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To elucidate blood-nerve barrier function and tight-junction protein expression in the dorsal root ganglion (DRG), we analyzed the vascular permeability in the rat DRG by i.v. administration of fluorescent Evans-blue albumin (EBA) and compared it with the localization of claudin-1, claudin-5, and occludin by immunoconfocal microscopy. In the cell body-rich area within the DRG, extravascular leakage of EBA was noted and claudin-5 but neither claudin-1 nor occludin was detected. Conversely, in the nerve fiber-rich area within the DRG, no extravascular leakage of EBA was observed and both claudin-5 and occludin but no claudin-1 were detected in the blood vessel. These results demonstrate regional differences in the blood-nerve barrier function and tight-junction protein expression within the DRG.

The blood-nerve barrier in peripheral nerves is important for maintaining the environment for axons. Breakdown of the barrier by nerve injury causes various pathologies. We hypothesized that the breakdown and recovery of the blood-nerve barrier after injury are associated with the changes in the expression of intercellular junctional proteins. To test this hypothesis, we induced crush injuries in the rat sciatic nerve by ligation and analyzed spatiotemporal changes of claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 by immunoconfocal microscopy and morphometry and compared them with changes in the permeability of the blood-nerve barrier by intravenous and local administration of Evans blue-albumin (EBA). On day 1 after removal of the ligature EBA leaked into the connective tissue in the endoneurium and then the leakage gradually decreased and disappeared on day 7. On day 1 claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 had totally disappeared from the perineurium and endoneurium. Thereafter, claudin-1, claudin-5, occludin, and VE-cadherin recovered from day 2, whereas connexin43 was redetected on day 5. These results indicate that the breakdown and following recovery of the blood-nerve barrier are closely associated with changes in the expression of claudins, occludin, VE-cadherin, and connexin43 and that the recovery time course is similar but nonidentical.

This study investigated sensory nerve distribution in 27 human coracoacromial ligaments by immunohistochemical methods using antiprotein gene product 9.5 antibody and anticalcitonin gene related peptide antibody. Mean nerve densities were compared among three areas (acromion side, center, and coracoid side) in two groups (patients with rotator cuff tears and patients with shoulder dislocations). In all three areas of both groups, many nerve fibers immunoreactive to antiprotein gene product 9.5 antibody were observed in the periligamentous bursal tissue. However, in the ligament parenchyma, nerve fibers immunoreactive to antiprotein gene product 9.5 antibody were recognized only around blood vessels. Nerve fibers immunoreactive to anticalcitonin gene related peptide antibody were recognized in the periligamentous bursal tissue. However, in the ligament parenchyma, there were no nerve fibers immunoreactive to anticalcitonin gene related peptide antibody. Nerve density of the rotator cuff tear group, as revealed by both immunostainings, showed a significant increase compared with that of the shoulder dislocation group in all three areas. The results of this study show that it is possible the increase in sensory nociceptive nerve fibers in the coracoacromial ligaments may be one of the causes for pain in patients with rotator cuff tears.

The morphological study of cubital tunnel, carpal tunnel and the ulnar (Guyon) canal of adult cadavers was done using stereoscopy after injection of the resin into the brachial artery. MRI (Magnetic Resonance Imaging) of the carpal tunnel was also performed for normal volunteers. In the area of sulcus nervi ulnaris at the elbow, the amount of fibrofatty tissue was extremely small in the nerve trunk, and the nerve was superficially located and found to run close to the hard bony tissue. At the distal margin of the bony sulcus of the ulnar nerve, the nerve ran on the base of hard collateral ligament, where it was covered with a hard ligamentous aponeurosis. The nutrient vessel to the epineurium of the nerve did not exist in the tunnel under the cover of aponeurosis and the nerve was fed by vessels at the entrance of the tunnel and by the recurrent vessels ascending from the musculature at the distal end of the tunnel. The thickness of the flexor retinaculum from the inlet to the midst of carpal tunnel increases and the thickness became maximum at the outlet. The images of MRI of the carpal tunnel were similar to the corresponding levels, and demonstrated the accuracy of imaging with the anatomical relationships. The ulnar canal itself had few bone elements, and there were neither tight connective tissue nor tendons on the palmar side except for the inlet portion.

