High resolution structural changes of Schwann cell and endothelial cells in peripheral nerves across leprosy spectrum.

A systematic ultrastructure of peripheral nerves across the spectrum of leprosy was studied with an aim to better understanding the pathogenesis of nerve involvement in leprosy using light and electron microscope. The pathogenesis of nerve destruction varies in leprosy considerably along the spectrum. The study has begun to shed new light on some aspects of the infection of Mycobacterium leprae (M. lepare) and phenomenon has opened new avenue of research and possible mechanism of pathogenesis in TT/BT/BL/LL leprosy. In tuberculoid type (TT) and borderline tuberculoid (BT) leprosy, the degenerative changes of Schwann cells (SCs) and presence of perineural and perivascular cuffing by mononuclear cells. The endoneurial blood vessel (EBV) showed thickening of basement membrane with hypertrophy of EC leading to narrowing or complete occlusion of lumen and causing ischemia. However, borderline lepromatous (BL) and lepromatous leprosy (LL) foamy macrophages and vacuolated SC contain numerous small dense materials, irregular in shape and size was prominent and, considered to be degenerated and fragmented M. Leprae. The dense materials were also found in the cytoplasm of vascular EC. It was revealed that besides SC, the EC of EBV frequently harbor M. leprae in LL. The lumen of the EBV was wide open with enlarged nucleus. In the present study, the ultrastructural characteristics suggest that hypersensitivity mechanisms are possibly responsible for nerve damage in TT/BT leprosy. However, the study indicates that the mechanisms of nerve damage in BL/LL are basically different wherein hypersensitivity appears to play a very limited role.

Microfasciculation: a morphological pattern in leprosy nerve damage.

AIMS: To study Microfasciculation, a perineurial response found in neuropathies, emphasizing its frequency, detailed morphological characteristics and biological significance in pure neural leprosy (PNL), post-treatment leprosy neuropathy (PTLN) and non-leprosy neuropathies (NLN). METHODS AND RESULTS: Morphological characteristics of microfascicles were examined via histological staining methods, immunohistochemical expression of neural markers and transmission electronmicroscopy. The detection of microfasciculation in 18 nerve biopsy specimens [12 PNL, six PTLN but not in the NLN group, was associated strongly with perineurial damage and the presence of a multibacillary inflammatory process in the nerves, particularly in the perineurium. Immunoreactivity to anti-S100 protein, anti-neurofilament, anti-nerve growth receptor and anti-myelin basic protein immunoreactivity was found within microfascicles. Ultrastructural examination of three biopsies showed that fibroblast-perineurial cells were devoid of basement membrane despite perineurial-like NGFr immunoreactivity. Morphological evidence demonstrated that multipotent pericytes from inflammation-activated microvessels could be the origin of fibroblast-perineurial cells. CONCLUSIONS: A microfasciculation pattern was found in 10% of leprosy-affected nerves. The microfascicles were composed predominantly of unmyelinated fibres and denervated Schwann cells (SCs) surrounded by fibroblast-perineurial cells. This pattern was found more frequently in leprosy nerves with acid-fast bacilli (AFB) and perineurial damage while undergoing an inflammatory process. Further experimental studies are necessary to elucidate microfascicle formation.

Axonal spherical bodies in the peripheral nerves of leprosy patients.

Spherical bodies, roughly 10 micro m in diameter, which have not been reported before, were found in the peripheral nerve axons of specimens collected during post-mortem examination of leprosy patients. These bodies were found in the fascicles of all peripheral nerves of the extremities examined (median, radial, ulnar, peroneal and sciatic nerves). Their incidence was not related to the type of leprosy. The area immediately below the thickened perineurium, a feature associated with leprosy, often showed a large number of spherical bodies. When observed under a transmission electron microscope, the spherical lesions often showed a lamellar structure, although some of them were amorphous. No structure resembling organelles was seen within the bodies. Observation with the merge technique showed a clearly lamellar structure in most of the spherical bodies. These bodies and the surrounding myelin sheaths were partially polarized. The axonal spherical bodies observed in our study seem to represent lesions gradually formed due to glycoprotein denaturation over long periods of time and to be associated with leprosy-caused thickening of the perineurium of peripheral nerves.

