Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.     

Functional Recovery Occurs Even After Partial Remyelination of Axon-Meshed Median and Ulnar Nerves in Mice

Upper limb nerve injuries are common, and their treatment poses a challenge for physicians and surgeons. Experimental models help in minimum exploration of the functional characteristics of peripheral nerve injuries of forelimbs. This study was conducted to characterize the functional recovery (1, 3, 7, 10, 14, and 21 days) after median and ulnar nerve crush in mice and analyze the histological and biochemical markers of nerve regeneration (after 21 days). Sensory–functional impairments appeared after 1 day. The peripheral nerve morphology, the nerve structure, and the density of myelin proteins [myelin protein zero (P0) and peripheral myelin protein 22 (PMP22)] were analyzed after 21 days. Cold allodynia and fine motor coordination recovery occurred on the 10th day, and grip strength recovery was observed on the 14th day after injury. After 21 days, there was partial myelin sheath recovery. PMP22 recovery was complete, whereas P0 recovery was not. Results suggest that there is complete functional recovery even with partial remyelination of median and ulnar nerves in mice.

     

Neuroregenerative effects of olfactory ensheathing cells transplanted in a multi-layered conductive nanofibrous conduit in peripheral nerve repair in rats

Background

The purpose of this study was to evaluate the efficacy of a multi-layered conductive nanofibrous hollow conduit in combination with olfactory ensheathing cells (OEC) to promote peripheral nerve regeneration. We aimed to harness both the topographical and electrical cues of the aligned conductive nanofibrous single-walled carbon nanotube/ poly (_L-lactic acid) (SWCNT/PLLA) scaffolds along with the neurotrophic features of OEC in a nerve tissue engineered approach.

Results

We demonstrated that SWCNT/PLLA composite scaffolds support the adhesion, growth, survival and proliferation of OEC. Using microsurgical techniques, the tissue engineered nerve conduits were interposed into an 8 mm gap in sciatic nerve defects in rats. Functional recovery was evaluated using sciatic functional index (SFI) fortnightly after the surgery. Histological analyses including immunohistochemistry for S100 and NF markers along with toluidine blue staining (nerve thickness) and TEM imaging (myelin sheath thickness) of the sections from middle and distal parts of nerve grafts showed an increased regeneration in cell/scaffold group compared with cell-free scaffold and silicone groups. Neural regeneration in cell/scaffold group was very closely similar to autograft group, as deduced from SFI scores and histological assessments.

Conclusions

Our results indicated that the tissue engineered construct made of rolled sheet of SWCNT/PLLA nanofibrous scaffolds and OEC could promote axonal outgrowth and peripheral nerve regeneration suggesting them as a promising alternative in nerve tissue engineering.     

A differential stain for neuronal nucleoli in unfixed cryostat sections.

The Klüver-Barrera procedure, using luxol fast blue and cresyl violet for a combined nissl and myelin stain, was adapted to unfixed cryostat sections. Neuronal nucleoli appeared as distinct dark blue structures. The color contrast between violet Nissl substance and the nucleoli facilitated their recognition in human and in rat central nervous systems. This modified staining procedure enabled us to combine a counting of nerve cells with a histochemical investigation by applying each technique to a different set of sections cut from the same block of unfixed, frozen brain tissue.     

Identification and Validation of Suitable Housekeeping Genes for Normalizing Quantitative Real-Time PCR Assays in Injured Peripheral Nerves

Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs.     

Transplantation of adipose derived stem cells for peripheral nerve regeneration in sciatic nerve defects of the rat

Tissue engineering approaches for promoting the repair of peripheral nerve injuries have focused on cell-based therapies involving Adipose-derived stem cells (ASCs). The authors evaluated the effects of undifferentiated ASCs and of neurally differentiated ASCs on the regenerating abilities of peripheral nerves. We hope that this would demonstrate the feasibility of using adipose derived stem cells for peripheral nerve regeneration and provide clues regarding the use of adipose- derived stem cells. ASCs were isolated and cultured. Then the cells were cultured with neuronal induction agents for neural differentiation. ASCs and neurally differentiated ASCs were transplanted into sciatic nerve defects. After 12 weeks, the number and diameter of the myelinated fibers were measured and nerve conduction study was done. The extent of regeneration of myelinated fibers in the neurally differentiated ASCs transplanted group was greater than that in the ASCs transplanted group or the control group. However, thickness of myelin sheath and diameter of nerve fibers in the ASCs transplanted group were greater than those in the neutrally differentiated ASCs transplanted group or the control group. Nerve conduction study showed good recovery in the neurally differentiated ASCs transplanted groups. Muscles can atrophy and contract if denervation has started. It would be difficult to recover muscle function even if the nerve was reinnervated. Therefore, although neurally differentiated ASCs were found to have a greater functional effect than non-differentiated ASCs, time constraint is important when considering a method of ASCs transplantation.     

