In this case report, the role of a myocardial bridge of the left anterior descending artery (LAD) and recent use of cannabis in a sudden death of a drug user is discussed, also considering the relevance of histopathological pulmonary findings. A 37-year-old man with a history of drug abuse was found dead in his house. External and autoptic examination were performed, as well as histologic and toxicologic analyses of tissues, organs and body fluids samples. Autoptic examination revealed signs of previous endovenous drugs administration and a 0.5 cm deep intramyocardial bridging of a 1.5 cm long segment of the left anterior descending (LAD) artery. Histo-pathologic examination revealed the presence of diffuse alveolar hemorrhage and multiple alveolar macrophages containing brownish pigments. Toxicological analysis of post-mortem blood revealed recent use of ketamine (<0.5 ng/ml) and cannabis derivatives (8.8 ng/ml for THC and 2.2 ng/ml for 11-OH-THC) in a subject with a history of previous abuse of cocaine, MDMA, and ketamine, confirmed through hair analysis. The analysis of forensic literature, thoroughly examined in this paper, along with a comprehensive evaluation of all post-mortem data, indicates that cannabis likely contributed to the cause of death, in conjunction with myocardial bridging. This suggests a dual mechanism involving both cardiac and pulmonary factors. This case could add to the limited body of evidence in the literature and provide insights into the potential contributory role of cannabis in sudden deaths.
One of the emerging concepts on the reduction of animal use in non-clinical studies is the use of virtual control group (VCG) to replace concurrent control group (CCG). The VCG involves the generation of a control data from historical control data to match a specific study design. This review focuses on two recently published proof-of-concept (POC) studies conducted in rats. One major issue that was consistently seen across these POC studies was the non-reproducibility of some quantitative endpoints between the CCG and the VCG, with clinical pathology parameters being the most affected. The inconsistencies observed with the clinical pathology parameters when using VCGs may lead to: (1) misconception about the accuracy and sensitivity of traditional clinical pathology biomarkers and its implications on safety monitoring in the clinic; (2) inability to correctly identify and characterize organ dysfunctions; (3) interference with the weight-of-evidence approach used in identifying hazards in toxicologic clinical pathology and toxicology studies at large; and (4) wrong interpretations and data reproducibility issues. Other alternatives to reduce animal use in toxicology studies are also discussed including blood microsampling for toxicokinetics, scientifically justified use of recovery animals, and appropriate use and continuous investments in new alternative methods.
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.
The INHAND Project is a joint initiative of the societies of toxicologic pathology from Europe, the United Kingdom, Japan, and North America to standardize diagnostic nomenclature and criteria used in toxicologic studies. The INHAND initiative includes recommended nomenclature for evaluating histologic specimens from nonclinical studies involving laboratory animals including rodents, non-human primates, dogs, minipigs, rabbits, and fish. Specific terminology and criteria are derived from the consensus opinions of senior toxicologic pathologists and subject matter experts who have expertise in the different species of interest. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included government databases, including the Registry of Tumors in Lower Animals (RTLA), academia, and industrial laboratories throughout the world. This introduction provides context for 14 chapters, arranged by organ system, that define the INHAND nomenclature and diagnostic criteria for fish used in nonclinical studies. Additionally, the current introductory chapter describes both general features of INHAND methodology as well as elements characteristic of toxicologic fish studies. The latter includes fish study design and conduct; euthanasia, sampling, the histologic processing of fish tissues, and a general approach to evaluating fish studies.
The FVB/N mouse strain is widely used in transgenic studies and as a model for autoimmune diseases. Although spontaneous lesions have been reported in aged FVB/N mice, information regarding younger FVB/N mice is lacking. This study aimed to investigate the spontaneous lesions in young FVB/N mice. Ten males and 10 females were necropsied at 10 and 26 weeks of age. All tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin. Histopathological examination revealed atrophy of the outer retina in all mice of both ages, with atrophy of the inner nuclear layer at 26 weeks. This ocular lesion is consistent with an autosomal recessive disorder in FVB/N mice. Decreased cellularity in the epiphyseal cartilage plate, reduced bone in the primary spongiosa of the femur, increased cellularity of lymphocytes in the thymus, dilatation of ducts in the mammary glands, and foveolar hyperplasia in the stomach were observed, all of which were indicative of age-related changes. These findings provide valuable background data for future studies using FVB/N mice.
Bladder cancer is treated by surgical removal of the tumor followed by injection of anticancer drugs or the Bacillus Calmette-Guerin vaccine. However, there are insufficient effective drug options depending on the risk category of bladder cancer. One of the reasons for this is the limited number of suitable experimental models that reproduce the pathology of bladder cancer for each risk category. There has been increasing interest in the patient-derived xenograft model as an experimental model to reproduce the original nature of the tumor in a patient. However, there are unresolved problems regarding its practical use, such as the low success rate of engraftment, variation in the growth rate between experiments, and the lack of a reliable method to prepare a patient-derived xenograft model from cryopreserved tumor tissue. In this study, the effect of scaffold material on the preparation of a bladder cancer patient-derived xenograft model was investigated and it was found that gelatin/polyethylene glycol-based hydrogel offers advantages for engraftment of cryopreserved bladder cancer tissue. It was shown that the proliferation of cryopreserved bladder cancer cells was promoted with less necrosis and thrombi around the tissue when transplanted into immunodeficient animals with glycol-based hydrogel compared to transplantation with Matrigel or without any scaffold. This study proposes a new method to generate patient-derived xenograft models from cryopreserved bladder cancer tissue, which is expected to have improved proliferation activity after transplantation.
