experimental mice for sale

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Are you at a point in your research where you need a new custom mouse model? Are you unable to find an off-the-shelf model that is suited to your research? A better mouse model can be the next step for your research no matter what you study. If you’re wondering where to buy transgenic mice, look no further than ingenious targeting laboratory. We understand that purchasing an animal model may seem like a complicated process, which is why we’ve put together this guide to simplify it for you.

Frequently asked questions about transgenic mice

1) what is a transgenic mouse model.

A transgenic mouse is a type of mouse model with an extra genetic sequence inserted into its genome. This sequence, also known as a transgene, varies from model to model as each transgenic mouse model is specifically designed with certain experiments in mind. Many off-the-shelf transgenic models are readily available but if you can’t find a suitable line it may be necessary to generate a custom transgenic mouse. This is crucial to keep in mind when you’re looking to buy transgenic mice for your next research project. A simple genetic design for a transgenic construct is shown here:

Knocking in a constitutive promoter and cDNA will give strong expression across all tissues. Different knockins can be used to achieve tissue-specific expression or drug-inducible expression. Transgenic mice became widely available in the 1980s including the famous oncomouse published in 1984 for cancer studies. It was this model, as well as other transgenic mice that were generated in the next few years, that helped the field of cancer research grow into what we know it as today. That’s because oncogene transgenic mice allowed scientists to observe the development of cancer in certain organs that could not be replicated in tissue cultures. Now, it is possible to buy transgenic mice that model many types of organ-specific cancers and the particular characteristics that come with each one. [ 1 ]

Why Use Transgenic Mouse Models?

2) Where can I buy a mouse model?

ingenious targeting laboratory offers custom mouse models that are tailored to our clients’ specifications and research needs. We established ourselves as one of the very first mouse gene targeting companies in 1998. Since then, our models have been featured in over 600 client publications including studies published in Science , Nature , and Cell . Our clients choose to buy transgenic mice and work with us again and again because of the quality of our work and the strong relationships we build with them. Once you decide to partner with us, our scientific consultants will guide you through the process of buying transgenic mice. We begin with a quote consultation that will help us determine what type of model you’d like to generate and what strategies are available. This is a good first step before you order a mouse model online.

3) How much do transgenic mice cost?

For pricing information, please download ingenious’ Pricing Guide or contact us for a custom quote. What sets us apart from other companies is that when you buy transgenic mice from ingenious, you are guaranteed to get your model, not just your money back. Because we’re backed by over 20 years in business we are proficient in working with both classic and cutting-edge technologies. Our team of experts is standing by to help get your new project off the ground quickly. We will be there with you every step of the way to make sure your mice are accurately targeted and arrive safely at your facility. You can also elect for our other available mouse generation services, such as cohort generation, colony management consultation, cryopreservation, genotyping, and more to help you get the most out of your new custom mouse line.

4) Why should I use targeted transgenics?

When buying transgenic mice, it’s important to consider whether you may need targeted insertion over random insertion. With targeted transgenics you can expect more predictable and consistent results. That’s because your construct is inserted into a specific genomic location, such as a safe harbor locus like Rosa26. While ingenious specializes in working with the Rosa26 locus, other safe harbor locus options are available. Some examples of models that can be generated by targeting the Rosa26 locus are constitutive expression, tissue-specific expression, and conditional expression. Targeted transgenics are by no means limited to the use of safe harbor loci however. The advantage of this powerful method is that a sequence can be inserted almost anywhere, for example to modify the expression pattern or protein product of a gene of interest. Random insertion, on the other hand, results in your transgene being inserted somewhere in the genome with no control over its location. While this may be suitable for some studies, you may find that you require your construct to be placed in a specific location in order to accurately study your gene of interest. Otherwise, your research may be left up to chance. Today the methods of the 1980s are still used to generate random insertion transgenic lines because founder mice can be produced quickly and at low cost. These advantages are greatly reduced when you consider that each potential founder has the transgene integrated at a different site. Offspring from multiple founders must be carefully screened to find mice where the transgene functions as planned. For more information, please read our blog post 4 Reasons To Use Targeted Transgenic Mice Over Random Insertion .

5) How do you make transgenic mice?

Transgenic knockin to your gene of interest.

With a targeted cDNA knockin it’s possible to alter a gene in the mouse genome and make it express a reporter gene or other alternate cDNA sequence, for example the sequence of a human gene. The sequence of Cre recombinase can be inserted using this method to create a new tissue-specific expression line. This is important to consider the next time you’re looking to buy transgenic mice. Targeted knockins often inactivate the mouse gene and result in the transgene being expressed in its place. However, the knockin strategy is flexible and it’s possible to co-express the cDNA along with your target gene. This makes it possible, for example, to generate a line that expresses Cre along with the targeted gene.

Safe-Harbor Transgenic Knockin (Rosa26)

ingenious targeting laboratory developed proprietary technology for generating transgenic models using the Rosa26 locus: Rapid-Rosa26™ Targeting . The advantage of using a safe harbor locus is that your overexpression construct can be introduced into a location that won’t interfere with surrounding genes, thus isolating it. This results in targeted expression that is more predictable.

Rapid-Rosa26™ Targeting

With Rapid-Rosa26™ targeting, when you buy transgenic mice, you can expect shorter timelines and a reliable, high-quality model. One concern researchers may have about targeted insertion is that it may take longer to generate, and at a higher cost. However, ingenious’ Rapid-Rosa26™ technology allows us to generate a custom transgenic model with accurate targeting on shorter timelines, enabled by vectors that have been created with thoroughly validated strategies. An example of a standard strategy is shown here, for conditional expression of a specific cDNA. Tissue-specific expression is achieved by crossing the Rosa26 transgenic line to one of the hundreds of available Cre-expressing lines.

6) How can I buy transgenic mice from ingenious?

If you’re ready to buy transgenic mice for your next research project, get in touch with ingenious targeting laboratory. Fill out our quote form and one of our scientific experts will provide you with a custom quote within 1 business day.

1) Hanahan D, Wagner EF, Palmiter RD. 2007. The origins of oncomice: a history of the first transgenic mice genetically engineered to develop cancer . Genes & Development 21 : 2258-2270.

Notable Client Publications

Mlynarczyk C, Teater M, Pae J, Chin CR, Wang L, Arulraj T, Barisic D, Papin A, Hoehn KB, Kots E, Ersching J, Bandyopadhyay A, Barin E, Poh HX, Evans CM, Chadburn A, Chen Z, Shen H, Isles HM, Pelzer B, Tsialta I, Doane AS, Geng H, Rehman MH, Melnick J, Morgan W, Nguyen DTT, Elemento O, Kharas MG, Jaffrey SR, Scott DW, Khelashvili G, Meyer-Hermann M, Victora GD, Melnick A. 2023. BTG1 mutation yields supercompetitive B cells primed for malignant transformation. Science 379 (6629): eabj7412.

Gleixner AM, Verdone BM, Otte CG, Anderson EN, Ramesh N, Shapiro OR, Gale JR, Mauna JC, Mann JR, Copley KE, Daley EL, Ortega JA, Cicardi ME, Kiskinis E, Kofler J, Pandey UB, Trotti D, Donnelly CJ. 2022. NUP62 localizes to ALS/FTLD pathological assemblies and contributes to TDP-43 insolubility. Nat Commun 13 (1): 3380.

Kasatkina LA, Ma C, Matlashov ME, Vu T, Li M, Kaberniuk AA, Yao J, Verkhusha VV. 2022. Optogenetic manipulation and photoacoustic imaging using a near-infrared transgenic mouse model. Nat Commun 13 (1): 2813.

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An Introduction to the Laboratory Mouse: Mus Musculus

JoVE Science Education Database. Model Organisms II: Mouse, Zebrafish, and Chick. An Introduction to the Laboratory Mouse: Mus musculus. Journal of Visualized Experiments, Cambridge, MA, doi: 10.3791/5129 (2014).

Mice (Mus musculus) are an important research tool for modeling human disease progression and development in the lab. Despite differences in their size and appearance, mice share a distinct genetic similarity to humans, and their ability to reproduce and mature quickly make them efficient and economical candidate mammals for scientific study.

This video provides a brief overview of mice, both as organisms and in terms of their many advantages as experimental models. The discussion features an introduction to common laboratory mouse strains, including the nude mouse, whose genetic makeup renders them both hairless and immunodeficient. A brief history of mouse research is also offered, ranging from their first use in genetics experiments to Nobel prize-winning discoveries in immunology and neurobiology. Finally, representative examples of the diverse types of research that can be performed in mice are presented, such as classic behavioral tests like the Morris water maze and in-depth investigations of mammalian embryonic development.

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Basic Mouse Care and Maintenance Mice (Mus musculus) are small rodents that breed and sexually mature quickly, making them perfectly suited to generating large animal colonies for biological research. As compared to other mammalian species, mice are simple and inexpensive to maintain in the laboratory. Nevertheless, mouse colonies do have specific husbandry needs that are critical to preserving animal health and safety as well as experimental reproducibility.

This video demonstrates standard practices that ensure mice are treated as humanely as possible within the laboratory animal facility, or vivarium. The discussion begins by reviewing a typical mouse housing setup, consisting of a plastic cage equipped with a layer of soft bedding and nesting material. The preformulated food pellets (also known as chow) that comprise the typical mouse diet are also introduced. In order to facilitate experiments performed on mice, safe animal handling practices are demonstrated, including common restraint techniques like “scruffing,” and the strategies used by researchers to keep track of individual mice within the facility. Finally, experimental manipulations of mouse housing and diet are discussed, in addition to one of the most common applications of the scruffing technique — performing injections.

