Endoneurial Extracellular Matrix Research Articles

Gli1 Regulates the Postnatal Acquisition of Peripheral Nerve Architecture.

Peripheral nerves are organized into discrete compartments. Axons, Schwann cells (SCs), and endoneurial fibroblasts (EFs) reside within the endoneurium and are surrounded by the perineurium, a cellular sheath comprised of layers of perineurial glia (PNG). SC secretion of Desert Hedgehog (Dhh) regulates this organization. In Dhh nulls, the perineurium is deficient and the endoneurium is subdivided into small compartments termed minifascicles. Human Dhh mutations cause a neuropathy with similar defects. Here we examine the role of Gli1, a canonical transcriptional effector of hedgehog signaling, in regulating peripheral nerve organization in mice of both genders. We identify PNG, EFs, and pericytes as Gli1-expressing cells by genetic fate mapping. Although expression of Dhh by SCs and Gli1 in target cells is coordinately regulated with myelination, Gli1 expression unexpectedly persists in Dhh null EFs. Thus, Gli1 is expressed in EFs noncanonically (i.e., independent of hedgehog signaling). Gli1 and Dhh also have nonredundant activities. Unlike Dhh nulls, Gli1 nulls have a normal perineurium. Like Dhh nulls, Gli1 nulls form minifascicles, which we show likely arise from EFs. Thus, Dhh and Gli1 are independent signals: Gli1 is dispensable for perineurial development but functions cooperatively with Dhh to drive normal endoneurial development. During development, Gli1 also regulates endoneurial extracellular matrix production, nerve vascular organization, and has modest, nonautonomous effects on SC sorting and myelination of axons. Finally, in adult nerves, induced deletion of Gli1 is sufficient to drive minifascicle formation. Thus, Gli1 regulates the development and is required to maintain the endoneurial architecture of peripheral nerves.SIGNIFICANCE STATEMENT Peripheral nerves are organized into distinct cellular/ECM compartments: the epineurium, perineurium, and endoneurium. This organization, with its associated cellular constituents, is critical for the structural and metabolic support of nerves and their response to injury. Here, we show that Gli1, a transcription factor normally expressed downstream of hedgehog signaling, is required for the proper organization of the endoneurium but not the perineurium. Unexpectedly, Gli1 expression by endoneurial cells is independent of, and functions nonredundantly with, Schwann Cell-derived Desert Hedgehog in regulating peripheral nerve architecture. These results further delineate how peripheral nerves acquire their distinctive organization during normal development, and highlight mechanisms that may regulate their reorganization in pathologic settings, including peripheral neuropathies and nerve injury.

Morphometric analysis of the human endoneurial extracellular matrix components during aging

The aim of this study was to analyze the expression of extracellular matrix (ECM) proteins in human endoneurium during aging. We harvested 15 cadaveric sural nerves, distributed in 3 age groups (I: 25-44, II: 45-64, III: 65-86 years old). Histological sections were stained immunohistochemically for the presence of collagen type I, type IV and laminin, and the ImageJ processing program was used in morphometrical analysis to determine the percentages of these endoneurial proteins. In two younger groups, the endoneurial matrix of the sural nerve was composed from about equal proportions of these proteins, which may be considered a favorable microenvironment for the regeneration of nerve fibers. Linear regression analysis showed a significant increase in endoneurial collagen type IV with age, while collagen type I and laminin significantly decreased during the aging process. In cases older than 65 years, remodeling of the endoneurial matrix was observed to be significantly higher for the presence of collagen type IV, and lower for the expression of collagen type I and laminin. This age-related imbalance of ECM proteins could represent a disadvantageous microenvironment for nerve fiber regeneration in older adults. Our findings contribute to the development of therapeutic approaches for peripheral nerve regeneration.

