Local chemical sympathectomy of rat bone marrow and its effect on marrow cell composition.

Existing experimental studies of the effect of sympathetic nerve fibers on bone marrow cells are based on the systemic administration of neurotoxic 6-hydroxydopamine. The method of global chemical sympathectomy has some serious disadvantages and could lead to questionable results. We describe a new method of local chemical sympathectomy of rat femoral bone marrow using guanethidine (Ismelin) delivery using an osmotic mini pump. Local guanethidine treatment for 14days led to complete elimination of sympathetic fibers in femoral bone marrow in contrast to bone marrow of contralateral or naïve femurs. Ablation of sympathetic fibers was associated with a loss of rat endothelial cell marker (RECA) indicating immunophenotype changes in blood vessel endothelial cells, but no significant effect of guanethidine was found on the survival of endothelial cells and mesenchymal stem cells in vitro. Moreover, local guanethidine treatment also elicited a significant reduction of Nestin+/SDF1+ mesenchymal stem cells and c-Kit+/CD90+ hematopoietic stem cells in femoral bone marrow. Tissue-specific chemical sympathectomy of rat bone marrow by guanethidine overcomes some of the drawbacks of systemic administration of neurotoxic compounds like 6-hydroxydopamine and delivers unequivocal evidence on the effects of sympathetic innervation on the cell content of bone marrow.

Intraepidermal nerve-fibre density as a biomarker of the course of neuropathy in patients with Type 2 diabetes mellitus.

AIMS: This paper aims to investigate whether intraepidermal nerve-fibre density (IENFD) may be used as a marker of the course of neuropathy in patients with Type 2 diabetes mellitus. METHODS: Skin biopsies from the distal leg were serially evaluated in a group of 30 patients with Type 2 diabetes mellitus (median age 60 years, 17 men) with a short duration of diabetes (< 3 years) and good glucose control, and in 23 age- and sex-matched controls. The time intervals between biopsies were > 2 years (median 33.8 months). Eighteen patients with Type 2 diabetes mellitus had symptoms or signs of distal symmetrical diabetic polyneuropathy, 12 had no neuropathy. RESULTS: At first skin biopsy, IENFD was normal in all controls and in patients without neuropathy (mean 9.5 and 7.9 fibres/mm, respectively) compared with abnormal IENFD in 77.8% in patients with polyneuropathy (mean 3.4 fibres/mm). The annual rate of intraepidermal nerve-fibre (IENF) loss expressed as a percentage of the first IENFD value in patients with diabetic polyneuropathy was significantly higher [mean (se), 11.95 (3.82)%] compared with controls [1.92 (1.81)%, P < 0.001] and similar to patients without polyneuropathy [12.16 (4.38)%]. The rate of IENF loss did not correlate with degree of glucose control. CONCLUSIONS: The annual rate of IENF loss in patients with Type 2 diabetes mellitus was several times higher than that of healthy participants, irrespective of the presence of signs or symptoms of diabetic polyneuropathy at initial evaluation. The change in IENFD is not linear and should be expressed as a proportion of initial IENFD to serve as a marker of the course of diabetic neuropathy.

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.

Endoneurial extracellular matrix influences regeneration and maturation of motor nerve axons--a model of acellular nerve graft.

Clinical results of functional reinnervation after application of autogeneous nerve grafts obtained from cutaneous nerves have not always been satisfactory. A foreign extracellular condition especially for regeneration of motor axons is assumed to be one of the reasons explaining these unsuccessful results. The role of endoneurial extracellular matrix in regeneration and maturation of motor axons was studied using acellular nerve segment prepared from muscular or cutaneous nerves applied between stumps of transected motor branch of the femoral nerve. No differences were found in the numbers of regenerated axons and related motoneurons through the motor and cutaneous nerve grafts 1 month after operation. Two months from grafting, however, the numbers of motoneurons and regenerated axons were increased significantly in the motor grafts while these were decreased after regeneration through the cutaneous grafts compared with 1 month. Axons' diameter and thickness of their myelin sheaths were similar in the cutaneous grafts 1 and 2 months after grafting. In comparison to 1 month, axons had larger diameter and thicker myelin in the motor than cutaneous nerve grafts 2 months from their application. Results of morphometric analysis indicate more beneficial extracellular conditions for regeneration and maturation of myelinated motor nerve axons in the acellular motor than cutaneous nerve graft. Generally, the results revealed that the endoneurial extracellular matrix of motor fibers has a positive effect on regeneration and maturation of motor nerve axons after lesion.