Development and Reproduction of the Laboratory Mouse Successful breeding of the laboratory mouse (Mus musculus) is critical to the establishment and maintenance of a productive animal colony. Additionally, mouse embryos are frequently studied to answer questions about developmental processes. A wide variety of genetic tools now exist for regulating gene expression during mouse embryonic and postnatal development, which can help scientists to understand more about heritable diseases affecting human development.

This video provides an introduction to the reproduction and development of mice. In addition to clarifying the terminology used to describe developmental progression, the presentation reviews key stages of the mouse life cycle. First, major development events that take place in utero are described, with special attention given to the unique layout of early rodent embryos. Next, husbandry protocols are provided for postnatal mice, or pups, including the process of weaning, or removal of pups from their mother’s cage. Since males and females must be separated at this stage to prevent unscheduled mating, the demonstration also reveals how to determine mouse sex. Subsequently, instructions are given for carrying out controlled mouse breeding, including screening for the copulatory plug, which is useful for precisely timed embryonic development. Finally, the video highlights strategies used to investigate the complex processes that govern mouse development, including the generation of genetically altered “knockout” mice.

Mouse Genotyping Even though the human genome was mapped over 10 years ago, scientists are still far from understanding the function of every human gene! One way to evaluate how a gene functions is to disrupt the sequence encoding it and then evaluate the impact of this change (the phenotype) on the animal’s biology. This approach is commonly used in the mouse (Mus musculus), since it shares a high degree of genetic similarity with humans. To track the animals bearing genetic changes over several generations, it is necessary to screen the DNA of each mouse in a process known as genotyping.

This video provides an overview of the theory and practice behind genotyping mice. The discussion begins with the basic principles of mouse genetics, including a review of the terms homozygote, heterozygote, wildtype, mutant, and transgenic. Next, step-by-step instructions are supplied for extracting and purifying genomic DNA from mouse tissue. Examples are provided demonstrating how to interpret genotyping results, as well as how to keep track of mice with the desired genotype. Finally, some representative applications of the genotyping procedure will be presented in order to demonstrate why this common technique is so essential to mouse research.

Introducing Experimental Agents into the Mouse Many investigations performed in mice (Mus musculus) require the administration of an experimental agent to the animal. For example, it may be of interest to test the efficacy of a specific therapy, to induce a pathologic condition, or to administer anesthesia or palliative care. In order to ensure safe and efficient delivery, it is important to consider a variety of factors prior to the administration of the treatment.

This video, which reviews agent administration in the mouse, begins by highlighting properties to consider, such as viscosity, dose, and palatability, when planning the administration of an experimental agent. The subsequent discussion focuses on injection methods, including delineation of the structural components of the syringe and needle, how to interpret needle gauge, and safe mouse restraint methods for common injection sites. Detailed instructions are provided for performing subcutaneous (SC/SubQ), intraperitoneal (IP), and tail vein (IV) injections in mice. Furthermore, applications of these techniques as well as alternative administration routes are discussed.

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High density housing of mice with up to 180 individually ventilated cages ( IVC’s) per system. RAIR HD Super Mouse 750™ Racks hold mouse populations of up to 900 adult mice in one mobile unit, with 2 different cages sizes that can be mixed and matched as needed. The design delivers low velocity HEPA filtered air to each cage while capturing cage effluent air. Cage air is prevented from entering the room and room air from entering cages, protecting both animals and personnel.

House Super Mouse 750™ Micro-Isolator® and Econo-Cage® Disposable Micro-Isolator® systems on each rack at the same time, without any changes to the rack. These innovative cages are designed with minimal components for simplifying handling, reducing inventory and increasing productivity. Observe animals with an unobstructed view of cage interiors, without having to remove cages from a rack.

These cages feature the same micro-isolation integrity the research community has come to expect from Lab Products, llc., with effective micro-isolation of static or individually ventilated caging, for almost 40 years. Every product we offer is designed to minimize experimental variables, meet your research requirements and help ensure the well being of your animals and personnel.

Be sure to download your copy of the Ventilated Products Guide and review information about some of our most popular ventilated animal housing systems.

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Health Evaluation of Experimental Laboratory Mice

Good science and good animal care go hand in hand. A sick or distressed animal does not produce the reliable results that a healthy and unstressed animal produces. This unit describes the essentials of assessing mouse health, colony health surveillance, common conditions, and determination of appropriate endpoints. Understanding the health and well-being of the mice used in research enables the investigator to optimize research results and animal care.

INTRODUCTION

Both investigative and veterinary staffs monitor the health and well-being of mice that are used in research. Indeed, this level of responsibility and care is mandated by the Public Health Service based on the Guide for the Care and Use of Laboratory Animals ( National Research Council. 2011 ). The Guide is “intended to assist investigators in fulfilling their obligation to plan and conduct animal experiments in accord with the highest scientific, humane, and ethical principles.” All investigators should become familiar with the Guide ( http://grants.nih.gov/grants/olaw/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf ).

Careful observation of mice in their home cage can provide a wealth of information about the health and welfare of the animals. Activity, nest building, interaction with cage mates and general appearance are indicators of general health and well-being. Hands-on physical examination provides an assessment of the animal’s hydration, body condition, observable abnormalities, and the presence of palpable anomalies in the abdomen. We will outline details that will be not only useful to the investigator, but quick and easy to perform as well.

Several infectious agents have been identified over the years that have either adverse affects on animal health and/or research outcome. For this reason, most mouse colonies are maintained as Specific Pathogen Free (SPF), free of defined infectious agents. SPF status is generally monitored by exposing sentinel animals to dirty bedding of colony animals and testing the sentinel animals. Tremendous effort and expense is expended maintaining SPF colonies and it is critical that all staff entering colonies and handling animals understand disease transmission and the importance of good practices to prevent pathogens from being introduced or spread.

Laboratory mice develop a number of common clinical conditions which will be described, together with recommended treatments and suggested endpoints. For instance, dermatitis is a poorly understood condition that is common and problematic, especially in animals with a C57BL/6 background. Some congenital conditions such as hydrocephalus or microphthalmia are also seen with a C57BL/6 background. Behavioral differences exist between strains and lines of animals with some prone to fighting when males are co-housed. BALB/c, FVB and SJL strains are particularly troublesome in this regard although management practices can help reduce fighting.

The Guide is based on three principles: Replace, Reduce and Refine. Replace animals whenever possible, reduce the number to lowest possible that will produce accurate conclusions, and refine the experimental paradigm to improve the science and the care of the animal. These principles were introduced by British investigators William Russell and Rex Burch in 1959 in response to the moral and ethical concerns associated with the use of animals in research ( Russell and Burch, 1959 ; Flexnell, 2002). Refinement is a principle that directly relates to the topic at hand, and can be as simple as adding palatable food on the cage floor or as sophisticated as utilizing telemetry to monitor physiology and activity.

The 8 th Guide published in 2011 also emphasizes the refinement of end points and states that “The use of humane endpoints contributes to refinement by providing an alternative to endpoints that result in unrelieved or severe animal pain and distress.” In other words, an animal should be euthanized at the earliest possible point that will provide experimental data in order to minimize suffering. A variety of refined endpoints for multiple species have been published and they include data-based systems for assessing animals, drops in core body temperature as an alternative endpoint, endpoints for tumors and ascites production, and changing from an awake sepsis model to an anesthetized sepsis model, among others. ( Hendriksen and Steen, 2000 ; Morton, 2000 ; Olfert and Godson, 2000 ; Minecci et al., 2007 ; Paster et al., 2009 ; Sass, 2000 ; Stokes, 2000 ; Stokes, 2002 ; Toth, 1997 ; Toth, 2000 ; Toth and Gardiner, 2000 ; Wallace, 2000 ).We discuss endpoints in mice for the consideration of the investigator.

COLONY HEALTH SURVEILLANCE

Colony health surveillance is typically part of the overall veterinary care program to ensure the SPF status of the animal facility. Many microbial outbreaks are subclinical in mice. Therefore, microbiological surveillance of colonies is required for detection and to ensure the appropriate health status of the colony and individual mouse. Sentinels are animals that are free of excluded microorganisms and are exposed to dirty bedding of colony animals to determine if excluded microorganisms are present in the colony animals. Health surveillance testing of sentinel and / or colony (non-sentinel) animals can include the following: gross, histopathology, and parasitology assessments, testing of serology samples for antibodies or antigens, culture or isolation of microorganisms, and molecular diagnostics such as the polymerase chain reaction (PCR) test. The diagnostic procedures utilizing samples collected from live animals for colony health surveillance include those used to assess the health of individual mice. For example, collection of: a fur sample for an ectoparasitology exam, fecal material for endoparasitology and PCR testing, and blood for serology testing can help determine the health status of an individual animal. Additional details regarding surveillance programs can be found in the text, Fox, et al., 2002 . “Laboratory Animal Medicine,” 2 nd ed. An example of commercial laboratory services for health surveillance can be found at: http://www.criver.com/enUS/ProdServ/ByType/ResAnimalDiag/Pages/home2.aspx .

Enormous expense is involved with maintaining SPF colonies. The protective personal equipment (PPE) used and the regular purchase and testing of sentinel animals is a significant investment by the facility/institution. Outbreaks of disease agents exponentially increase that cost. To contain the disease outbreak extensive additional testing is necessitated. For this reason facility staff and investigative staff should do their utmost to follow facility procedures, traffic patterns and standard operating procedures. For instance, in a facility where animals are changed in hoods using appropriate micro-isolator techniques then investigative staff should follow those same practices (e.g., not open cages outside of the hood). Another important consideration in this same vein is following appropriate procedures for introducing animals from outside facilities. Typically, there is a gatekeeper through who all imports of animals are managed. This is to ensure that their health status is known prior to arrival and confirmed after arrival before introduction into the main animal colony.

