Myelination key factor krox-20 is downregulated in Schwann cells and murine sciatic nerves infected by Mycobacterium leprae.

Schwann cells (SCs) critically maintain the plasticity of the peripheral nervous system. Peripheral nerve injuries and infections stimulate SCs in order to retrieve homeostasis in neural tissues. Previous studies indicate that Mycobacterium leprae (ML) regulates the expression of key factors related to SC identity, suggesting that alterations in cell phenotype may be involved in the pathogenesis of neural damage in leprosy. To better understand whether ML restricts the plasticity of peripheral nerves, the present study sought to determine the expression of Krox-20, Sox-10, c-Jun and p75NTR in SC culture and mice sciatic nerves, both infected by ML Thai-53 strain. Primary SC cultures were stimulated with two different multiplicities of infection (MOI 100:1; MOI 50:1) and assessed after 7 and 14 days. Sciatic nerves of nude mice (NU-Foxn1nu ) infected with ML were evaluated after 6 and 9 months. In vitro results demonstrate downregulation of Krox-20 and Sox-10 along with the increase in p75NTR-immunolabelled cells. Concurrently, sciatic nerves of infected mice showed a significant decrease in Krox-20 and increase in p75NTR. Our results corroborate previous findings on the interference of ML in the expression of factors involved in cell maturation, favouring the maintenance of a non-myelinating phenotype in SCs, with possible implications for the repair of adult peripheral nerves.

Are human peripheral nerves sensitive to X-ray imaging?

Diagnostic imaging techniques play an important role in assessing the exact location, cause, and extent of a nerve lesion, thus allowing clinicians to diagnose and manage more effectively a variety of pathological conditions, such as entrapment syndromes, traumatic injuries, and space-occupying lesions. Ultrasound and nuclear magnetic resonance imaging are becoming useful methods for this purpose, but they still lack spatial resolution. In this regard, recent phase contrast x-ray imaging experiments of peripheral nerve allowed the visualization of each nerve fiber surrounded by its myelin sheath as clearly as optical microscopy. In the present study, we attempted to produce high-resolution x-ray phase contrast images of a human sciatic nerve by using synchrotron radiation propagation-based imaging. The images showed high contrast and high spatial resolution, allowing clear identification of each fascicle structure and surrounding connective tissue. The outstanding result is the detection of such structures by phase contrast x-ray tomography of a thick human sciatic nerve section. This may further enable the identification of diverse pathological patterns, such as Wallerian degeneration, hypertrophic neuropathy, inflammatory infiltration, leprosy neuropathy and amyloid deposits. To the best of our knowledge, this is the first successful phase contrast x-ray imaging experiment of a human peripheral nerve sample. Our long-term goal is to develop peripheral nerve imaging methods that could supersede biopsy procedures.

Contact-dependent demyelination by Mycobacterium leprae in the absence of immune cells.

Demyelination results in severe disability in many neurodegenerative diseases and nervous system infections, and it is typically mediated by inflammatory responses. Mycobacterium leprae, the causative organism of leprosy, induced rapid demyelination by a contact-dependent mechanism in the absence of immune cells in an in vitro nerve tissue culture model and in Rag1-knockout (Rag1-/-) mice, which lack mature B and T lymphocytes. Myelinated Schwann cells were resistant to M. leprae invasion but undergo demyelination upon bacterial attachment, whereas nonmyelinated Schwann cells harbor intracellular M. leprae in large numbers. During M. leprae-induced demyelination, Schwann cells proliferate significantly both in vitro and in vivo and generate a more nonmyelinated phenotype, thereby securing the intracellular niche for M. leprae.

Sciatic nerve of normal and T200x5R Swiss white mice fails to support multiplication of intraneurally injected M. leprae.

In a preliminary study we have shown that freshly harvested Mycobacterium leprae, when injected into the sciatic nerve in normal and immunosuppressed (TR) mice, induce massive but localized epithelioid and macrophage granuloma, respectively, in 3-4 weeks. In order to determine the fate of M. leprae injected intraneurally into normal and TR mice, in the present study we measured sequentially the viability, fold increase and clearance, if any, using semi-quantitative methods. The average M. leprae yield per nerve assessed at regular intervals, beginning at 24 hr and including 72 hr, 1 week, 2, 3, 4, 12, 24 and 48 weeks, showed neither a significant increase nor a decrease in either the normal or the TR mice. The viability of M. leprae, assessed using the standard mouse foot pad method, showed a significant decrease as compared to baseline growth effective at 24 hr and remained static until approximately 4 weeks. A further decline and total loss of viability was noted by 12 months. The results show that injection of M. leprae via the intraneural route in both normal and TR mice failed to sustain the viability and failed to support the multiplication of the organisms.

Inflammatory pseudotumor of nerve.

