An 8-year-old male ferret was presented with dyspnea and large, palpable cervical masses. Pathological examination revealed multiple exophytic nodular masses arising from the atlas and axis. Those vertebrae were almost completely replaced by neoplastic tissue. Histologically, the masses were composed of physaliferous cells with foamy cytoplasm embedded within a prominent chondro-osseous matrix. The diagnosis of chordoma was confirmed by immunohistochemistry, as tumor cells were positive for pan-cytokeratin, vimentin, S-100, and neuron-specific enolase. Notably, some chondrocytes and osteocytes adjacent to the tumor cells also expressed these markers, suggesting a neoplastic origin rather than a reactive process. Ki-67 labeling was confined to a subset of cytokeratin-positive cells at the periphery of the nodules, indicating that peripheral proliferation contributes to the exophytic growth pattern. This report describes a case of cervical chordoma with a unique exophytic presentation in a ferret.
Albino rats lack melanin production due to a defect in the tyrosinase gene. Melanomas arising in albino rats, which originate from neural crest cells, are non-melanin-producing and are referred to as amelanotic melanomas (AM). AM in albino rats represents one of the most challenging tumors to diagnose pathologically using light microscopy, primarily because of the absence of melanin pigment. Although electron microscopy remains the gold standard for AM diagnosis, PNL2 serves as a practical alternative, particularly in toxicologic studies. To date, only eight reports have definitively diagnosed AM in albino rats by confirming the presence of premelanosomes through ultrastructural analysis. This review aims to provide toxicologic pathologists with information to aid in diagnosing melanomas by analyzing eight previous reports along with data from our own case studies.
Epithelial components of the rectum and anal canal comprise the rectal mucosa, anal canal mucosa, and anal glands. The clinicopathological characteristics of 111 cases of canine epithelial tumors in these regions were reviewed. Histopathological examination was performed on normal rectal and anal canal tissues and on 64 tumors. All signet-ring cell carcinomas and anal gland adenomas, as well as approximately half of the adenomas, were classified as anal canal tumors and were located within 1.5 cm of the anus. Acinar adenocarcinomas, papillary adenocarcinomas, and mucinous adenocarcinomas were predominantly classified as rectal tumors. Immunohistochemically, rectal adenomas, acinar adenocarcinomas, papillary adenocarcinomas, and mucinous adenocarcinomas frequently exhibited a CDX2+CK20+SOX2-CK7- immunophenotype, consistent with that of the proximal rectal mucosa. In contrast, adenomas in the anal canal frequently showed a CDX2-CK20-SOX2+CK7± immunophenotype, consistent with that of the rectal mucosa at the recto-anal junction. Signet-ring cell carcinomas and anal gland adenomas exhibited a CDX2-CK20-SOX2+CK7+ immunophenotype, consistent with that of the anal glands. In a hierarchical cluster analysis of tumor immunophenotypes, Group 1 (mostly anal canal adenomas) and Group 2 (rectal adenomas, acinar adenocarcinomas, papillary adenocarcinomas, and mucinous adenocarcinomas) formed one cluster, whereas Group 3 (signet-ring cell carcinomas) and Group 4 (anal gland adenomas) formed another distinct cluster. Based on these results, canine epithelial tumors in the rectum and anal canal may be categorized into a rectal mucosa-like immunophenotype (including adenomas, acinar adenocarcinomas, papillary adenocarcinomas, and mucinous adenocarcinomas) and anal gland-like immunophenotype (including signet-ring cell carcinomas and anal gland adenomas).
Acute toxicity and 14-day repeated-dose toxicity studies were performed via oral gavage to elucidate the effects of oral exposure to carbon tetrachloride (CCl4) on the liver of zebrafish. In the acute toxicity studies, the lethal dose 50% (LD50) was 386 μL/kg (614 mg/kg, based on density conversion) for both males and females when using a 1% Tween aqueous solution vehicle, and 5,045 μL/kg (8,036 mg/kg) for males and 6,419 μL/kg (10,206 mg/kg) for females when using a corn oil vehicle (cf., rats: 10,054 mg/kg; mice: 13,000 mg/kg, according to known LD50 values). The doses in the repeated toxicity study were set at 0, 200, and 300 μL/kg using a 1% Tween aqueous solution as the vehicle. The survival rate was 87% on Day 7 and 50% on Day 14 in the 200 μL/kg CCl4-treated group, while it was 10% on Day 7 in the 300 μL/kg CCl4-treated group. Histopathological findings, including focal bile duct proliferation, focal macrophage aggregation, and focal fibrosis, were detected in both acute and repeated-dose toxicity studies. However, no significant differences were observed in the incidence of these lesions between the control and CCl4-treated groups. Therefore, the present study demonstrates that the acute lethal dose of CCl4 administered via oral gavage in zebrafish is nearly equivalent to that observed in rodents. However, zebrafish exhibit markedly low sensitivity to CCl4-induced liver injury in a species-specific manner.
