normalize in sham controls surviving for 14 days. Activated microglial cells significantly increased bilaterally in the TSC of both sham- and IONL-operated animals with a marked but gradual increase in the ipsilateral TSC from 1 to 7 days followed by a decrease by day 14. In contrast, robust activation of astrocytes was found bilaterally in the TSC of IONL-operated rats from 3 to 14 days with a transient activation in the ipsilateral TSC of sham-operated animals. Cellular distribution of CCL2 varied with survival time. CCL2 immunofluorescence was detected in neurons within 3 days and in astrocytes at later time points. In contrast, CCR2 was found only in astrocytes at all time points with CCR2 intensity being dominant in the ipsilateral TSC. In summary, our results reveal bilateral activation of microglial cells and astrocytes as well as changes in the cellular distribution of CCL2 and its receptor CCR2 in the TSC during the development and maintenance of orofacial neuropathic pain.

Interleukin-6 contributes to initiation of neuronal regeneration program in the remote dorsal root ganglia neurons after sciatic nerve injury.

To assess the potential role of IL-6 in sciatic nerve injury-induced activation of a pro-regenerative state in remote dorsal root ganglia (DRG) neurons, we compared protein levels of SCG-10 and activated STAT3, as well as axon regeneration in IL-6 knockout (IL-6ko) mice and their wild-type (WT) counterparts. Unilateral sciatic nerve compression and transection upregulated SCG-10 protein levels and activated STAT3 in DRG neurons not only in lumbar but also in cervical segments of WT mice. A pro-regenerative state induced by prior sciatic nerve lesion in cervical DRG neurons of WT mice was also shown by testing for axon regeneration in crushed ulnar nerve. DRG neurons from IL-6ko mice also displayed bilaterally increased levels of SCG-10 and STAT3 in both lumbar and cervical segments after sciatic nerve lesions. However, levels of SCG-10 protein in lumbar and cervical DRG of IL-6ko mice were significantly lower than those of their WT counterparts. Sciatic nerve injury induced a lower level of SCG-10 in cervical DRG of IL-6ko than WT mice, and this correlates with significantly shorter regeneration of axons distal to the crushed ulnar nerve. These results suggest that IL-6 contributes, at the very least, to initiation of the neuronal regeneration program in remote DRG neurons after unilateral sciatic nerve injury.

Bilateral activation of STAT3 by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and its nuclear translocation in primary sensory neurons following unilateral sciatic nerve injury.

Unilateral sciatic nerve compression (SNC) or complete sciatic nerve transection (CSNT), both varying degrees of nerve injury, induced activation of STAT3 bilaterally in the dorsal root ganglia (DRG) neurons of lumbar (L4-L5) as well as cervical (C6-C8) spinal cord segments. STAT3 activation was by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and was followed by their nuclear translocation. This is the first evidence of STAT3(S727) activation together with the well-known activation of STAT3(Y705) in primary sensory neurons upon peripheral nerve injury. Bilateral activation of STAT3 in DRG neurons of spinal segments anatomically both associated as well as non-associated with the injured nerve indicates diffusion of STAT3 activation inducers along the spinal cord. Increased levels of IL-6 protein in the CSF following nerve injury as well as activation and nuclear translocation of STAT3 in DRG after intrathecal injection of IL-6 shows that this cytokine, released into the subarachnoid space can penetrate the DRG to activate STAT3. Previous results on increased bilateral IL-6 synthesis and the present manifestation of STAT3 activation in remote DRG following unilateral sciatic nerve injury may reflect a systemic reaction of the DRG neurons to nerve injury.

CD200R1 agonist attenuates glial activation, inflammatory reactions, and hypersensitivity immediately after its intrathecal application in a rat neuropathic pain model.

