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1.  Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves 
The FASEB Journal  2012;26(3):1064-1076.
Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed at junctions between adjacent endothelial and epithelial cells and implicated in multiple inflammatory and vascular responses. In addition, we recently reported on the expression of JAM-C in Schwann cells (SCs) and its importance for the integrity and function of peripheral nerves. To investigate the role of JAM-C in neuronal functions further, mice with a specific deletion of JAM-C in SCs (JAM-C SC KO) were generated. Compared to wild-type (WT) controls, JAM-C SC KO mice showed electrophysiological defects, muscular weakness, and hypersensitivity to mechanical stimuli. In addressing the underlying cause of these defects, nerves from JAM-C SC KO mice were found to have morphological defects in the paranodal region, exhibiting increased nodal length as compared to WTs. The study also reports on previously undetected expressions of JAM-C, namely on perineural cells, and in line with nociception defects of the JAM-C SC KO animals, on finely myelinated sensory nerve fibers. Collectively, the generation and characterization of JAM-C SC KO mice has provided unequivocal evidence for the involvement of SC JAM-C in the fine organization of peripheral nerves and in modulating multiple neuronal responses.—Colom, B., Poitelon, Y., Huang, W., Woodfin, A., Averill, S., Del Carro, U., Zambroni, D., Brain, S. D., Perretti, M., Ahluwalia, A., Priestley, J. V., Chavakis, T., Imhof, B. A., Feltri, M. L., Nourshargh, S. Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves.
PMCID: PMC3370675  PMID: 22090315
adhesion molecules; tight junctions; peripheral nerves
2.  Expression and Function of Junctional Adhesion Molecule–C in Myelinated Peripheral Nerves 
Science (New York, N.Y.)  2007;318(5855):1472-1475.
JAM-C is an adhesion molecule that is expressed on cells within the vascular compartment and epithelial cells and, to date, has been largely studied in the context of inflammatory events. Using immunolabeling procedures in conjunction with confocal and electron microscopy, we show here that JAM-C is also expressed in peripheral nerves and that this expression is localized to Schwann cells at junctions between adjoining myelin end loops. Sciatic nerves from JAM-C–deficient [having the JAM-C gene knocked out (KO)] mice exhibited loss of integrity of the myelin sheath and defective nerve conduction as indicated by morphological and electrophysiological studies, respectively. In addition, behavioral tests showed motor abnormalities in the KO animals. JAM-C was also expressed in human sural nerves with an expression profile similar to that seen in mice. These results demonstrate that JAM-C is a component of the autotypic junctional attachments of Schwann cells and plays an important role in maintaining the integrity and function of myelinated peripheral nerves.
PMCID: PMC3299566  PMID: 18048693
3.  Knockout of TLR4 and TLR2 impair the nerve regeneration by delayed demyelination but not remyelination 
Knockout of either toll-like receptor 4 (TLR4) or 2 (TLR2) had been reported to delay the Wallerian degeneration after peripheral nerve injury by deterring the recruitment of the macrophages and clearance of myelin debris. However, the impact on the remyelination process is poorly understood. In this study, the effect of TLR2 and TLR4 knockout on the nerve regeneration and on the remyelination process was studied in a mouse model of sciatic nerve crush injury.
A standard sciatic nerve crush injury by a No. 5 Jeweler forcep for consistent 30 seconds was performed in Tlr4−/− (B6.B10ScN-Tlr4lps-del/JthJ), Tlr2−/− (B6.129-Tlr2tm1Kir/J) and C57BL/6 mice. One centimeter of nerve segment distal to the crushed site was harvested for western blot analysis of the myelin structure protein myelin protein zero (Mpz) and the remyelination transcription factors Oct6 and Sox10 at day 0, 3, 7, 10, 14, 17, 21, 28. Nerve segment 5-mm distal to injured site from additional groups of mice at day 10 after crush injury were subjected to semi-thin section and toluidine blue stain for a quantitative histomorphometric analysis. With less remyelinated nerves and more nerve debris, the histomorphometric analysis revealed a worse nerve regeneration following the sciatic nerve crush injury in both Tlr4−/− and Tlr2−/− mice than the C57BL/6 mice. Although there was a delayed expression of Sox10 but not Oct6 during remyelination, with an average 4-day delay in the demyelination process, the subsequent complete formation of Mpz during remyelination was also delayed for 4 days, implying that the impaired nerve regeneration was mainly attributed to the delayed demyelination process.
Both TLR4 and TLR2 are crucial for nerve regeneration after nerve crush injury mainly by delaying the demyelination but not the remyelination process.
PMCID: PMC3765918  PMID: 23984978
Toll-like receptor 4 (TLR4); Toll-like receptor 2 (TLR2); Peripheral nerve regeneration; Sciatic nerve crush injury
4.  Beneficial effects of treadmill training in experimental diabetic nerve regeneration 
Clinics  2010;65(12):1329-1337.
We investigated the effects of treadmill training (10 weeks) on hindlimb motor function and nerve morphometric parameters in diabetic rats submitted to sciatic nerve crush.
Wistar rats (n = 64) were divided into the following groups: non-diabetic; trained non-diabetic; non-diabetic with sciatic nerve crush; trained non-diabetic with sciatic nerve crush; diabetic; trained diabetic; diabetic with sciatic nerve crush or trained diabetic with sciatic nerve crush. Diabetes was induced by streptozotocin injection (50 mg/kg, iv). Hindlimb motor function was evaluated weekly by assessing sciatic functional indices, and the proximal and distal portions of the sciatic nerve were used for morphometric analysis.