The European hedgehog (Erinaceus europaeus), common in rural and suburban environments, is perceived as declining within its native range, prompting conservation concerns. This study of hedgehogs admitted to a French rehabilitation center aimed to identify causes of death, detect potential emerging diseases, and systematically screen dead hedgehogs for toxicants and hemopathogens. Using clinical information, necropsy examination, and ancillary testing in 159 dead hedgehogs, we identified the primary cause of death of 92% of them. The leading causes of death were impaired general condition (30%); bacterial infections (26%), frequently caused by Salmonella Enteritidis; and trauma (20%). Additional factors contributing to death were identified in 78%, including a high proportion with significant parasite infestations. Toxicologic screening revealed that 42% of hedgehogs had been exposed to anticoagulant rodenticides, including 6.4% hedgehogs with levels compatible with acute intoxication. These hedgehogs were also exposed to trace metals, especially lead, but no pesticides residues were detected. Hemopathogen screening revealed a low diversity and prevalence of blood pathogens with 3.6, 2.9, and 2.9% individuals infected with Anaplasma phagocytophilum, Mycoplasma turicensis, and Mycoplasma wenyonii, respectively. The variety of diagnoses suggests that there is no unique prominent or emerging disease responsible for the mortality of these hedgehogs. This study also highlighted the high exposure of hedgehogs to various toxicants. To further investigate the extent and causes of population declines, health information issued from several centers should be combined with demographic data as well as ecologic assessments on the availability and quality of natural resources.
Pulmonary emphysema is primarily attributable to prolonged exposure to cigarette smoke. Novel tobacco substitutes, such as heated tobacco products, have emerged as healthier alternatives to cigarettes. The effects of short-term inhalation of a heated tobacco product, Ploom TECH+ (PT+), on the lungs of mice were compared with those of 3R4F reference cigarettes. Male 10-week-old C57BL mice were exposed to clean air (control), 3R4F, or PT+ for 1 h/d, 5 d/week for two or four weeks. After four weeks of exposure, the number of inflammatory cells and proportion of neutrophils and lymphocytes in the bronchoalveolar lavage fluid and the number of macrophages in the lung tissue increased significantly in mice exposed to 3R4F but not in those exposed to PT+. Changes in the expression of genes related to inflammation-related factors were observed in the lung tissues of mice exposed to 3R4F for two and four weeks. Chemokine (C-C motif) ligand 17, resistin-like alpha, and lipocalin 2 were among the upregulated genes. In our previous short-term tobacco inhalation study, these genes were identified as useful markers of emphysema effects induced by exposure to cigarette smoke from Peace cigarettes, detectable before pulmonary histological changes appeared. These effects were not observed in the PT+-exposed mice. These data suggest that PT+ caused less damage to the lungs of mice than 3R4F, particularly regarding the induction of emphysema.
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.
Chimeric mice with humanized liver are considered a useful tool to predict drug pharmacokinetics and in vivo toxicity in humans. The PXB-mouse is one of such chimeric (humanized) mouse models with more than 70% of human hepatocytes in their liver, which can produce human albumin with human-type bile secretion and express human xenobiotic metabolizing enzymes. However, data are limited regarding the properties of such humanized mice in hepatotoxicity studies. This study aimed to explore the distinctive characteristics of chimeric PXB-mice with humanized liver that can influence susceptibility to hepatotoxicity. Morphologically, the PXB-mice have a diffuse hepatic macrovesicular and microvesicular steatosis in the transplanted human hepatocytes, which can be suppressed after human growth hormone treatment. The humanized liver of the PXB-mice has a metabolic zonation of glutamine synthetase, cytochrome P450 2E1, and argininosuccinate synthase 1, similar to normal liver in rodents and humans. The transplanted human hepatocytes in the PXB liver have a markedly decreased N-cadherin expression compared with normal human liver. Scanning electron microscopy revealed formation of septum-like structures encircling the transplanted human hepatocytes in the PXB liver, which consists of an accumulation of fibers in the space of Disse under transmission electron microscopy and is immunolabeled for laminin. Overall, the present report demonstrated the morphological and immunohistochemical characteristics of the PXB-mice with humanized liver along with some abnormalities in the cell adhesion of the transplanted human hepatocytes. These findings would be useful for hepatotoxicity studies using humanized animal models.
