Jorge J. Vilches

Influence of aging on peripheral nerve function and regeneration

Abstract   Aging deeply influences several morphologic and functional features of the peripheral nervous system (PNS). Morphologic studies have reported a loss of myelinated and unmyelinated nerve fibers in elderly subjects, and several abnormalities involving myelinated fibers, such as demyelination, remyelination and myelin balloon figures. The deterioration of myelin sheaths during aging may be due to a decrease in the expression of the major myelin proteins (P0, PMP22, MBP). Axonal atrophy, frequently seen in aged nerves, may be explained by a reduction in the expression and axonal transport of cytoskeletal proteins in the peripheral nerve. Aging also affects functional and electrophysiologic properties of the PNS, including a decline in nerve conduction velocity, muscle strength, sensory discrimination, autonomic responses, and endoneurial blood flow. The age‐related decline in nerve regeneration after injury may be attributed to changes in neuronal, axonal, Schwann cell and macrophage responses. After injury, Wallerian degeneration is delayed in aged animals, with myelin remnants accumulated in the macrophages being larger than in young animals. The interaction between Schwann cells and regenerative axons takes longer, and the amount of trophic and tropic factors secreted by reactive Schwann cells and target organs are lower in older subjects than they are in younger subjects. The rate of axonal regeneration becomes slower and the density of regenerating axons decrease in aged animals. Aging also determines a reduction in terminal and collateral sprouting of regenerated fibers, further limiting the capabilities for target reinnervation and functional restitution. These age‐related changes are not linearly progressive with age; the capabilities for axonal regeneration and reinnervation are maintained throughout life, but tend to be delayed and less effective with aging.

Genetics of congenital insensitivity to pain with anhidrosis (CIPA) or hereditary sensory and autonomic neuropathy type IV: Clinical, biological and molecular aspects of mutations in TRKA(NTRK1) gene encoding the receptor tyrosine kinase for nerve growth factor

Abstract Three new silicone-based impression materials manufactured for use in dentistry (Silasoft®, CutterSil® and Xantopren®) have been evaluated for the silicone mold sweat test in humans and mice and compared with the well-known silicone material Elasticon®. The new materials produced more translucent molds and the sweat impressions show less contrast than Elasticon. Molds made of Xantopren and CutterSil retained air bubbles that make counting of sweat impressions more difficult than with Elasticon. The density of sweat droplets from human skin varied depending on the silicone used, but differences were not significant. In the mouse hindpaw, total counts of sweat droplets were similar in Elasticon, Silasoft and CutterSil molds but slightly higher in Xantopren molds. There was good correlation between the number of sweat droplet impressions recorded with Elasticon and that recorded with the new materials, with the exception of CutterSil, both in humans and mice. From these results we recommend Silasoft as a valid substitute of Elasticon for the evaluation of sudomotor function with the silicone impression technique.

Physiological and immunohistochemical characterization of cisplatin-induced neuropathy in mice.

We investigated the neuropathic effects of cisplatin in two groups of mice treated with 5 or 10 mg/kg/week of cisplatin for 7 or 8 weeks. Peripheral nerve functions were evaluated by sweat imprints, and electrophysiological, rotarod, and nociceptive tests. Protein gene product 9.5 (PGP), calcitonin gene‐related peptide (CGRP), and vasoactive intestinal peptide (VIP) were immunohistochemically localized in footpads. Tibial nerves were analyzed morphometrically. Functional deficits developed progressively with higher cumulative doses, more markedly in mice treated with high than in those with low doses. From cumulative doses of 10 mg/kg, significant declines in sensory nerve conduction velocity and sudomotor responses were found, whereas motor and nociceptive functions were involved later. There were no morphometrical changes in tibial nerves. A marked decrease of CGRP‐ and VIP‐immunoreactive nerves occurred in samples from treated mice, whereas PGP‐labeled profiles decreased mildly at late stages. Impairment of the content of neuropeptides with neurosecretor role was detectable earlier than functional abnormalities. Immunohistochemical analysis of skin biopsies offers a useful diagnostic tool for peripheral neuropathies.

Influence of physical parameters of nerve chambers on peripheral nerve regeneration and reinnervation.