Two cases of inflammatory pseudotumor are described. The first patient, a 35-year-old white man, developed a progressive sensorimotor deficit in the right leg associated with a fusiform sciatic nerve mass in the posterior thigh. The lesion, compressive in nature and situated entirely within the epineurium, was totally resected. Histology revealed lymphocytic and plasmacellular inflammation as well as extensive fibrosis and collagen deposition. The patchy infiltrate consisted equally of CD2, CD3, CD5, and CD7 positive T-lymphocytes as well as CD20-and CD22-positive B-lymphocytes expressing both kappa and lambda immunoglobulin light chains. A selective biopsy of the encompassed and compressed nerve fascicles demonstrated both myelin loss and axonal injury. The second case was that of an 18-year-old woman with focal enlargement of a radial nerve by an epineurial infiltrate of multinucleate histiocytes and T as well as occasional B lymphocytes. No etiology was apparent in either case. The differential diagnosis includes non-neoplastic processes (amyloidoma and tuberculoid leprosy) as well as tumors (benign and malignant peripheral nerve sheath tumors, lymphoma). Although rare, inflammatory pseudotumors must be included in the differential diagnosis of tumor-like lesions of peripheral nerve.

Effect of T-cell depletion on bacterial multiplication and pattern of nerve damage in M. leprae-infected mice.

Various mechanisms for nerve damage in tuberculoid leprosy have been proposed. A common feature amongst them is the crucial role played by T-cells. Therefore, the present study was designed to determine the role of T-cells in the induction of nerve damage in leprosy using two different protocols for obtaining graded levels of T-cell depletion: (i) Cyclosporine A, for depletion of T-helper cells and (ii) Anti Thy 1.2, for total depletion of T-cells. The findings indicate that the early changes seen in the unmyelinated fibres may not involve T-cells. However, the later stages of nerve damage associated with demyelination are dependent on T-cell responses.

A morphological and functional assessment of Mycobacterium leprae-induced nerve damage in a guinea-pig model of leprous neuritis.

Nerve damage, resembling that caused by Mycobacterium leprae in man, was created by the injection of cobalt-irradiated M. leprae organisms into the tibial nerve of guinea-pigs. Assessment of nerve damage was made by clinical, electrophysiological and morphometric means at intervals up to 13 weeks after injection. Quantitative immunohistochemical analysis of neuropeptide-containing fibres in the skin of the foot was also carried out. Significant nerve damage occurred 3 weeks after injection of M. leprae organisms. Motor and sensory functional loss peaked at 5 weeks after injection, and there was a significant decrease of peptide-immunoreactive nerves in all skin compartments. The nerve damage was self-limiting and functional recovery had occurred by 13 weeks. The model shows many of the features found in the nerve damage of treated leprosy patients.

Animal model to study the mechanism of nerve damage in leprosy--a preliminary report.

Intraneural injection of 10-20 x 10(6) viable Mycobacterium leprae into the sciatic nerve of normal, unsensitized, Swiss white mice gives rise to a tuberculoid type of granulomatous response in 2 weeks. The same dose of viable M. leprae when injected into the sciatic nerves of unsensitized immunosuppressed mice (T200 x 5R) elicited a macrophage response. When macrophages were systemically immobilized using an intraperitoneal injection of silica quartz dust in normal mice, the lesion produced was of the lepromatous type, suggesting a role for the macrophage in the induction of the tuberculoid type of granulomatous response. In all of these in situ experiments, M. leprae failed to enter the Schwann cells.

Nerve lesions induced by macrophage activation.

The neuropathies associated with infectious processes, including leprosy, retroviral infections and Chagas' disease, represent the largest group of neuropathies in the world. Segmental demyelination and axonal degeneration of nerve fibres are associated with inflammatory infiltrates which contain a large number of mononuclear phagocytes. In order to learn more about the role played by macrophage activation in the nerve lesions observed in inflammatory neuropathies, we have performed a morphological study of nerves injected with products of activation of macrophages including proteolytic enzymes and cytokines (tumour necrosis factor and alpha beta-interferon). We have also studied the effects on nerve fibres of macrophages activated by ingestion of proteose-peptone, a foreign protein, and in the course of a delayed-type hypersensitivity (DTH) reaction. We have found that proteases and urokinase were potent demyelinating agents and that activated macrophages were also able to induce significant demyelination of neighbouring fibres. In contrast, injection of TNF alpha induced more severe nerve lesions consisting of axonal degeneration of the majority of nerve fibres. We thus conclude that infected macrophages which penetrate the endoneurium and macrophages activated in a DTH reaction can both cause neuropathy.

Reaction of peripheral nerves to vascular and bacterial injuries.