At 13 weeks post-injury, the distal nerve portion of all injured groups and the proximal nerve portion of the diabetic with sciatic nerve crush group presented altered morphometric parameters such as decreased myelinated fiber diameter (∼7.4±0.3µm vs ∼4.8±0.2µm), axonal diameter (∼5±0.2µm vs ∼3.5±0.1µm) and myelin sheath thickness (∼1.2±0.07µm vs ∼0.65±0.07µm) and an increase in the percentage of area occupied by endoneurium (∼28±3% vs ∼60±3%). In addition, in the non-diabetic with sciatic nerve crush group the proximal nerve portion showed a decreased myelinated fiber diameter (7.4±0.3µm vs 5.8±0.3µm) and myelin sheath thickness (1.29±0.08µm vs 0.92±0.08µm). The non-diabetic with sciatic nerve crush, trained non-diabetic with sciatic nerve crush, diabetic with sciatic nerve crush and trained diabetic with sciatic nerve crush groups showed normal sciatic functional index from the 4th, 4th, 9th and 7th week post-injury, respectively. Morphometric alterations in the proximal nerve portion of the diabetic with sciatic nerve crush and non-diabetic with sciatic nerve crush groups were either prevented or reverted to values similar to the non-diabetic group by treadmill training.
Diabetic condition promoted delay in sciatic nerve regeneration. Treadmill training is able to accelerate hindlimb motor function recovery in diabetic injured rats and prevent or revert morphometric alterations in proximal nerve portions in non-diabetic and diabetic injured rats.
PMCID: PMC3020345  PMID: 21340223
Diabetes; Sciatic nerve crush; Motor function; Nerve morphometry; Treadmill training
5.  Traumatic neuroma in continuity injury model in rodents: a preliminary report 
Selected abstracts delivered at the 8th Annual AOSpine North America Fellows Forum
Consistent with EBSJ's commitment to fostering quality research, we are pleased to feature some of the most highly rated abstracts from the 8th Annual AOSpine North America Fellows Forum in Banff Canada. Enhancing the quality of evidence in spine care means acknowledging and supporting the efforts of young researchers within our AOSpine North America network. We look forward to seeing more from these promising researchers in the future.
Study type: Basic science research report
Introduction: Spinal nerve-injury management and prevention constitute a substantial proportion of a spinal surgeon's practice. Functional recovery after peripheral nerve injuries is often unsatisfactory and to optimize the outcomes, an intimate understanding of these injuries is required. Sunderland classified peripheral nerve injuries into five grades.1 Grade 1 (neurapraxia) and grade 2 (axonal disruption) injuries usually recover with no or insignificant functional deficits within weeks to a few months, respectively. Injuries that are most difficult to manage clinically are the often mixed grade 3 (endoneurial disruption) and grade 4 (perineurial disruption) lesions where spontaneous functional recovery is limited or absent, resulting in neuroma in continuity (NIC). Traumatic NIC is characterized by aberrant intra- and extra- fascicular axonal regeneration and scar formation within an unsevered injured nerve, resulting in impaired and erroneous end-organ reinnervation.2,3 Animal models reproducing grade 1, 2, 3, and 5 lesions have been developed, but to our knowledge a clinically relevant rodent model of NIC has not been developed.4,5,6,7,8 The effective peripheral nerve regeneration and resilience of rodents make it challenging to recreate the NIC scenario.
Objective: Our goal was to develop a practical rodent model for focal traumatic NIC, demonstrating the characteristic histological features, supported by concordant functional deficits. Such a model may help us to identify this injury pattern earlier and allow development of intervention strategies to reduce neuronal misdirection, scar formation, and enhance regeneration for improved functional recovery.
Methods: Various injury techniques were tested on freshly harvested Lewis rat sciatic nerves ex vivo, and examined histologically before inflicting more refined injuries in vivo. The optimal experimental injuries combined a 50 g traction force applied with a spring scale hooked around the sciatic nerve, and focal three second maximal compression using a malleus nipper (Figure 1). Nerves were harvested at 0, 5, 13, 21, and 65 days, and processed for longitudinal 8 micron cryostat sectioning, H&E, laminin, neurofilament, and Masson's trichrome staining. Skilled locomotion (tapered beam, ladder rung) and flat plane locomotion for ground reaction force (GRF) analysis were performed serially up to 9 weeks with the experimental (n = 4) and simple (control) crush (n = 1) injuries by blinded animal behavior experts, using methods as recently described.9
Photograph illustrating the experimental injury. Fifty grams of traction is applied in a direction orthogonal to the native nerve course after external neurolysis, simultaneously, three second maximal compression is applied at the sciatic trifurcation, just distal to a mesoneurial suture. Malleus nipper with tip detail and 100 g spring scale in bottom left. In situ sciatic nerve immediately after injury (top right).
Results: Disruption of the endoneurium and perineurium with aberrant intra- and extrafascicular axonal regeneration and progressive fibrosis was consistently demonstrated histologically in ten out of ten nerves with experimental injuries. In contrast, crush injuries showed only signs of Wallerian degeneration (Figure 2). At 8 weeks, experimental animals made more errors during skilled locomotion as compared to nerve crush animals. GRFs revealed impaired vertical and fore-aft force generation by the injured limbs at week 9 in the experimental group, whereas GRFs from the simple crush animal revealed recovery at the same time point (Figure 3).
Injury zones at five days (a–d, bar = 200 µm) and 65 days (e–h, bar = 50 µm), comparing crush (top) to experimental (bottom) injuries; Masson's trichrome and neurofilament. Note the aberrant axonal sprouting and regeneration in the experimental injury group, associated with increased intrafascicular collagen, in contrast to orderly regeneration and lack of scar in the simple crush group.
