In Defense of Parity,Females,and Pathology in Research

In “The Pathology of Aging in NOD scid gamma Female Mice,” Santagostino et al ¹⁸ report and discuss pathology findings and assign causes of death in 125 female retired breeder NOD.Cg-Prkdc^scid Il2rg^tm1Wjl /SzJ (NSG) mice. The mice had been maintained in a barrier production colony, as well as in experimental conditions that included experimental exposures to immune-sufficient mice, parasites, and/or amoxicillin. Mice were subjected to gross examination and to histopathology examination of more than 30 tissues. Further characterization of selected findings included immunohistochemistry, electron microscopy, or microbial assessment by culture and/or polymerase chain reaction (PCR).

Pathology assessments of mortality in immune-deficient and immune-sufficient production animals (breeders) offer quality assurance to the users and aim to identify conditions that can affect production and research. Pathology assessments of morbidity or mortality in experimental animals can identify problems that may complicate a study by reducing n (ie, if the identified conditions cause death of animals) and identify conditions and unintended variables that can complicate research by affecting data and conclusions. As descriptive studies, rather than hypothesis-driven “science,” these reports may be shunned from the scientific literature. But in the interest of research rigor, reproducibility, and validity, such reports contribute to the data on spontaneous or background findings in a strain or “model” and enhance its value in research.

Historical control data from nonclinical (preclinical) chronic studies (up to 2 years) are available for mouse and rat strains that are used in toxicologic pathology. ¹⁴ These studies usually involve reproductively and experimentally naive (or vehicle-treated) mice or rats maintained in single-sex housing, with defined specific pathogen–free (SPF) microbial status. Best practices for the reporting and use of historical control data are published, ¹² as is frequently extensive characterization of spontaneous lesions to aid in understanding background changes and in discriminating them from intervention-related changes. ^8,11 Such data are highly informative and especially relevant to these strains under well-defined study conditions. Breeding mice (and most adult humans) are not so socially and reproductively naive, and in many research settings, mice are not so well defined or naive in terms of microbial status.

Pathology data are less accessible for the inbred strains that are more common in academic preclinical research. ^4,17 Such data can facilitate interpretation of findings in research involving related mice, and are appreciated by the community of pathologists that evaluates research mice and is aware of the complex genotypes, background genetics, and microbial status of often locally sourced research subjects. The mouse phenome database (MPD; phenome.jax.org) ¹⁰ makes available a variety of clinical pathology, some anatomic pathology, and other phenotype data from selected strains, usually under clean and well-defined conditions. These efforts (investments) enhance the utility of these strains to the research community. In addition, the Mouse Tumor Biology database (MTB; http://tumor.informatics.jax.org) usefully curates published reports of tumors and tumor models in mice. ⁵

Published pathology data are limited for NSG mice (stock No. 05557; The Jackson Laboratory, Bar Harbor, ME, https://www.jax.org/strain/005557), and for similarly immune-deficient NOD.Cg- Prkdc^scid Il2rg^tm1Sug/JicTac (CIEA NOG mouse [NOG]; Taconic Biosciences, Hudson, NY; https://www.taconic.com/mouse-model/ciea-nog-mouse) mice, which were developed on a slightly different NOD scid background and carry a different Il2rg mutation. ¹⁹

NSG and NOG mice are used widely as recipients of patient-derived xenograft (PDX) tumors and as viable hosts of human tissues or immune system components (humanized mice). Reports of the pathology of unexpected findings in these models offer important insights into recognizing potential complications such as graft vs host disease (GVHD). Reports of Epstein-Barr virus (EBV)–related lymphoproliferative disease in these severely immunodeficient host animals should also serve as a warning about the risks of anthroponotic infections to the studies, as well as to contact personnel. ¹⁶