Tribbles pseudokinase 3 (Trib3) is an inactive protein kinase whose expression increases in response to various stresses. Our previous work showed that Trib3 may play a role in myelin destruction induced by oxidative and endoplasmic reticulum stress in demyelination (dmy) rats. The dmy rat exhibits hind limb ataxia and severe myelin breakdown in the central nervous system. To elucidate how Trib3 contributes to oxidative stress-mediated injury in organs other than the central nervous system, we used two models: an acute liver injury model induced by thioacetamide injection and an acute kidney injury model induced by cisplatin injection. Trib3 mRNA expression increased concurrently with tissue injury and declined during the repair phase. TRIB3 was detected in damaged areas, mainly in degenerated cells and infiltrating macrophages. These results suggest that Trib3 is upregulated in tissues damaged by oxidative stress and may serve as an indicator of tissue injury.
Tumor clonality is determined by somatic mutations in genes that regulate cell proliferation, and this can be either monoclonal or multiclonal. Assays based on X-chromosome inactivation that exploit the random inactivation of one of the two X chromosomes in female embryos have been used to evaluate tumor clonality. However, these methods require technically complex procedures and are not easily applicable to various types of tumors. Here, we visualized the clonality of tumors induced by chemical substances in vivo using X-linked LacZ heterozygous transgenic female mice that displayed a blue or white mosaic pattern of tissue on X-gal staining. In a model of colorectal tumors induced by 1, 2-dimethylhydrazine dihydrochloride and dextran sulfate sodium salt, 18 blue, 20 unstained (white), and seven mixed-colored tumors in intestinal tissues from 20 mice were observed after X-gal staining. Similarly, in a model of diethylnitrosamine-induced liver tumors, multiple blue or white nodules were observed. These findings demonstrated that this is a simple and effective method for visualizing tumor clonality in vivo. This approach may be readily applicable to models of chemically induced carcinogenesis and useful for evaluating the clonality of multifocal lesions.
In aged F344/DuCrlCrlj rats, we observed that all animals with group atrophy of the biceps femoris muscle also had islet cell tumors, suggesting that spontaneous islet cell tumors may induce peripheral neuropathy and muscle atrophy. Among 220 aged male F344/DuCrlCrlj rats examined, 12.3% (27/220) had islet cell tumors, and of these, 22.2% (6/27) had neurogenic muscular atrophy. Sciatic nerve degeneration was observed in 3.2% (7/220) of cases, and all animals with neurogenic muscular atrophy had sciatic nerve degeneration. Notably, no neurogenic muscular atrophy was observed in rats without islet cell tumors. In contrast, rats with neurogenic muscular atrophy tended to have larger islet cell tumors. Although spinal nerve root degeneration was prevalent (90.8%, 198/218), two of the six rats with neurogenic muscular atrophy did not exhibit this pathology. Immunohistochemically, insulin was positive in all islet cell tumors, although glucagon- and somatostatin-positive reactions showed no association with neurogenic muscular atrophy. Experimentally induced hyperinsulinemia in rats is a known cause of neurogenic muscular atrophy, and similar associations have been reported in humans and spontaneous cases of pet rats with islet cell tumors. A complete coincidence between the occurrence of neurogenic muscular atrophy and islet cell tumors in our investigation suggests that some islet cell tumors in F344/DuCrlCrlj rats may be functionally active, and that hyperinsulinemia may contribute to the pathogenesis of neurogenic muscular atrophy.