Clinicopathologic analysis of 124 biopsy-proven peripheral nerve diseases.

We reviewed dinical, histological and ultrastructural findings of 124 cases of sural nerve biopsy specimens to delineate the trends of peripheral nerve diseases in our institute. Eighty-one were men and 43 were women. We categorized them into five groups: specific diagnosis (66 cases, 53.2%), axonal degeneration type (47 cases, 37.9%), demyelinating type (4 cases, 3.2%), mixed axonal degeneration-demyelinating type (6 cases, 4.8%) and normal (1 case, 0.9%). Cases with specific diagnosis included 21 inflammatory demyelinating polyneuropathy (15 chronic inflammatory demyelinating polyradiculoneuropathy, 6 Guillain-Barre disease), 13 hereditary motor and sensory neuropathy (7 Charcot-Marie-Tooth type I, 6 Charcot-Marie-Tooth type II), 10 vasculitis, 6 toxic neuropathy, 4 leprosy, 3 diabetic neuropathy, 2 alcoholic neuropathy, 1 Fabry's disease and other specific diseases (5 cases). In our cases, the proportion of specific diagnoses was higher, while the proportion of demyelinating peripheral neuropathies and normal were lower than those of Western series. The results of this study indicate that 1) a dose clinicopathologic correlation is important to make a precise diagnosis of peripheral nerve biopsy, 2) Biopsy under strict indication may reduce unnecessary histologic examination, 3) There is no difference in disease pattern of peripheral neuropathy between Western people and Koreans.

Value of nerve biopsy in the diagnosis and follow-up of leprosy: the role of vascular lesions and usefulness of nerve studies in the detection of persistent bacilli.

Nerve biopsy specimens from 53 patients with leprosy and neuropathy were taken from the sural, the dorsal branch of the ulnar, or the superficial radial nerves and processed for light and electron microscopy. There was inflammation in 40 cases (75%), 7 with a granulomatous reaction, various stages of fibrosis in 35 (66%), and endoneurial vascular neoformation in 7. In two cases, small focal infarcts were associated with marked endoneurial inflammation compressing the vessels, in addition to endoneurial lymphocytic vasculitis. Most had an axonal neuropathy of varying degree, some with total fibre loss, others with predominant small myelinated and unmyelinated fibre loss. Signs of demyelination and remyelination were the main findings in 9 cases (17%). Bacilli were present in endothelial, perineurial, Schwann cells and in macrophages. On two occasions, they lost their alcohol acid resistance, were suspected in semithin sections, and confirmed ultrastructurally. The biopsy was decisive for the diagnosis of leprosy in 15 cases (28%), most without skin lesions. We evaluated the effectiveness of the treatment in 20 (37.7%), 12 without and 8 with bacilli, despite negativity in the skin. The diagnosis of leprosy based on skin lesions was confirmed with the nerve biopsy in 9 cases, 6 had an inflammatory neuropathy suggestive of leprosy in the absence of bacilli, and 3 had nonspecific changes in the sural nerve since the neuropathy was in the upper limbs. We conclude that nerve biopsy is indicated for the diagnosis of leprosy in cases without clinically visible skin lesions and to evaluate the effectiveness of the treatment. In these cases the ultrastructural studies are important for recognition of the bacilli. Vascular lesions may play an important role in the progression of the nerve damage, including the occurrence of focal nerve infarcts which, to our knowledge, have not been previously reported in association with leprosy.