Urokinase Plasminogen Receptor and the Fibrinolytic Complex Play a Role in Nerve Repair after Nerve Crush in Mice, and in Human Neuropathies

Remodeling of extracellular matrix (ECM) is a critical step in peripheral nerve regeneration. In fact, in human neuropathies, endoneurial ECM enriched in fibrin and vitronectin associates with poor regeneration and worse clinical prognosis. Accordingly in animal models, modification of the fibrinolytic complex activity has profound effects on nerve regeneration: high fibrinolytic activity and low levels of fibrin correlate with better nerve regeneration. The urokinase plasminogen receptor (uPAR) is a major component of the fibrinolytic complex, and binding to urokinase plasminogen activator (uPA) promotes fibrinolysis and cell movement. uPAR is expressed in peripheral nerves, however, little is known on its potential function on nerve development and regeneration. Thus, we investigated uPAR null mice and observed that uPAR is dispensable for nerve development, whereas, loss of uPAR affects nerve regeneration. uPAR null mice showed reduced nerve repair after sciatic nerve crush. This was a consequence of reduced fibrinolytic activity and increased deposition of endoneurial fibrin and vitronectin. Exogenous fibrinolysis in uPAR null mice rescued nerve repair after sciatic nerve crush. Finally, we measured the fibrinolytic activity in sural nerve biopsies from patients with peripheral neuropathies. We showed that neuropathies with defective regeneration had reduced fibrinolytic activity. On the contrary, neuropathies with signs of active regeneration displayed higher fibrinolytic activity. Overall, our results suggest that enforced fibrinolysis may facilitate regeneration and outcome of peripheral neuropathies.

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.

Advanced Glycation End Products in Extracellular Matrix Proteins Contribute to the Failure of Sensory Nerve Regeneration in Diabetes

OBJECTIVEThe goal of this study was to characterize glycation adducts formed in both in vivo extracellular matrix (ECM) proteins of endoneurium from streptozotocin (STZ)-induced diabetic rats and in vitro by glycation of laminin and fibronectin with methylglyoxal and glucose. We also investigated the impact of advanced glycation end product (AGE) residue content of ECM on neurite outgrowth from sensory neurons.RESEARCH DESIGN AND METHODSGlycation, oxidation, and nitration adducts of ECM proteins extracted from the endoneurium of control and STZ-induced diabetic rat sciatic nerve (3–24 weeks post-STZ) and of laminin and fibronectin that had been glycated using glucose or methylglyoxal were examined by liquid chromatography with tandem mass spectrometry. Methylglyoxal-glycated or unmodified ECM proteins were used as substrata for dissociated rat sensory neurons as in vitro models of regeneration.RESULTSSTZ-induced diabetes produced a significant increase in early glycation Nε-fructosyl-lysine and AGE residue contents of endoneurial ECM. Glycation of laminin and fibronectin by methylglyoxal and glucose increased glycation adduct residue contents with methylglyoxal-derived hydroimidazolone and Nε-fructosyl-lysine, respectively, of greatest quantitative importance. Glycation of laminin caused a significant decrease in both neurotrophin-stimulated and preconditioned sensory neurite outgrowth. This decrease was prevented by aminoguanidine. Glycation of fibronectin also decreased preconditioned neurite outgrowth, which was prevented by aminoguanidine and nerve growth factor.CONCLUSIONSEarly glycation and AGE residue content of endoneurial ECM proteins increase markedly in STZ-induced diabetes. Glycation of laminin and fibronectin causes a reduction in neurotrophin-stimulated neurite outgrowth and preconditioned neurite outgrowth. This may provide a mechanism for the failure of collateral sprouting and axonal regeneration in diabetic neuropathy.

Immunohistochemical labelling of components of the endoneurial extracellular matrix of intact and rhizotomized dorsal and ventral spinal roots of the rat – a quantitative evaluation using image analysis

The endoneurial extracellular matrix (ECM) molecules are involved in cell signalling during nervous system development and regeneration. Quantitative differences of immunofluorescence labelling for chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), tenascin-C (TN-C), and thrombospondin (TSP) were evaluated in intact rat dorsal and ventral roots and dorsal and ventral roots 2 and 4 weeks after rhizotomy using image analysis. The distal stumps of spinal roots displayed increased immunolabelling for the molecules with higher immunofluorescence in dorsal than in ventral roots up to 2 weeks from transection. Four weeks after rhizotomy, the immunoreactivity for CSPG, TN-C and TSP decreased in dorsal and increased in ventral root stumps, although a higher level of immunofluorescence for FN remained in both dorsal and ventral root stumps 4 weeks after injury in comparison to 2 weeks after injury. We suggest that the amount of some ECM molecules changed differentially 2 and 4 weeks after rhizotomy to create an appropriate environment in the endoneurium for early and later regrowth of sensory and motor axons. The results presented here are the first report of differences between the endoneurial ECM content of damaged afferent and motor nerve fibers. In addition, the immunohistochemical detection of individual ECM molecules indicated that final extrinsic conditions stimulating the regrowth of regenerating axons probably arise from a balance of both growth-promoting and -inhibiting molecules in the endoneurium.