We compared reinnervation of target organs after sciatic nerve section leaving gaps of 2, 4, 6, or 8 mm or gaps repaired with silicone tubes in different groups of mice. Functional reinnervation was assessed by noninvasive methods to determine recovery of sweating, nociceptive, and muscular functions in the hindpaw repeatedly during 3 months postoperation. The increase of gap length between nerve stumps delayed the beginning and reduced the degree of functional recovery achieved either with or without repair. When lesions were left unrepaired, functional reinnervation was only noticeable with a 2-mm gap and practically absent with longer gaps. With tube repair, reinnervation started earlier and achieved higher values than in the corresponding unrepaired groups. Tubulization was most effective with 4-mm gaps and comparatively less with shorter and longer gaps. With 4-mm gaps, recovery was higher when the silicone tube had a cross-sectional area 2.5 times that of the sciatic nerve than with narrower or wider tubes and when the wall was the thinnest available. In all cases muscle reinnervation showed a lower progression than sweating and nociceptive recovery.

Neurophysiological, histological and immunohistochemical characterization of bortezomib-induced neuropathy in mice

Bortezomib, a proteasome inhibitor, is an antineoplastic drug to treat multiple myeloma and mantle cell lymphoma. Its most clinically significant adverse event is peripheral sensory neuropathy. Our objective was to characterize the neuropathy induced by bortezomib in a mouse model. Two groups were used; one group received vehicle solution and another bortezomib (1mg/kg/twice/week) for 6weeks (total dose as human schedule). Tests were performed during treatment and for 4weeks post dosing to evaluate electrophysiological, autonomic, pain sensibility and sensory-motor function changes. At the end of treatment and after washout, sciatic and tibial nerves, dorsal ganglia and intraepidermal innervation were analyzed. Bortezomib induced progressive significant decrease of sensory action potential amplitude, mild reduction of sensory velocities without effect in motor conductions. Moreover, it significantly increased pain threshold and sensory-motor impairment at 6weeks. According to these data, histopathological findings shown a mild reduction of myelinated (-10%; p=0.001) and unmyelinated fibers (-27%; p=0.04), mostly involving large and C fibers, with abnormal vesicular inclusion body in unmyelinated axons. Neurons were also involved as shown by immunohistochemical phenotypic switch. After washout, partial recovery was observed in functional, electrophysiological and histological analyses. These results suggest that axon and myelin changes might be secondary to an initial dysfunctional neuronopathy.

GAL3 receptor KO mice exhibit an anxiety-like phenotype

Significance In the modern world, stress-related diseases, including depression and anxiety disorders, are rapidly increasing. Neuropeptides are important modulators of these diseases. The neuropeptide galanin (GAL) has already been implicated in anxiety- and depression-related behaviors, but the relevant receptor subtypes remain to be elucidated. In the present work, we are the first, to our knowledge, to examine the role of the GAL3 receptor in anxiety- and depression-related behaviors in GAL3 receptor-deficient mice. We provide evidence that this receptor subtype is involved in stress-related diseases, and we propose this receptor as a target for alternative treatment strategies for mood disorders. The neuropeptide galanin (GAL) is widely distributed in the central and peripheral nervous systems. It is a modulator of various physiological and pathological processes, and it mediates its effects via three G protein-coupled receptors (GAL1–3 receptors). A role for GAL as a modulator of mood and anxiety was suggested, because GAL and its receptors are highly expressed in limbic brain structures of rodents. In recent years, numerous studies of animal models have suggested an involvement of GAL and GAL1 and GAL2 receptors in anxiety- and depression-related behavior. However, to date, there is sparse literature implicating GAL3 receptors in behavioral functions. Therefore, we studied the behavior of GAL3 receptor-deficient (GAL3-KO) mice to elucidate whether GAL3 receptors are involved in mediating behavior-associated actions of GAL. The GAL3-KO mouse line exhibited normal breeding and physical development. In addition to behavioral tests, phenotypic characterization included analysis of hematology, amino acid profiles, metabolism, and sudomotor function. In contrast to WT littermates, male GAL3-KO mice exhibited an anxiety-like phenotype in the elevated plus maze, open field, and light/dark box tests, and they were less socially affiliated than WT animals to a stranger mouse in a social interaction test. In conclusion, our data suggest involvement of GAL3 receptors in GAL-mediated effects on mood, anxiety, and behavior, making it a possible target for alternative treatment strategies for mood disorders.