EXAMINATION AND ASSESSMENT OF THE MOUSE

An overall assessment of the health and welfare of a research mouse includes an evaluation of the animal in its home cage and a hands-on exam. Because mice are easily stressed by handling, the cage side exam should be performed first. Observing the mouse in its home cage will provide information about the animal’s overall appearance and activity level, the interaction with the environment, including nest building, and its behavior with respect to its cage mates. The hands-on examination allows assessment of observable abnormalities, hydration status, body condition, and the presence of abnormalities in the bones, genitals and abdomen.

Home cage evaluation

Mice are inquisitive and active and will generally be observed moving around the cage, grooming, eating, drinking and interacting with cage mates; particularly after being stimulated by having their cage picked up and moved from the shelf or rack.

Behavioral indicators of a welfare issue can be obvious including wounds and limping, hunched posture, dull or sluggish movements, large or open tumors, or a mouse that does not move when the cage is manipulated. Many behaviors are more subtle and non specific and will take practice and time to evaluate. Among these is nest building. All mice will build a nest if given suitable material ( Hess et al., 2008 ). A mouse that is placed in a new cage with nesting material and has not built a nest by the next day warrants veterinary attention and possibly euthanasia. Similarly, the absence of feces in a cage after the mouse has been housed several hours suggests the animal has not been eating.

Mice are prey species and will generally mask signs of pain. If evaluating mice for pain after surgery or a procedure that has the potential to be painful, the observer needs to be quiet and monitor the mice without moving the home cage. Mice, like all animals express emotion including pain through facial expressions. Many of the expressions are identical to those expressed by humans in pain including squinting the eyes and contracting the skin around the nose and mouth. Mice may also pull their ears back ( Langford et al., 2010 ; Figure 1 ). Mice in pain are less active than normal, although this type of qualitative assessment is difficult to evaluate on cage side exam unless it is severe ( Clark et al., 2004 ; Roughan et al., 2009 ).

Facial expressions in mice indicating pain and/or distress include squinted eyes, contracted skin around nose and ears pulled back.

Developing and assigning a study specific pain scale can be an effective tool to facilitate communication between observers and can serve as a convenient indication when an animal’s condition is deteriorating or has reached a clinical end point. Table 1 is an example of a general clinical pain score.

Assessing Pain and Distress in Mice

Handling and Hands on evaluation

Laboratory mice are generally docile but will move quickly or jump away from the person trying to restrain them, and some strains may bite. Mice can be moved a short distance for examination by being picked up at the base of the tail and placed onto the top of their cage allowing the mouse to grasp the bars with the forefeet and direct its effort away from the handler. Very obese or pregnant mice should have a hand placed under the abdomen to prevent the heavy abdominal contents from compressing the diaphragm and limiting respiration. Retain control of the tail to prevent the mouse from escaping and potentially harming itself.

• Restraint . With the animal restrained on top of the cage, run a finger over the animal’s coat to feel for wounds that may be covered with fur and feel for masses that may not have been obvious when the mouse was moving around the cage.
• Hydration. Severely dehydrated mice will be weak and often will look paralyzed in their rear legs. These mice may also have trouble gripping the cage bars with their forefeet. Other symptoms of severe dehydration include sunken or recessed eyes and fuzzy facial fur, which results due to piloerection. More moderate dehydration can be detected by pinching the skin over the shoulder blades. In a well hydrated mouse the skin will quickly return to its original shape. The skin remaining bunched up is an indication of dehydration.

Body Condition Scoring (BCS) is a quick, easy and reliable method for assessing mouse health. It utilizes a scoring system of 1 to 5 with 3 being the optimal condition, 1 being emaciated and 5 being obese.

Restraint and physical examination

While holding the animal by the tail with the little finger, use the forefinger and thumb to gently pin down the head and grasp the loose skin over the back of the neck. The remaining fingers can be used to scruff the skin along the back of the mouse, allowing the mouse to be picked up and restrained. Once restrained, examine the animal from its nose to the tail. Perform the exam in the same order each time an animal is examined to establish a pattern that will help prevent overlooking a more subtle abnormality.

• Face and mouth. Evaluate the eyes, ears, face and neck for abnormalities. Examine the incisors by gently moving the lips using a cotton tipped applicator. Evaluate the color of the mucous membranes, which should be pink. The mucous membranes around the eyes and the skin of albinos appearing pale, bluish, or brick red in coloration indicate conditions such as anemia, hypoxia, or systemic infection and circulatory failure. These are near terminal conditions and indications for euthanasia. Note that a greenish hue may be due to genetic constructs with green fluorescent protein and are expected in some strains.
• Feet and limbs. Next, examine the feet and limbs. Explore any gait abnormalities observed during the cage side exam. Lameness in one limb may be due to a wound on the foot or bony tumor that can be felt along the length of the bone. Pinch the toes to distinguish between weakness in the limbs and paralysis. Weak mice will generally pull away when the toes are pinched.
• Genital abnormalities . Evaluate the female mammary chain for masses, abnormalities around the nipples or irregularities around the vulva. Examine the male penis by gently sliding back the prepuce. A purple or distended penis during exam likely indicates a urinary obstruction, which is a painful and life threatening condition. Check the rectal area for swelling, trauma or prolapsed tissue. Swellings adjacent to the rectum in older male mice likely indicate cysts in the reproductive glands and are generally benign.
• Abdominal palpation . Palpate the abdomen by gently compressing the contents between the fingers from just under the ribs down to the hips. Common abnormalities palpated in the cranial aspect of the abdomen include tumors of the liver and spleen. In male mice, the most common abnormality felt in the middle part of the abdomen is due to enlargements of the kidneys secondary to urinary obstruction. In pregnant females, the distended uterus can be palpated in the mid abdomen and can extend up under the rib cage. In older females or retired breeders, masses in the mid abdomen are generally due to uterine tumors. The bladder can be felt in the caudal abdomen. Mice will generally urinate as a stress response when picked up. A large or distended bladder can indicate an obstruction. In males, the glands of the reproductive organs can become enlarged and distended with fluid as the mice age which can be palpated in the caudal abdomen.

See http://www.ors.od.nih.gov/sr/dvr/od/Documents/Post_Op_Form.docx

COMMON CLINICAL HEALTH CONDITIONS OF MICE

While mice are valuable research tools, they are also living animals with characteristics and health issues that may influence or be of concern while conducting a study. A holistic approach to evaluation of clinical conditions, including influences on the research and the welfare of the animals will allow the investigative and veterinary teams to make appropriate decisions. The discussion here will cover the most frequently seen conditions, but is not an exhaustive listing. The photographs included will illustrate many of these conditions. Treatment suggestions are based on the author’s clinical experience but should always be discussed with the veterinary team at your institution. Also, keep in mind that the physiology of the animal may be affected by responses to the condition itself and by any treatments administered. Finally, euthanasia may be the most appropriate response for both humane and research related reasons.

Skin lesions

Skin lesions are one of the most commonly observed clinical problems in mice and arise from many different causes. Often these can be clinically managed to keep the mouse on study or retain it for breeding, but strain predilections must also be considered. The immune system of the mouse will be activated to fight these lesions, which must be considered in light of the study protocol.

• Fight wounds : Most commonly seen in co-housed male mice, especially from strains such as BALB/c, SJL, and FVB, however this behavior can also occur between females or in mixed sex groups. The typical presentation is a cluster of wounds on the rump, hips, and/or genital region, which may extend to the trunk of the body or forelegs. Often there is one aggressor in the cage which can be removed while beginning treatment of the other mice with antibiotics and analgesics systemically and/or topically. If the wounds are not severe, mice generally heal well. If wounds are severe, humane endpoint criteria should be discussed with your veterinary staff. Fighting can be minimized by housing only littermate males together, or single housing particularly aggressive strains.
• Ear dermatitis : Often related to ear tags used for identification. Although tags are normally very well accepted by the animals, problems may occur due to tag placement, sensitivity to the tag metals, or secondary to fighting. If irritation is noted, it is advisable to remove the tag if still present, and to use antibiotic and analgesic therapies to treat secondary infection and pruritis. Clipping of the hind toenails helps reduce trauma due to scratching.
• Alopecia : Especially when seen in patches around the face or in one location on several mice within a group, is a sign of what is termed “barbering”. The skin is generally healthy, and short stubby hairs may be seen in the alopecic area. In some cases the whiskers or eyelashes may be missing. This condition is caused by over grooming by an animal’s cage mates or itself. It may be a dominance action or an obsessive compulsive grooming disorder. Unless there is secondary ulceration or inflammation of the skin, no medical treatment is necessary, but increased environmental enrichment has potential benefit.
• Dermatitis : Includes ulcerative dermatitis (common in C57BL/6 background mice), miliary dermatitis, muzzle dermatitis involving the hair follicles, (referred to as furunculosis or botryomycosis), and contact dermatitis. Erosions of the skin or small raised scabbed lesions may be seen. If lesions are deep, large, or bleeding then aggressive care with analgesics, anti-inflammatory medications, and antibiotics, or euthanasia are indicated. For less severe lesions, control of infection by skin bacteria relieves the irritation and may allow healing; treatment may be systemic or topical. Many different treatment regimens have been used for ulcerative dermatitis including nutritional supplements and NK1 receptor antagonists (Lawson, G.W., et al. 2011).
• Hyperkeratosis : Thickening of the skin without shedding of the surface dead epithelium is often a sign of irritation. It may also be a sign of neoplasia, bacterial skin infection, or mite infestation. Skin scrapings, fur plucks, or biopsy may be used diagnostically to guide treatment decisions. In nude mice Corynebacterium bovis has been noted to cause a flaky white dandruff-like skin condition which may be responsive to antibiotic treatment. Sterile caging is beneficial to reduce incidence.
• Otitis : Head tilt or circling behavior can be associated with inflammation within the inner or outer ear. External lesions often respond to topical antibiotic, while inner ear conditions with nerve damage are more complicated and may lead to indications for euthanasia if the animal cannot maneuver to reach feed and water.
• Tail lesions : May present as dermatitis, as fight wounds, or as concentric rings with hyperkeratosis. The latter has historically been related to very low humidity and is known as “ringtail”. Granulomas may form after tail biopsies for genotyping of mice. Treatments are the same as similar lesions found on the body. Small nicks used for collection of blood from the tail vein(s) will scab and generally heal without medical treatment.