Regeneration and repair of peripheral nerves

Posttraumatic nerve repair continues to be a major challenge in restorative medicine and microsurgery. Although progress has been made in surgical techniques over the last 30 years, functional recovery after a severe lesion of a major nerve trunk is often incomplete and often unsatisfactory. Functional recovery after surgical repair of mixed nerves is even more disappointing. Functional recovery after peripheral nerve lesion is dependent upon accurate regeneration of axons to their original target tissues. Thus, in order to enhance regeneration, a better understanding of the cellular and molecular biology of selective nerve regeneration is required. Schwann cells and their endoneurial extracellular matrix play pivotal roles in the selective promotion of motor and sensory axon regeneration. Knowledge of these mechanisms allows for the better development of biocompatible nerve grafting material.

Schwann cells and endoneurial extracellular matrix molecules as potential cues for sorting of regenerated axons: A review

Besides very well elaborated microsurgical management of severed peripheral nerves, the clinical results of functional recovery following surgical repair of mixed nerves are disappointing. An improvement of functional recovery after peripheral nerve lesion requires the accurate regeneration of axons to their original target tissues and structures. Therefore, better clinical results could be obtained by a greater understanding of the cellular and molecular biology of selective nerve regeneration. The studies concerning Schwann cells and their endoneurial extracellular matrix as potent cues for selective promotion and influence of regenerating motor and sensory axons are reviewed. Knowledge of the sorting mechanisms of regenerated motor and sensory axons is needed not only for improvement of functional recovery, but also for the development of biocompatible nerve prostheses.

Monocyte chemoattractant protein 1 and chemokine receptor CCR2 productions in Guillain–Barré syndrome and experimental autoimmune neuritis

Infiltration of activated lymphocytes and monocytes is a key phenomenon in the pathogenesis of Guillain–Barré syndrome (GBS) and experimental autoimmune neuritis (EAN). To investigate the role of chemokines, we determined the blood and nerve tissue expression of monocyte chemoattractant protein 1 (MCP-1), a major chemoattractant of monocytes and activated lymphocytes, and its receptor CCR2 in GBS and EAN. MCP-1 circulating levels (ng/ml) in GBS were increased at the time of progression, peaked at the time of plateau and normalized with recovery. MCP-1 circulating levels were the highest in the most disabled patients. The number of circulating CCR2 positive cells was lower in patients with GBS than in healthy subjects ( p<0.004). In GBS, MCP-1 expression was observed in epineurial and endoneurial vessels, on infiltrating cells, Schwann cells and in the endoneurial extracellular matrix. Some CCR2 positive cells were observed in nerve biopsies of GBS patients. In EAN, a slight positivity for MCP-1 was observed in the sciatic nerve. There was no circulating CCR2 positive cells. However, at the time of plateau, a conspicuous infiltration of CCR2 positive cells was observed in the sciatic nerve that was no longer observed at the time of recovery. These results suggest that MCP-1 and CCR2 may participate to the recruitment of circulating mononuclear cells in nerve tissue in EAN and GBS.

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

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

Changes in expression and localization of hemopexin and its transcripts in injured nervous system: a comparison of central and peripheral tissues

The recent demonstration of hemopexin synthesis in the adult rat sciatic nerve[48]and its accumulation after injury[24]has raised the question of the possible role of this acute phase protein during the process of nerve repair. To gain insight into its function, we have compared the distribution of both hemopexin and its messenger RNA in the peripheral and the central nervous systems. We find that hemopexin is present in all types of peripheral nerves and ganglia, confined to the extracellular matrix and basement membranes of the endoneurium, blood vessels and connective tissues. After injury, hemopexin messenger RNA is overexpressed by Schwann cells, fibroblasts and invading macrophages. The content in hemopexin protein increases in all nerves studied, without changes in localization. Therefore, hemopexin does not appear to be associated with the fate of myelin or with the regeneration of a particular type of nerve fibre. In the central nervous system, hemopexin messenger RNA cannot be detected and the protein is only found in basement membranes of the vascular system (capillaries, meninges and choroid plexus). Furthermore, hemopexin and its messenger RNA remain absent from the distal part of the injured optic nerves.Our results further support the idea that hemopexin plays specific roles during nerve repair, and that it may be associated with the endoneurial extracellular matrix.