Mean vertical and fore-aft ground reaction forces at both baseline and 9 weeks from representative animals. Compared to baseline and crush-injured animal at 9 weeks, animals in the experimental group bear less weight on both their right (surgical) hind limb (solid line), and fore limb (dotted line) at 9 weeks. Comparable with historical data, the crush animal have improved braking (*) and propulsive (#) forces in fore and hind limbs (injured side) compared to the experimental group, though these have not returned to baseline values.
Conclusions: We have demonstrated histological features and poor functional recovery consistent with NIC formation in a rodent model. The injury mechanism employed combines traction and compression forces akin to the physical forces at play in clinical nerve injuries. Additional validating experiments are in progress.
PMCID: PMC3623102  PMID: 23637668
Locomotion; nerve regeneration; Sunderland grade 4 nerve injury
6.  ATF3 upregulation in glia during Wallerian degeneration: differential expression in peripheral nerves and CNS white matter 
BMC Neuroscience  2004;5:9.
Many changes in gene expression occur in distal stumps of injured nerves but the transcriptional control of these events is poorly understood. We have examined the expression of the transcription factors ATF3 and c-Jun by non-neuronal cells during Wallerian degeneration following injury to sciatic nerves, dorsal roots and optic nerves of rats and mice, using immunohistochemistry and in situ hybridization.
Following sciatic nerve injury – transection or transection and reanastomosis – ATF3 was strongly upregulated by endoneurial, but not perineurial cells, of the distal stumps of the nerves by 1 day post operation (dpo) and remained strongly expressed in the endoneurium at 30 dpo when axonal regeneration was prevented. Most ATF3+ cells were immunoreactive for the Schwann cell marker, S100. When the nerve was transected and reanastomosed, allowing regeneration of axons, most ATF3 expression had been downregulated by 30 dpo. ATF3 expression was weaker in the proximal stumps of the injured nerves than in the distal stumps and present in fewer cells at all times after injury. ATF3 was upregulated by endoneurial cells in the distal stumps of injured neonatal rat sciatic nerves, but more weakly than in adult animals. ATF3 expression in transected sciatic nerves of mice was similar to that in rats. Following dorsal root injury in adult rats, ATF3 was upregulated in the part of the root between the lesion and the spinal cord (containing Schwann cells), beginning at 1 dpo, but not in the dorsal root entry zone or in the degenerating dorsal column of the spinal cord. Following optic nerve crush in adult rats, ATF3 was found in some cells at the injury site and small numbers of cells within the optic nerve displayed weak immunoreactivity. The pattern of expression of c-Jun in all types of nerve injury was similar to that of ATF3.
These findings raise the possibility that ATF3/c-Jun heterodimers may play a role in regulating changes in gene expression necessary for preparing the distal segments of injured peripheral nerves for axonal regeneration. The absence of the ATF3 and c-Jun from CNS glia during Wallerian degeneration may limit their ability to support regeneration.
PMCID: PMC400733  PMID: 15113454
7.  Detection of subtle neurological alterations by the Catwalk XT gait analysis system 
A new version of the CatWalk XT system was evaluated as a tool for detecting very subtle alteration in gait based on higher speed sample rate; the system could also demonstrate minor changes in neurological function. In this study, we evaluated the neurological outcome of sciatic nerve injury intervened by local injection of hyaluronic acid. Using the CatWalk XT system, we looked for differences between treated and untreated groups and differences within the same group as a function of time so as to assess the power of the Catwalk XT system for detecting subtle neurological change.
Peripheral nerve injury was induced in 36 Sprague–Dawley rats by crushing the left sciatic nerve using a vessel clamp. The animals were randomized into one of two groups: Group I: crush injury as the control; Group II: crush injury and local application with hyaluronic acid. These animals were subjected to neurobehavior assessment, histomorphology evaluation, and electrophysiology study periodically. These data were retrieved for statistical analysis.
The density of neurofilament and S-100 over the distal end of crushed nerve showed significant differences either in inter-group comparison at various time points or intra-group comparison from 7 to 28 days. Neuronal structure architecture, axon counts, intensity of myelination, electrophysiology, and collagen deposition demonstrate significant differences between the two groups. There was significant difference of SFI and angle of ankle in inter- group analysis from 7 to 28 days, but there were no significant differences in SFI and angle of ankle at time points of 7 and 14 days. In the Cat Walk XT analysis, the intensity, print area, stance duration, and swing duration all showed detectable differences at 7, 14, 21, and 28 days, whereas there were no significant difference at 7 and 14 days with CatWalk 7 testing. In addition, there were no significant differences of step sequence or regularity index between the two versions.
Hyaluronic acid augmented nerve regeneration as early as 7 days after crush injury. This subtle neurological alteration could be detected through the CatWalk XT gait analysis but not the SFI, angle of ankle, or CatWalk 7 methods.
PMCID: PMC3997750  PMID: 24739213
8.  Expression, Localization, and Function of Junctional Adhesion Molecule-C (JAM-C) in Human Retinal Pigment Epithelium 
To determine the localization of JAM-C in human RPE and characterize its functions.
Immunofluorescence, Western blot, and PCR was used to identify the localization and expression of JAM-C, ZO-1, N-cadherin, and ezrin in cultures of human fetal RPE (hfRPE) with or without si-RNA mediated JAM-C knockdown and in adult native RPE wholemounts. A transepithelial migration assay was used to study the migration of leukocytes through the hfRPE monolayer.
