Bilateral changes of IL-10 protein in lumbar and cervical dorsal root ganglia following proximal and distal chronic constriction injury of peripheral nerve.

Interleukin-10 prevents transition of a physiological inflammatory reaction to a pathological state that may result in neuropathic pain. We studied bilateral changes of IL-10 protein levels in L4-L5 and C7-C8 dorsal root ganglia (DRG) after a chronic constriction injury (CCI) of either L4-L5 spinal nerves (pCCI) or the sciatic nerve (dCCI). Rats undergoing pCCI or dCCI were left to survive for 1, 3, 7 or 14 d, sham-operated rats for 3 or 14 d. After the survival time, C7-C8 and L4-L5 DRG were removed bilaterally from naïve, operated, and sham-operated rats and IL-10 protein was detected by immunohistochemical staining and measured using ELISA analysis. Unilateral pCCI and dCCI induced a transient bilateral elevation in IL-10 protein level not only in the homonymous lumbar DRG but also in the heteronymous cervical DRG nonassociated with the spinal segments of constricted nerve. Sham operations also induced bilateral elevation of IL-10 protein in both homonymous and heteronymous DRG. Our experiments revealed that the more proximal is a nerve injury the more rapid is the initial increase and slower the subsequent decrease of IL-10 protein level in DRG. Changes of IL-10 protein in DRG nonassociated with damaged nerve could be related to a general neuroinflammatory reaction of the nervous system to injury and thereby promote potential of the DRG neurons for regenerating their axons following a conditioning lesion.

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.

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.

Laser-tissue interaction in endovenous laser treatment.

The authors present the results of recorded changes in the endothelium in rabbit's veins following photocoagulation by laser diode. This study concentrates mainly on the detailed description of individual changes in the rabbit's venous system in the area of pelvis following laser therapy. The aim of our experiments was to reach the obliteration of rabbits' lateral saphenous vein using 980 nm laser diode with 200 micron fibre. The intensity of discharge was 3, 5, 6 and 7 watts. We examined the relationship between changes and the amount of joules that affect the endothelium of the rabbit's veins. The operation was conducted under general anaesthesia. All animals survived treatment and were returned to breeders. Within the time span of one, three, six and eight weeks the laser treated veins were removed. They were fixed in formaldehyde and sent for microscopic examination. We were interested in how long it will take for fibrous changes to occur in the endothelium of the vein and thus also the subsequent occlusion of the vein depending on the amount of joules applied per one centimetre of the vein (Tab. 1, Fig. 4, Ref 6).

Intra- and extraneuronal changes of immunofluorescence staining for TNF-alpha 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-alpha 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-alpha and TNFR1 proteins. The TNF-alpha 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-alpha and ED-1. The immunofluorescence brightness was measured by image analysis system (LUCIA-G v4.21) to quantify immunostaining for TNF-alpha 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-alpha and TNFR1 when compared with DRG harvested from naive or sham-operated rats. The TNFalpha 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-alpha after sciatic nerve transection for 1 and 2 weeks. In addition, the affected DRG were invaded by ED-1 positive macrophages which displayed simultaneously TNFalpha 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-alpha 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-alpha molecules. Moreover, the different cellular distribution of TNFR1 in the ipsi- and contralateral DRG may reflect different pathways by which TNF-alpha effect on the primary sensory neurons can be mediated following nerve injury.

[Heel pain]. / Bolest paty.

Heel pain is quite frequent clinical symptom in our population. Successful therapy derives from the problem aetiology. The most frequent source of pain is the mechanical basis, both on dorsal and plantar side of calcaneum. Therapy includes a variety of procedures, from routine measures to surgical intervention.

An anatomical study of the anterior interosseous nerve and its innervation of the pronator quadratus muscle.

This anatomical study of 40 upper limbs from cadavers investigated the branching pattern of the anterior interosseous nerve in its distal part using the operating microscope. An articular branch to the wrist joint and/or the distal radioulnar joint was only found in seven of the 40 specimens and was always a small terminal continuation of the anterior interosseous nerve after the nerve had passed through the pronator quadratus and innervated it. Therefore, we do not recommend division of the anterior interosseous nerve from the dorsal approach through the interosseous membrane before it gives off its muscular branches to the pronator quadratus. This risks damage of the innervation of this muscle of importance for initiation of hand pronation.

