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.

Denatured muscle grafts for nerve repair in an experimental model of nerve damage in leprosy. 2. Recovery of peripheral peptide-containing nerves assessed by quantitative immunohistochemical study.

A marked depletion of neuropeptide-immunoreactive nerves, a consequence of the nerve damage which is commonly found in leprosy, has been reported in peripheral tissues of leprosy patients and of a leprosy animal model. The aim of this study was to investigate peripheral reinnervation following a denatured autologous muscle graft in an animal model of leprosy nerve damage. Possible reinnervation of the foot-pad skin was studied by immunohistochemistry using antisera to the neuronal marker protein gene product 9.5 (PGP), the neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP), and the C-flanking peptide of neuropeptide Y (CPON). The extent of the reinnervation process was assessed by image analysis quantification at different time points. At 8 weeks after muscle grafting, there were small numbers of immunoreactive nerves (p < 0.05). At 12, 16, and 20 weeks postoperatively there was a gradual increase in all immunostaining. At 20 weeks, no significant difference was found for PGP-, CGRP-, and SP-immunoreactive nerves in the epidermal and subepidermal layers compared to control (contralateral) tissue. In experimental tissue the recovery of immunoreactive nerves around sweat glands took longer (up to 12 weeks) than in other skin compartments, but after that time the recovery was rapid and at 20 weeks no difference was measured for VIP-immunoreactive nerves in comparison with controls. Around blood vessels, the recovery of CGRP- and CPON-immunoreactive fibers was slow, and at 20 weeks a difference with control samples (p < 0.01) was noted. In the same area, there was no significant difference for PGP immunoreactivity between controls and tissues at 20 weeks. In contrast, the immunoreactive nerve bundles in the dermis showed a faster recovery than nerves in other skin areas, with amounts similar to controls at 20 weeks. The significant recovery of immunoreactive nerves, in particular of those containing sensory neuropeptide, is consistent with the described functional recovery.

Time-related decrease of substance P and CGRP in central and peripheral projections of sensory neurones in Mycobacterium leprae infected nude mice: a model for lepromatous leprosy in man.

We have previously shown the depletion of cutaneous calcitonin gene-related peptide (CGRP)- and substance P-containing nerves in human leprosy. The aims of this study were to investigate the temporal effects of leprosy on nerves in skin and spinal cord. Tissues were taken from nude mice, 6 and 12 months after inoculation of Mycobacterium leprae into the hind footpads, and from age-matched controls. Sections were immunostained with antisera to substance P or CGRP. After 6 months of infection, substance P- and CGRP-immunoreactive nerves were reduced in skin from all body areas; by 12 months, the reduction was substantially greater. In the spinal cord, sensory fibres immunoreactive for substance P had decreased compared with controls at 6 and 12 months [by 60 per cent (0.022 mm2) and 80 per cent (0.048 mm2), respectively, P less than 0.001], as with CGRP [30 per cent (0.018 mm2) (P less than 0.02) and 40 per cent (0.028 mm2) (P less than 0.01), respectively]. CGRP immunoreactivity was completely absent in motor neurones after 12 months of infection. Loss of CGRP- and substance P-immunoreactive fibres in skin and spinal cord, and CGRP in motor neurones is in accord with impaired pain sensation and muscle weakness in leprosy.

Changes in nerves and neuropeptides in skin from 100 leprosy patients investigated by immunocytochemistry.

The cutaneous innervation is now known to contain neuropeptides including substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory nerves, and vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), principally in autonomic nerves. Skin biopsies from 100 leprosy patients and equivalent areas from 50 non-leprosy controls were fixed in p-benzoquinone solution for immunofluorescence staining and in Bouin's fluid for classification of leprosy type. Antisera to the neural markers, neurofilaments, and protein gene product 9.5 (PGP 9.5), and to neuropeptides were used. Cutaneous nerves and nerve endings immunoreactive for neuropeptides, neurofilaments, and PGP 9.5 were seen in all non-leprous control cases. In leprosy, PGP 9.5- and neurofilament-immunoreactive nerve fibres were seen in all 14 cases of the indeterminate (early) type and in the majority (33/43) of lepromatous cases, but in a smaller proportion (15/43) of tuberculoid cases. Neuropeptide immunoreactivity was seen in only 2/14 of the indeterminate leprosy specimens and was completely absent in other types. This early disappearance may be of diagnostic significance. Thus, cutaneous sensory and autonomic dysfunctions in leprosy are well reflected by changes in nerve fibres and neuropeptides.