JAM-C localized at the tight junctions of cultured hfRPE cells and adult native RPE. During initial junction formation JAM-C was recruited to the primordial cell– cell contacts and after JAM-C knockdown, the organization of N-cadherin and ZO-1 at those contacts was disrupted. JAM-C knockdown caused a delay in the hfRPE cell polarization, as shown by reduced apical staining of ezrin. JAM-C inhibition significantly decreased the chemokine-induced transmigration of granulocytes but not monocytes through the hfRPE monolayer.
JAM-C localizes specifically in the tight junctions of hfRPE and adult native RPE. It is important for tight junction formation in hfRPE, possibly by regulating the recruitment of N-cadherin and ZO-1 at the cell– cell contacts, and has a role in the polarization of hfRPE cells. Finally, JAM-C promotes the basal-to-apical transmigration of granulocytes but not monocytes through the hfRPE monolayer.
PMCID: PMC2752302  PMID: 19060272
9.  Cis-Dimerization Mediates Function of Junctional Adhesion Molecule A 
Molecular Biology of the Cell  2008;19(5):1862-1872.
Junctional adhesion molecule-A (JAM-A) is a transmembrane component of tight junctions that has been proposed to play a role in regulating epithelial cell adhesion and migration, yet mechanistic structure–function studies are lacking. Although biochemical and structural studies indicate that JAM-A forms cis-homodimers, the functional significance of dimerization is unclear. Here, we report the effects of cis-dimerization–defective JAM-A mutants on epithelial cell migration and adhesion. Overexpression of dimerization-defective JAM-A mutants in 293T cells inhibited cell spreading and migration across permeable filters. Similar inhibition was observed with using dimerization-blocking antibodies. Analyses of cells expressing the JAM-A dimerization-defective mutant proteins revealed diminished β1 integrin protein but not mRNA levels. Further analyses of β1 protein localization and expression after disruption of JAM-A dimerization suggested that internalization of β1 integrin precedes degradation. A functional link between JAM-A and β1 integrin was confirmed by restoration of cell migration to control levels after overexpression of β1 integrin in JAM-A dimerization-defective cells. Last, we show that the functional effects of JAM dimerization require its carboxy-terminal postsynaptic density 95/disc-large/zonula occludins-1 binding motif. These results suggest that dimerization of JAM-A regulates cell migration and adhesion through indirect mechanisms involving posttranscriptional control of β1 integrin levels.
PMCID: PMC2366836  PMID: 18272784
10.  Inflammatory Profiling of Schwann Cells in Contact with Growing Axons Distal to Nerve Injury 
BioMed Research International  2014;2014:691041.
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-1β, 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.
PMCID: PMC4022316  PMID: 24877128
11.  Myelination and nodal formation of regenerated peripheral nerve fibers following transplantation of acutely prepared olfactory ensheathing cells 
Brain research  2006;1125(1):1-8.
Transplantation of olfactory ensheathing cells (OECs) into injured spinal cord results in improved functional outcome. Mechanisms suggested to account for this functional improvement include axonal regeneration, remyelination and neuroprotection. OECs transplanted into transected peripheral nerve have been shown to modify peripheral axonal regeneration and functional outcome. However, little is known of the detailed integration of OECs at the transplantation site in peripheral nerve. To address this issue cells populations enriched in OECs were isolated from the olfactory bulbs of adult green fluorescent protein (GFP)-expressing transgenic rats and transplanted into a sciatic nerve crush lesion which transects all axons. Five weeks to six months after transplantation the nerves were studied histologically. GFP-expressing OECs survived in the lesion and distributed longitudinally across the lesion zone. The internodal regions of individual teased fibers distal to the transection site were characterized by GFP expression in the cytoplasmic and nuclear compartments of cells surrounding the axons. Immuno-electron microscopy for GFP indicated that the transplanted OECs formed peripheral type myelin. Immunostaining for sodium channel and Caspr revealed a high density of Nav1.6 at the newly formed nodes of Ranvier which were flanked by paranodal Caspr staining. These results indicate that transplanted OECs extensively integrate into transected peripheral nerve and form myelin on regenerated peripheral nerve fibers, and that nodes of Ranvier of these axons display proper sodium channel organization.
PMCID: PMC2673087  PMID: 17112480
Regeneration; olfactory ensheathing cells; peripheral nervous system; transplantation; nodal formation
12.  Blockade but Not Overexpression of the Junctional Adhesion Molecule C Influences Virus-Induced Type 1 Diabetes in Mice 
PLoS ONE  2013;8(1):e54675.
Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing beta-cells in the pancreas. Recruitment of inflammatory cells is prerequisite to beta-cell-injury. The junctional adhesion molecule (JAM) family proteins JAM-B and JAM–C are involved in polarized leukocyte transendothelial migration and are expressed by vascular endothelial cells of peripheral tissue and high endothelial venules in lympoid organs. Blocking of JAM-C efficiently attenuated cerulean-induced pancreatitis, rheumatoid arthritis or inflammation induced by ischemia and reperfusion in mice. In order to investigate the influence of JAM-C on trafficking and transmigration of antigen-specific, autoaggressive T-cells, we used transgenic mice that express a protein of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen in the β-cells of the islets of Langerhans under the rat insulin promoter (RIP). Such RIP-LCMV mice turn diabetic after infection with LCMV. We found that upon LCMV-infection JAM-C protein was upregulated around the islets in RIP-LCMV mice. JAM-C expression correlated with islet infiltration and functional beta-cell impairment. Blockade with a neutralizing anti-JAM-C antibody reduced the T1D incidence. However, JAM-C overexpression on endothelial cells did not accelerate diabetes in the RIP-LCMV model. In summary, our data suggest that JAM-C might be involved in the final steps of trafficking and transmigration of antigen-specific autoaggressive T-cells to the islets of Langerhans.