BACKGROUND: Interaction of CD200 with its receptor CD200R has an immunoregulatory role and attenuates various types of neuroinflammatory diseases. METHODS: Immunofluorescence staining, western blot analysis, and RT-PCR were used to investigate the modulatory effects of CD200 fusion protein (CD200Fc) on activation of microglia and astrocytes as well as synthesis of pro- (TNF, IL-1ß, IL-6) and anti-inflammatory (IL-4, IL-10) cytokines in the L4-L5 spinal cord segments in relation to behavioral signs of neuropathic pain after unilateral sterile chronic constriction injury (sCCI) of the sciatic nerve. Withdrawal thresholds for mechanical hypersensitivity and latencies for thermal hypersensitivity were measured in hind paws 1 day before operation; 1, 3, and 7 days after sCCI operation; and then 5 and 24 h after intrathecal application of artificial cerebrospinal fluid or CD200Fc. RESULTS: Seven days from sCCI operation and 5 h from intrathecal application, CD200Fc reduced mechanical and thermal hypersensitivity when compared with control animals. Simultaneously, CD200Fc attenuated activation of glial cells and decreased proinflammatory and increased anti-inflammatory cytokine messenger RNA (mRNA) levels. Administration of CD200Fc also diminished elevation of CD200 and CD200R proteins as a concomitant reaction of the modulatory system to increased neuroinflammatory reactions after nerve injury. The anti-inflammatory effect of CD200Fc dropped at 24 h after intrathecal application. CONCLUSIONS: Intrathecal administration of the CD200R1 agonist CD200Fc induces very rapid suppression of neuroinflammatory reactions associated with glial activation and neuropathic pain development. This may constitute a promising and novel therapeutic approach for the treatment of neuropathic pain.

Dynamic response to peripheral nerve injury detected by in situ hybridization of IL-6 and its receptor mRNAs in the dorsal root ganglia is not strictly correlated with signs of neuropathic pain.

BACKGROUND: IL-6 is a typical injury-induced mediator. Together with its receptors, IL-6 contributes to both induction and maintenance of neuropathic pain deriving from changes in activity of primary sensory neurons in dorsal root ganglia (DRG). We used in situ hybridization to provide evidence of IL-6 and IL-6 receptors (IL-6R and gp130) synthesis in DRG along the neuraxis after unilateral chronic constriction injury (CCI) of the sciatic nerve as an experimental model of neuropathic pain. RESULTS: All rats operated upon to create unilateral CCI displayed mechanical allodynia and thermal hyperalgesia in ipsilateral hind paws. Contralateral hind paws and forepaws of both sides exhibited only temporal and nonsignificant changes of sensitivity. Very low levels of IL-6 and IL-6R mRNAs were detected in naïve DRG. IL-6 mRNA was bilaterally increased not only in DRG neurons but also in satellite glial cells (SGC) activated by unilateral CCI. In addition to IL-6 mRNA, substantial increase of IL-6R mRNA expression occurred in DRG neurons and SGC following CCI, while the level of gp130 mRNA remained similar to that of DRG from naïve rats. CONCLUSIONS: Here we evidence for the first time increased synthesis of IL-6 and IL-6R in remote cervical DRG nonassociated with the nerve injury. Our results suggest that unilateral CCI of the sciatic nerve induced not only bilateral elevation of IL-6 and IL-6R mRNAs in L4-L5 DRG but also their propagation along the neuraxis to remote cervical DRG as a general neuroinflammatory reaction of the nervous system to local nerve injury without correlation with signs of neuropathic pain. Possible functional involvement of IL-6 signaling is discussed.

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.

BACKGROUND: Current research implicates interleukin (IL)-6 as a key component of the nervous-system response to injury with various effects. METHODS: We 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. RESULTS: The 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. CONCLUSIONS: Bilateral 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.

Ciliary neurotrophic factor promotes motor reinnervation of the musculocutaneous nerve in an experimental model of end-to-side neurorrhaphy.