[Partial denervation of the wrist by excision of the interosseous nerves from the dorsal approach]. / Parciální denervace karpu resená excizí interoseálních nervu z dorzálního prístupu.

PURPOSE OF THE STUDY: The aim of the study was to optimize, on the basis of an anatomical study, the technique of partial denervation of the wrist in terms of safety for preserving motor innervation of the quadrate pronator muscle, and to include this technique in the range of reconstruction operations for the treatment of degenerative carpal diseases. MATERIAL AND METHODS: The technique of partial denervation of the wrist, using excision of the sensitive branches of the dorsal and volar interosseous nerves, carried out by one-stage surgery from the dorsal approach is presented in a group of 28 patients. The partial denervation was always performed in addition to reconstructive surgery on the proximal carpals and the distal radioulnar joint. On the basis of an anatomical study involving 40 cadaverous upper extremities, the authors determined the location for resection of the sensitive branch of the volar interosseous nerve that is not associated with the risk of damaged motor innervation of the quadrate pronator muscle. RESULTS: A reliable identification of the motor branches of the volar interosseous nerve was achieved when an approximately 2-cm incision in the interosseous membrane was made 1 cm distal to the passing anterior branch of the interosseous artery that was clearly seen in the operating field. This corresponded to an incision in the skin, leading in the proximal direction at a length of about 7 cm, made 2 cm proximal to the distal radio-ulnar joint. DISCUSSION: The identification of motor branches is often difficult and therefore the authors recommend resection of the sensitive branch of the volar interosseous nerve to be performed in a safe zone that was determined by a large number of anatomical dissections. Partial denervation carried out according to the principles of a physiological procedure does not pose a burden for the patient and can markedly enhance the effect of reconstructive surgery. CONCLUSION: Partial denervation of the wrist performed from the dorsal approach is a simple procedure easy to combine with other reconstructive operations on the wrist.

Laminin molecules in freeze-treated nerve segments are associated with migrating Schwann cells that display the corresponding alpha6beta1 integrin receptor.

Isolated acellular nerve segments protected from migration of Schwann cells and the acellular nerve segments joined with the distal nerve stumps were prepared by a repeated freeze-thaw procedure in the rat sciatic nerves. The presence of laminin-1 and -2, as well as alpha6 and beta1 integrin chains, was detected by indirect immunohistochemistry in the sections through acellular nerve segments at 7 and 14 days after cryotreatment. The position of basal laminae and Schwann cells was identified by immunostaining for collagen IV and S-100 protein, respectively. The isolated cryo-treated segment without living Schwann cells (S-100-) did not display immunoreactivity for laminins and integrin chains, while the basal lamina position was verified through the whole segment by immunostaining for collagen IV. The absence of immunostaining for laminin-1 and -2 in cryo-treated nerve segment was verified by Western blot analysis. A crucial diminution of laminin-1 and -2 in the cryo-treated nerve segment of 10-mm length did not abolish the growth and maturation of axons. The greater part of nerve segment connected with the nerve stump displayed no immunohistochemical staining for S-100, corresponding with absence of Schwann cells. The border region of the nerve segment contained Schwann cells (S-100+) migrating from the near-freeze undamaged part of the distal nerve stump. In addition to immunostaining for S-100 protein, the migrating Schwann cells displayed immunostaining for laminins (-1, and -2) and integrin chains (alpha6 and beta1). The results indicate that the presence of laminin molecules in the acellular nerve segments prepared by the repeated freeze-thaw procedure is related with the migrating Schwann cells. The immunostaining for laminins and integrin chains, which constitute one of integrin receptor, suggests an autocrine and/or paracrine utilization of laminin molecules in the promotion of Schwann cell migration.

Immunohistochemical localization of laminin-1 in the acellular nerve grafts is associated with migrating Schwann cells which display corresponding integrin receptors.

The presence of laminin-1, collagen-IV, alpha6 and beta1 integrin chains was detected by indirect immunohistochemistry using biotin/streptavidin/HRP or gold-conjugated secondary antibody at the light and electron microscope level, respectively. Cryo-treated segment of the peripheral stump without living Schwann cells (S-100-) did not display immunoreactivity for laminin-1 and integrin's chains, while the migrating Schwann cells in the marginal regions were immunostained for the antigens. Isolated acellular nerve segments protected from migration of Schwann cells (S-100-) exhibited laminin-1-, beta1-, and alpha6- integrin chains immunoreactivities. Position of the basal lamina was verified by collagen-IV+ immunoreactivity. Results indicate that presence of the laminin in the peripheral nerve is related with living Schwann cells.