Lumps and Bumps

“Lumps and Bumps” require further diagnostic evaluation to determine the cause and significance. Common causes are tumors (spontaneous or study induced), abscesses, cysts, lymphadenopathy, salivary gland hyperplasia, and reactions to injections especially if adjuvants are used.

• Mammary tumors : A common problem in female mice which may be observed almost anywhere on the trunk of the body due to the extensive distribution of mammary tissue. These are subcutaneous, may be smooth or rough, and are usually easily moveable under the skin. In mice these tend to be malignant. Treatment is not advised.
• Tumors : These should be evaluated with consideration of humane endpoints. Their size, location, secondary effects, etc. will be considered in decisions to observe, surgically remove, or recommend euthanasia.
• Abscesses : Can occur in any location, but the most common are related to bite wounds, necrotic tumors, or blocked ducts to normal exocrine glands such as the preputial glands of male mice. They are usually a soft to firm swelling which may or may not be inflamed. Needle aspirate allows drainage of pus or caseous exudate which can be submitted for culture and antibiotic sensitivity testing to guide treatment plans. In some cases the abscess ruptures at the skin surface spontaneously. Treatments include draining and/or flushing the abscess, antibiotic therapy, and clean soft bedding if located ventrally. If not responsive to therapy, euthanasia must be considered.
• Lymphadenopathy : Usually noted under the legs or in the neck region, but may occur anywhere lymph nodes are found, including within the abdomen. It may indicate a primary lymphoma or be an indication of systemic inflammatory responses. Evaluation of the inciting cause will help in determining treatment options or indications for euthanasia.
• Reactions to injections : Seen with use of some adjuvants, result in small subcutaneous lumps at the local site, which may ulcerate to a small dry, open lesion in the skin. In most cases no treatment is necessary; however guidelines should be established in studies for treatment if these lesions become larger or deeper than expected. They are usually sterile, so the primary concern is to prevent secondary infection.

Eyes and surrounding tissues

Problems of the eyes and surrounding tissues are commonly seen in clinical evaluation of mice. These may involve the structures in front of the eye, eyelids, conjunctiva, tear production, or tear drainage; the eye itself including the cornea, lens, and deep structures; or the orbit behind the eye. Blepharospasm (squinting), discharge from the eye, or buphthalmia (bulging) are the most common initial presentations.

• Microphthalmia or anophthalmia : Congenital conditions (common in C57BL/6) which present as a partially opened or closed eye. Often tear production continues with poor drainage resulting in a mild watery or waxy ocular discharge. Most mice are stable and groom to keep the area clean, precluding the need for treatment. Occasionally treatment for conjunctivitis may be needed.
• Conjunctivitis : Presents as swollen pink to red tissue under the eyelid and often a thick ocular discharge. It may be caused by foreign bodies such as a piece of bedding or an aberrant eyelash, or be related to trauma to the conjunctiva or the globe. Often this can be treated by gentle flushing of the eye with a saline eye wash and topical application of an antibiotic lubricant ointment. Systemic antibiotics are also beneficial.
• Keratitis : Inflammation of the cornea, presents as a cloudy or vascular surface of the eye, and is often combined with conjunctivitis. Lack of tear production or inability to close the eyelids properly can lead to drying of the corneal surface. Ulceration of the cornea may be secondary to drying, or a result of trauma or displacement of the lens of the eye. A dent in the cornea may be visible and a tissue plug may be present if all layers of the cornea have been penetrated. The cornea is well innervated, so corneal lesions have potential to be quite painful. Treatment with an analgesic either topically or systemically combined with lubrication and antibiotics leads to healing of most such lesions. In severe cases the globe may collapse or be lost; however healing of the orbit can occur and the mouse itself can be maintained.
• Cataracts : Develop in some strains of mice, such as C57BL/6. They are seen as a central white material behind a clear cornea. Generally they do not cause any problem for the animal, however occasionally the lens will luxate leading to inflammation within the eye, glaucoma, or even expulsion of the lens. In such cases treatment is the same as for keratitis. White scars on the cornea may be confused with cataracts.
• Retro-orbital tumors , blood clots, or abscesses: Result in bulging of the eye forward of the normal position, and often difficulty in closing the eyelids. These are difficult to treat, and euthanasia of the mouse is usually the preferred option.

Mobility issues

Mobility issues may be due to injuries, central nervous system disorders, or degenerative conditions.

• Injuries :The most common include catching a foot or leg in some part of the cage apparatus and fight wounds. Each instance needs to be evaluated with the veterinary team for appropriate pain management, treatment, or euthanasia.
• Neurologic conditions : Present in many ways including ataxia, head tilt, spinning when lifted by the tail, circling with inability to straighten out the path, and seizures. The primary clinical concern is ability to reach feed and water. Supportive care includes placing a soft diet on the floor of the cage, soft bedding, support of bodily functions such as urination, etc. Maintenance of such mice should be justified in the study proposal, or they should be euthanized.
• Arthritis : Presents as swelling and often redness of joints, with favoring of the affected limb or a reluctance to move. This may be transient, related to the strain or study, or a result of trauma. Analgesia is usually indicated, and if pain is not resolved then euthanasia may be necessary.
• Pododermatitis : Irritation on the bottoms of the feet may be part of a study model using foot pad injections, or may be caused by the floor of the cage (wire floors) or a wet cage. Treatment will depend on the specifics of the case but may include antibiotics, analgesics, and soft absorbent bedding.

Respiratory issues

Respiratory problems are seen as changes in the breathing pattern or nasal discharge. Signs of respiratory distress include dyspnea, shallow rapid breathing, gasping, or abdominal effort in breathing. Nasal discharge may be seen dried on the nostrils, or more commonly as crusty material on the forelegs from self grooming.

Immunosuppressed animals are highly susceptible to pulmonary infections similar to those seen in humans with AIDS. Use of sterile caging greatly reduces the risk for these animals. Preventive strategies are most effective; once symptoms develop there is little chance for successful treatment and euthanasia is the preferred option.

Congenital deformities

Congenital deformities are generally seen shortly after birth or at the time of weaning. These conditions may also lead to difficulty with pregnancy or pup delivery by the dam. If not part of the research model, it is best not to use affected mice or their parents for future breeding of a research line. These problems include:

• Hydrocephalus : A condition in which fluid builds up in the ventricles of the brain and does not distribute normally between the brain and spinal cord. Visibly these mice have a large rounded head and shortened muzzle. They are smaller than littermates, and with time develop lethargy and neurologic abnormalities. Supportive care with special feed may be provided short term, but these animals rarely survive to adulthood. Euthanasia is the most humane option.
• Malocclusion : Mouse teeth grow throughout life. The teeth should meet in such a way that they grind on each other and on the feed to keep the teeth a normal length. When this does not happen teeth may grow into the palate or out of the mouth making eating or drinking difficult for the animal. Deformities may be caused by congenitally defective jaw structure, damage to the developing teeth, or trauma to the mouth or jaw. Treatment short term involves trimming of the incisor teeth, however care for crooked or maloccluded teeth is a lifelong process. Euthanasia should be strongly considered and breeding is not recommended.
• Runt pups : Very small, poorly developing pups usually indicate a genetic abnormality, or competitive disadvantage. In very large litters or if the dam is a poor milk producer, supportive care with soft dough or gel diets may provide sufficient support for runts to catch up to normal mice. However, if a dam produces runts in subsequent litters, it is best to retire her from breeding.
• Imperforate vagina : A defect that may occur in the maturing young female mouse. It is produced by lack of opening of the vaginal membrane, and appears as swelling between the anus and genital papilla giving the appearance of a male mouse. Female mice have nipples while males do not, which helps in determining the sex of these mice. Although the vaginal canal can be surgically opened, breeding performance is usually poor, so this defect also is an indication for euthanasia.
• Other common congenital deformities : Extra limbs or toes, lack of one or more limbs, an outwardly curved sternum (breast bone), small or absent eyes, abnormal organ development which may lead to difficulty breathing or a distended abdomen, closed rectum leading to inability to defecate, and others. Each should be evaluated by the veterinary staff with the investigator to determine the significance to the mouse, the line, and the best clinical approach.

Reproductive associated conditions

Reproductive-associated conditions are a common reason for veterinary care in mouse colonies. Clinical success requires rapid identification; thus observations by investigators as well as the animal care team can be very helpful in addressing these situations.