Mouse sciatic nerves were subjected to devascularization, M. leprae inoculation, and combined insult of devascularization + footpad inoculation (FPI). Changes were seen in FPI nerves only after eight months, but in cases of combined insult, changes were evident in hours. Both the groups showed initial loss of small myelinated fibres. No proliferation of Schwann cells was in FPI nerves, but in combined insult it was maximum after two weeks. Presence of M. leprae seems to be arresting Schwann cell activity after two weeks. Blood vessels showed increased endothelial cell cytoplasm, basement membrane proliferation and villi formation. These changes seem to be specific of endoneurial blood vessels of leprosy nerves. Increased number of mast cells seems to be specific of devascularized and FPI nerves. Increased number of macrophages expressed low immunity of devascularized nerves. Eosinophils migrated to endoneurium as a result of leakage of axoplasm.

Nerve damage induced by mycobacterial granulomas in guinea pig sciatic nerves.

A possible model for nerve damage in leprosy has been developed in the sciatic nerve of the guinea pig. Intraneural injection of 10(7) BCG organisms into an unsensitized animal induces an epithelioid cell granuloma in 2 weeks similar to that found in tuberculoid leprosy patients. In contrast, intraneural injection of 10(9) cobalt-irradiated Mycobacterium leprae organisms induces a macrophage granuloma in 5 weeks, similar to that found in lepromatous leprosy patients. Histological, immunohistochemical, electron microscopical and electrophysiological studies have demonstrated that the lesions induced in the experimental animals show many of the features documented in studies of nerve damage in leprosy patients.

Sciatic nerve in experimental leprosy.

Swiss albino mice were inoculated with Mycobacterium leprae obtained from untreated lepromatous patients. Histopathological study of sciatic nerves showed no abnormality. However a few free acid fast bacilli (AFB) were detected in the sciatic nerves taken from the inoculated limbs during the early stages of infection, suggesting the nerve-fibre route of travel as seen in humans in experimental leprosy, too.

The surgical management of nerve lesions in the lower limbs. Clinical evaluation, surgical technique and results.

The author present his experience of the repair of nerve lesions in the lower limb. The cases are classified into four main groups: traumatic lesions; acute and chronic compression; tumours and leprosy. The traumatic lesions include those associated with a clean wound, missile injuries, traction injuries and a miscellaneous group. The diagnosis and treatment of the different lesions are discussed. The results of nerve grafting using microsurgical techniques are presented. The results are good enough, even in severe traction injuries, to recommend the repair of nerve lesions of the lower limbs in the circumstances which are outlined.

Manifestations of experimental leprosy in the armadillo.

Three experiments, using different routes and doses of infection, were conducted using 42 armadillos. Thirty-six of them developed generalized disease. There is no significant sex or age difference in susceptibility. Route and dose of infection make very little difference in the disease prevalence except that the intravenous administration of a large dose reduces the period of development of generalized disease. It is quite possible that in armadillos the resistance to the disease is partly genetic. Although a majority of the armadillos developed lepromatous disease, borderline leproma is fairly common. In skin nodules large colonies of extracellular bacilli are demonstrated. Bacilli are also demonstrated in liver parenchymal cells.

Study of the involvement of the sciatic nerve following inoculation with M. leprae and other mycobacteria in the mouse foot pad.

In order to determine whether Mycobacterium leprae alone produce the typical damage in the sciatic nerves of foot pad inoculated mice as demonstrated earlier, a comparative study was undertaken using various other mycobacteria inoculated into the hind foot pads of normal Swiss white mice. The findings indicate that FMR isolates No. 51 and No. 75 and M. avium showed multiplication in the foot pads of the mice throughout the 4th, 6th or 8th postinoculation months and these infections were associated with neural changes in the sciatic nerves. The type of nerve involvement in the case of M. avium differs from M. leprae in being predominantly an axonal degeneration at the 8th post-inoculation month, that is, degeneration of the complete axon and myelin debris remnants; whereas in M. leprae infection, where segmental demyelination predominates, the axons are intact and it is the Schwann cell that is affected. The neural changes in the case of FMR isolates No. 51 and No. 75 were similar to those seen in mice inoculated with M. leprae obtained directly from human biopsies. Other mycobacteria, HI-75 (Skinsnes) and M. scrofulaceum, showed growth in the foot pad initially which persisted in the case of M. scrofulaceum until the 20th post-inoculation month, but no ultrastructural changes were observed in the sciatic nerves of these mice. In ICRC-inoculated mice, nerve lesions were seen much later (at the 16th post-inoculation month) and the changes were similar to those seen with M. leprae. M. vaccae, M. smegmatis, M. phlei, and M. intracellulare showed almost no growth in the foot pads of the mice, and there were no detectable changes in the sciatic nerves. M. lepraemurium showed growth in the foot pad but no lesions were seen in the sciatic nerve.(ABSTRACT TRUNCATED AT 250 WORDS)