Ultrastructural study of the dermal nerves in the cutaneous macular lesions of patients with early leprosy

Thirteen biopsies of macular lesions of early leprosy patients were studied ultrastructurally with transmission electron microscopy (TEM). All of the biopsies displayed at least one dermal nerve partially or completely encircled by mononuclear cells in the conventional histopathological study with light microscopy. The patients'diagnosis varied from indeterminate leprosy to borderline tuberculoide (BT). In the ultrastructural study, twenty-seven dermal nerve branches were found in the thirteen biopsies. Twenty dermal nerve branches in eleven biopsies were found to display no inflammatory involvement. Seven nerves in seven biopsies were morphologically associated with mononuclear leukocytic cells. Four biopsies exhibited nerves with and without inflammatory involvement concomitantly. Three nerves showed morphological evidence of endoneurial fibrosis, not morphologically associated with the inflammatory process at least in the sections examined. No detectable axional and Schwann cell ultra strutural changes even in the twenty-seven nerves were found. The sensorial loss exhibited by the patients before the institution of treatment was completely reversed in eight patients after the end of multidrug therapy regimen. These findings suggest that sensory loss in the early stages of leprosy may be caused by reversible pathological mechanisms, rather than anatomical damage. It is also possible, concerning the mechanisms of nerve damage in leprosy, to speculate on the existence of a pathological process which may precede the inflammation.

Viability of Mycobacterium leprae in skin and peripheral nerves and persistence of nerve destruction in multibacillary patients after 2 years of multidrug therapy.

The pathological changes, bacterial load, and viability of Mycobacterium leprae in the skin and nerves of nine lepromatous leprosy patients who had undergone 2 years of multidrug therapy (MDT) were studied. M. leprae and varying amounts of their remnants were present in the nerves and skin of all but one patient. M. leprae isolated from skin biopsies of six patients and nerve biopsies of nine patients were inoculated into mouse foot pads. No growth was obtained from any one of them. During the electron-microscopic examination of three nerve biopsies, only one specimen showed a small number of solid-staining M. leprae. These findings would explain the low relapse rate in patients treated with 2 years of fix-duration MDT. Results of a long-term follow up of patients is awaited with interest. The possibility of nerve paralysis due to intraneural microreaction and fibrosis consequent to the continued presence of dead bacterial remnants should be seriously considered.

Electron microscopic observations of the relationship between peripheral nerve tissue proper and an endoneurial capillary in a dermal lesion of relapsed lepromatous patient.

The origin of relapse in clinically cured leprosy patients and the dissemination of Mycobacterium leprae in such patients are hitherto little understood phenomena. A detailed electron microscopical examination of a small dermal nerve in a lepromatous lesion of a presently relapsed patient was carried out. Our observations showed the presence of M. leprae in Schwann cells, perineurial cells, vacuolar spaces located in axoplasm and elsewhere. The course of a capillary, entering the endoneurium from the epineurium via the perineurium, suggested the possibility of hematogenous spread of M. leprae which had been tucked away in the dermal peripheral nerve. At the time of relapse, bacilli may multiply in the nerve, may enter the bloodstream, and thus disseminate from the nerve into other nerves and other tissues via hematogenous spread.

Histological methods for assessing myelin sheaths and axons in human nerve trunks.

Although there are many histological techniques for assessing myelin sheaths and axons in paraffin embedded or frozen sections of the peripheral nervous system, modern approaches usually use plastic embedded material. Although plastic embedding is superior for small cutaneous branches, this method has limited value for histological assessment of nerve trunks. We report three methods which together yield a comprehensive approach for thorough and detailed investigation of human nerve trunks. The rapid osmication method permitted assessment of myelinated nerve fibers from frozen sections at operation, thus providing the surgeon with guidance on the extent of nerve resection. The modification presented here resulted in permanent slides, allowing comparison of results with those of the other two procedures. The new osmium-hematoxylin technique could be performed on paraffin embedded nerves. Paraffin, unlike plastic, permitted the study of the whole cross sectional area of the nerve in single sections. Moreover, the sharp image of the myelin permitted computerized morphometry. The significantly modified axonal silver impregnation technique was performed on frozen sections mounted on glass slides, as opposed to the time-consuming impregnation of free-floating sections. The latter technique had a high success rate and permitted semiquantitative assessment of axons in nerve trunks. These methods can be performed in any routine histology laboratory and resulted in greater accuracy compared to conventional methods.

