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.

Regeneration at the predilective damage sites of nerve trunks in treated leprosy.

Superficially located large and medium sized mixed peripheral limb nerves in active leprosy have previously been shown to have well-recognized fusiform swellings. It is generally agreed that these are the sites of predilective nerve involvement where the severest degeneration and fibrosis occur. A semiquantitative histopathological study on one of these sites, the flexor retinaculum region of the posterior tibial nerve, has been carried out on 14 treated leprosy patients who suffered from total sensory loss to the foot for between 2 and 40 years. The following observations were made: (1) large-scale nerve regeneration was present as characterized by numerous Schwann cells and unmyelinated axons which formed regeneration clusters; (2) thick myelinated axons were either absent or present only in very low numbers; (3) the intraneural fibrosis was usually not severe; (4) the presence of active inflammation probably interfered with nerve regeneration; (5) it appeared that this regeneration started shortly after the onset of therapy and persisted for decades; (6) lepromatous cases were characterized by evenly distributed pathology, whereas borderline tuberculoid cases had an unevenly distributed pathology; (7) the massive nerve regeneration observed was functionally ineffective--these findings indicate that the total nerve damage may affect the more peripheral nerve branches.

Damage and regeneration of peripheral nerves in advanced treated leprosy.

Despite the rapidly falling prevalence of leprosy, the disability and handicap resulting from loss of protective sensation, due to irreversible nerve damage, will remain a huge medical problem for many years. To elucidate the location and consequences of permanent nerve damage in treated leprosy, a prospective study involving nine patients who underwent leg amputation was conducted. Full-length nerves dissected from amputated legs were studied with histological and immunohistochemical methods. Our main findings were that: in both lepromatous and tuberculoid leprosy nerve damage increased distally, culminating in total destruction of dermal nerves and sensory nerve endings; after the therapy-related decrease of inflammation large-scale nerve regeneration took place; and that regenerating axons persisted for decades and in tuberculoid leprosy they might reach the subcutaneous fat of the plantar skin. We conclude that nerve regeneration was blocked by fibrous replacement of the distal-most nerves and nerve endings, and that the theoretical basis of nerve grafting in leprosy is in need of further clarification. In some patients, autologous transplantation of skin flaps, probably irrespective of the duration of loss of sensation, might help in regaining protective sensation.

Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES.

In contrast to other bacterial species, mycobacteria were thus far considered to contain groEL and groES genes that are present on separate loci on their chromosomes, Here, by screening a Mycobacterium leprae lambda gt11 expression library with serum from an Ethiopian lepromatous leprosy patient, two DNA clones were isolated that contain a groEL gene arranged in an operon with a groES gene. The complete DNA sequence of this groESL operon was determined. The predicted amino acid sequences of the GroES and GroEL proteins encoded by this operon are 85-90% and 59-61% homologous to the sequences from previously characterized mycobacterial GroES and GroEL proteins. Southern blotting analyses with M. leprae groES- and groEL-specific probes demonstrate that similar groESL homologous DNA is present in the genomes of other mycobacteria, including Mycobacterium tuberculosis. This strongly suggests that mycobacteria contain a groESL operon in addition to a separately arranged second groEL gene. Using five T-cell clones from two leprosy patients as probes, expression of the M. leprae GroES protein in Escherichia coli after heat shock was demonstrated. Four of these clones recognized the same M. leprae-specific GroES-derived peptide in a DR2-restricted fashion. No expression of the groEL gene from this operon was detected in E. coli after heat shock, as tested with a panel of T-cell clones and monoclonal antibodies reactive to previously described GroEL proteins of mycobacteria.