A 10-year-old male toy poodle presented with hypoglycemia. An insulinoma was suspected and a surgical excision of two pancreatic masses was performed. White-gray, demarcated, soft masses were identified in the pancreas. Histopathologically, two types of growth patterns were observed in the same neoplasm: nest and glandular. To investigate cellular differentiation, we performed immunohistochemical and transmission electron microscopy analyses. Both types of neoplastic cells were immunopositive for INSM1, Nkx2.2 and insulin. However, the neoplastic cells exhibiting the nest pattern contained exocrine granules, whereas those with the glandular pattern were immunopositive for cytokeratin. Both types of neoplastic cells showed not only neuroendocrine but also exocrine differentiation in the same neoplastic cell. To the best of our knowledge, this is the first report describing the morphology and immunophenotype of the canine insulinoma with amphicrine differentiation: showing both neuroendocrine and exocrine features.
This study focused on the histological characterization of age-related intramuscular collagen accumulation in different skeletal muscle fiber types, specifically fast- and slow-twitch fibers, in young and middle-aged male rats, in relation to the number of nuclei between muscle fibers. The extensor digitorum longus (EDL) and soleus (SOL) muscles from male Sprague-Dawley (SD) rats were collected and sectioned. Hematoxylin and eosin staining was performed for histological examination, while Picrosirius Red and hematoxylin staining were used for morphometric analyses. The SOL, a slow-twitch dominant muscle, tended to have a more distinct and thicker interstitium, as well as more collagen fibers and nuclei between muscle fibers, than the EDL, a fast-twitch dominant muscle. The degree of collagen accumulation between muscle fibers was positively correlated with the number of nuclei. Intramuscular collagen fibers increased with age in both the EDL and SOL, particularly in the latter. The number of nuclei remained unchanged with age. These results suggest that the increase in intramuscular collagen fibers with age is due to increased collagen production by existing fibroblasts rather than fibroblast proliferation. Given that middle-aged male SD rats fed ad libitum were obese, their slow-twitch muscles may have become susceptible to sarcopenic obesity accompanied by intramuscular collagen accumulation.
Drug-induced liver injury is a major reason for the discontinuation of drug development. Autophagy is a self-digestive process in the cell and can suppress cell death by removing damaged organelle from the cell. It is known that autophagy can modify drug-induced liver injury; however, details of the effects of autophagy modulation on chemically-induced hepatotoxicity are unclear. In this study, we investigated the influence of autophagy induction by rapamycin or inhibition by chloroquine on carbon tetrachloride (CCl4)- or allyl alcohol (AA)-induced acute liver injury. Ten- to eleven-week-old male F344 rats were administrated with CCl4 or AA after pretreatment by rapamycin or chloroquine, and were sampled 18 hours after the hepatotoxicant administration. Hepatic expression of the autophagosomal membrane protein LC3-II was significantly suppressed after CCl4 administration by rapamycin pretreatment, compared with that in vehicle (DMSO) pretreatment. Expression of autophagy cargo protein p62, were significantly decreased after rapamycin treatment with AA administration. Hepatic p62 expression increased by chloroquine pretreatment. Serum AST and ALT were decreased after CCl4 exposure in both rapamycin- and chloroquine-pretreated rats. On the other hand, regardless of pretreatment, pathological changes were mild in rats with AA exposure. These results showed that pretreatment with rapamycin or chloroquine can attenuate CCl4-induced acute liver injury in rats.
Hepatic iron overload is a common complication of human chronic liver diseases, including liver cirrhosis; however, the underlying mechanisms remain unclear. In the present study, we investigated the temporal changes in iron metabolism and the expression of iron-regulatory molecules during thioacetamide-induced liver cirrhosis in rats. Histopathological and biochemical analyses revealed that iron overload develops concurrently with the suppression of hepcidin expression in advanced cirrhosis. Hepatic expression of genes involved in cellular iron intake, storage, and export increased persistently in cirrhotic livers. The IL6-STAT3 and BMP6-SMAD pathways, which are the major intracellular mechanisms that induce hepcidin transcription, were inactivated in advanced cirrhosis. Furthermore, microRNA-135b-5p (miR-135b-5p), which targets JAK2 and SMAD5, key molecules of the IL6-STAT3 and BMP6-SMAD pathways, respectively, was highly upregulated in parallel with the progression of cirrhosis. These results indicate that inactivation of multiple hepcidin pathways, possibly mediated by miR-135b-5p upregulation, is responsible for hepatic iron overload in advanced cirrhosis. Our findings provide new insights into the mechanisms underlying iron dysregulation in liver cirrhosis.