PMCID: PMC3556033  PMID: 23372751
13.  Low-Level Laser Irradiation Improves Functional Recovery and Nerve Regeneration in Sciatic Nerve Crush Rat Injury Model 
PLoS ONE  2014;9(8):e103348.
The development of noninvasive approaches to facilitate the regeneration of post-traumatic nerve injury is important for clinical rehabilitation. In this study, we investigated the effective dose of noninvasive 808-nm low-level laser therapy (LLLT) on sciatic nerve crush rat injury model. Thirty-six male Sprague Dawley rats were divided into 6 experimental groups: a normal group with or without 808-nm LLLT at 8 J/cm2 and a sciatic nerve crush injury group with or without 808-nm LLLT at 3, 8 or 15 J/cm2. Rats were given consecutive transcutaneous LLLT at the crush site and sacrificed 20 days after the crush injury. Functional assessments of nerve regeneration were analyzed using the sciatic functional index (SFI) and hindlimb range of motion (ROM). Nerve regeneration was investigated by measuring the myelin sheath thickness of the sciatic nerve using transmission electron microscopy (TEM) and by analyzing the expression of growth-associated protein 43 (GAP43) in sciatic nerve using western blot and immunofluorescence staining. We found that sciatic-injured rats that were irradiated with LLLT at both 3 and 8 J/cm2 had significantly improved SFI but that a significant improvement of ROM was only found in rats with LLLT at 8 J/cm2. Furthermore, the myelin sheath thickness and GAP43 expression levels were significantly enhanced in sciatic nerve-crushed rats receiving 808-nm LLLT at 3 and 8 J/cm2. Taken together, these results suggest that 808-nm LLLT at a low energy density (3 J/cm2 and 8 J/cm2) is capable of enhancing sciatic nerve regeneration following a crush injury.
PMCID: PMC4131879  PMID: 25119457
14.  Integration of engrafted Schwann cells into injured peripheral nerve: Axonal association and nodal formation on regenerated axons 
Neuroscience letters  2005;387(2):85-89.
Transplantation of myelin-forming cells can remyelinate axons, but little is known of the sodium channel organization of axons myelinated by donor cells. Sciatic nerve axons of female wild type mice were transected by a crush injury and Schwann cells (SCs) from green fluorescence protein (GFP)-expressing male mice were transplanted adjacent to the crush site. The male donor cells were identified by GFP fluorescence and fluorescence in situ hybridization (FISH) for Y chromosome. In nerves of GFP-expressing mice, GFP was observed in the axoplasm and in the cytoplasmic compartments of the Schwann cells, but not in the myelin. Following transplantation of GFP-SCs into crushed nerve of wild type mice, immuno-electron microscopic analysis indicated that GFP was observed in the cytoplasmic compartments of engrafted Schwann cells which formed myelin. Nodal and paranodal regions of the axons myelinated by the GFP-SCs were identified by Nav 1.6 sodium channel and Caspr immunostaining, respectively. Nuclear identification of the Y chromosome by FISH confirmed the donor origin of the myelin-forming cells. These results indicate that engrafted GFP-SCs participate in myelination of regenerated peripheral nerve fibers and that Nav 1.6 sodium channel, which is the dominant sodium channel at normal nodes, is reconstituted on the regenerated axons.
PMCID: PMC2605373  PMID: 16084645
Schwann cells; Transplantation; Peripheral nerve
15.  Junctional Adhesion Molecule 2 Mediates the Interaction between Hatched Blastocyst and Luminal Epithelium: Induction by Progesterone and LIF 
PLoS ONE  2012;7(4):e34325.
Junctional adhesion molecule 2 (Jam2) is a member of the JAM superfamily. JAMs are localized at intercellular contacts and participated in the assembly and maintenance of junctions, and control of cell permeability. Because Jam2 is highly expressed in the luminal epithelium on day 4 of pregnancy, this study was to determine whether Jam2 plays a role in uterine receptivity and blastocyst attachment in mouse uterus.
Methodology/Principal Findings
Jam2 is highly expressed in the uterine luminal epithelium on days 3 and 4 of pregnancy. Progesterone induces Jam2 expression in ovariectomized mice, which is blocked by progesterone antagonist RU486. Jam2 expression on day 4 of pregnancy is also inhibited by RU486 treatment. Leukemia inhibitory factor (LIF) up-regulates Jam2 protein in isolated luminal epithelium from day 4 uterus, which is blocked by S3I-201, a cell-permeable inhibitor for Stat3 phosphorylation. Under adhesion assay, recombinant Jam2 protein increases the rate of blastocyst adhesion. Both soluble recombinant Jam2 and Jam3 can reverse this process.
Jam2 is highly expressed in the luminal epithelium of receptive uterus and up-regulated by progesterone and LIF via tyrosine phosphorylation of Stat3. Jam2 may play a role in the interaction between hatched blastocyst and receptive uterus.
PMCID: PMC3325240  PMID: 22511936
16.  Evidence for cross-reactivity of JAM-C antibodies: implications for cellular localization studies 
Background information
JAM-C (junctional adhesion molecule C) has been implicated in the regulation of leukocyte migration, cell polarity, spermatogenesis, angiogenesis and nerve conduction. JAM-C has been also reported to concentrate at TJs (tight junctions) and desmosomes, although detailed localization studies remain incomplete.