[Vascular endothelial growth factor]. / Vaskulární endoteliální rustový faktor.

VEGF, vascular endothelial growth factor, is a substance firstly described in 1983 as a tumor-secreted protein which causes the development of ascitic fluid in case of abdominal tumors. Its influence on angiogenesis was ascertained by many studies. The strongest stimulus for its production is hypoxia, which leads to higher secretion of VEGF and new angiogenesis of so affected tissue. The neurogenic effect was firstly mentioned in 1999. Its protective and proliferative influence both on CNS and peripheral nerves is now widely accepted. It was demonstrated that VEGF has more wide ranging effect than previously thought.

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.

An experimental animal model of spinal root compression syndrome: an analysis of morphological changes of myelinated axons during compression radiculopathy and after decompression.

The treatment of radicular pain is mainly empirical because there are only few experimental studies dealing with morphological changes during compression radiculopathy. The goal of the study was to investigate changes in the morphology of myelinated axons during spinal root compression and the influence of decompression in a new rat model. The number of myelinated axons and their diameter were measured at 1, 2, 5, and 8 weeks during compression of the dorsal spinal root. The same approach was applied for 1-week compression followed by decompression for 1 or 2 weeks and compression for 5 weeks followed by 3-week decompression. A decrease in the number of myelinated axons (particularly those of large diameters) occurred after compression for 1 week. Continued compression for up to 8 weeks resulted in centripetal increase in the number of myelinated axons and the persistence of a small fraction of large myelinated axons at the site of compression. After that time, a decreased number of axons and a reduced fraction of large myelinated axons occurred again. Decompression after 1-week compression caused a rapid increase in the number of both small and large myelinated axons within the spinal root including the site of compression. A small fraction of regenerated axons was found after 5-week compression followed by 3-week decompression. Finally, we investigated the time course of the temporary increase in the number of regenerated myelinated axons during dorsal root compression for up to 8 weeks. The efficacy of decompression was superior when applied one week after compression or after regress of the acute phase of aseptic inflammation associated with fragility of spinal root. The results of the study verify the need for early surgical decompression to prevent irreversible damage of the spinal roots.

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.

Reinnervation of the rat musculocutaneous nerve stump after its direct reconnection with the C5 spinal cord segment by the nerve graft following avulsion of the ventral spinal roots: a comparison of intrathecal administration of brain-derived neurotrophic factor and Cerebrolysin.

Experimental model based on the C5 ventral root avulsion was used to evaluate the efficacy of brain-derived neurotrophic factor (BDNF) and Cerebrolysin treatment on motor neuron maintenance and survival resulted in the functional reinnervation of the nerve stump. In contrast to vehicle, BDNF treatment reduced the loss and atrophy of motor neurons and enhanced the regrowth axon sprouts into the distal stump of musculocutaneous nerve. However, the axon diameter of the myelinated fibers was smaller than those of control rats. The morphometric results were related to a low score in behavioral test similar to vehicle-treated rats. Cerebrolysin treatment greatly protected the motor neurons against cell death. Moreover, morphometric features of myelinated axons were better than those of rats treated with vehicle or BDNF. The mean score of grooming test suggested better results of the functional motor reinnervation than after BDNF administration. The majority of rescued motor neurons regenerating their axons through nerve graft in both BDNF- and Cerebrolysin-treated rats expressed choline acetyltransferase immunostaining. The results demonstrate that BDNF has more modest effects in preventing the death of motor neurons and functional recovery of injured motor nerve after root avulsion than Cerebrolysin.