Regenerating Sciatic Nerve Does Not Utilize Circulating Cholesterol

The rapid accumulation of myelin in the peripheral nervous system during the early postnatal period requires large amounts of cholesterol, a major myelin lipid. All of the cholesterol accumulating in the developing rat sciatic nerve is synthesized locally within the nerve, rather than being derived from the supply in lipoproteins in the systemic circulation (Jurevics and Morell, J. Lipid Res. 5:112–120; 1994). Since this lack of utilization of circulating cholesterol may relate to exclusion by the blood-nerve barrier, we examined the sources of cholesterol needed for regeneration following nerve injury, when the blood-nerve barrier is breached. One sciatic nerve was crushed or transected, and at various times later, the rate of cholesterol accumulation was compared with the rate of local in vivo synthesis of cholesterol within the nerve, utilizing intraperitoneally injected ³H₂O as precursor. The accumulation of additional cholesterol in nerve during regeneration and remyelination could all be accounted for by that locally synthesized within the nerve. There was also an increase in cholesterol esters in injured nerve segments; in crushed nerves, these levels decreased during regeneration and remyelination, consistent with reutilization of cholesterol originally salvaged by phagocytic macrophages and Schwann cells. Thus, regeneration and remyelination following injury in sciatic nerve utilizes both salvaged cholesterol and cholesterol synthesized locally within the nerve, but not cholesterol from the circulation.     

Form birefringence as applied to biopolymer and inorganic material supraorganization

Abstract

I review here form, or textural, birefringence (Δ_F) in the context of advances in the field, as well as with regard to findings and applications in the physics of photonic devices, fibers maintaining polarization, photonic crystal fibers, and in biopolymers present in extracellular matrices and the myelin sheath. Some advantages of applying knowledge of Δ_F to biological fields involving biopolymers, especially collagen fibers, are considered in more detail. Tendon and cartilage collagen fibers have been regarded as a model of dense, highly aggregated biopolymers with preferential orientations. Owing to their supramolecular organization, such materials may be used to study molecular order by using anisotropic optical properties, especially Δ_F. Differences between collagen type I- and collagen type II-rich structures, and similarities between collagen crimp and second harmonic generation images are reported. Based on data reported here, it is possible to deduce that collagen type I supramolecular organization has nonlinear optical properties and that tendon segments can conduct red laser light. With respect to nerve fibers, the detection and measurements of Δ_F have allowed the myelin sheath to be considered a smectic liquid crystal.     

Axonal Regeneration and Remyelination Evaluation of Chitosan/Gelatin-Based Nerve Guide Combined with Transforming Growth Factor-β1 and Schwann Cells

Despite efforts in peripheral nerve injury and regeneration, it is difficult to achieve a functional recovery following extended peripheral nerve lesions. Even if artificial nerve conduit, cell components and growth factors can enhance nerve regeneration, integration in peripheral nerve repair and regeneration remains yet to be explored. For this study, we used chitosan/gelatin nerve graft constructed with collagenous matrices as a vehicle for Schwann cells and transforming growth factor-β1 to bridge a 10-mm gap of the sciatic nerve and explored the feasibility of improving regeneration and reinnervation in rats. The nerve regeneration was assessed with functional recovery, electrophysiological test, retrograde labeling, and immunohistochemistry analysis during the post-operative period of 16 weeks. The results showed that the internal sides of the conduits were compact enough to prevent the connective tissues from ingrowth. Nerve conduction velocity, average regenerated myelin area, and myelinated axon count were similar to those treated with autograft (p > 0.05) but significantly higher than those bridged with chitosan/gelatin nerve graft alone (p < 0.05). Evidences from retrograde labeling and immunohistochemistry analysis are further provided in support of improving axonal regeneration and remyelination. A designed graft incorporating all of the tissue-engineering strategies for peripheral nerve regeneration may provide great progress in tissue engineering for nerve repair.     