Monoclonal (LUCA14, MAB1189, Gi11, and PACA4) and polyclonal (40–9000) antibodies were employed to evaluate JAM-C expression/localization in various epithelial cell lines. However, RT–PCR (reverse transcription– PCR) assays revealed no JAM-C mRNA in SK-CO15, HeLa and HPAF-II cells, whereas abundant mRNA was detected in platelets, Caco-2 and ARPE cells. Interestingly, immunofluorescence localization in all cells revealed strong intercellular junctional staining with all of the above antibodies, except PACA4. Given the positive staining results in cells lacking JAM-C mRNA, immunoblot analyses were performed. Western blots revealed a prominent protein band at 52 kDa in all cells tested with all antibodies except PACA4. However, the correct size of JAM-C (37 kDa) was only detected in cells containing JAM-C mRNA. Immunofluorescence staining of JAM-C mRNA-expressing Caco-2 cells using mAb PACA4 revealed co-localization with occludin and ZO-1 (zonula occludens 1) at TJs. Analyses by MS identified the cross-reactive 52 kDa protein band as K8 (keratin 8). Furthermore, siRNA (small interfering RNA)-mediated downregulation of K8 in JAM-C mRNA-negative cells resulted in diminished junctional staining along with a reduction in the intensity of the 52 kDa protein band. Using an antibody specific for K8 phosphorylated at Ser73, the 52 kDa protein was identified as this phosphorylated form of K8.
The results from the present study demonstrate that a majority of available anti-human JAM-C antibodies cross-react with phosphorylated K8 and suggest that cellular localization studies using these reagents should be interpreted with caution. Of the JAM-C antibodies tested, only mAb PACA4 is monospecific for human JAM-C. Analyses using PACA4 reveal that JAM-C expression is variable in different epithelial cell lines with co-localization at TJs.
PMCID: PMC2877264  PMID: 19143587
antibody specificity; epithelial cell; junctional adhesion molecule C (JAM-C); keratin; tight junction
17.  Axonal neuregulin 1 is a rate limiting but not essential factor for nerve remyelination 
Brain  2013;136(7):2279-2297.
Neuregulin 1 acts as an axonal signal that regulates multiple aspects of Schwann cell development including the survival and migration of Schwann cell precursors, the ensheathment of axons and subsequent elaboration of the myelin sheath. To examine the role of this factor in remyelination and repair following nerve injury, we ablated neuregulin 1 in the adult nervous system using a tamoxifen inducible Cre recombinase transgenic mouse system. The loss of neuregulin 1 impaired remyelination after nerve crush, but did not affect Schwann cell proliferation associated with Wallerian degeneration or axon regeneration or the clearance of myelin debris by macrophages. Myelination changes were most marked at 10 days after injury but still apparent at 2 months post-crush. Transcriptional analysis demonstrated reduced expression of myelin-related genes during nerve repair in animals lacking neuregulin 1. We also studied repair over a prolonged time course in a more severe injury model, sciatic nerve transection and reanastamosis. In the neuregulin 1 mutant mice, remyelination was again impaired 2 months after nerve transection and reanastamosis. However, by 3 months post-injury axons lacking neuregulin 1 were effectively remyelinated and virtually indistinguishable from control. Neuregulin 1 signalling is therefore an important factor in nerve repair regulating the rate of remyelination and functional recovery at early phases following injury. In contrast to development, however, the determination of myelination fate following nerve injury is not dependent on axonal neuregulin 1 expression. In the early phase following injury, axonal neuregulin 1 therefore promotes nerve repair, but at late stages other signalling pathways appear to compensate.
PMCID: PMC3692042  PMID: 23801741
injury; Nrg1; regeneration; remyelination; Schwann
18.  Targeting of Junctional Adhesion Molecule-C Inhibits Experimental Choroidal Neovascularization 
JAM-C blockade may be useful for CNV suppression by inhibiting macrophage transmigration, RPE cell migration, and monolayer RPE barrier malfunction. These data reveal a novel function of JAM-C and demonstrate that JAM-C may be a compelling target for CNV therapy.
To identify the expression of junctional adhesion molecule-C (JAM-C) in choroidal neovascularization (CNV) and evaluate the effect of JAM-C targeting on CNV formation and on cellular functions relevant to CNV in vitro, such as macrophage transmigration, human retinal pigment epithelial (hRPE) cell migration, and monolayer RPE permeability.
JAM-C expression in CNV was analyzed by real-time PCR, immunoblot analysis, and immunofluorescence staining. CNV area and blood vessel leakage were quantified using isolectin B4 staining and fluorescein angiography, respectively, 1 week after laser treatment. Macrophage infiltration within the CNV area was measured by immunofluorescence, and transmigration through monolayer RPE was analyzed using a transepithelial migration assay. After JAM-C shRNA transfection, human RPE cell migration was quantified using a transwell assay, and monolayer RPE permeability was determined by measuring the apical-to-basolateral movements of sodium fluorescein.
JAM-C expression was upregulated during CNV formation after laser treatment in a time-dependent manner. However, no change in JAM-C expression was found in the retina up to 14 days after laser treatment. JAM-C targeting by intravitreal injection of JAM-C Fc chimera inhibited CNV, blood vessel leakage, and macrophage infiltration. JAM-C Fc chimera inhibited basolateral-to-apical transmigration in vitro through a monolayer of hRPE of macrophages from patients with wet AMD. In addition, shRNA-mediated JAM-C knockdown inhibited hRPE cell migration and hRPE permeability.
JAM-C blockade may prove useful for CNV suppression by inhibiting macrophage transmigration, RPE cell migration, and monolayer RPE barrier malfunction.