• Dystocia : Difficulty in delivery of pups is one of the most common and clinically difficult conditions in mice. Signs of dystocia include a pup visible in the vaginal canal but not passing, immobility and dehydration, distension of the abdomen with little muscle tone, or labor for an extended period of time (more than a couple hours). Mice often deliver their pups during the night, so early morning health checks are the most common time to discover dystocia. Causes may include congenitally deformed or dead pups, very large pups, breach presentation, non-dilated vaginal canal, or exhaustion of the dam. A stuck pup may be removed by gently applying a lubricant around the pup, grasping the pup with gauze, and exerting gentle traction on the pup. However, if the mother is weak, she is unlikely to push out additional pups. Caesarian delivery of the pups and fostering to another breeder with pups the same age or slightly older is recommended for valuable lines. Provision of warm fluids subcutaneously, soft diets, and treatment with oxytocin and calcium may be of benefit.
• Prolapse of vaginal or uterine tissue : May be secondary to vaginal hyperplasia, or excessive abdominal contractions. If minor, the exposed tissue may be cleaned, treated with a hyperosmotic solution to reduce swelling, and replaced via the vagina. A suture may be used to close the vaginal opening for a few days (mice have a separate external urethral opening so closure of the vagina temporarily is okay). However, if the amount of tissue is large or there is evidence of necrosis or self mutilation then euthanasia is indicated.
• Prolapsed penis (paraphimosis) : Occurs in male mice when the penis is not retracted into the surrounding prepuce. The usual presentation is a swollen, dragging penis, often with secondary trauma to the surface skin. Blockage of the urethra may also be noted. If the mouse is able to urinate, lubrication and placement on a soft bedding surface may allow the swelling to decrease and the penis to return to the normal position.
• Perineal cysts : The bulbo-urethral glands may become filled with fluid giving the appearance of a severely enlarged scrotum or perineum. Needle aspiration yields a clear slightly yellow fluid. Generally no treatment is needed.

General conditions

Other general conditions that may be noted include:

• Diarrhea : Noted as liquid feces when the animal is picked up or seen in the cage bedding. Diarrhea can lead to dehydration, so treatment is similar. Antibiotic therapy may also be beneficial.
• Ascites : The buildup of fluid in the abdomen may be induced by a study; in which case the inclusion of endpoint guidelines is critical. It can also indicate organ failure of the heart or liver, neoplasia, or lymphatic malfunction. There is no long term treatment for this condition.
• Anasarca : The buildup of fluid in the subcutaneous space, also indicative of organ failure, usually renal or lymphatic, is another indication for euthanasia. In examining a mouse this may initially be confused with obesity. Pitting edema, lethargy, and distribution of subcutaneous fluid will aid in differentiation of these conditions.
• Rectal prolapse : A bulging of the distal colon out of the rectum, common in mice affected by Helicobacter spp. or intestinal parasites; but also caused by straining, constipation, or unspecific reasons. The health status of the colony will help in determining possible causes. Some respond well to hyperosmotic soaks, but many remain chronic and are managed by cleansing the exposed tissue, providing soft bedding, and in some cases using antibiotic and lubricant treatments as well.
• Anorexia : While not directly observable, this is indicated when there is a lack of feces in a cage that has not just been cleaned, when there is no evidence of mice chewing on the chow, or when mice appear too thin or dehydrated. The first thing to check is the animal’s teeth for possible malocclusion then palpate for any masses in the abdomen and generally examine the mouse. Supportive care includes fluid therapy and feed on the floor.

Background characteristics

Background strain/stock characteristics include clinical presentations such as deafness, blindness, and hyperactivity. When choosing a research model or establishing a new genetically engineered line, these need to be considered. See Table 2 .

Clinical Presentations Associated with Strain or Background

Developed from information in: Hedrich, H.J., Bullock, G., and Petrusz, P., 2004 ; Percy, D.H., and Barthold, S.W., 2007

DEFINING AND REFINING ENDPOINTS

Important for refining the way we conduct animal research is identifying appropriate endpoints, and facilitating ways to adhere to those endpoints in practice. Endpoints are meant to minimize or eliminate pain or distress, when possible, and enhance animal well being. The Guide defines a humane endpoint as “(T)he point at which pain or distress in an experimental animal is prevented, terminated or relieved” ( National Research Council, 2011 ).

Frequent health evaluations may be required to identify animals approaching a study’s endpoint, and these observations play an important role in assuring humane animal research. In some instances, the endpoint described in animal study proposals is a compromise between the humane endpoint and the experimental endpoint, the time at which scientific aims and objectives are met ( National Research Council, 2011 ). The Principal Investigator, in collaboration with a veterinarian and the Institutional Animal Care and Use Committee (IACUC), is responsible for identifying a study endpoint that is both scientifically relevant and humane before animal studies commence( Morton and Griffiths, 1985 ).

Morbidity endpoints are preferred and considered more humane than moribundity or death endpoints because they allow interventions or treatments that prevent pain and distress. However, in cases when veterinary intervention interferes with experimental results, moribundity/death as an endpoint may be required to reach experimental goals. Some examples of scientifically justified moribundity/death models include: metastatic tumor models, infectious disease/vaccine challenge, pain modeling, trauma, production of monoclonal antibodies, toxicology, organ/system failure, and cardiovascular shock ( National Research Council, 2011 ).

Identifying Animals Nearing Endpoint

Multiple criteria may indicate an animal has reached its study endpoint. While it is possible to describe an exhaustive list, Table 3 presents commonly referenced categories and criteria that can be used when planning a study and deciding on an appropriate study endpoint. It is not uncommon for an animal to show multiple clinical signs listed in the protocol while remaining below a study’sdescribed endpoint. In such cases, evaluation of the five criteria of an animal's condition as described by Morton and Griffiths is useful. These are: body weight, physical appearance, measurable clinical signs, unprovoked behavior and response to external stimuli ( Morton and Griffiths,1985 ). These criteria, when considered with objective measurements and in consultation with a veterinarian, can help identify animals at the earliest endpoint. It is also important to note that clinical signs may arise spontaneously that are not described in an approved study protocol. Attending veterinarians may, based on the animal’s overall condition, consider an animal to be at its humane endpoint and recommend euthanasia.

Clinical Signs and Evaluation Criteria Used to Determine Humane Endpoints

Special Considerations of Endpoints

Endpoint evaluation criteria presented in Table 3 is a good foundation for evaluating laboratory mice. Some conditions, such as aging studies warrant extra consideration when defining study endpoints. Aging mice often exhibit a host of clinical signs that would indicate disease in younger mice (Office of Animal Care and Use, National Institutes of Health, 2011), including decreased body condition, increased respiratory rate, and pallor; but these may be considered normal in an aging mouse. More subjective criteria, such as quality of life and general health may be used.

Genetically Modified Animals (GMA) is an example of studies where unexpected experimental outcomes may occur. Small pilot studies and additional screening/monitoring are recommended since the effects of the genetic modifications can be largely unknown ( Stokes, 2000 ).One should also consider the use of body condition scores, rather than body weight, in the determination of endpoints in models involving tumors, ascites, and other diseases that cause organomegally. These conditions all can falsely increase body weight, despite concurrent muscle wasting and cachexia.

Importance of Training and Monitoring

Adequate training of staff to recognize clinical signs and identify animals at endpoint is key to minimizing pain and distress. When the nature or severity of an animal’s condition is in question, consultation with a veterinarian is useful not only in determining a diagnosis but in training oneself and the staff to recognize various disease states. Additionally, there are numerous training resources available to help staff become familiar with these clinical signs, such as the Charles River Handbook of Clinical Signs in Rodents and Rabbits ( Pritchett-Corning, 2010 ) .

The use of study specific records that detail both the assessment criteria for endpoints as well as the animal’s condition can play a key role in the effective monitoring of animals. Such a document would ideally include: the definition of the study endpoint and assessment criteria, frequency of observation, and the response required when the animal has reached the study endpoint (Office of Animal Care and Use, National Institutes of Health, 2011). Animal users and animal care staff can communicate more efficiently, and animals can be identified for intervention/euthanasia at the earliest possible time point, preventing or alleviating unnecessary pain or distress--a refinement by definition.

In summary, when we choose endpoints thoughtfully and use trained staff to monitor animals frequently, we improve the welfare of laboratory mice by minimizing pain or distress. Using training resources, key references on endpoints, and study-specific monitoring records lend to our mission of refining the way we do research on animals.

Fight wounds. (A) Typical pattern of miliary wounds on the side of the body. (B) Deep wound on the caudal portion of the rump. (C) Wounds and associated swelling on the tail.

Ear Dermatitis. Crusty lesions on and below the ear associated with loss of an eartag.

Barbering. (A) Thin coat with short stubby hairs on the head and neck. (B) Two mice, one with minor and one with extensively barbered fur. Note that the skin is healthy in these cases.

Ulcerative dermatitis, deep ulcerative lesion with redness and moist surface at the base of the neck.

Ringtail. Circular constriction is noted with normal skin coloration. There may be one ring as shown or a series of rings around the tail.

Tumor. A subcutaneous irregular mass is shown caudal to the front leg of this nude mouse.

Conjunctivitis. The left eye demonstrates swelling of the eyelids, redness of conjunctiva, and serous discharge. Compare to the normal right eye.

Keratitis. The left cornea is white, opaque, and dry. Compare to the normal right eye.

Retrobulbar abscess. Swelling and discoloration are noted below and caudal to the orbit of the left eye. Compare to the normal right eye.