BACKGROUND: It is difficult to repair nerve if proximal stump is unavailable or autogenous nerve grafts are insufficient for reconstructing extensive nerve damage. Therefore, alternative methods have been developed, including lateral anastomosis based on axons' ability to send out collateral sprouts into denervated nerve. The different capacity of a sensory or motor axon to send a sprout is controversial and may be controlled by cytokines and/or neurotrophic factors like ciliary neurotrophic factor (CNTF). The aim of the present study was to quantitatively assess collateral sprouts sent out by intact motor and sensory axons in the end-to-side neurorrhaphy model following intrathecal administration of CNTF in comparison with phosphate buffered saline (vehiculum) and Cerebrolysin. The distal stump of rat transected musculocutaneous nerve (MCN) was attached in an end-to-side fashion with ulnar nerve. CNTF, Cerebrolysin and vehiculum were administered intrathecally for 2 weeks, and all animals were allowed to survive for 2 months from operation. Numbers of spinal motor and dorsal root ganglia neurons were estimated following their retrograde labeling by Fluoro-Ruby and Fluoro-Emerald applied to ulnar and musculocutaneous nerve, respectively. Reinnervation of biceps brachii muscles was assessed by electromyography, behavioral test, and diameter and myelin sheath thickness of regenerated axons. RESULTS: Vehiculum or Cerebrolysin administration resulted in significantly higher numbers of myelinated axons regenerated into the MCN stumps compared with CNTF treatment. By contrast, the mean diameter of the myelinated axons and their myelin sheath thickness in the cases of Cerebrolysin- or CNTF-treated animals were larger than were those for rats treated with vehiculum. CNTF treatment significantly increased the percentage of motoneurons contributing to reinnervation of the MCN stumps (to 17.1%) when compared with vehiculum or Cerebrolysin treatments (at 9.9 or 9.6%, respectively). Reduced numbers of myelinated axons and simultaneously increased numbers of motoneurons contributing to reinnervation of the MCN improved functional reinnervation of the biceps brachii muscle after CNTF treatment. CONCLUSION: The present experimental study confirms end-to-side neurorrhaphy as an alternative method for reconstructing severed peripheral nerves. CNTF promotes motor reinnervation of the MCN stump after its end-to-side neurorrhaphy with ulnar nerve and improves functional recovery of the biceps brachii muscle.

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

BACKGROUND: There 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. METHODS: We studied levels of tumor necrosis factor alpha (TNF-alpha) 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. RESULTS: The 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-alpha and IL-10 protein were measured by ELISA in the lumbar than cervical DRG of naïve rats. Bilateral elevation of TNF-alpha 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-alpha, 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. CONCLUSIONS: The results of our experiments show a bilateral elevation of TNF-alpha 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-alpha 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.

Subarachnoid Hemorrhage Induces Dynamic Immune Cell Reactions in the Choroid Plexus.

Subarachnoid hemorrhage (SAH) is a specific form of hemorrhagic stroke that frequently causes intracranial hypertension. The choroid plexus (CP) of the brain ventricles is responsible for producing cerebrospinal fluid and forms the blood - cerebrospinal fluid barrier. The aim of the current study was to determine whether SAH induces an immune cell reaction in the CP and whether the resulting increase in intracranial pressure (ICP) itself can lead to cellular changes in the CP. SAH was induced by injecting non-heparinized autologous blood to the cisterna magna. Artificial cerebrospinal fluid (ACSF) instead of blood was used to assess influence of increased ICP alone. SAH and ACSF animals were left to survive for 1, 3, and 7 days. SAH induced significantly increased numbers of M1 (ED1+, CCR7+) and M2 (ED2+, CD206+) macrophages as well as MHC-II+ antigen presenting cells (APC) compared to naïve and ACSF animals. Increased numbers of ED1+ macrophages and APC were found in the CP only 3 and 7 days after ACSF injection, while ED2+ macrophage number did not increase. CD3+ T cells were not found in any of the animals. Following SAH, proliferation activity in the CP gradually increased over time while ACSF application induced higher cellular proliferation only 1 and 3 days after injection. Our results show that SAH induces an immune reaction in the CP resulting in an increase in the number of several macrophage types in the epiplexus position. Moreover, we also found that increased ICP due to ACSF application induced both an immune reaction and increased proliferation of epiplexus cells in the CP. These findings indicate that increased ICP, and not just blood, contributes to cellular changes in the CP following SAH.

Bilateral changes in IL-6 protein, but not in its receptor gp130, in rat dorsal root ganglia following sciatic nerve ligature.