Reconstruction of elbow flexion, wrist and finger extension by transposition of pedicled latissimus dorsi muscle and flexor carpi ulnaris muscle.

The technique of restoring extension of the hand and fingers is described in an inveterate injury of the brachial plexus. The insertion of m. flexor carpi ulnaris was transferred with an intact nervous and vascular supply to different tendons of the m. extensor digitorum closely above the retinaculum extensorum. The muscle strength was 4--using Janda's muscle test. Dorsal flexion of the hand was possible from the basic position, with extension of the fingers to their full extent. Anatomical investigation revealed that the mean length of the caput humerale m. flexor carpi ulnaris is 26.5 cm, the number of final nerve branches 2-3, the mean length of the nerves varies between 1.3-2.3 cm, the vascular supply is in 90% directly from the ulnar artery. In 10% of dissections the vascular supply is from the anterior ulnar recurrent artery. The length of the vascular bundle is 3.2 cm. In 10% of upper extremities examined an additional vascular bundle was present which was 2 cm distal from the main hilus, also from the a. ulnaris. The pattern of the neurovascular supply is no impediment for the transposition of the insertion tendon into the regio antebrachii posterior. The transfer of the insertion tendon of the m. flexor carpi ulnaris in inveterate injuries of the brachial plexus is a useful alternative for the reconstruction of nerves to restore the extension of the hand and fingers.

Morphometric analysis of early regeneration of motor axons through motor and cutaneous nerve grafts.

Peripheral nerve damage is a frequent consequence of trauma, tumor surgery or diseases. Clinical results of functional reinnervation after the application of cutaneous grafts are still unsatisfactory. Differences in the extracellular matrix are considered to be one of the factors responsible for poor results of motor axon reinnervation through the cutaneous graft. To verify these differences, we compared morphological features of the motor axons regenerating through the graft prepared from the saphenous nerve and the motor branch of the femoral nerve. Eighteen female adult rats (Wistar) were used in experiments. The saphenous nerve, the femoral nerve, and its main motor branch were exposed under deep anesthesia with ketamine and xylazine. The nerve graft (10 mm) prepared from the saphenous nerve was applied between the stumps of the transected motor branch of the femoral nerve in the 6 rats. In the next 6 rats, the nerve graft (10 mm) harvested from the motor branch of the femoral nerve was inserted between stumps of the transected motor branch of the femoral nerve on the contralateral side. All rats were perfused with Zamboni's fixative solution 14 days after grafting. The samples of grafts and the intact motor branch (n = 6) were dissected and embedded in Durcupan ACM. Semithin sections stained with Toluidine Blue were used for morphometric analysis of myelinated axons by means of computer-assisted image analysis system. Ultrathin sections counterstained with uranyl acetate were viewed and photographed in an electron microscope. The number of myelinated motor axons showing early regeneration under conditions of the cutaneous and motor nerve grafts was similar. The diameter of axons and thickness of their myelin sheaths were significantly smaller when the axons regenerated into the saphenous nerve in contrast to the motor graft. Morphometric analysis of early regeneration of myelinated motor axons suggests that the cutaneous and motor branches of the femoral nerve provide different conditions not for the growth but for the maturation of motor axons.

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.

Restoration of lamellar structures in adult rat Pacinian corpuscles following their simultaneous freezing injury and denervation.