An ultrastructural study of Schwann cells in peripheral nerves of leprosy patients.

An ultrastructural study of peripheral nerves in leprosy patients was carried out of ascertain the changes in Schwann cells containing myelinated and nonmyelinated axons. Axonal multiplication was noticed in nonmyelinated axons in specimens from both tuberculoid and lepromatous leprosy. The Schwann cells in tuberculoid nerves were devoid of M. leprae in contrast to those in lepromatous nerves in which large number of bacilli were seen. These observations suggest that the Schwann cells containing nonmyelinated axons may be affected more frequently in either type of leprosy.

Mycobacteria in nerve trunks of long-term treated leprosy patients.

Mycobacteria were present in 4 out of 8 mixed peripheral nerve trunks from patients (3 BT and 1 BL) treated with DDS and/or MDT for periods ranging from 21 months to 8 years. Most of the bacilli appeared to be 'whole'. Nerve destruction with areas of granulomatous infiltration appeared more active than expected. Possible reasons for a continued presence of bacilli in treated nerves and its implications in 'relapse' are discussed.

Light- and electron-microscopic study of M. leprae-infected armadillo nerves.

Lesions in peripheral nerves of armadillos experimentally infected with Mycobacterium leprae were studied by light- and electron-microscopy. Bacilli could be found clearly inside axons of unmyelinated nerve fibers. Heavily bacillated Schwann cells were seen embracing unmyelinated axons with interrupted cytoplasmic membranes. This indicated the initiation of rupture of those cells which were responsible for the liberation of bacilli into the axons. The nerve lesions were divided into three grades according to their severity: grade I showed lesions focalized in the perineurium; grade II lesions were scattered inside nerve tissue; and in grade III lesions the nerve tissues were diffusely affected. No regressive changes, such as fibrosis or scar formation, were seen in the nerve lesions. Bacillated macrophages were not as foamy as those of human lesions, indicating that these bacillated cells were younger or more easily disrupted with a higher turnover than the cells in human lesions. This would promote the spread of lesions in armadillos, and would explain the less foamy appearance of the cells. We found bacilli inside lymphatics surrounding the nerves, substantiating the opinion that lesions spread to peripheral nerves not only by a hematogenous route but also by the lymphatics.

Light and electron-microscopic study of M. leprae infected Armadillo Neves

Lesions in peripheral nerves of armadillos experimentally infected with Mycobacterium leprae were studied by light- and electron-microscopy. Bacilli could be found clearly inside axons of unmyelinated nerve fibers. Heavily bacillated Schwann cells were seen embracing unmyelinated axons with interrupted cytoplasmic membranes. This indicated the initiation of rupture of those cells which were responsible for the liberation of bacilli into the axons. The nerve lesions were divided into three grades according to their severity: grade I showed lesions focalized in the perineurium; grade II lesions were scattered inside nerve tissue; and in grade III lesions the nerve tissues were diffusely affected. No regressive changes, such as fibrosis or scar formation, were seen in the nerve lesions. Bacillated macrophages were not as foamy as those of human lesions, indicating that these bacillated cells were younger or more easily disrupted with a higher turnover than the cells in human lesions. This would promote the spread of lesions in armadillos, and would explain the less foamy appearance of the cells. We found bacilli inside lymphatics surrounding the nerves, substantiating the opinion that lesions spread to peripheral nerves not only by a hematogenous route but also by the lymphatics

Ultrastructural studies of peripheral nerves in lepromatous leprosy patients.

Ultrathin sections of the peripheral nerves taken from three lepromatous leprosy patients (One untreated, other treated and third in ENL reaction) was examined in the electronmicroscope. In the untreated patient, solid M. leprae organism inside the schwann cell and the degeneration of schwann cell was seen. In contrast, the treated patient showed the degeneration of bacilli and myelinated fibres. However, the characteristics of cells in the ENL reaction showed close similarities with the untreated case.