Local intracellular signaling cascades following peripheral nerve injury lead to robust axon regeneration and neuropathic pain induction. Cytokines are classic injury-induced mediators. We used sciatic nerve ligature (ScNL) to investigate temporal changes in IL-6 and its receptor gp130 in both ipsilateral and contralateral lumbal (L4-L5) dorsal root ganglia (DRG). Rats were operated aseptically on unilateral ScNL and allowed to survive for 1, 3, 7, and 14 days. Immunohistochemistry and Western blot analysis were used to determine levels of IL-6 and gp130 in DRG. A distinct increase in immunostaining for IL-6 was found in the neuronal cell bodies of sections through both ipsilateral and contralateral DRG at 1 and 3 days after operation. After 7 and 14 days, the DRG sections displayed only a moderate elevation in immunostaining when compared with sections of naïve DRG. The levels of IL-6 protein increased in both ipsilateral and contralateral lumbal DRG following peripheral nerve injury. The elevation of IL-6 protein was significant in both ipsilateral and contralateral DRG 1, 3, 7, and 14 days after operation. On the other hand, the levels of gp130 receptor did not change significantly. The data provide evidence for changes in IL-6 levels not only in the DRG associated with the damaged nerve but also in those unassociated with nerve injury during the experimental neuropathic pain model.

A Conditioning Sciatic Nerve Lesion Triggers a Pro-regenerative State in Primary Sensory Neurons Also of Dorsal Root Ganglia Non-associated With the Damaged Nerve.

The primary sensory neurons of dorsal root ganglia (DRG) are a very useful model to study the neuronal regenerative program that is a prerequisite for successful axon regeneration after peripheral nerve injury. Seven days after a unilateral sciatic nerve injury by compression or transection, we detected a bilateral increase in growth-associated protein-43 (GAP-43) and superior cervical ganglion-10 (SCG-10) mRNA and protein levels not only in DRG neurons of lumbar spinal cord segments (L4-L5) associated with injured nerve, but also in remote cervical segments (C6-C8). The increase in regeneration-associated proteins in the cervical DRG neurons was associated with the greater length of regenerated axons 1 day after ulnar nerve crush following prior sciatic nerve injury as compared to controls with only ulnar nerve crush. The increased axonal regeneration capacity of cervical DRG neurons after a prior conditioning sciatic nerve lesion was confirmed by neurite outgrowth assay of in vitro cultivated DRG neurons. Intrathecal injection of IL-6 or a JAK2 inhibitor (AG490) revealed a role for the IL-6 signaling pathway in activating the pro-regenerative state in remote DRG neurons. Our results suggest that the pro-regenerative state induced in the DRG neurons non-associated with the injured nerve reflects a systemic reaction of these neurons to unilateral sciatic nerve injury.

Expression of Regeneration-Associated Proteins in Primary Sensory Neurons and Regenerating Axons After Nerve Injury-An Overview.

Peripheral nerve injury results in profound alterations of the affected neurons resulting from the interplay between intrinsic and extrinsic molecular events. Restarting the neuronal regenerative program is an important prerequisite for functional recovery of the injured peripheral nerve. The primary sensory neurons with their cell bodies in the dorsal root ganglia provide a useful in vivo and in vitro model for studying the mechanisms that regulate intrinsic neuronal regeneration capacity following axotomy. These studies frequently need to indicate the regenerative status of the corresponding neurons. We summarize the critical issues regarding immunohistochemical detection of several regeneration-associated proteins as markers for the initiation of the regeneration program in rat primary sensory neurons and indicators of axon regeneration in the peripheral nerves. This overview also includes our own results of GAP43 and SCG10 expression in different DRG neurons following double immunostaining with molecular markers of neuronal subpopulations (NF200, CGRP, and IB4) as well as transcription factors (ATF3 and activated STAT3) following unilateral sciatic nerve injury. Anat Rec, 301:1618-1627, 2018. © 2018 Wiley Periodicals, Inc.

Activation of Astrocytes and Microglial Cells and CCL2/CCR2 Upregulation in the Dorsolateral and Ventrolateral Nuclei of Periaqueductal Gray and Rostral Ventromedial Medulla Following Different Types of Sciatic Nerve Injury.