PMCID: PMC3339919  PMID: 22323465
19.  A role for apolipoprotein E, apolipoprotein A-I, and low density lipoprotein receptors in cholesterol transport during regeneration and remyelination of the rat sciatic nerve. 
Journal of Clinical Investigation  1989;83(3):1015-1031.
Recent work has demonstrated that apo E secretion and accumulation increase in the regenerating peripheral nerve. The fact that apoE, in conjunction with apoA-I and LDL receptors, participates in a well-established lipid transfer system raised the possibility that apoE is also involved in lipid transport in the injured nerve. In the present study of the crushed rat sciatic nerve, a combination of techniques was used to trace the cellular associations of apoE, apoA-I, and the LDL receptor during nerve repair and to determine the distribution of lipid at each stage. After a crush injury, as axons died and Schwann cells reabsorbed myelin, resident and monocyte-derived macrophages produced large quantities of apoE distal to the injury site. As axons regenerated in the first week, their tips contained a high concentration of LDL receptors. After axon regeneration, apoE and apoA-I began to accumulate distal to the injury site and macrophages became increasingly cholesterol-loaded. As remyelination began in the second and third weeks after injury, Schwann cells exhausted their cholesterol stores, then displayed increased LDL receptors. Depletion of macrophage cholesterol stores followed over the next several weeks. During this stage of regeneration, apoE and apoA-I were present in the extracellular matrix as components of cholesterol-rich lipoproteins. Our results demonstrate that the regenerating peripheral nerve possesses the components of a cholesterol transfer mechanism, and the sequence of events suggests that this mechanism supplies the cholesterol required for rapid membrane biogenesis during axon regeneration and remyelination.
PMCID: PMC303779  PMID: 2493483
20.  JAM-A regulates permeability and inflammation in the intestine in vivo 
The Journal of Experimental Medicine  2007;204(13):3067-3076.
Recent evidence has linked intestinal permeability to mucosal inflammation, but molecular studies are lacking. Candidate regulatory molecules localized within the tight junction (TJ) include Junctional Adhesion Molecule (JAM-A), which has been implicated in the regulation of barrier function and leukocyte migration. Thus, we analyzed the intestinal mucosa of JAM-A–deficient (JAM-A−/−) mice for evidence of enhanced permeability and inflammation. Colonic mucosa from JAM-A−/− mice had normal epithelial architecture but increased polymorphonuclear leukocyte infiltration and large lymphoid aggregates not seen in wild-type controls. Barrier function experiments revealed increased mucosal permeability, as indicated by enhanced dextran flux, and decreased transepithelial electrical resistance in JAM-A−/− mice. The in vivo observations were epithelial specific, because monolayers of JAM-A−/− epithelial cells also demonstrated increased permeability. Analyses of other TJ components revealed increased expression of claudin-10 and -15 in the colonic mucosa of JAM-A−/− mice and in JAM-A small interfering RNA–treated epithelial cells. Given the observed increase in colonic inflammation and permeability, we assessed the susceptibility of JAM-A−/− mice to the induction of colitis with dextran sulfate sodium (DSS). Although DSS-treated JAM-A−/− animals had increased clinical disease compared with controls, colonic mucosa showed less injury and increased epithelial proliferation. These findings demonstrate a complex role of JAM-A in intestinal homeostasis by regulating epithelial permeability, inflammation, and proliferation.
PMCID: PMC2150975  PMID: 18039951
The Journal of Cell Biology  1962;12(2):361-383.
Wallerian degeneration was produced in guinea pig sciatic nerves by a crush injury. At intervals of 2, 12, 24, 36, 48, 72, and 96 hours after the crush, the nerves were fixed in osmium tetroxide, and blocks from the distal, degenerating segment identified topographically prior to embedding in Araldite or Epon. Phase and electron microscopic study of serial cross- and longitudinal sections reveals a striking, localized accumulation of axonal mitochondria which precedes or accompanies the swelling and fragmentation previously reported by others. These focal accumulations of mitochondria are transient and are most frequently observed in the paranodal axoplasm of large myelinated fibers 24 to 36 hours after crush injury, but are also occasionally identified in small myelinated fibers and unmyelinated axons. Migration and proliferation of axonal mitochondria are considered as possible explanations of these observations.
PMCID: PMC2106025  PMID: 14005486
22.  Expression of JAM-A in the Human Corneal Endothelium and Retinal Pigment Epithelium: Localization and Evidence for Role in Barrier Function 
Junctional adhesion molecules (JAMs) are a family of adhesion proteins found in intercellular junctions. Evidence suggests that JAM-A is important for the regulation of tight junction assembly and epithelial barrier function. The authors recently reported that JAM-A is expressed in rabbit corneal endothelium and that antibody to JAM-A produces corneal swelling. In the present study, they investigate JAM-A expression in the human corneal endothelium and retinal pigment epithelium (RPE) and examine the effect of a function-blocking antibody to JAM-A on the permeability of cultured RPE cell monolayers.
Expression of JAM-A in human corneal endothelium, human RPE tissue, and cultured ARPE-19 monolayers was assessed by immunofluorescence confocal microscopy. Localization of JAM-A was compared with the tight junction-associated protein zonula occludens-1 (ZO-1). To investigate JAM-A function in ARPE-19 cells, ARPE-19 monolayers were subjected to a calcium switch protocol to disrupt cell junctions and treated with a function-blocking antibody to JAM-A or an isotype-matched control. Dextran flux assays were performed to assess the effect of JAM-A antibody on ARPE-19 monolayer permeability.
