Combination of pathological, biochemical and behavioral evaluations for peripheral neurotoxicity assessment in isoniazid-treated rats.

In drug development, assessment of non-clinical peripheral neurotoxicity is important to ensure human safety. Clarifying the pathological features and mechanisms of toxicity enables the management of safety risks in humans by estimating the degree of risk and proposing monitoring strategies. Published guidelines for peripheral neurotoxicity assessment do not provide detailed information on which endpoints should be monitored preferentially and how the results should be integrated and discussed. To identify an optimal assessment method for the characterization of peripheral neurotoxicity, we conducted pathological, biochemical (biomaterials contributing to mechanistic considerations and biomarkers), and behavioral evaluations of isoniazid-treated rats. We found a discrepancy between the days on which marked pathological changes were noted and those on which biochemical and behavioral changes were noted, suggesting the importance of combining these evaluations. Although pathological evaluation is essential for pathological characterization, the results of biochemical and behavioral assessments at the same time points as the pathological evaluation are also important for discussion. In this study, since the measurement of serum neurofilament light chain could detect changes earlier than pathological examination, it could be useful as a biomarker for peripheral neurotoxicity. Moreover, examination of semi-thin specimens and choline acetyltransferase immunostaining were useful for characterizing morphological neurotoxicity, and image analysis of semi-thin specimens enabled us to objectively show the pathological features.

Spontaneous ovarian choriocarcinoma in a young ICR mouse.

This paper describes the spontaneous ovarian choriocarcinoma observed in a young female Crl:CD1 (ICR) mouse. The mouse was sacrificed at 8 weeks of age after oral administration of a compound for 2 weeks. The left ovary was found to be cystically enlarged with dark red hemorrhaging. The cystic mass contained abundant blood plasma and erythrocytes. At the peripheral regions of the mass, large pleomorphic tumor cells with bizarre shaped nuclei were detected. Tumor cells contained a single large nucleus and abundant eosinophilic to amphophilic cytoplasm. Histopathology of the tumor cells resembled that of trophoblastic giant cells. Therefore, the observed ovarian lesion was diagnosed as a choriocarcinoma. No microscopic lesions were observed in the right ovary or other reproductive organs. Ovarian choriocarcinoma was considered to be of non-gestational origin. This is the first report of ovarian choriocarcinoma in a young ICR mouse.

Karnovsky's fixative prevents artifacts appearing as vacuolation derived from tissue processing in kidneys treated with antisense oligonucleotide.

Antisense oligonucleotide (ASO) therapies have been identified as a new treatment modality for intractable diseases. In kidneys treated with ASOs, vacuoles, in addition to basophilic granules, are often observed in the proximal tubules. Some reports have described that these vacuoles are likely to be a secondary phenomenon resulting from the extraction of ASOs during tissue processing. In this study, we compared renal morphology after fixation with Karnovsky's fixative or 4% paraformaldehyde phosphate buffer (PFA) with that of 10% neutral-buffered formaldehyde solution (NBF). Female Sprague-Dawley rats, intravenously treated four times with 50 mg/kg locked nucleic acid containing antisense oligonucleotides (LNA-ASOs) for 1 or 2 weeks, were examined. Microscopically, vacuoles and basophilic granules in the proximal tubules were observed in the kidneys fixed with NBF. Basophilic granules are indicative of the accumulation of ASOs. Moreover, some of the vacuoles also contained faint basophilic granules, suggesting that the vacuoles were relevant to the accumulation of ASOs. Although moderate vacuolation was observed in the proximal tubules, the majority of the vacuolated epithelia were negative for kidney injury molecule-1 on immunohistochemical staining. Vacuoles in the proximal tubules were not observed in samples subjected to Karnovsky's fixation, although basophilic granules were observed. In samples subjected to PFA fixation, vacuoles and basophilic granules were observed in the proximal tubules, similar to those in samples subjected to NBF fixation. Overall, our findings demonstrated the possibility of overestimation of vacuolation due to artifacts during tissue processing when using conventional NBF fixation. Karnovsky's fixative is considered a useful alternative for distinguishing artificial vacuoles from true nephrotoxicity.

Spontaneous hyaline glomerulopathy in a young Slc:ICR mouse.

Hyaline glomerulopathy is a type of glomerular lesion that occurs in aging mice. Spontaneous hyaline glomerulopathy is rare in young mice. Here, we report spontaneous hyaline glomerulopathy in a young adult (15-week-old) ICR mouse. Necropsy revealed discoloration and roughness of the kidney surface. Microscopically, diffuse glomerular lesions were prominent. Amorphous, eosinophilic materials were deposited globally in the glomeruli. The mesangial region was expanded; however, the mesangial cells showed no proliferation. Thickening of the Bowman's capsule with proliferation of parietal epithelial cells was observed. Glomerular deposits were strongly positive for anti-IgM, anti-IgG, and periodic acid-Schiff stain and were stained red by Masson's trichrome stain. The deposits were negative for anti C3 and stained negatively with Congo red stain. Periodic acid methenamine silver and electron microscopy revealed glomerular deposits limited to intraglomerular capillaries. Based on the histological features, we diagnosed this lesion as hyaline glomerulopathy. This case could improve our understanding of spontaneous lesions in toxicological and pharmacological studies.

Quantification of histopathological findings using a novel image analysis platform.

Digital pathology, including image analysis and automatic diagnosis of pathological tissue, has been developed remarkably. HALO is an image analysis platform specialized for the study of pathological tissues, which enables tissue segmentation by using artificial intelligence. In this study, we used HALO to quantify various histopathological changes and findings that were difficult to analyze using conventional image processing software. Using the tissue classifier module, the morphological features of degeneration/necrosis of the hepatocytes and muscle fibers, bile duct in the liver, basophilic tubules and hyaline casts in the kidney, cortex in the thymus, and red pulp, white pulp, and marginal zone in the spleen were learned and separated, and areas of interest were quantified. Furthermore, using the cytonuclear module and vacuole module in combination with the tissue classifier module, the number of erythroblasts in the red pulp of the spleen and each area of acinar cells in the parotid gland were quantified. The results of quantitative analysis were correlated with the histopathological grades evaluated by pathologists. By using artificial intelligence and other functions of HALO, we recognized morphological features, analyzed histopathological changes, and quantified the histopathological grades of various findings. The analysis of histopathological changes using HALO is expected to support pathology evaluations.

Natriuretic peptide signaling is involved in the expression of oxidative metabolism-related and muscle fiber constitutive genes in the gastrocnemius muscle.

Natriuretic peptides regulate cyclic guanosine monophosphate (cGMP) levels via their receptors and have various physiological effects. Natriuretic peptide receptor C (NPR-C) increases cGMP signaling by functioning as a clearance receptor. We analyzed the role of natriuretic peptides in the skeletal muscle, which increases in mass with bone elongation, of NPR-C- mice. High-fat diet (HFD)-fed NPR-C- mice exhibited obesity resistance and higher oxygen consumption. PGC1α gene expression was upregulated in the gastrocnemius muscle of HFD-fed NPR-C- mice compared with HFD-fed NPR-C+ (wild-type) mice. Gene expression of proliferator-activated receptor delta and estrogen-related receptor α, which upregulate oxidative metabolism, was increased in the gastrocnemius muscle of NPR-C- mice, irrespective of diet. Expression of myosin heavy chain 7, a component of type I slow-twitch fiber, was enhanced. Natriuretic peptide signaling may influence oxidative metabolism-related and slow-twitch fiber constitutive gene expression in the fast-twitch gastrocnemius muscle but not in slow-twitch muscles such as the soleus.