Peripheral nerve injuries (PNIs) may result in cellular and molecular changes in supraspinal structures possibly involved in neuropathic pain (NPP) maintenance. Activated glial cells in specific supraspinal subregions may affect the facilitatory role of descending pathways. Sterile chronic compression injury (sCCI) and complete sciatic nerve transection (CSNT) in rats were used as NPP models to study the activation of glial cells in the subregions of periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Molecular markers for activated astrocytes (glial fibrillary acidic protein, GFAP) and microglial cells (OX42) were assessed by quantitative immunohistochemistry and western blotting. The cellular distribution of CCL2/CCR2 was monitored using immunofluorescence. sCCI induced both mechanical and thermal hypersensitivity from day 1 up to 3 weeks post-injury. Unilateral sCCI or CSNT for 3 weeks induced significant activation of astrocytes bilaterally in both dorsolateral (dlPAG) and ventrolateral PAG (vlPAG) compared to naïve or sham-operated rats. More extensive astrocyte activation by CSNT compared to sCCI was induced bilaterally in dlPAG and ipsilaterally in vlPAG. Significantly more extensive activation of astrocytes was also found in RVM after CSNT than sCCI. The CD11b immunopositive region, indicating activated microglial cells, was remarkably larger in dlPAG and vlPAG of both sides from sCCI- and CSNT-operated rats compared to naïve or sham-operated controls. No significant differences in microglial activation were detected in dlPAG or vlPAG after CSNT compared to sCCI. Both nerve injury models induced no significant differences in microglial activation in the RVM. Neurons and activated GFAP+ astrocytes displayed CCL2-immunoreaction, while activated OX42+ microglial cells were CCR2-immunopositive in both PAG and RVM after sCCI and CSNT. Overall, while CSNT induced robust astrogliosis in both PAG and RVM, microglial cell activation was similar in the supraspinal structures in both injury nerve models. Activated astrocytes in PAG and RVM may sustain facilitation of the descending system maintaining NPP, while microglial activation may be associated with a reaction to long-lasting peripheral injury. Microglial activation via CCR2 may be due to neuronal and astrocytal release of CCL2 in PAG and RVM following injury.

Mitochondrial Damage-Associated Molecular Patterns of Injured Axons Induce Outgrowth of Schwann Cell Processes.

Activated Schwann cells put out cytoplasmic processes that play a significant role in cell migration and axon regeneration. Following nerve injury, axonal mitochondria release mitochondrial damage-associated molecular patterns (mtDAMPs), including formylated peptides and mitochondrial DNA (mtDNA). We hypothesize that mtDAMPs released from disintegrated axonal mitochondria may stimulate Schwann cells to put out cytoplasmic processes. We investigated RT4-D6P2T schwannoma cells (RT4) in vitro treated with N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) or cytosine-phospho-guanine oligodeoxynucleotide (CpG ODN) for 1, 6 and 24 h. We also used immunohistochemical detection to monitor the expression of formylpeptide receptor 2 (FPR2) and toll-like receptor 9 (TLR9), the canonical receptors for formylated peptides and mtDNA, in RT4 cells and Schwann cells distal to nerve injury. RT4 cells treated with fMLP put out a significantly higher number of cytoplasmic processes compared to control cells. Preincubation with PBP10, a selective inhibitor of FPR2 resulted in a significant reduction of cytoplasmic process outgrowth. A significantly higher number of cytoplasmic processes was also found after treatment with CpG ODN compared to control cells. Pretreatment with inhibitory ODN (INH ODN) resulted in a reduced number of cytoplasmic processes after subsequent treatment with CpG ODN only at 6 h, but 1 and 24 h treatment with CpG ODN demonstrated an additive effect of INH ODN on the development of cytoplasmic processes. Immunohistochemistry and western blot detected increased levels of tyrosine-phosphorylated paxillin in RT4 cells associated with cytoplasmic process outgrowth after fMLP or CpG ODN treatment. We found increased immunofluorescence of FPR2 and TLR9 in RT4 cells treated with fMLP or CpG ODN as well as in activated Schwann cells distal to the nerve injury. In addition, activated Schwann cells displayed FPR2 and TLR9 immunostaining close to GAP43-immunopositive regenerated axons and their growth cones after nerve crush. Increased FPR2 and TLR9 immunoreaction was associated with activation of p38 and NFkB, respectively. Surprisingly, the growth cones displayed also FPR2 and TLR9 immunostaining. These results present the first evidence that potential mtDAMPs may play a key role in the induction of Schwann cell processes. This reaction of Schwann cells can be mediated via FPR2 and TLR9 that are canonical receptors for formylated peptides and mtDNA. The possible role for FPR2 and TLR9 in growth cones is also discussed.