Immunohistochemical localization of some extracellular molecules and their integrin receptors in the rat Pacinian corpuscles.

The present results suggest that laminin-1 and 3 are localized in the specialized Schwann cells of Pacinian corpuscles, in spite of incomplete deposition of the basal lamina on the surface of their cytoplasmic processes. In addition, laminin-3 is concentrated and probably function as a stop protein not only in the neuromuscular junction, but also in the specialized Schwann cells enveloping the dendritic zone of the afferent axon. No significant changes of immunostaining for both laminins and their integrin receptors following denervation of Pacinian corpuscles indicate that their synthesis is independent to afferent axon as a prerequisite for successful reinnervation.

Denervated skeletal muscle stimulates migration of Schwann cells from the distal stump of transected peripheral nerve: an in vivo study.

We have tested the stimulation of Schwann cell migration from the distal stump of a 1 week transected sciatic nerve of adult rats by denervated skeletal muscle. Migrating Schwann cells were distinguished by the presence of non-specific cholinesterase (nChE) activity and glial fibrillary acidic protein (GFAP) at a distance of about 6 mm among denervated muscle fibres 4 weeks after insertion of the distal stump. In addition, the distal stump was introduced into the open end of a silicone chamber packed with artificial fibrin sponge (Gelaspon) soaked in homogenate from intact or denervated muscles. A larger amount of migrated Schwann cells was observed in the chambers filled with homogenate from denervated muscles. An alteration in the amounts of Schwann cells migrating into the silicone chambers observed after histochemical staining (nChE or GFAP) was supported by biochemical measurements of the nChE activity. The biochemical assessment of the nChE activity revealed the increased amounts of migrated Schwann cells in proportion to the protein contents of homogenates from the denervated muscles. In addition, heating of homogenate from the denervated muscles resulted in a diminution of Schwann cell migration. Bromodeoxyuridine incorporation did not show an increased proliferation of Schwann cells inside the chambers following application of homogenate from the denervated muscles in comparison with the homogenate from the innervated muscles. Our results suggest a stimulation of Schwann cell migration from the distal stump of the transected sciatic nerve by soluble factor(s) produced by denervated skeletal muscles.

Structural and histochemical similarities in three principal sites of sensory axons with the presence of excitable activities.

In the present paper the ultrastructural similarities among the terminal portions of Pacinian corpuscles, the nodes of Ranvier, and the initial segments of primary sensory neurons are pointed out. Our conclusion is based on our observations of cat Pacinian corpuscles and other general knowledge of the node of Ranvier and the initial segment published elsewhere. The morpho-functional similarities of three principal excitable regions of the sensory nerve fibres (the initial segments, the node of Ranvier, and the terminal portions of sensory nerve formations) are illustrated by identical distribution of the enzymes which are associated with ionic transport (alkaline phosphatase, Mg(2+)-ATPase), and non-specific cholinesterase. Furthermore, the polyanionic material revealed by Alcian blue staining in three excitable sites of the sensory axon confirms the supposition that excitable axolemma cannot be considered in the isolation of its surroundings produced by Schwann cells.

Migration of Schwann cells from the distal stump of the sciatic nerve 1 week after transection: the effects of insulin and cytosine arabinoside.

We have examined the migratory capacity of Schwann cells from the distal stump of a 1-week transected sciatic nerve of adult rat for a distance of 10 mm. The distal stump was introduced into the open end of a silicone chamber packed with artificial fibrin sponge (Gelaspon) soaked in phosphate-buffered saline (control chambers), cytosine arabinoside (Ara-C) (0.05 mM), or insulin (40 U/ml). Migrating Schwann cells were distinguished from fibroblasts by the presence of non-specific cholinesterase (nChE) activity and glial fibrillary acidic protein (GFAP). The cells of distal stumps including Schwann cells accepted Gelaspon as a suitable adhesive substratum. In the chambers filled with Gelaspon soaked in phosphate-buffered saline alone Schwann cells were outnumbered by fibroblasts. The addition of Ara-C resulted in greater numbers of Schwann cells, which migrate longer distances into the chambers. The application of insulin enhanced Schwann cell migration as well. These morphologic observations were further supported by biochemical measurements of nChE activity. The results suggest an influence on Schwann cell migration by fibroblasts of connective tissue sheaths and a stimulation of Schwann cell migration by insulin.

