Corneal and Epidermal Nerve Quantification in Chemotherapy Induced Peripheral Neuropathy.

Chemotherapy-induced neurotoxicity is an increasingly recognized clinical issue in oncology. in vivo confocal microscopy (IVCM) of corneal nerves has been successfully used to diagnose peripheral neuropathies, including diabetic neuropathy. The purpose of this study was to test if the combination of corneal nerve density and morphology assessed by IVCM is useful to monitor the neurotoxic effects of chemotherapy compared to epidermal nerve quantification. Overall, 95 adult patients with different cancer types were recruited from the oncology and hematology departments of the San Raffaele Hospital. Neurological examination, including clinical Total Neuropathy Score, and in vivo corneal confocal microscopy (IVCM), were performed before and after chemotherapy. In a group of 14 patients, skin biopsy was performed at the first and last visit. In the group of 14 patients who underwent both skin biopsy and corneal nerve imaging, clinical worsening (+69%, p = 0.0018) was paralleled by corneal nerve fiber (CNF) density reduction (-22%, p = 0.0457). Clinical Total neuropathy score significantly worsened from the first to the last visit (+62%, p < 0.0001). CNF length was not significantly reduced overall. However, CNF density/tortuosity ratio significantly decreased after therapy. Correlation analysis showed that the CNF density/tortuosity ratio was also correlated with the number of chemotherapy cycles (r = -0.04790, P = 0.0009). Our data confirm that in vivo corneal confocal microscopy is a helpful, non-invasive tool which shows promise for the diagnosis of chemotherapy-induced peripheral neuropathies. IVCM could allow a rapid, reproducible and non-invasive quantification of peripheral nerve pathology in chemotherapy-associated neuropathy.

Laser capture microdissection for transcriptomic profiles in human skin biopsies.

BACKGROUND: The acquisition of reliable tissue-specific RNA sequencing data from human skin biopsy represents a major advance in research. However, the complexity of the process of isolation of specific layers from fresh-frozen human specimen by laser capture microdissection, the abundant presence of skin nucleases and RNA instability remain relevant methodological challenges. We developed and optimized a protocol to extract RNA from layers of human skin biopsies and to provide satisfactory quality and amount of mRNA sequencing data. RESULTS: The protocol includes steps of collection, embedding, freezing, histological coloration and relative optimization to preserve RNA extracted from specific components of fresh-frozen human skin biopsy of 14 subjects. Optimization of the protocol includes a preservation step in RNALater® Solution, the control of specimen temperature, the use of RNase Inhibitors and the time reduction of the staining procedure. The quality of extracted RNA was measured using the percentage of fragments longer than 200 nucleotides (DV200), a more suitable measurement for successful library preparation than the RNA Integrity Number (RIN). RNA was then enriched using the TruSeq® RNA Access Library Prep Kit (Illumina®) and sequenced on HiSeq® 2500 platform (Illumina®). Quality control on RNA sequencing data was adequate to get reliable data for downstream analysis. CONCLUSIONS: The described implemented and optimized protocol can be used for generating transcriptomics data on skin tissues, and it is potentially applicable to other tissues. It can be extended to multicenter studies, due to the introduction of an initial step of preservation of the specimen that allowed the shipment of biological samples.

The brachial plexus branches to the pectoral muscles in adult rats: morphological aspects and morphometric normative data.

Animal models provide an important tool to investigate the pathogenesis of neuromuscular disorders. In the present study, we analyze fiber composition of the brachial plexus branches to the pectoral muscles: the medial anterior thoracic nerve (MATN) and the lateral anterior thoracic nerve (LATN). The morphological and morphometric characteristics and the percentage of motor fibers within each nerve are here reported, adding information to microscopic anatomy knowledge of the rat brachial plexus. As control, we employed the quadriceps nerve, commonly used for the evaluation of motor fibers at hindlimbs. We demonstrated that the MATN and the LATN are predominantly composed of large motor fibers and therefore could be employed to evaluate the peripheral nervous system (PNS) involvement at forelimbs in neurological diseases models, predominantly affecting the motor fiber compartment.

Foot pad skin biopsy in mouse models of hereditary neuropathy.

Numerous transgenic and knockout mouse models of human hereditary neuropathies have become available over the past decade. We describe a simple, reproducible, and safe biopsy of mouse skin for histopathological evaluation of the peripheral nervous system (PNS) in models of hereditary neuropathies. We compared the diagnostic outcome between sciatic nerve and dermal nerves found in skin biopsy (SB) from the hind foot. A total of five animal models of different Charcot-Marie-Tooth neuropathies, and one model of congenital muscular dystrophy associated neuropathy were examined. In wild type mice, dermal nerve fibers were readily identified by immunohistochemistry, light, and electron microscopy and they appeared similar to myelinated fibers in sciatic nerve. In mutant mice, SB manifested myelin abnormalities similar to those observed in sciatic nerves, including hypomyelination, onion bulbs, myelin outfolding, redundant loops, and tomacula. In many strains, however, SB showed additional abnormalities--fiber loss, dense neurofilament packing with lower phosphorylation status, and axonal degeneration-undetected in sciatic nerve, possibly because SB samples distal nerves. SB, a reliable technique to investigate peripheral neuropathies in human beings, is also useful to investigate animal models of hereditary neuropathies. Our data indicate that SB may reveal distal axonal pathology in mouse models and permits sequential follow-up of the neuropathy in an individual mouse, thereby reducing the number of mice necessary to document pathology of the PNS.