Direct communication of the spinal subarachnoid space with the rat dorsal root ganglia.

The anatomical position of the subarachnoid space (SAS) in relation to dorsal root ganglia (DRG) and penetration of tracer from the SAS into DRG were investigated. We used intrathecal injection of methylene blue to visualize the anatomical position of the SAS in relation to DRG and immunostaining of dipeptidyl peptidase IV (DPP-IV) for detecting arachnoid limiting the SAS. Intrathecal administration of fluorescent-conjugated dextran (fluoro-emerald; FE) was used to demonstrate direct communication between the SAS and DRG. Intrathecal injection of methylene blue and DPP-IV immunostaining revealed that SAS delimited by the arachnoid was extended up to the capsule of DRG in a fold-like recess that may reach approximately half of the DRG length. The arachnoid was found in direct contact to the neuronal body-rich area in the angle between dorsal root and DRG as well as between spinal nerve roots at DRG. Particles of FE were found in the cells of DRG capsule, satellite glial cells, interstitial space, as well as in small and medium-sized neurons after intrathecal injection. Penetration of FE from the SAS into the DRG induced an immune reaction expressed by colocalization of FE and immunofluorescence indicating antigen-presenting cells (MHC-II+), activated (ED1+) and resident (ED2+) macrophages, and activation of satellite glial cells (GFAP+). Penetration of lumbar-injected FE into the cervical DRG was greater than that into the lumbar DRG after intrathecal injection of FE into the cisterna magna. Our results demonstrate direct communication between DRG and cerebrospinal fluid in the SAS that can create another pathway for possible propagation of inflammatory and signaling molecules from DRG primary affected by peripheral nerve injury into DRG of remote spinal segments.

Cellular reactions of the choroid plexus induced by peripheral nerve injury.

The choroid plexus (CP) of brain ventricles forms the blood-cerebrospinal fluid (blood-CSF) barrier that is involved in many diseases affecting the central nervous system (CNS). We used ED1 and ED2 immunostaining to investigate epiplexus cell changes in rat CP after chronic constriction injury (CCI). In contrast to naïve CP, the CP of sham-operated rats showed an increase in the number of ED1+ cells of a similar magnitude during all periods of survival up to 3 weeks, while the number of ED2+ increased only at 3 days from operation. In comparison to naïve and sham-operated animals, the number of ED1+ and ED2+ cells in the epiplexus position increased with the duration of nerve compression. We detected no or negligible cell proliferation in the CP after sham- or CCI-operation. This suggests that increased number of ED1+ and ED2+ cells in the epiplexus position of the CP is derived from peripheral monocytes passing through altered blood-CSF barrier. The changes in epiplexus cells indicate that the CP reacts to tissue injury after the surgical approach itself and that the response to peripheral nerve lesion is greater. This suggests a role for an altered blood-CSF barrier allowing for propagation of signal molecules from damaged tissue and nerve to the CNS.

Computer-assisted quantitative analysis of immunofluorescence staining of the extracellular matrix in rat dorsal and ventral spinal roots.

The endoneurial extracellular matrix (ECM) is produced by Schwann cells and fibroblasts under the control of axons. Dorsal and ventral spinal roots contain different types of axons, but information is not available on differences in the composition of their ECM. A comparison was made of the intensity of immunofluorescence staining of chondroitin sulfate proteoglycan, fibronectin, tenascin and thrombospondin in the endoneurial ECM of rat dorsal and ventral spinal roots. Sections of dorsal and ventral roots were incubated simultaneously for indirect immunofluorescence detection of the epitopes studied. Brightness of immunofluorescence staining was assessed by computer-assisted image analysis using interactive segmentation of digitized images to select areas to be analyzed. Our results revealed quantitative differences in the composition of endoneurial ECM of spinal dorsal and ventral roots, probably due to the presence of different types of axons. The ECM composition of the endoneurium in dorsal and ventral roots may be related with the creation of extrinsic conditions that support differential regeneration of afferent and motor axons after injury.