Determination in the system of the neuromuscular relations of the coronary arteries at stages in human postnatal development

By means of micromorphometric method in transversal sections of electively stained right and left coronary arteries the data, characterizing the area of the middle and external sheaths, amount of myelin fibers in nerves, accompanying the artery have been obtained in men of 8 age groups (from birth up to old age), who died from causes not connected with the cardiovascular pathology. The summary amount of all classes of myelin fibers increases synchronously with an increase of the muscle sheath area from newborn up to 35-45 years of age. After this age the amount of the myelin fibers decreases together with decreasing area of the middle sheath. Minimal values of these indices are observed in the old age (85-90 years). The external sheath area increases permanently from birth up to the old age. The correlative analysis reveals a positive and significant connection of various degree between the myelin fibers number and value of the middle arterial sheath in persons of juvenile, mature and elderly age: amount of the muscular layer is adequate to the myelin fibers amount. In other age groups the correlative connections are either small, or not significant. Alternative, corresponding to each age quantitative gradations of the nervous connections are determined by means of the peripheral substrate. Its role in the vessels investigated is performed, to a definite degree, by the muscle sheath.     

Diabetic complications in a new animal model (TSOD mouse) of spontaneous NIDDM with obesity.

The TSOD mouse has been established as an inbred strain with spontaneous development of diabetes mellitus as the first clinical signs of diabetes. Polydipsia and polyuria are observed at about 2 months old only in male mice, after which hyperglycemia and hyperinsulinemia are detected. Following these symptoms obesity gradually develops until about 12 months old. In histopathological examination of the pancreas, severe hypertrophy of pancreatic islets was observed due to proliferation and swelling of B cells. In the kidney, thickening of the basement membrane in glomeruli and an increase of the mesangial area were observed at 18 months old. Motor neuropathy in TSOD mice began to appear at 14 months old and most male mice at 17 months old showed weakness of front and hind paws caused by neuron degeneration in the peripheral nerve. In sensory neuropathy, the threshold in the tail pressure test decreased significantly at 12 months old. Light microscopic and electron microscopic examination of sciatic nerves showed a decrease in the density of nerve fibers by the endoneural fibrosis and loss of these fibers. Degenerative changes of myelinated fibers, separation of myelin sheaths with intralamellar edema and remyelination were frequently observed. In the severely affected nerve fibers, the lamellar structure was completely destroyed and macrophages migrated around the myelin sheath or invaded the intramyelin space. Considering these findings similar to non-insulin dependent diabetes mellitus (NIDDM) in humans, the TSOD mouse should be a useful model for the pathogenic study of diabetic complications, especially of peripheral neuropathy.     

ULTRASTRUCTURAL STUDY OF REMYELINATION IN AN EXPERIMENTAL LESION IN ADULT CAT SPINAL CORD

This report presents ultrastructural observations on the cytological events that attend myelin formation occurring in the wake of demyelination in adult cat spinal cord. Lesions were induced in subpial cord by cerebrospinal fluid (c.s.f.) exchange (1, 2). Tissue from eleven cats at nine intervals from 19 to 460 days was fixed in situ by replacing c.s.f. with buffered OsO₄ and embedded in Araldite. After demyelination, axons are embraced by sheet-like glial processes. An occasional myelin sheath is first seen at 19 days; by 64 days, all axons are at least thinly myelinated. The cytoplasm of the myelin-forming cells, unlike that of either oligodendrocyte or fibrous astrocyte in normal cord, is dense with closely packed organelles and fine fibrils. Many of the myelinogenic cells become scarring astrocytes and at 460 days the lesion teems with their fibril-filled processes. Oligodendrocytes appear in the lesion after remyelination is under way. Phagocytes disappear gradually. A myelin sheath is formed by spiral wrapping of a sheet-like glial process around an axon. Where the first turn of the spiral is completed, a mesaxon is formed. As cytoplasm is lost from the process, the plasma membrane comes together along its outer and cytoplasmic surfaces to form compact myelin. Only a small amount of cytoplasm is retained; it is confined to the paramesaxonal region and, on the sheath exterior, to a longitudinal ridge which appears in profile as a small loop. This outer loop has the same rotational orientation as the inner mesaxon. These vestiges of spiral membrane wrapping are also found in normal adult and new-born cat cord. Nodes are present in all stages of remyelination and in normal adult cat and kitten cord. These observations suggest that myelin is reformed in the lesion in the same way it is first formed during normal development. The mechanism of myelin formation is basically similar to that proposed for peripheral nerve and amphibian and mammalian optic nerve; it does not agree with present views on the mechanism of myelinogenesis in mammalian brain and cord. This is the first demonstration of remyelination in adult mammalian central nervous tissue.     