Amyloidosis is characterized by the extracellular deposition of insoluble protein fibrils that cause cellular damage and dysfunction in organs and tissues. Multiple types of amyloidosis and their causative precursor proteins have been identified in humans and animals. In toxicological studies, a high incidence of spontaneous amyloidosis has been reported in CD-1 mice; however, the precursor protein responsible remains unclear. In contrast, B6C3F1 mice have a low incidence of amyloidosis. This study aimed to identify the types of amyloidosis and causative precursor proteins in CD-1 mice and investigate the role of copy number variations (CNVs) in genes encoding precursor proteins in different mouse species. Histopathological examination revealed amyloids in multiple organs, which were confirmed by direct fast scarlet staining. Immunohistochemistry and liquid chromatography-tandem mass spectrometry analyses revealed that the deposition was derived from serum amyloid A (SAA1 and 2), suggesting that the CD-1 mice had AA amyloidosis. Copy number variation assays demonstrated higher copy numbers of SAA1 and SAA2 in CD-1 mice with amyloidosis than in C3H/He mice (the parent strain of B6C3F1 mice). These findings suggest that the high copy numbers of SAA1 and SAA2 may contribute to the high incidence of AA amyloidosis in CD-1 mice. This study examined spontaneous amyloidosis in CD-1 mice and revealed the correlation between SAA1 and SAA2 CNVs in the pathogenesis of the disease and the genetic factors influencing amyloidosis in mice.
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.
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).
This technical report presents a collection of illustrative images and concise descriptions of non-neoplastic microscopic findings noted in transgenic CByB6F1-Tg(HRAS)2Jic (Tg.rasH2) mice from 26-week-carcinogenicity studies. A unique finding in the Tg.rasH2 strain was the skeletal muscle myopathy observed in nearly all animals, particularly affecting the femoralis and pectoralis muscles, diaphragm, and subcutaneous muscles. Pigment was noted in various organs, particularly in the spleen due to the C57BL/6J background. Mononuclear and/or mixed cell inflammatory infiltrates occurred in various tissues, with or without secondary changes, similar to other rodent and non-rodent laboratory species. Vascular anomalies were sporadically noted, mainly in the uterus. Other notable findings included extramedullary hematopoiesis in the spleen; alveolar macrophage infiltrate (often with eosinophilic crystals) in the lung; and proliferative findings in several tissues, such as the lung (bronchiolo-alveolar hyperplasia), adrenal cortex (subcapsular hyperplasia), and uterus (cystic-endometrial hyperplasia). This paper also includes illustrations of other less frequently incidental findings. The information presented in this manuscript aims to serve as a valuable reference for pathologists and researchers and expected to offer contextual insights for carcinogenicity and other toxicological studies utilizing this animal model.
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 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.
Acute kidney injury induced by stings from multiple wasps is a medical emergency and is a driving factor of acute renal dysfunction. Numerous studies have shown that mitochondrial reactive oxygen species (mtROS) play a key role in ischemia-reperfusion injury-, cisplatin-, and sepsis-induced acute kidney injury. However, the role of mtROS and its underlying mechanisms in wasp-venom-induced acute kidney injury remain inconclusive. In this study, we investigated the role and mechanisms of mtROS in mitochondrial damage and inflammation in a mouse model of acute kidney injury induced using wasp venom. Changes in mitochondrial function, transcription factor A (TFAM) expression, and DNA maintenance levels, renal function, stimulator of interferon gene (STING) expression, and inflammatory mediator levels in model mice with or without the mtROS scavenger Mito-Tempo were analyzed in vivo. Downregulation of mtROS levels reversed renal damage and mitochondrial dysfunction, and reduced STING expression and inflammation in the kidneys of model mice. The suppression of mtROS levels also improved the decrease in TFAM levels and mitochondrial DNA copy numbers in the kidneys of the model mice. In summary, the existing evidence in this study shows that mtROS contribute significantly to mitochondrial damage and inflammation in acute kidney injury induced by wasp venom.
The Cause of Death in Non-Rodents (CODN) Working Group is an initiative under the Scientific and Regulatory Policy Committee (SRPC) of the Society of Toxicologic Pathology (STP), focused on understanding existing practices and expectations among pharmaceutical companies, academic entities, and contract research organizations (CROs) when it comes to identifying and reporting the "Cause of Death" (COD) or moribundity for early or unplanned necropsies in non-rodent animal species (mainly non-human primates [NHP] and dogs) within both GLP (Good Laboratory Practice) and non-GLP toxicity studies. A survey was sent out to STP members to collect data on industry practices for determining COD in animals that underwent unscheduled euthanasia or were found deceased. Other non-rodent animals (such as pigs and rabbits) were also included to evaluate different approaches taken with various species. The insights obtained led to the development of "Points to Consider" for establishing and documenting the COD in large animal toxicity studies. Four key considerations include utilizing information from both control and treated animals in the study, consideration of COD for cohabiting or co-shipped non-study animals, including additional evaluations to help rule-in or rule-out specific causes, and recording the COD consistently in pathology databases or reports as a standard practice.
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.