Arthritis. The mouse on the left has swelling of the hock (ankle) joint associated with arthritis. A normal mouse is shown on the right.

Malocclusion. The incisor teeth are unequal in length and the shorter tooth angles inward more than normal. Teeth may be observed to grow inward or outward, and may be very curved or broken as shown.

Runts. A runt or small mouse is shown on the left compared with its normal littermate on the right.

Imperforate vagina. Both are female mice. The mouse on the left lacks the normal vaginal opening and shows subcutaneous perineal swelling due to accumulated secretions which are unable to drain. Normal mouse is shown on the right.

Bulbourethral gland cyst. Swelling in the right side of the scrotum is caused by a cystic bulbourethral gland. This can be confirmed by performing a needle aspiration, with fluid indicating a cyst while no fluid may be indicative of a tumor.

Ascites. Fluid accumulation within the abdomen leads to a potbellied appearance with prominent spine.

Rectal prolapse. Red, edematous mucosal tissue is seen bulging from the rectal orifice.

Internet Resources with Annotations:

http://phenome.jax.org Mouse phenome database maintained by The Jackson Laboratory with detailed phenotype strain survey data. The Jackson Laboratory site www.jax.org has links to many valuable resources such as mouse nomenclature, resource manuals, and specific strain information.

http://www.ors.od.nih.gov/sr/dvr/od/Documents/Post_Op_Form.docx A link to a post-op monitoring form that may be useful to either the investigative team and/or the veterinary team.

An example of commercial laboratory services for health surveillance can be found at: http://www.criver.com/en-US/ProdServ/ByType/ResAnimalDiag/Pages/home2.aspx

File name: Mouse Health Evaluation Video.mov

Title: “Health Evaluation of Experimental Laboratory Mice”

Legend: Instructional video detailing normal behavior, home cage observation, restraint, body conditioning, and physical examination of experimental laboratory mice.

File name: Circling in Laboratory Mice.mov

Title: “Circling in Laboratory Mice”

Legend: An example of circling in a laboratory mouse.

File name: Ataxia in Laboratory Mice.mov

Title: “Ataxia in Laboratory Mice”

Legend: An example of ataxia in a laboratory mouse.

LITERATURE CITED

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Key References with Annotations

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August 27, 2024

Keto diet enhances experimental cancer therapy in mice

At a glance.

• Researchers showed how a ketogenic diet can enhance the effects of an experimental anti-cancer drug and starve pancreatic tumors in mice.
• The findings suggest that diet might be paired with drugs to block the growth of certain types of cancerous tumors.

Cancer cells need fuel to survive and thrive. The energy they need usually comes from glucose in the blood. Some studies have shown that intermittent fasting or a ketogenic diet—high in fat and low in carbohydrates—can help to protect against cancer. These cause the body to break down fat to form molecules called ketones, which can serve as the body’s main energy source while glucose is scarce. Fasting and ketogenic diets likely work by limiting the amount of glucose available to feed cancer cells. But some cancers, such as pancreatic cancer, can also use ketones as an energy source.

A research team led by Dr. Davide Ruggero of the University of California, San Francisco, set out to better understand the underlying gene activities and metabolic pathways affected by diet and fasting. They hope to use this knowledge to enhance cancer therapies. The team focused on a protein called eIF4E (eukaryotic translation initiation factor), which is often hijacked by cancer cells. Results appeared in Nature on August 14, 2024.

The researchers found that chemical tags called phosphates are added to eIF4E as mice transition from fed to fasting. Further analyses showed that this phosphorylated eIF4E (P-eIF4E) plays an important role in coordinating the activity of genes involved in processing fats for energy during fasting. When mice were placed on a ketogenic diet instead of fasting, the P-eIF4E protein similarly triggered a shift to using fat for energy.

The scientists next asked how fasting activated eIF4E. They found that free fatty acids, the small molecules released by fat shortly after fasting begins, activated a chain of events leading to eIF4E phosphorylation. This suggests that free fatty acids have a dual role, serving both as an energy source and as signaling molecules that boost fat-based energy production during fasting.

To assess the relevance of these findings to cancers that can thrive on fat, the researchers combined a ketogenic diet with an experimental anti-cancer drug that blocks P-eIF4E. The drug is called eFT508 (or tomivosertib). They found that giving eFT508 alone did not slow the growth of pancreatic tumors in mice, likely because the tumors could survive with energy from carbohydrates. But when mice were given the drug while on a ketogenic diet, the cancer cells no longer had ready access to glucose or fat for energy. The cells then starved, and growth declined.

"Our findings open a point of vulnerability that we can treat with a clinical inhibitor that we already know is safe in humans,” Ruggero says. “We now have firm evidence of one way in which diet might be used alongside pre-existing cancer therapies to precisely eliminate a cancer.”

—by Vicki Contie

Related Links

• Research in Context: Obesity and Metabolic Health
• An mRNA Vaccine To Treat Pancreatic Cancer
• Keto Molecule Offers Clue for Preventing Colorectal Cancer
• Low-Fat Diet Compared to Low-Carb Diet
• Nutrition in Cancer Care
• Pancreatic Cancer

References:  Remodelling of the translatome controls diet and its impact on tumorigenesis. Yang H, Zingaro VA, Lincoff J, Tom H, Oikawa S, Oses-Prieto JA, Edmondson Q, Seiple I, Shah H, Kajimura S, Burlingame AL, Grabe M, Ruggero D. Nature . 2024 Aug 14. doi: 10.1038/s41586-024-07781-7. Online ahead of print. PMID: 39143206.

Funding:  NIH’s National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Cancer Institute (NCI), and National Institute of General Medical Sciences (NIGMS); American Heart Association; American Cancer Society; Howard Hughes Medical Institute.

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• Published: 28 August 2024

Single-cell transcriptomic analysis of retinal immune regulation and blood-retinal barrier function during experimental autoimmune uveitis

• Joel Quinn 1 ,
• Ahmed Salman 1 ,
• Christopher Paluch 2 , 3 , 4 ,
• Matthew Jackson-Wood 2 ,
• Michelle E. McClements   ORCID: orcid.org/0000-0002-2941-4464 1 ,
• Jian Luo 5 ,
• Simon J. Davis 3 , 4 ,
• Richard J. Cornall 6 , 7 ,
• Robert E. MacLaren   ORCID: orcid.org/0000-0002-3096-4682 1 , 8 ,
• Calliope A. Dendrou 9 , 10 &
• Kanmin Xue   ORCID: orcid.org/0000-0003-0065-9131 1 , 8 , 11  

Scientific Reports volume  14 , Article number:  20033 ( 2024 ) Cite this article

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• Autoimmunity
• Neuroimmunology

Uveitis is characterised by breakdown of the blood-retinal barrier (BRB), allowing infiltration of immune cells that mediate intraocular inflammation, which can lead to irreversible damage of the neuroretina and the loss of sight. Treatment of uveitis relies heavily on corticosteroids and systemic immunosuppression due to limited understanding of disease pathogenesis. We performed single-cell RNA-sequencing of retinas, as well as bulk RNA-sequencing of retinal pigment epithelial (RPE) cells from mice with experimental autoimmune uveitis (EAU) versus healthy control. This revealed that the Th1/Th17-driven disease induced strong gene expression changes in response to inflammation in rods, cones, Müller glia and RPE. In particular, Müller glia and RPE cells were found to upregulate expression of chemokines, complement factors, leukocyte adhesion molecules and MHC class II, thus highlighting their contributions to immune cell recruitment and antigen presentation at the inner and outer BRB, respectively. Additionally, ligand-receptor interaction analysis with CellPhoneDB revealed key interactions between Müller glia and T cell / natural killer cell subsets via chemokines, galectin-9 to P4HB/TIM-3, PD-L1 to PD-1, and nectin-2/3 to TIGIT signalling axes. Our findings elucidate mechanisms contributing to breakdown of retinal immune privilege during uveitis and identify novel targets for therapeutic interventions.

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Introduction.

Uveitis is a major cause of blindness, contributing to 10–15% of severe visual impairment, particularly in the working age 1 , 2 . It encompasses a group of disease entities that present with inflammation of the iris, ciliary body, vitreous, retina or choroid, which is associated with breakdown of the blood-retinal barrier (BRB) and infiltration of immune cells. While some cases of uveitis are linked to infections (such as Mycobacterium tuberculosis ), many appear to be autoimmune or autoinflammatory in nature and associated with systemic diseases such as sarcoidosis, HLA-B27-associated spondyloarthropathies, Behçet’s disease, and multiple sclerosis. Current treatment options for non-infectious and posterior uveitis (inflammation centred on the retina and choroid) rely heavily on systemic immunosuppression, which is hampered by variable efficacy and severe adverse effects 3 . This is in part due to a poor understanding of disease pathogenesis.

Retinal glial cells (mainly Müller glia and some astrocytes) and retinal pigment epithelial (RPE) cells are important constituents of the inner and outer BRB, respectively. These physical barriers tightly regulate the movement of immune cells and molecules in and out of the neuroretina, thus reducing the risk of inflammatory damage of the non-regenerating neurons 4 , 5 . Previous work has suggested that Müller glia and RPE have direct suppressive effects on lymphocyte activation 6 , 5 , 8 , although this biological aspect of the BRB remains poorly understood. Characterization of changes in gene expression in the BRB and retinal cells between the physiological state and ocular inflammation could help reveal key immune interactions and identify therapeutic targets for immune interventions.