This study investigates the protective effects of roselle aqueous extract (RAE) against aristolochic acid (AA)-induced developmental nephrotoxicity in zebrafish embryos. Fluorescence imaging, immunohistochemistry, and real-time polymerase chain reaction (PCR) experiments were conducted. The results showed that pre-treatment with RAE significantly improved survival rates and reduced kidney malformations in AA-exposed zebrafish. Specifically, survival rates in the AA-treated group decreased to 81.11% by 72 hours post-fertilisation, whereas those in the AA+RAE (30 and 60 ppm) groups increased to 86.67-90%. Additionally, RAE pre-treatment reduced kidney malformation rates from 98.89% in the AA group to 75.56% and 63.33% in the AA+RAE (30 and 60 ppm) groups, respectively. Histological analysis revealed that RAE mitigated structural damage in the pronephric tubes, restoring their compact architecture. Molecular analysis using real-time reverse transcription (RT)-PCR further indicated that RAE reduced the expression of inflammatory markers, suggesting that its protective effects are mediated through the attenuation of AA-induced inflammation. These findings demonstrate that RAE exerts a protective role against AA-induced developmental kidney malformations in zebrafish, likely through its antioxidant and anti-inflammatory properties.
In March 2024, a health hazard associated with the consumption of food products containing red yeast rice (beni-koji), which could lead to renal dysfunction, was reported in Japan. Puberulic acid (PA) was identified as an unintentional contaminant in these products; however, information on PA toxicity remains limited. The toxicological profile of PA was evaluated in a 28-day subacute toxicity study in rats. Synthesized PA was administrated by gavage to 6-week-old Crl:CD(SD) rats at 0, 1, 3, or 10 mg/kg/day (male) or 0, 0.3, 1, or 3 mg/kg/day (female) over 28 days, and satellite groups were used to evaluate the reversibility over a 14-day period. Male rats in the 10 mg/kg group exhibited increased urinary glucose and serum creatinine levels compared to controls. Histopathological examination revealed vacuolation, necrosis, and regeneration of proximal tubules in kidneys of all rats in the male 10 mg/kg and female 3 mg/kg groups. After the 14-day recovery period, focal interstitial fibrosis was observed in one male rats from the high-dose group, whereas no renal lesions were detected in the remaining rats of either sex. These results suggest that PA-induced nephrotoxicity is largely reversible under the conditions studied, although residual chronic lesions may occur in severe cases. Apoptosis/necrosis and diffuse hyperplasia of the glandular stomach mucosa were observed in male 3 and 10 mg/kg and female 3 mg/kg groups but were absent after the recovery period. These results indicate that, under the study conditions, the no-observed-adverse-effect level for PA was 1 mg/kg/day for both sexes.
This study investigated the effects on ovarian development in adult rats irradiated with γ-rays at fetal, neonatal, weaning, and early sexual maturation. Female Fischer-344 (F344) rats mated with male rats and their F1 offspring were irradiated with a single dose of 0.5 or 2 Gy of γ-rays on gestation day 15 or 19 (GD15 or 19), or postnatal day 5, 20, or 49 (PND5, 20, or 49). F1 females were reared until 27 weeks of age and necropsied. HE-stained specimens of the reproductive organs were prepared for histological examination (n=10-22 per group). The corpus luteum and follicle numbers were also counted in all ovaries. In addition, PCNA-stained specimens were used to count the primordial follicles. At 2 Gy, corpora lutea and follicle depletion was observed in the GD15, PND5, and PND20 irradiation groups. Instead of lost follicles consisting of granulosa cells, numerous tubular structures composed of Sertoli-like cells similar to those found in the testes were noted. In the GD19 group, the ovaries showed less sensitivity to γ-rays. In the PND49 irradiation group, the number of corpora lutea was normal; however, the number of follicles, including primordial follicles, decreased. At 0.5 Gy, the ovaries appeared histologically normal in all the groups; however, the number of follicles decreased in the GD15 and PND5 irradiation groups. In conclusion, we found that the timing of γ-ray irradiation significantly affected subsequent ovarian development, and the degree of change depended on the γ-ray dose.