Isolation of peripheral nerve collagen

A new method for the isolation of high yields of collagen from human peripheral nerve is described. A major technique adopted in the present work is sedimentation of the tissue homogenates in a sucrose density gradient. The defatted fibrous material isolated after the removal of myelin was shown to be a relatively pure collagenous substance by amino acid analysis, indicating that the removal of noncollagenous proteins, especially glycoproteins, from collagen fibrils was effectively achieved by this method. The yield of collagen at this step was more than 90% of the total collagen in peripheral nerve. Subsequent extractions with solutions of neutral saline and sodium citrate were found to give further purification of the collagenous protein. The collagens from embryonic peripheral nerves were composed of Type I and III collagens, while Type III collagen was found to be less abundant in adult peripheral nerves.     

Fibrin inhibits peripheral nerve remyelination by regulating Schwann cell differentiation

Remyelination is a critical step for functional nerve regeneration. Here we show that fibrin deposition in the peripheral nervous system after injury is a key regulator of remyelination. After sciatic nerve crush, fibrin is deposited and its clearance correlates with remyelination. Fibrin induces phosphorylation of ERK1/2 and production of p75 NGF low-affinity receptor in Schwann cells and maintains them in a nonmyelinating state, suppresses fibronectin production, and prevents synthesis of myelin proteins. In mice depleted of fibrin(ogen), remyelination of myelinated axons is accelerated due to the faster transition of the Schwann cells to a myelinating state. Regulation of fibrin clearance and/or deposition could be a key regulatory mechanism for Schwann differentiation after nerve damage.     

Faster nerve regeneration after sciatic nerve injury in mice over-expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF-2) is expressed in the peripheral nervous system and is up-regulated after nerve lesion. It has been demonstrated that administration of FGF-2 protects neurons from injury-induced cell death and promotes axonal regrowth. Using transgenic mice over-expressing FGF-2 (TgFGF-2), we addressed the importance of endogenously generated FGF-2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild-type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF-2. Morphometric evaluation of intact nerves from TgFGF-2 mice revealed no difference in number and size of myelinated fibers compared to wild-type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF-2 over-expression on Schwann cell proliferation during the early regeneration process, we used BrdU-labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild-types. We propose that endogenously synthesized FGF-2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination.     

Myelin movements in mature mammalian peripheral nerve fibers

Mature mouse and cat peripheral nerve fibers have been examined in vitro by time-lapse photography. Some Schmidt-Lanterman clefts which were open at the start closed later; other were seen to open and then to close, some of them more than once. The implications of these movements are considered, especially in regard to the question of the passage of materials from the endoneurial connective tissue spaces to the axon. Myelin movements other than those occurring at the Schmidt-Lanterman clefts consisted primarily of the development and frequent regression of indentations of the myelin sheath. A single evagination was seen to develop and then to recede. These myelin movements suggest that previously described invaginations and evaginations of the myelin sheath, including flaps of “redundant myelin”, are not static but rather that they are in a state of movement, forming and regressing at intervals. The possible functional significance of the development and regression of myelin sheath indentations in relationship to axoplasmic flow is discussed.     

Alterations in Gene Expression Associated with Primary Demyelination and Remyelination in the Peripheral Nervous System

Primary demyelination is an important component of a number of human diseases and toxic neuropathies. Animal models of primary demyelination are useful for isolating processes involved in myelin breakdown and remyelination because the complicating events associated with axonal degeneration and regeneration are not present. The tellurium neuropathy model has proven especially useful in this respect. Tellurium specifically blocks synthesis of cholesterol, a major component of PNS myelin. The resulting cholesterol deficit in myelin-producing Schwann cells rapidly leads to synchronous primary demyelination of the sciatic nerve, which is followed by rapid synchronous remyelination when tellurium exposure is discontinued. Known alterations in gene expression for myelin proteins and for other proteins involved in the sequence of events associated with demyelination and subsequent remyelination in the PNS are reviewed, and new data regarding gene expression changes during tellurium neuropathy are presented and discussed.     

Mediators of oligodendrocyte differentiation during remyelination

Myelin, a dielectric sheath that wraps large axons in the central and peripheral nervous systems, is essential for proper conductance of axon potentials. In multiple sclerosis (MS), autoimmune-mediated damage to myelin within the central nervous system (CNS) leads to progressive disability primarily due to limited endogenous repair of demyelination with associated axonal pathology. While treatments are available to limit demyelination, no treatments are available to promote myelin repair. Studies examining the molecular mechanisms that promote remyelination are therefore essential for identifying therapeutic targets to promote myelin repair and thereby limit disability in MS. Here, we present our current understanding of the critical extracellular and intracellular pathways that regulate the remyelinating capabilities of oligodendrocyte precursor cells (OPCs) within the adult CNS.