The advent of single-cell RNA-sequencing (scRNA-seq) as a tool for studying gene expression in individual cells has greatly improved our understanding of tissue heterogeneity and enabled in-depth characterization of novel cellular subpopulations. Moreover, differential gene expression analysis of specific cell types in response to disease or stimulus is greatly improved by scRNA-seq, as contaminating populations can be identified and filtered out. This facilitates discovery of key genes and cell types that are important to tissue function or disease pathogenesis. Here, we use a combination of scRNA-seq of the retina and bulk RNA-sequencing of the RPE to study changes in the local immune environment of the murine retina in the most faithful mouse model of human endogenous uveitis. In this model, immunisation against a retinal antigen, interphotoreceptor retinoid-binding protein (IRBP), leads to experimental autoimmune uveitis (EAU) which typically onsets between 10–14 days and peaks between 17–22 days in C57BL/6 mice 9 , 8 , 9 , 12 . Through differential expression and ligand-receptor interaction analyses, we found that both Müller glia and RPE undergo wide-ranging transcriptional changes and upregulate genes that contribute to immune cell recruitment from the circulation. In addition, both cell types may be capable of antigen-presentation through upregulation of MHC Class II genes which we also confirmed at the protein level. This supports a hypothesis where, during inflammation, BRB cells shift from an anti-inflammatory to a complex state of anti- and pro-inflammatory gene expression, that not only permits but actively promotes leukocyte entry into the retina. Thus, targeting these acquired BRB-leukocyte interactions may represent an attractive local treatment approach for posterior uveitis, and may be applicable to other neuroinflammatory diseases of the central nervous system (CNS) where the similar blood–brain barrier exists.

Single-cell transcriptomic analysis of EAU retinas

To explore the transcriptomes of inflamed versus healthy retinas at single cell resolution, 6–10 week-old female C57BL6/J mice were immunised against the retinal antigen peptide IRBP and imaged for the presence of uveitis at day 14 and day 21 post-immunisation (Fig.  1 A). At day 21, one healthy retina from an untreated mouse and two retinas with grade 2 EAU 13 (Fig.  1 B,C) from two immunised mice were harvested and dissociated into single cells. Droplet-based scRNA-seq (10X Genomics Chromium system) captured a total of 11,516 cells following quality control (Fig.  1 D, Supplementary Table S1 ). Clusters corresponding to retinal cell types were identified using marker genes, including rod photoreceptors ( Rho, Nrl, Nr2e3; n  =  6376 cells ), cone photoreceptors ( Arr3, Opn1mw; n = 1189 cells), Müller glia ( Slc1a3, Rlbp1, Glul; n  =  622 cells ), rod bipolar cells ( Prkca, Sebox, Trpm1; n  =  919 cells ), cone bipolar cells ( Lhx4, Vsx2, Scgn; n  =  1309 cells ), amacrine cells ( Calb2, Slc6a9; n = 452 cells), a small cluster of retinal pigment epithelial (RPE) cells ( Rpe65, Rdh5, Rlbp1; n  =  35 cells ), as well as a cluster of immune cells ( Ptprc; n  =  614 ) (Fig.  1 E, Supplementary Table S2 ). Annotated retinal cell types were found in similar proportions in the two EAU samples, while non-microglial immune cells came almost exclusively from EAU retinae, as expected (Supplementary Fig. S1 , Supplementary Table S3 ).

Single-cell transcriptomic analysis of experimental autoimmune uveitis (EAU) versus healthy retinas in mice. ( A ) Study outline. 1 retina per mouse from 2 mice with grade 2 EAU and 1 control littermate mouse (all females) were dissociated 21 days post-immunisation. ( B ) In vivo optical coherence tomography (OCT) of the mouse retinas immediately prior to tissue harvesting for scRNA-seq analysis. Arrows indicate vitreous immune cells; arrowheads indicate subretinal immune infiltrates associated with structural disruption of retinal layers. ( C ) Immunostaining of healthy and EAU retinas for CD45 (in red), showing infiltrating immune cells on the inner retinal surface and within the outer retina in EAU. Asterisk (*) indicates an aggregate of immune cells, likely within an inflamed deep capillary vessel, in the outer plexiform layer (OPL). Arrow indicates immune cells in the vitreous. Arrowhead indicates subretinal immune cells. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS/OS, inner segment-outer segment junction of photoreceptors. ( D ) Integrated UMAP of 11,516 cells from 1 healthy and 2 EAU retinas with annotated cell types. ( E ) Dot plot of marker genes used to identify each cell type.

Focused data analysis on the retinal immune cell population further separated this into 10 clusters (Fig.  2 A, Supplementary Table S3 ). An additional cell cluster of 66 cells containing presumed low quality cells and possible DCs was removed for downstream analysis. Unbiased marker gene identification showed the presence of microglia ( P2ry12 , Tmem119; n  =  115 cells from all retinas ), monocytes ( Cd14 , Fn1; n  =  65 cells ), neutrophils ( Csf3r , S100a8; n  =  34 ), plasmacytoid dendritic cells ( Klk1 , Tcf4; n  =  28 cells ), Th1 cells ( Cd3d , Cd4, Ifng; n  =  42 cells ), Th17/γδ cells ( Cd4 , Il17a, Rorc, Tcrg-C1; n  =  29 cells ), CD8 + T cells ( Cd8a , Gzmb; n  =  70 cells ), Treg cells ( Foxp3 , Il2ra; n  =  46 cells ), naïve CD4 + T cells ( Cd3d , Cd4 , S1pr1; n  =  73 cells ) and natural killer (NK) cells ( Klrb1c , Ncr1; n  =  41 cells ) (Fig.  2 B).

Single-cell analysis of infiltrating immune cells in inflamed retinas. ( A ) UMAP of the 548 immune cells captured in the two retinas with autoimmune uveitis. ( B ) Dot plot of selected immune cell marker genes. Black arrow highlights Tcrg-C1 as a marker in Th17/γδ T cells. ( C ) Stacked violin plots showing expression of all TCR γ genes detected in the dataset. Greater expression of Tcrg-C1 was present in the Th17/γδ cluster, suggesting γδ T cells are significant IL-17 producing cells in EAU retinas. ( D ) IHC of EAU retinas confirming presence of intraretinal CD3 + TCR γ/δ + T cells (inset). Scale bar = 50 μm.

Interestingly, from unbiased marker gene identification Tcrg-C1 was identified as a marker gene for the Th17 cell cluster (Fig.  2 C), and immunostaining confirmed the presence of intraretinal γδ T cells (Fig.  2 D), suggesting that γδ T cells are present and contribute to IL-17 production in the retina during uveitis. In addition, the T cell receptor (TCR) β gene Trbv3 was defined as a significant marker gene for the Th1 cell cluster, and was also expressed in the Th17 cluster (Supplementary Fig. S2 ). However, it should be noted that the 10X 5’ kit is not able to distinguish between nascent and recombined TCR chains without specific TCR-seq.

Differential gene expression analysis of retinal cells

To examine the immunological roles played by retinal cells during intraocular inflammation, we identified differentially expressed genes between the EAU and healthy retinas by pseudobulk differential expression analysis with DESeq2. All cell types displayed a number of differentially expressed genes apart from RPE, which was likely due to the small number of cells captured, as the number of differentially expressed genes appeared to correlate with the number of cells in the cluster (Supplementary Fig. S3 ; Supplementary Dataset 1 ). Preranked Gene Set Enrichment Analysis (GSEA) was then performed on differentially expressed genes with the Molecular Signatures Database (MSigDB) Hallmark gene sets. We found a general enrichment for inflammation-associated gene sets among all annotated retinal cell types, with rods, cones and Müller glia showing the greatest number of significantly enriched gene sets (Fig.  3 A; Supplementary Dataset 2 ). Interferon-gamma (IFN-γ) and interferon-alpha (IFN-α) response gene sets were significantly upregulated by rods, cones and Müller glia, as well as showing positive enrichment in other retinal cell types, suggesting signalling through these cytokines is one of the major mechanisms by which retinal cells respond to inflammatory conditions during autoimmune uveitis directed against IRBP. Leading edge genes for inflammatory response gene sets in rods and cones were largely involved in upregulation of MHC Class I proteins ( H2-K1, H2-D1, B2m, Tap1 ; Supplementary Fig. S4 A) and the interferon regulatory factors ( Irf1, Irf8, Irf9 ; Supplementary Fig. S4 B). Interestingly, the rods and cones also demonstrate significant alterations in the expression of oxidative phosphorylation gene set, which was downregulated in rods but upregulated in cones. Upregulated oxidative phosphorylation leading edge genes in cones included cytochrome c oxidase subunits ( Cox4i1 , Cox5b , Cox6b1 ) and ATP synthase subunits ( Atp5h , Atp5g1 , Atp5e ; Supplementary Fig. S4 C). Downregulated oxidative phosphorylation leading edge genes in rods were primarily from the Mitochondrial Complex I ( Ndufa1 , Ndufs2 , Ndufa4; Supplementary Fig. S4 D). This observation provides a potential mechanistic link between retinal inflammation and impaired visual function via changes in photoreceptor ATP production.