NSG and NOG mice are expensive, but they breed quite well. Thus, many programs breed them for use within the institution, where the microbial status, housing conditions, and handling practices are less stringent than at the original vendor sources. In Santagostino et al, ¹⁸ the heterogeneous conditions could be considered a weakness, but there is merit in the effort to collect, analyze, and report these findings. While concerns for sex bias in research are real, a practical reason to use female mice in research is that cohoused females are less likely than male mice to kill each other and be lost from the study. ²⁰ Furthermore, in defense of a study limited to breeding females, should we also be concerned about a virginity bias or discriminating against parity in preclinical research? The use of breeding rodents is not frequently reported (or not disclosed) in the peer-reviewed scientific literature, and the majority of mice intended for research are maintained in single-sex groups after weaning and while on study. ³

Under the conditions of this study, infectious conditions such as pneumonia and corynebacterial skin lesions are unsurprising. An unusual finding is a fungal skin lesion consistent with candidiasis, as useful models of cutaneous or mucocutaneous candidiasis have been challenging to develop, even in immune-deficient mice. ⁷

Mammary tumors were the most common neoplasm, identified in 31% of these mice, which exceeds reported findings in virgin CD-1 (Swiss) mice. ^4,9 Interestingly, in another Swiss mouse (FVB/N), far more mammary proliferative lesions, including squamous components, are reported, with more lesions in multiparous than in nulliparous mice. ^13,15

Liver tumors are not surprising in older male Swiss mice. They are unusual in female mice, ⁹ but primary hepatocellular tumors were identified in 5% of these female mice, similar to the number of primary lung tumors, and exceeding the number of hematopoietic neoplasms.

Hematopoietic neoplasms were uncommon in these mice. Their scarcity reflects an important feature of NSG (and NOG) compared with the NOD scid parental strains, in which thymic lymphomas are an expected cause of death beginning at about 6 months of age. ⁶

Parietal yolk sac carcinomas are uncommon in humans or rodents ⁸ but were identified in 3 mice and were characterized by histopathology and immunohistochemistry. Two mesometrial neuroendocrine carcinomas also were identified and characterized by histopathology and immunohistochemistry.

In terms of nonneoplastic conditions, renal tubule intranuclear inclusion bodies associated with tubule degenerative changes have been reported in immune-deficient and immune-sufficient mice, without evidence of an infectious cause. These authors characterize 2 types of renal tubule intranuclear inclusion bodies by histopathology and electron microscopy, and they found the mice and tissues to be negative for likely infectious agents. It is interesting that similar kidney changes were not identified in cohoused immune-sufficient mice but were identified in another cohoused immune-deficient strain. Also interesting and appreciated are joint, spine, and skull changes identified using the authors’ standard necropsy protocol. Arthritis and vertebral changes are reported in studies on aging mice, but cranial hyperostoses, impinging on the brain in some cases, are a new finding.

In research involving animals (human and nonhuman), pathology to characterize and to validate a model and research outcomes seems mission critical, but often pathology assessments are absent (or are unclear or incomplete) in peer-reviewed scientific literature. In the current climate of concern for research validity and reproducibility, pathology seems more essential than ever.

Concerns have been expressed regarding the relevance of microbially naive research mice to the human population. Recent studies show that a single strain of laboratory mice received from the (SPF) vendor source indeed has different immune responses from pet store mice or wild mice. ^1,2 That should not be a surprise. Similar concerns could be raised regarding the relevance of socially and reproductively naive research mice to the human population. This report illustrates how pathology provides mission-critical information regarding infectious complicators of research, as well as how pathology identified causes of morbidity and mortality that were not related to experimental interventions and should be accounted for in research data. Study design involving “clean,” single-sex, virgin populations eliminates variables (or controls controllable variables) to enable refinements in the interrogation of a scientific question. If funders and reviewers determine that more complex (messier) models are more relevant to important research questions, will the necessary controls and pathology assessments be funded too?

Characterizing and reporting spontaneous background findings of a common research “model” animal under real research conditions is an investment that improves understanding of the model, and increases its value to research. This report offers additional context for the use of NSG mice and sets a welcome standard for quality assurance for a breeding colony of immune-deficient mice, as well as for pathology of unexpected mortality in the course of a study.