Non-specific cholinesterase activity in mouse spinal ganglia. The usefulness of histochemical study and image analysis for simple characterization of neuron subclasses.

Non-specific cholinesterase (ChE) activity was studied histochemically at light and electron microscopical levels in dorsal root ganglia (DRG) of adult mice. The reaction staining and diameter of neuron cells perykaria were measured by using an image analysis system. The methodological approach enable to distinguish 8 subclasses of primary sensory neurons. The proportion of individual subclasses was mapping in three subsequent cervical, thoracal and lumbar DRG. The populations of small-sized neurons increased towards lumbar level similarly as medium and small neurons exhibiting high ChE reactivity. The variations in ChE-containing neurons among DRG from different area may reflect differences in modality-specific primary sensory neurons at each spinal cord level. In addition, the effect of 3 week sciatic nerve transection on the percentage of the subclasses in L4-L6 DRG has been investigated. The number of large neurons was reduced and a decrease of ChE reactivity in medium-size neurons was found in DRG on the operated side. Thus, the present results demonstrate a selective affectation of primary sensory neurons in mouse DRG by the peripheral nerve transection. Different amounts of the reaction product corresponding with ChE activity were found in the nuclear envelope and the cisternae of rough endoplasmic reticulum.

Recovery of non-specific cholinesterase activity in sensory corpuscles of mouse toe skin after irreversible inhibition of this enzyme and cold injury.

Mouse digital corpuscles, located in the dermal papillae of toe pad skin, consist of the sensory axon terminals enveloped by the cytoplasmic processes of Schwann-derived cells forming the so-called inner core. The inner core cells are capable to synthetize nCHE molecules which are released into the interlamellar spaces filled by the basal lamina, collagenous microfibrils, and amorphous matrix. In the present study, the histochemical detection of the nCHE activity was investigated in the sensory corpuscles after sciatic and saphenous nerve transections and subsequent application of irreversible nCHE inhibitor (iso-OMPA) or cryo-treatment of toe pad skin. The recovery of the nCHE reaction product in both intact and denervated corpuscles revealed the resynthesis of the nCHE molecules by the inner core cells without assistance of sensory terminals, as well. The cellular constituents of corpuscles were degraded while extracellular matrix appeared to be undamaged after freezing injury. The molecules of nCHE attached to the extracellular matrix components disappeared in coincidence with the disintegration of Schwann-derived cells. After about 5 d of survival, the Schwann cells exhibiting the nCHE reactivity migrated through the basal lamina tubes as guidance of regrowing axons or alone. After 7 d from the treatment, immature Schwann cells marked by the nCHE reaction product occupied the scaffolds of old damaged sensory corpuscles. During further days of surviving, the Schwann cells entering the extracellular matrix of degraded corpuscles were differentiated to the inner core cells. The re-differentiation of the Schwann cells into the inner core cells was observed not only in the presence but also in the absence of sensory terminals. These findings suggest certain trophic independence of inner core cells upon sensory terminals in the sensory corpuscles of adult animals.

Contribution to histochemical distribution of non-specific cholinesterase activity in sensory ganglia of some mammals.

The activity of non-specific cholinesterase was demonstrated histochemically in satellite cells of the spinal ganglia from adult rat, cat, rabbit and baboon. The spinal ganglia of newborn rats displayed distinct intraneuronal reactivity for non-specific cholinesterase while a low reactivity was observed in satellite cells. The spinal and trigeminal ganglia of adult mice contained satellite cells with non-specific cholinesterase reactivity only sporadically. Most of reaction product for non-specific cholinesterase activity (from low to high intensity) was found in perikarya of the neurons. Spinal and trigeminal ganglia of the same mice embryo exhibited diffuse staining for non-specific cholinesterase activity remaining in the spinal ganglia of newborn mice. The trigeminal ganglia of newborn mice exhibited, however, more differentiated pattern of the positive reaction for non-specific cholinesterase like adult animals. The pattern of histochemical distribution of non-specific cholinesterase activity in trigeminal and spinal ganglia from mice of various ages corresponds with morphological differentiation and maturation undergoing in a rostrocaudal wave. Intraneuronal presence of non-specific cholinesterase activity in sensory ganglia during development and in adult animals gives a new possibilities for explanation of the functional involvement of this enzyme in the nervous system.