Inflammatory profiling of Schwann cells in contact with growing axons distal to nerve injury.

Activated Schwann cells distal to nerve injury upregulate inflammatory mediators, including cytokines. The goal of the present study was to investigate expression of proinflammatory (IL-1ß, TNFα) and anti-inflammatory cytokines (IL-4, IL-10) in activated Schwann cells in relation to growing axons distal to crush injury of rat sciatic nerves. Seven days from sciatic nerve crush, transverse cryostat sections were cut 5 mm distal to lesion and incubated for double immunostaining to indicate Schwann cells (GFAP or S100b) and individual investigated cytokines or to demonstrate growing axons (GAP43). The Schwann cells of naïve sciatic nerves and those removed from sham-operated rats displayed similar weak immunoreactivity for the investigated cytokines. In contrast, increased intensity of cytokine immunofluorescence was found in Schwann cells distal to crush lesion. The cytokine-positive Schwann cells were found in close contact with growing axons detected by immunostaining for GAP43. The results of immunohistochemical analysis distal to nerve crush injury suggest that inflammatory profiling of Schwann cells including upregulation of both pro- and anti-inflammatory cytokines does not prevent growth of axons distal to nerve crush injury.

Bilateral changes of cannabinoid receptor type 2 protein and mRNA in the dorsal root ganglia of a rat neuropathic pain model.

Cannabinoid receptor type 2 (CB2R) plays a critical role in nociception. In contrast to cannabinoid receptor type 1 ligands, CB2R agonists do not produce undesirable central nervous system effects and thus promise to treat neuropathic pain that is often resistant to medical therapy. In the study presented here, we evaluated the bilateral distribution of the CB2R protein and messenger RNA (mRNA) in rat dorsal root ganglia (DRG) after unilateral peripheral nerve injury using immunohistochemistry, western blot, and in situ hybridization analysis. Unilateral chronic constriction injury (CCI) of the sciatic nerve induced neuropathic pain behavior and bilateral elevation of both CB2R protein and mRNA in lumbar L4-L5 as well as cervical C7-C8 DRG when compared with naive animals. CB2R protein and mRNA were increased not only in DRG neurons but also in satellite glial cells. The fact that changes appear bilaterally and (albeit at a lower level) even in the remote cervical DRG can be related to propagation of neuroinflammation alongside the neuraxis and to the neuroprotective effects of CB2R.

Expression of growth-associated protein 43 in the skin nerve fibers of patients with type 2 diabetes mellitus.

The growth-associated protein 43 (GAP-43) is known as a marker of regenerating nerve fibers and their continuous remodeling in the adult human skin. The purpose of this pilot study was to investigate a possible role for GAP-43 in the detection of the early stages of small-fiber neuropathy in patients with type 2 diabetes mellitus (DM2) as compared with a well- established and validated parameter - intra-epidermal nerve fiber density (IENFD) of protein gene product 9.5 (PGP 9.5) immunoreactive intra-epidermal C fibers. In a group of 21 patients with DM2 within three years of diagnosis (13 men, 8 women; mean age 53.9±12.8; range 30-74) and a group of 17 healthy volunteers (8 men, 9 women; mean age 55.8±8.5; range 45-70 years), skin punch biopsies were taken from a distal calf and double immunostained with both PGP 9.5 and GAP-43. In healthy controls, 96.8% of 629 PGP 9.5 immunoreactive fibers were immunostained with GAP-43; the proportion of PGP 9.5 intra-epidermal nerve fibers immunoreactive for GAP-43 in control subjects ranged from 86.5 to 100%. In DM2 patients, IENFD was significantly lower compared to controls (median, 1.5 vs. 11.2/mm; p<0.001). The proportion of GAP-43 immunoreactive intraepidermal nerve fibers was significantly lower in DM2 patients compared to healthy controls (73.6% of 337 PGP 9.5 positive fibers; p<0.001); ranged from 0 to 98.1%. In conclusion, these results show that impaired regeneration of intra-epidermal C fibers in the early stages of type 2 diabetes mellitus, as indicated by GAP-43, might be a marker of incipient diabetic neuropathy.