Canine generalized ceroid lipofuscinosis (GCL) is a rare disease characterized by the deposition of lipofuscin in systemic organs and tissues. In this case report, we encountered a dog with GCL and performed a detailed histopathological examination. A 7-year-old male beagle was euthanized due to progressive weight loss and loose or bloody stools, without any neurological symptoms. Histopathologically, deposition of lipofuscin was observed in the parenchymal cells of systemic organs, particularly in the pancreatic acini and intestinal smooth muscle, accompanied by interstitial infiltration of macrophages. No neuronal loss was observed in the central nervous system, despite such findings and neurological symptoms being commonly associated with GCL. However, some lipofuscin deposition was evident in systemic organs, so the present case was diagnosed as GCL characterized by predominant deposition in the pancreatic acini and intestinal smooth muscle. This detailed description of the morphological features may contribute to a deeper understanding of lipofuscinosis.
Ectopic intestinal cysts are extremely rare in the rat liver. Here, we report a case of a spontaneous ectopic intestinal cyst in the liver of an 8-week-old male Crl:CD (SD) rat. Necropsy revealed a solitary white, firm nodule, approximately 3 mm in diameter on the diaphragmatic surface near the porta hepatis of the medial lobe of the liver. Histologically, the lesion exhibited a cystic structure lined with tissue resembling intestinal mucosa, located on the liver capsule. Periodic acid-Schiff and Alcian blue (pH 1.0) staining-positive mucous cells, similar to goblet cells, and Paneth cell-like cells containing eosinophilic granules were observed in the mucosal epithelium. Immunohistochemically, the mucosal epithelium demonstrated low proliferative activity, as confirmed by Ki-67 staining. The thin outer layer of the mucosa was positive for alpha-smooth muscle actin, suggesting the presence of the lamina muscularis or a poorly developed muscular layer. Based on the lesion's location and histological features, this case was diagnosed as an ectopic intestinal cyst, likely resulting from persistence of the vitelline duct. To the best of our knowledge, there are no previous reports of ectopic intestinal cysts in the rat liver that include such detailed histochemical and immunohistochemical findings. This report provides valuable insights into congenital lesions of the rat hepatobiliary system.
In recent years, the development of Artificial Intelligence (AI) technology has led to the introduction and use of AI-based histopathological evaluation (AI pathology) by various companies and organizations. The AI Pathology Task Force of the Non-clinical Evaluation Expert Committee within the Drug Evaluation Committee of the Japan Pharmaceutical Manufacturers Association (JPMA) recognizes the importance of understanding the current use and needs surrounding AI pathology in Japan. This includes its role in non-clinical research fields, such as toxicity evaluation, drug efficacy evaluation, and basic research. In addition, assessing needs and challenges related to pathology image databases is essential. Between October and November 2023, with the cooperation of the Japanese Society of Toxicologic Pathology (JSTP), we conducted a questionnaire survey on non-clinical pathology image databases to explore these issues among JPMA-affiliated and JSTP-affiliated organizations. The questionnaire survey consisted of three items: (1) implementation and utilization of whole slide images, (2) use of AI pathology in non-clinical research fields, and (3) needs and feasibility of establishing a precompetitive pathology image database (repository) and AI pathology in the non-clinical pathology field. This report summarizes the survey results and serves as a foundation for guiding future directions in the use of AI pathology in non-clinical studies in Japan.
The use of artificial intelligence (AI) in non-clinical pathology is rapidly expanding. In this study, we conducted a literature survey of articles published after 2017 that used AI to analyze the histopathological images of experimental animals. We identified 44 articles that used AI for various purposes, including the detection of abnormal sites, determination and quantification of normal tissues, and classification of normal/abnormal images. AI systems or applications were either custom-built, commercially available, or a combination of both. Rats and mice were mainly used, and the liver was the most frequently analyzed organ. Our findings suggest that AI can be useful in non-clinical pathology and that collaboration between pharmaceutical companies or cooperation with IT experts can be a potential approach to further advance the utilization of AI in this field.
Lymphoblastic lymphoma (LBL) is an aggressive neoplasm characterized by the proliferation of undifferentiated lymphocytes. It primarily spreads to immune organs such as the thymus, spleen, lymph nodes, bone marrow, and liver. Although well-documented in humans, spontaneous LBL cases in laboratory animals are exceedingly rare. This study reports a case of T cell-derived LBL in a young adult ICR mouse, notably without bone marrow metastasis. This case provides valuable insights into the spontaneous occurrence of LBL in laboratory rodents by contributing to comparative oncology and preclinical research.