Expression of JAM-A was observed in human corneal endothelium, and its distribution correlated with the tight junction-associated protein ZO-1. In addition, expression of JAM-A was observed in human RPE and in intercellular junctions of ARPE-19 monolayers. The localization pattern of JAM-A in the RPE and ARPE-19 monolayers was similar to that of ZO-1. ARPE-19 monolayers treated with antibody to JAM-A demonstrated a 33% increase in permeability to 10,000 MWt dextran compared with monolayers treated with control antibody.
Results of this study provide new information about JAM-A expression in tight junctions of the human corneal endothelium and human RPE. The observation that antibodies to JAM-A increase ARPE-19 monolayer permeability is consistent with previous findings of JAM-A function in epithelial tight junctions and suggests JAM-A may have a role in the regulation of RPE barrier function.
PMCID: PMC2074894  PMID: 17724169
23.  Dual Interaction of JAM-C with JAM-B and αMβ2 Integrin: Function in Junctional Complexes and Leukocyte AdhesionD⃞ 
Molecular Biology of the Cell  2005;16(10):4992-5003.
The junctional adhesion molecules (JAMs) have been recently described as interendothelial junctional molecules and as integrin ligands. Here we show that JAM-B and JAM-C undergo heterophilic interaction in cell-cell contacts and that JAM-C is recruited and stabilized in junctional complexes by JAM-B. In addition, soluble JAM-B dissociates soluble JAM-C homodimers to form JAM-B/JAM-C heterodimers. This suggests that the affinity of JAM-C monomers to form dimers is higher for JAM-B than for JAM-C. Using antibodies against JAM-C, the formation of JAM-B/JAM-C heterodimers can be abolished. This liberates JAM-C from its vascular binding partner JAM-B and makes it available on the apical side of vessels for interaction with its leukocyte counterreceptor αMβ2 integrin. We demonstrate that the modulation of JAM-C localization in junctional complexes is a new regulatory mechanism for αMβ2-dependent adhesion of leukocytes.
PMCID: PMC1237098  PMID: 16093349
24.  Activation of NF-κB in Axons and Schwann cells at Site of Sciatic Nerve Crush and Role in Modulating Axon Regeneration in Adult Rats: Studies with Etanercept 
An increasing weight of evidence implicates early inflammatory events as inhibitors of functional recovery in both peripheral and central neuropathologies. In this study, we investigated the role of the inflammatory TNF-α/NF-κB axis on events subsequent to sciatic nerve crush injury in rats. Electrophoretic mobility shift assays (EMSA) revealed that within 6 hours post-crush NF-κB DNA binding activity increased significantly in a 1 cm section of sciatic nerve, centered on the crush site. Immunofluorescent staining for NF-κB subunits verified increased nuclear localization of p50, but not p65 or c-Rel, in Schwann cells, with no evidence of immune cell infiltration. In rats injected s.c. with etanercept, a TNF-α receptor chimera which binds free cytokine, the injury-induced rise in NF-κB DNA-binding activity was inhibited. Immunofluorescent staining confirmed that nuclear localization of NF-κB subunit p50 in Schwann cells was significantly lower in etanercept treated vs. control nerves following nerve injury. Axonal growth determined 3 days after nerve crush with immunofluorescent staining for GAP43 demonstrated that regeneration distance of leading axons from the site of nerve crush was significantly greater in etanercept treated animals than saline-treated controls. These data indicate that TNF-α mediates rapid activation of injury-induced NF-κB DNA binding in Schwann cells, and inhibits post-injury axonal sprouting.
PMCID: PMC2891952  PMID: 19458540 CAMSID: cams797
25.  Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair 
The Journal of Cell Biology  1986;103(3):929-945.
Peripheral nerve injury results in short-term and long-term changes in both neurons and glia. In the present study, immunohistological and immunoblot analyses were used to examine the expression of the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) within different parts of a functionally linked neuromuscular system extending from skeletal muscle to the spinal cord after peripheral nerve injury. Histological samples were taken from 3 to 150 d after crushing or transecting the sciatic nerve in adult chickens and mice. In unperturbed tissues, both N-CAM and Ng-CAM were found on nonmyelinated axons, and to a lesser extent on Schwann cells and myelinated axons. Only N-CAM was found on muscles. After denervation, the following changes were observed: The amount of N-CAM in muscle fibers increased transiently on the surface and in the cytoplasm, and in interstitial spaces between fibers. Restoration of normal N-CAM levels in muscle was dependent on reinnervation; in a chronically denervated state, N-CAM levels remained high. After crushing or cutting the nerve, the amount of both CAMs increased in the area surrounding the lesion, and the predominant form of N-CAM changed from a discrete Mr 140,000 component to the polydisperse high molecular weight embryonic form. Anti-N-CAM antibodies stained neurites, Schwann cells, and the perineurium of the regenerating sciatic nerve. Anti-Ng- CAM antibodies labeled neurites, Schwann cells and the endoneurial tubes in the distal stump. Changes in CAM distribution were observed in dorsal root ganglia and in the spinal cord only after the nerve was cut. The fibers within affected dorsal root ganglia were more intensely labeled for both CAMs, and the motor neurons in the ventral horn of the spinal cord of the affected segments were stained more intensely in a ring pattern by anti-N-CAM and anti-Ng-CAM than their counterparts on the side contralateral to the lesion. Taken together with the previous studies (Rieger, F., M. Grumet, and G. M. Edelman, J. Cell Biol. 101:285-293), these data suggest that local signals between neurons and glia may regulate CAM expression in the spinal cord and nerve during regeneration, and that activity may regulate N-CAM expression in muscle. Correlations of the present observations are made here with established events of nerve degeneration and suggest a number of roles for the CAMs in regenerative events.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC2114294  PMID: 2427528

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