The capsule and inner core are multilamellar auxiliary structures enveloping the axon terminal of the Pacinian corpuscle. The freezing injury of the rat interosseal Pacinian corpuscles induced the destruction of all cellular components while the extracellular matrix including the basal laminae survive the treatment. Simultaneous denervation and the freezing treatment of the Pacinian corpuscles discovered an ability of the basal lamina and other components of the extracellular matrix to stimulate a differentiation of migrated Schwann cells and fibroblasts into multilamellar auxiliary structures. The restoration of inner core and capsule in the Pacinian corpuscles was independent of the presence of sensory axon terminals. The restored lamellar structures of Pacinian corpuscles in long-term surviving rat (4 to 8 months) displayed atrophic changes. The results suggest that the extracellular matrix of rat Pacinian corpuscles may contain molecules that are produced by Schwann cells and fibroblasts during maturation of the multilamellar auxiliary structures. The molecules deposited into the extracellular matrix are able to influence the redifferentiation of multilamellar auxiliary structures from immature cells.

Acellular nerve graft re-seeded by Schwann cells migrating from the nerve stump can stimulate spinal motoneurons for functional reinnervation of the rat muscle.

The acellular nerve graft was utilised to restore a functional reinnervation of the biceps brachii muscle from the motoneuron pool of the cervical spinal cord. The musculocutaneous nerve stump was sutured to an acellular nerve graft, the opposite end of which was inserted into the cervical spinal cord cranial to the avulsed C5 ventral root. The acellular nerve graft was repopulated by Schwann cells heavily immunostained for NGFr within 90 days. The Schwann cells migrating from the nerve stump reached the spinal cord grey matter, where they stimulated the motoneurons to send axonal sprouts. The functional reinnervation of the biceps brachii muscle was assessed by means of the behavioural (grooming) test and EMG, the presence of myelinated and unmyelinated axons was demonstrated by light and electron microscopy. The axonal reconnection of the musculocutaneous nerve stump was verified by horseradish peroxidase retrograde labelling of the spinal motoneurons. Moreover, the motoneurons on the operated side of the C5 spinal segment displayed increased immunostaining for GAP-43 in comparison to the contralateral side, whereas the pattern of AChE histochemical reaction was similar on both the operated and contralateral side, of the C5 segment 150 days after acellular graft implantation. The regenerated axons bridged a 4-cm long originally acellular nerve graft to reach and reinnervate the biceps brachii muscle. The reinnervation of the neuromuscular junctions was morphologically determined by immunofluorescence for neurofilaments. The number of myelinated axons in the acellular nerve graft was significantly higher than those growing over the cellular graft, but their diameter was smaller. The results of experiments presented here demonstrate functional recovery of the biceps muscle reinnervation through the acellular nerve graft repopulated by migrating Schwann cells. The process of reinnervation by acellular nerve graft is however delayed and worse in comparison with the cellular graft.

The extracellular matrix of rat pacinian corpuscles: an analysis of its fine structure.

The Pacinian corpuscle consists of a sensory axon terminal that is enveloped by two different structures, the inner core and the capsule. Since proteoglycans are extremely water soluble and are extracted by conventional methods for electron microscopy, the current picture of the structural composition of the extracellular matrix in the inner core and the capsule of the Pacinian corpuscle is incomplete. To study the structural composition of the extracellular matrix of the Pacinian corpuscles, cationic dyes (ruthenium red, alcian blue, acridine orange) and tannic acid were applied simultaneously with the aldehyde fixation. The interosseal Pacinian corpuscles of the rat were fixed either in 2% formaldehyde and 1.5% glutaraldehyde, with the addition of one of these cationic dyes or, in Zamboni's fixative, with tannic acid added. The cationic dyes and tannic acid revealed a different structural pattern of proteoglycans in the extracellular matrix in the inner core and in the capsule of the rat Pacinian corpuscles. The inner core surrounding the sensory axon terminal is a compartment containing proteoglycans that were distributed not only in the extracellular matrix but also in the cytoplasm of the lamellae. In addition, this excitable domain was separated from the capsular fluid by a thick layer of proteoglycans on its surface. An enlarged interlamellar space of the capsule contained large amounts of proteoglycans that were removed by digestion with chondroitinase-ABC. Ruthenium red and alcian blue provided only electron dense granules, probably corresponding to collapsed monomeric proteoglycan molecules. Acridine orange and tannic acid preserved proteoglycans very well and made it possible to visualize them as "bottlebrush" structures in the electron microscope. These results show that the inner core and the capsule of rat Pacinian corpuscles have different structural patterns of proteoglycans, which are probably involved in different functions.