Functional sudomotor responses to cholinergic agonists and antagonists in the mouse

This study evaluates the functional activity of the mouse sweat glands in response to cholinergic agonists and antagonists using the silicone imprint technique. In intact mice the response to acetylcholine, methacholine and pilocarpine did not differ significantly from control saline injection, indicating that immobilization induces high levels of sweating, masking the effects of cholinergic stimulation. Plantar emotional sweating was completely abolished by local anesthesia at the ankle. Under these conditions, administration of acetylcholine only provoked detectable sweating when injected locally into the sole skin. Methacholine activated an increasing number of sweat glands in a dose-dependent manner between 0.5 and 10 mg/kg; the response was maximal after 5-10 min of administration and decreased subsequently. With pilocarpine the maximum number of reactive sweat glands was observed at a dose of 2.5 mg/kg. The response was stable for 45 min with doses 2.5 and 5 mg/kg, but decreased exponentially with higher doses. The subtype of sweat gland muscarinic receptor was characterized by determining the inhibitory effect of different cholinergic antagonists on pilocarpine response. Atropine and 4-DAMP were equally potent inhibitors, showing a dose-related effect from 0.05 mg/kg. Pirenzepine only showed inhibitory effects with doses 10-times higher, whereas gallamine and hexamethonium did not induce inhibition at any of the doses tested. These findings suggest that the mouse eccrine sweat gland muscarinic receptors are predominantly M3.

Changes in mouse sudomotor function and sweat gland innervation with ageing.

Age-related changes in sudomotor neuroeffector function have been evaluated in mice aged 2 (young), 6, 12 (adult) and 18 (old) months. We evaluated sudomotor function by determining the number of sweat glands reactive to pilocarpine and the sweat output per gland on the plantar surface of the hindpaws with the impression mould technique. Protein gene product 9.5 (PGP) and vasoactive intestinal polypeptide (VIP) were immunohistochemically localised in footpads. A marked decrease (44%) in sweat output per gland was observed in old mice as well as a slight (17%), not significant decline in the number of secreting sweat glands. The sudomotor innervation, expressed as the area of sweat gland occupied by VIP and PGP immunoreactive nerve profiles, showed an initial increase from 2 to 6 months and a significant decline (35%) in 18- vs. 6-month-old mice. These results indicate that, in contrast to the number of secreting sweat glands, sweat output per gland does not reach the maximum in adult mouse until 6 months old and that sweating decreases in aged mice mainly due to a decline of sweat output per gland and to a lesser extent to a decrease in number of secreting glands. A reduction of sweat glands size in aged mice was also found, suggesting that the diminished sweat gland responsiveness with ageing may be attributed to sweat gland atrophy as well as to loss of innervation.

Sudomotor function and sweat gland innervation in galanin knockout mice

The presence of galanin and galanin binding sites in sweat gland has been demonstrated previously. In order to investigate whether galanin can influence sweat gland function, we compared sweating induced in footpads of wild type and galanin knockout mice by cholinergic and thermal stimulation using the silicone impression technique. Pilocarpine injections resulted in a similar number of reactive sweat glands and non-significant difference in the amount of sweat secretion in wild type and galanin knockout mice. However, thermal stimulation led to a significant increase in the number of secreting sweat glands in galanin knockout mice. To further evaluate possible differences in the innervation of sweat glands that could explain differences in their secretory activity, immunohistochemical labeling of cutaneous and sudomotor innervations against protein gene product 9.5, vasoactive intestinal polypeptide and choline acetyltransferase in plantar pads was performed. Immunohistochemical analysis revealed no significant differences in the distribution and intensity of the innervations between wild type mice and galanin knockout mice. Although our results indicate normal cholinergic responses and innervation of the sweat glands in galanin knockout mice, they also demonstrate that galanin plays a role in regulating the sudomotor activity in response to thermal stimulation.

Changes in cholinergic responses of sweat glands during denervation and reinnervation

Functional sudomotor responses have been studied in sweat glands reinnervated after sciatic nerve crush and partially denervated by cisplatin intoxication in the mouse. The sudomotor function mediated by the sciatic nerve was evaluated by silicone imprints on the plantar surface of the hindpaws. Five days after nerve crush, completely denervated sweat glands became unresponsive to cholinergic stimulation with pilocarpine. During the following weeks, the number of reinnervated, reactive sweat glands increased progressively to reach a maximum of 89% of preoperative control counts by 40 days after nerve crush. At this time, the mean volume of sweat secreted per gland was normal, but reinnervated glands showed a secretory activity abnormally sustained over time after pilocarpine stimulation and, on the other hand, had an increased resistance to the inhibition of secretion induced by atropine. The effects of cisplatin administration on sudomotor function were investigated in two groups of mice, one treated with high doses of cisplatin (10 mg/kg/week for 4 weeks) and another treated with low doses of cisplatin (5 mg/kg/week for 8 weeks). Cisplatin intoxication produced abnormal sudomotor responses indicative of denervation from cumulative doses of 10 mg/kg. The first abnormality found was a partial resistance of sweat glands to atropine, followed by a decrease in the sweat output per gland and finally a decline in the number of sweat glands activated by pilocarpine. These abnormalities in the sudomotor responses were more pronounced in mice treated with a high dose than in those with a lower dose regime.