Following crush injury of rat sciatic nerve, strong synaptophysin immunoreactivity was demonstrated in the regenerating sprouts that emerged from the proximal nodes of Ranvier and in their growth cones that extended through the space between Schwann cell basal lamina and myelin sheath of the parent axon. These findings suggest that synaptophysin is involved in the growth regulation of regenerating sprouts.

Sprouting from crush-injured rat sciatic nerves was studied by electron microscopy to clarify how regenerating sprouts are generated at the node of Ranvier and extended distally in the injured nerves. Three hours after injury, sprouting had already begun in a few nodes: part of the dense submembranous undercoat had disappeared from the nodal axolemma, and the axolemma was slightly evaginated where the undercoat was avoided. The sprout contained clear vesicles of about 50 nm in diameter in an amorphous axoplasm. In addition, vacuoles measuring 100-200 nm in diameter and multivesicular bodies were commonly found within or near these early sprouts. Six to 12 h after injury, nodes of Ranvier with sprouts increased in number in the region 1-1.5 mm proximal to the lesion. Sprouts grew toward the overlying Schwann cell basal lamina, and extended through the space between the basal lamina and Schwann cell plasmalemma (or myelin sheath). In the stem region continuous with the parent axon, regenerating sprouts displayed prominent neurofilaments, which were randomly arranged at 1 day post-injury, but oriented longitudinally by 5 days post-injury.

Limited perineurial injury, known as a perineurial window, can lead to neuropathic pain. This article hypothesizes that the recovery of the perineurium is associated with the intercellular junctional proteins. It analyzes the expressions of occludin, ZO-1, and connexin 43 by immunoconfocal microscopy. Seven days after injury, immunoreactivities for occludin and ZO-1 were observed, although there was no connexin 43 detected. Then, 21 days after injury, immunoreactivity for connexin 43 were observed. These results indicate that recovery of the perineurium is associated with the intercellular junctional proteins and that the recovery of gap junctions is delayed compared with that of tight junctions.

The ultrastructure of the early regenerative response in an end-to-side neurorrhaphy rat model was studied using transmission electron microscopy. The ipsilateral saphenous nerve was grafted to the sciatic nerve under the following conditions: Group 1, the epineurium and perineurium of the sciatic nerve remained intact; Group 2, an epineurial and perineurial window was created at the site of the lateral neurorrhaphy; Group 3, the same as in Group 2 and, in addition, the sciatic nerve sustained a partial neurectomy. Rats were perfused through the heart with fixative containing 2 percent paraformaldehyde and 2.5 percent glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4, 8, 12, 24 and 48 hr after surgery. In Group 1, no regenerating axons were observed and the myelin sheath in the donor nerve did not demonstrate any degenerative changes through 48 hr. In Group 2, an increased diameter of the unmyelinated axons and growth cones was observed in the donor nerve proximal to the coaptation site after 12 hr. Degenerative changes in the myelin sheath were observed after 12 hr within the several layers under the coaptation site. In Group 3, many growth cone-like structures were observed in the area proximal to the coaptation site after 12 hr. After 24 hr, proximal regenerating axons elongated to the coaptation site and, at 48 hr, many regenerating nerves grew inside the Schwann cell basement membrane of the graft nerve. These results indicate that the perineurial window and nerve graft are the critical conditions for inciting nerve regeneration in the donor nerve.

The nerve distribution to the knee joints was analyzed in 5 cadavers and 10 joint capsules specimens were resected during total knee arthroplasty. We found nerve fibers immunoreactive for anti-substance P antibody in the articular capsule. By confocal laser scanning microscopy, we evaluated the three-dimensional structures of the Ruffini's corpuscles and the free nerve endings, both of which were immunoreactive for anti-protein gene product 9.5.

