Protocol to image and analyze the morphology of mouse peripheral nerves using transmission electron microscopy.

Morphological analysis of peripheral nerves in mouse models can be used to characterize the pathophysiology of peripheral nerve disease, but obtaining high-quality electron micrographs can be challenging. Here, we present a protocol to obtain electron micrographs of mouse peripheral nerves. We detail the procedures of sampling, fixation, and embedding of peripheral nerves. We then outline the steps for ultrathin sectioning and transmission electron microscopy imaging. Finally, we describe morphological evaluation of nerve fibers in these images using ImageJ and AxonSeg. For complete details on the use and execution of this protocol, please refer to Nakai-Shimoda et al. (2021).

Deficiency of glucagon gene-derived peptides induces peripheral polyneuropathy in mice.

Although diabetic polyneuropathy (DPN) is the commonest diabetic complication, its pathology remains to be clarified. As previous papers have suggested the neuroprotective effects of glucagon-like peptide-1 in DPN, the current study investigated the physiological indispensability of glucagon gene-derived peptides (GCGDPs) including glucagon-like peptide-1 in the peripheral nervous system (PNS). Neurological functions and neuropathological changes of GCGDP deficient (gcg-/-) mice were examined. The gcg-/- mice showed tactile allodynia and thermal hyperalgesia at 12-18 weeks old, followed by tactile and thermal hypoalgesia at 36 weeks old. Nerve conduction studies revealed a decrease in sensory nerve conduction velocity at 36 weeks old. Pathological findings showed a decrease in intraepidermal nerve fiber densities. Electron microscopy revealed a decrease in circularity and an increase in g-ratio of myelinated fibers and a decrease of unmyelinated fibers in the sural nerves of the gcg-/- mice. Effects of glucagon on neurite outgrowth were examined using an ex vivo culture of dorsal root ganglia. A supraphysiological concentration of glucagon promoted neurite outgrowth. In conclusion, the mice with deficiency of GCGDPs developed peripheral neuropathy with age. Furthermore, glucagon might have neuroprotective effects on the PNS of mice. GCGDPs might be involved in the pathology of DPN.

Investigation of the ototoxicity of gadoteridol (ProHance) and gadodiamide (Omniscan) in mice.

CONCLUSION: In the mouse, when a tympanic perforation is present, gadoteridol does not seem to cause ototoxicity. Gadodiamide may cause mild ototoxicity other than toxicity to the outer hair cells of the cochlea. OBJECTIVES: Endolymphatic hydrops have been visualized through intra-tympanic injection of gadolinium-based contrast agents (GBCAs) and three-dimensional fluid-attenuated inversion recovery (3-D FLAIR) magnetic resonance imaging. However, reports on the safety of GBCAs are limited. This study aimed to assess ototoxicity of gadoteridol and gadodiamide. METHOD: In a prospective, randomized, controlled trial, myringotomies in the left ear were performed in 20 male C57 BL/6 mice. After testing the baseline auditory brainstem response (ABR) (range = 8-32 kHz), the test solution (gadoteridol, gadodiamide, saline, or cisplatin) was injected into the left ear. ABR testing was repeated 14 days after test solution application. In morphological experiments, images of post-mortem surface preparations were assessed for cochlear hair cell status. RESULTS: At 14 days following gadoteridol application, there was no significant change in ABR thresholds at 8, 16, or 32 kHz. Gadodiamide application caused a significant change in the ABR threshold at 8 kHz. Apparent cochlear hair cell loss was not observed in the surface preparation after gadoteridol or gadodiamide application.

Membranous nephropathy and pulmonary alveolar proteinosis.

A 47-year-old woman with a severe cough and high-grade fever demonstrated proteinuria of 3.2 g/day. Chest radiograph and CT scan revealed scattered small nodules and ground-glass opacities with interlobular septal thickening in both lungs. The serum levels of surfactant A, surfactant D, and KL-6 were increased to 190 ng/ml (normal: 0-43.8), 360 ng/ml (normal: 0-110), and 4850 U/ml (normal: 0-500), respectively. Video-assisted thoracoscopic lung biopsy revealed eosinophilic amorphous material within alveoli and thickened alveolar septa, which is compatible with pulmonary alveolar proteinosis. Kidney biopsy exhibited membranous nephropathy (Stage I-II) accompanied by granular IgG deposition along the glomerular basement membrane. Although the patient refused treatment with granulocyte macrophage colony stimulating factor (GM-CSF) for pulmonary alveolar proteinosis, her proteinuria and the pulmonary lesion gradually diminished and disappeared after one year.

Apoptosis induced by culturing MH134 cells in the presence of porcine skin gelatin in vitro.

Porcine skin (PS) gelatin showed antitumor effect in vitro on MH134 murine hepatoma cells. We analyzed the effect of PS gelatin on MH134 cells compared to the effect of Bovine bone (BB) gelatin, 5-Fluorouracil (5-FU) and 7-ethyl-10-hydroxycamptothecin (SN-38). We previously suggested that PS gelatin induces apoptosis in MH134 cells using flow cytometric analysis. We performed agarose gel electrophoresis and electron microscopic studies to ascertain apoptosis. Agarose gel electrophoresis showed the typical DNA ladder pattern and electron microscopic findings revealed characteristic features in the case of apoptosis on PS gelatin. SN-38 also showed DNA ladder pattern and ultrastructural changes in apoptosis. 5-FU didn't show DNA ladder pattern but electron microscope revealed changes in necrosis. On the other hand, BB gelatin didn't induce apoptosis or necrosis.