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The cutaneous nerves of rat, cat, guinea pig, pig, and man were studied by immunocytochemistry to compare the staining potency of general neural markers and to investigate the density of nerves containing peptides. Antiserum to protein gene product 9.5 (PGP 9.5) stained more nerves than antisera to neurofilaments, neuron-specific enolase (NSE), and synaptophysin or histochemistry for acetylcholinesterase (AChE). Peptidergic axons showed species variation in density of distribution and were most abundant in pig and fewest in man. However, the specific peptides in nerves innervating the various structures were consistent between species. Nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP) and/or vasoactive intestinal polypeptide (VIP) predominated in all the species; those immunoreactive to tachykinins (substance P and neurokinin A [NKA]) and neuropeptide tyrosine (NPY) were less abundant. Neonatal capsaicin, at the doses employed in this study, destroyed approximately 70% of CGRP- and tachykinin-immunoreactive sensory axons; whereas 6-hydroxydopamine (6-OHDA) at the doses employed resulted in a complete loss of NPY and tyrosine hydroxylase (TH) immunoreactivity without affecting VIP, CGRP, and tachykinins. Thus, this study confirms that antiserum to PGP 9.5 is the most suitable and practical marker for the demonstration of cutaneous nerves. Species differences exist in the density of peptidergic innervation, but apparently not for specific peptides. Not all sensory axons immunoreactive for CGRP and substance P/NKA are capsaicin-sensitive. However, all sympathetic TH- and NPY-immunoreactive axons are totally responsive to 6-OHDA; but no change was seen in VIP-immunoreactive axons, suggesting some demarcation of cutaneous adrenergic and cholinergic sympathetic fibers.

Chlorpyrifos (CPF) and parathion (PS), two common organophosphorus (OP) pesticides, exhibit higher acute toxicity in younger animals compared to adults. Maturational differences in detoxification via carboxylesterases (CEs) and A-esterases (AEs) have been suggested as contributors to the higher sensitivity of younger animals to OP toxicants. AEs (e.g., chlorpyrifos oxonase and paraoxonase) catalytically inactivate while CEs stoichiometrically eliminate OP anticholinesterases. While earlier studies have reported a relationship between the toxicity of some OP pesticides and the maturational profile of AEs and CEs, little information exists on the relative OP-toxicant sensitivity and detoxification capacities of aged animals. In the present study, we investigated the relationship between toxicity of CPF and PS and the activity of CEs and AEs in liver, plasma, and lung of neonatal (7 day), juvenile (21-day), adult (3-month), and aged (24-month) Sprague Dawley rats. CE sensitivity in vitro to chlopyrifos oxon and paraoxon was also evaluated across age groups. Neonatal and juvenile rats were more sensitive than adults to the acute lethality of both CPF and PS. Aged rats exhibited similar sensitivity to CPF but were markedly more sensitive than adults to PS. Levels of CEs and AEs in neonatal and juvenile rats were significantly lower than in adult tissues. Aged rats showed similar levels of AEs in all tissues and CEs in liver and lung, but plasma CE levels were significantly lower (50%) when compared to the adult rats. There were no significant age-related differences in in vitro sensitivity of CEs to either chlorpyrifos oxon or paraoxon in any tissues. In general, acute sensitivity (MTD) was highly correlated with age-related differences in both esterase activities across all 3 tissues with CPF, but only plasma carboxylesterase activity was highly correlated with sensitivity to parathion. The results suggest that both carboxylesterase and A-esterase activities can be correlated with acute sensitivity to CPF and PS, but that age-related differences in CE activity are probably more important in differential toxicity. Furthermore, plasma carboxylesterase activity may play a more pivotal role in the differential sensitivity to PS.

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Immunocytochemistry for the general neuronal marker protein gene product 9.5 and four neuropeptides (calcitonin gene-related peptide, substance P, vasoactive intestinal polypeptide and neuropeptide Y) was performed on 20 skin biopsy specimens from 19 diabetic patients, age range 20-75 years, 17 Type 2 (non-insulin-dependent) and 3 Type 1 (insulin-dependent). Fifteen specimens were from the lower limb, 3 from the upper limb and 2 from the abdominal wall. Seven subjects had lower limb neurophysiological tests. All but one specimen showed reduced protein gene product 9.5 and neuropeptide immunoreactivity. Reduced protein gene product 9.5 and neuropeptide immunoreactivity was found in specimens taken from the abdominal wall and hand as well as those from the leg, and also in specimens from patients undergoing amputation for peripheral vascular disease. In general, the greater the number of abnormal neurophysiological tests, the greater the extent of neuronal abnormalities. Three patients with normal tests had abnormalities of dermal innervation. While these changes are also found in other axonal neuropathies, in the absence of other causes of peripheral nerve disease and of macrovascular disease, immunocytochemistry of skin biopsies may have a role in the assessment of diabetic neuropathy and its response to treatment.

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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.

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