An alternative preparation of the acellular muscle graft for reconstruction of the injured nerve--morphological and morphometric analysis.

The application of cutaneous nerve grafts is accompanied by some disadvantages, including insufficient graft material for the reconstruction of a large mixed nerve. Recently, evacuated muscle autografts have been suggested as possible alternatives to cutaneous nerve grafts. In the present paper we have demonstrated a possible preparation of the evacuated muscle graft using an infiltration of Marcaine. The reinnervation of the distal stump of the rat median nerve was evaluated by morphological and morphometric analysis after application of the muscle acellular grafts prepared by three methods: an ordinary freeze-thawed muscle graft, a Marcaine evacuated muscle graft and a Marcaine treated graft with subsequent freezing and thawing. A comparison of the numbers and diameters of the myelinated axons in the distal nerve stumps revealed very similar conditions for axon regrowth and maturation in Marcaine evacuated and freeze-thawed muscle grafts. The best results with myelinated axon numbers and spectrum of their calibres were obtained when the Marcaine treated graft was repeatedly frozen and thawed. The pre-treatment of the muscle graft by Marcaine prevents it from shrinking and fragmenting, the main disadvantage during freeze-thawing of fresh muscle. The present results demonstrate that infiltration of striated muscles with a myotoxic compound, e.g. Marcaine, with subsequent freezing-thawing is the method of choice for the preparation of an acellular muscle graft used in peripheral nerve reconnection in the experimental model.

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.

Regeneration of tactile lamellar corpuscles of the rat after postnatal freeze injury.

Tactile lamellar corpuscles were studied after freeze injury of rat toe pads under normal conditions and following permanent denervation in 1- to 65-day-old animals. In the innervated skin, digital corpuscles redifferentiated in all age groups examined during development and maturation. Characteristic of the reinnervated skin was a great diversity in the shape and size of newly formed corpuscles. Small corpuscles with only 1-3 lamellae around their terminals and well-developed corpuscles of about normal size with up to 15 lamellae were sometimes found within the same sample of skin. The regenerated corpuscles were reduced in number; they reappeared in only 50% of dermal papillae in the toe pads after freeze injury in 7-week-old rats, compared with approximately 100% of dermal papillae that contained lamellar corpuscles in normal toe-pad skin. In denervated toes, occasional corpuscular lamellar structures appeared first after freeze injury applied to 34-day-old rats. In the toe pads denervated and injured by freezing in 42- and 49-day-old rats, lamellar structures redifferentiated in about 10% of the papillae, and in 23.5% after freeze injury applied to 2-month-old rats. Unsatisfactory preservation of basal laminae at the former sites of the corpuscles and in the acellular peripheral nerve stumps, and/or insufficient migration of Schwann cells, may be responsible for the absence or abortive regeneration of lamellar structures in denervated skin of food pads after freeze injury in young rats.

Growth-associated protein (GAP-43) in terminal Schwann cells of rat Pacinian corpuscles.

Growth-associated protein (GAP-43) immunoreactivity was examined in Pacinian corpuscles of intact neonatal and adult rats as well as after denervation and reinnervation in adult rats. All immature Pacinian corpuscles were GAP-43 immunoreactive (GAP-43+) in their inner cores while only 46 +/- 5.6% of the mature corpuscles exhibited GAP-43+ inner cores. The frequency of GAP-43+ inner cores increased to 90 +/- 7.2% after their permanent denervation. The expression of GAP-43 in the inner cores was reduced by contact with regrowing axons, but 38 +/- 5.3% of Pacinian corpuscles retained GAP-43+ in their inner cores following reinnervation. These results indicate that GAP-43 regulation is not confined only to axons but also involves some extra-axonal cues, and support a role for this protein in the process formation by terminal Schwann cells.