Rat sciatic nerve crush injury is one of the models commonly employed for studying the mechanisms of nerve regeneration. In this study, we analyzed the temporal change of gene expression after injury in this model, to elucidate the molecular mechanisms involved in nerve regeneration. First, a cDNA analysis method, Restriction Landmark cDNA Scanning (RLCS), was applied to cells in the ventral horn of the spinal cord during a 7-day period after the crush injury. A total of 1991 cDNA species were detected as spots on gels, and 37 of these were shown to change after the injury. Temporally changed patterns were classified into three categories: the continuously up-regulated type (10 species), the transiently up-regulated type (22 species), and the down-regulated type (5 species). These complex patterns of gene expression demonstrated after the injury suggest that precise regulation in molecular pathways is required for accomplishing nerve regeneration. Secondly, the rat homologue of uridine kinase gene was identified as one of the up-regulated genes. Northern blot analysis on rat ventral horn tissue and brain revealed that the UK gene had three transcripts with different sizes (4.3, 1. 4, and 1.35 kb, respectively). All of the transcripts, especially the 4.3 kb one, were up-regulated mainly in a bimodal fashion during the 28-day period after the injury. The RLCS method that we employed in the present study shows promise as a means to fully analyze molecular changes in nerve regeneration in detail.

We immunohistochemically and morphometrically examined the expression of gap junction protein connexin (Cx) in normal and crush-injured rat sciatic nerves using confocal laser scanning microscopy. Cx26 was localized in the perineurium and Cx43 was present in the perineurium and the epineurium, whereas Cx32 was confined to the paranodal regions of the nodes of Ranvier. Double labeling for connexins and laminin revealed that Cx43 was localized in multiple layers of the perineurium, whereas Cx26 was confined to the innermost layer. Double labeling for connexins and a tight junction protein, occludin, showed that occludin frequently coexisted with Cx43 but existed separately from Cx26 in the perineurium. After crush injury, the pattern of normal Cx32 expression was initially lost but recovered, whereas Cx43 rapidly appeared in the endoneurium and its expression was subsequently attenuated. Although crush injury produced no apparent alteration in Cx43 and occludin in the perineurium, a rapid increase and a subsequent decrease in the frequency of Cx26-positive spots during nerve regeneration were shown by morphometric analysis. These results indicate that Cx26, Cx32, and Cx43 are expressed differently in various types of cells in peripheral nerves and that their expressions are differentially regulated after injury. The expression of connexins and occludin in the perineurium suggests that perineurial cells are not uniform in type and that the regulation of gap junctions and tight junctions is closely related in the perineurium.

Normal intact and regenerating axon terminals up to 30 days after nerve crushing were studied in the rat flexor carpi ulnaris muscle by confocal laser scanning microscopy (CLSM) and electron microscopy using immunohistochemistry for protein gene product 9.5 (PGP 9.5). The motor axons were intensely and homogeneously stained along their entire length. "Three dimensional" organizations of elaborate axon terminals were clearly demonstrated by reconstructing serial optical images obtained by CLSM in normal neuromuscular junctions. In the injured nerve, the earliest regenerating axons could be identified at endplate regions six days after nerve crushing as intensely immunoreactive thin processes which bifurcated in T-shape and formed delicate lace-like terminals. Such lace-like terminals were composed of fine thread-like portions 0.2-0.8 microm in diameter and expanded portions of 2-3 microm in diameter. Electron microscopy revealed that all the axon terminals in the cytoplasm were stained almost homogeneously by PGP 9.5 immunohistochemistry up to their extreme tips. Axon terminals were in direct contact with the basal lamina of the postsynaptic folds, and showed occasional branching. The thin thread-like portions contained no mitochondria but only a few vesicles, where as the expanded portions, abundant mitochondria. And preterminal axons and some expanded portions were abutted by Schwann cells, while thin thread-like portions were exposed with no association with Schwann cells. Twenty to 30 days after crushing injury, regenerating motor axon terminals resumed their mature form in terms of branching elaborations and ultrastructural features. Thus, CLSM of PGP 9.5 immunocytochemistry combined with electron microscopy was able to demonstrate the "three-dimensional" organization of elaborate axon terminals at high resolution in the normal and regenerating neuromuscular junctions. Using this technique, extremely fine processes of axonal terminals were identifiable at the earliest stage of reinnervation.