Activation of inflammation-associated gene sets in retinal cells during experimental autoimmune uveitis. ( A ) Summary heatmap of Gene Set Enrichment Analysis (GSEA) using the Molecular Signatures Database (MSigDB) Hallmark Gene Sets. The Müller glia, rods and cones, in particular, demonstrate significant upregulation of a range of proinflammatory gene sets, as denoted by black dots (adjusted p value < 0.05). Inflammation-associated gene sets that were significantly upregulated in at least one retinal cell type are highlighted in red. ( B ) Gene Ontology (GO) Biological Process gene sets that were significantly enriched in Müller glia. Leading edge analysis of significantly enriched gene sets shows upregulation of ( C ) chemokines, ( D ) complement factors/receptors, ( E ) leukocyte adhesion molecules and ( F ) MHC Class II genes. (ns) Not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, DESeq2 Wald test. ( G ) Validation of MHC Class II protein expression by Müller glia in inflamed retina of EAU mice. Glutamine synthetase (GS) staining of Müller glia in yellow, MHC-II in cyan, scale bar = 50 μm. Inset images highlight an area showing co-staining of GS and MHC-II along Müller glia processes (arrows). Bright patches of confluent MHC-II high only staining (arrow heads) likely represent infiltrating blood-derived professional antigen presenting cells (APCs). ( H ) Immunostaining of EAU retina for CD45, CD4 and MHC-II. Four arrows indicate areas where CD45 + CD4 + T cells colocalize with CD45 - MHC-II + regions (corresponding to Müller glia processes as seen in panel ( G )). These colocalizations appear distinct from the interaction between CD45 + CD4 + T cells and the MHC-II high only blood-derived professional APCs (single arrowhead). Insets (i) and (ii): colocalization between CD45 + CD4 + T cells and CD45 - MHC-II + processes in the outer plexiform layer and ganglion cell layer, respectively, suggestive of antigen presentation by the Müller glia.

To focus on gene expression changes in the Müller glia, which are thought to play a critical role in the regulation of inner blood-retinal barrier function, we performed additional preranked GSEA with the Gene Ontology (GO) Biological Process gene sets (Fig.  3 B; Supplementary Dataset 3 ). This confirmed a predominant inflammatory response gene expression profile, with significantly enriched gene sets exclusively consisting of those associated with immunity. Leading edge analysis of the significantly upregulated GO Biological Process gene sets identified several chemokines ( Ccl2 , Cxcl10 , Cxcl1, Cxcl12 ) and leukocyte adhesion molecules ( Icam1 , Vcam1 ) as contributing to enrichment scores (Fig.  3 C,D). This suggests that Müller glia contribute to the recruitment and subsequent adhesion of circulating immune cells during autoimmune uveitis. Additionally, upregulation of complement factors and receptors ( C1ra , C3 , C4b ) and MHC Class II genes ( Cd74 , H2-Aa , H2-Ab1 , H2-Eb1 ) indicate their active roles in tissue damage and antigen presentation, respectively (Fig.  3 E,F). The latter was validated at the protein level by immunohistochemical co-staining of the Müller glia marker, glutamine synthetase (GS) and MHC-II in EAU but not healthy retina (Fig.  3 G). Additionally, non-leukocyte-mediated MHC-II expression likely attributed to Müller glia colocalized with CD45 + CD4 + cells, indicating possible antigen presentation (Fig.  3 H). However, this was relatively rare in comparison to leukocyte-mediated strong expression of MHC-II colocalizing with CD45 + CD4 + cells, the major antigen presenting cells during experimental autoimmune uveitis.

Predicted retinal cell-leukocyte interactions during EAU

Given that Müller glia are important regulators of the inner BRB and appear to increase expression of molecules associated with leukocyte recruitment and adhesion during EAU, we interrogated their potential ligand-receptor interactions with leukocytes using CellPhoneDB. Ligand-receptor interaction analysis across all annotated cell types during EAU suggested Müller glia were one of the main cell types interacting with immune cells in the retina (Fig.  4 A). In total, 594 interactions were predicted between Müller glia and leukocytes. Of interest, IFN-γ, lymphotoxin-α, lymphotoxin-β and TNF-α produced by lymphocytes were predicted to interact with their corresponding receptors on Müller glia (Fig.  4 B), further supporting the notion that these cytokines may drive the proinflammatory transcriptional changes. Müller glia also express TGF-β2, which is predicted to interact with its receptor primarily found on CD8 + and regulatory T cells (Fig.  4 C, Supplementary Fig. S5 ).

Immune ligand-receptor interactions between retinal cells and infiltrating immune cells. ( A ) Summary heatmap of ligand-receptor interactions between retinal cells and immune cells during experimental autoimmune uveitis using CellPhoneDB. ( B ) Extensive cytokine signalling between infiltrating lymphocytes and Müller glia was detected. Presence of a dot represents a significant interaction between two cell types, while the size of the dot is proportional to the mean expression level of the ligand-receptor pair. Ligands and ligand-expressing lymphocyte labels are coloured red, while receptors and receptor-expressing Müller glia are in black. IFN-γ produced by CD4 + T cells and NK cells is predicted to interact with the IFN-γ receptor expressed on Müller glia. CD4 + T cells also produce TNF-α and LT-α which may interact with their cognate receptors on Müller glia. ( C ) TGFB2 expressed by Muller glia was predicted to interact with TGFB receptors on CD8 + T cells, monocytes, neutrophils, pDCs, Th17/γδ T cells and Tregs. ( D ) Predicted chemokine-receptor interactions between Müller glia and lymphocytes. CXCL10, CXCL12 and CXCL16 expressed by Müller glia are predicted to interact with their receptors expressed by various immune cells. ( E ) Violin plots depicting expression of Cxcr6 , Cxcr4 and Cxcr3 on immune cell subsets. Cxcr6 was most highly expressed on Th1 and Th17/γδ T cells. Although Cxcr4 was expressed on several immune cell subsets, highest expression appeared to be on monocytes and neutrophils. Cxcr3 was most highly expressed on CD8 + T cells, Tregs, Th1 cells and pDCs. ( F ) Immune checkpoint ligand-receptor interactions between Müller glia and lymphocytes. Galectin-9 ( Lgals9 ) expressed by Müller glia is predicted to interact with a several range of receptors on immune cells, including TIM-3 ( Havcr2 ) expressed on Tregs. PD-L1 ( Cd274 ) expression is upregulated in Müller glia during EAU and predicted to interact with its receptor PD-1 expressed on T cells. Nectin-2 and Nectin-3 expressed by Müller glia are also predicted to interact with their receptor TIGIT expressed on T cells and NK cells. ( G ) Violin plots showing expression of the checkpoint receptors Tigit , Havcr2 and Pdcd1 . Tigit was most strongly expressed by T and NK cell subsets, while Pdcd1 showed higher expression on Tregs, Th1 and Th17/γδ T cells. Havcr2 expression by comparison was lower across immune cell subsets.

Müller glial expression of the chemokines CXCL10, CXCL12 and CXCL16 was predicted to interact with leukocytes via their receptors CXCR3, CXCR4 and CXCR6, respectively (Fig.  4 D,E). Cxcl10 was also found to be significantly upregulated by Müller glia by differential expression between EAU and healthy retinas, thus indicating an acquired interaction that only occurs during autoimmune uveitis. The CXCL12-CXCR4 interaction was predicted to occur between Müller glia and all leukocyte subsets apart from naïve CD4 + T cells, with highest expression of Cxcr4 occurring in neutrophils and monocytes (Fig.  4 E). This may reflect a broad leukocyte recruitment strategy used by Müller glia during inner blood-retinal barrier breakdown. Finally, the CXCL16-CXCR6 interaction was predicted to occur exclusively between Müller glia and T cells, with the highest expression of Cxcr6 occurring in the Th1 and Th17/γδ subsets (Fig.  4 E). Thus, this may be an important signalling axis for recruiting pathogenic T cells to the retina.

Interestingly, Müller glia appear to provide a complex range of costimulatory and coinhibitory signals to the infiltrating T cells and natural killer (NK) cells in the retina via a number of different immune checkpoint ligand-receptor interactions (Fig.  4 F,G). For instance, the inhibitory galectin-9-TIM-3 interaction was to occur between Müller glia and Th17 cells, Tregs, microglia and monocytes. Furthermore, inhibitory interactions between Müller glia and all T cell subsets were predicted to occur through the PD-L1-PD-1, nectin-2-TIGIT and nectin-3-TIGIT axes. In contrast, Müller glia-derived galectin-9 was also predicted to interact with the cell surface protein disulfide-isomerase, P4HB, on Th1, CD8 + T cells, naïve T cells, Tregs, NK cells, monocytes, microglia and pDCs. This interaction may enhance T cell migration in the retina 14 ; however, the role of this interaction in myeloid cells is unclear. Together, these results suggest that Muller glia may directly provide immunomodulatory signals to infiltrating immune cells during uveitis.

Transcriptomic analysis of the retinal pigment epithelium during EAU

The healthy RPE monolayer with tight junctions consisting of claudins, occludin and other junctional adhesion molecules (JAMs) between hexagonal cells constitute the physical outer blood-retinal barrier. Due to the technical limitation of capturing only a small number of RPE cells during retinal harvesting for single cell RNA-sequencing, we performed separate bulk RNA-seq analysis of pooled RPE isolated from control (n = 3) and EAU (n = 2) posterior eyecups (Fig.  5 A,B). Principal Component Analysis (PCA) revealed that the majority of variation between samples was driven by the condition, with EAU and healthy RPE showing good segregation (Fig.  5 C).

Bulk RNA-seq and immunohistochemical analysis of the retinal pigment epithelium (RPE) during homeostasis and experimental autoimmune uveitis. ( A ) Experimental outline of bulk RNA-seq of EAU RPE, with cells harvested from eyecups at 21 days post-immunisation. ( B ) OCT of eyes from each of two mice that provided the EAU RPE samples showing characteristic features of posterior uveitis. ( C ) PCA plot of analysed samples showing variation between samples is primarily by condition. ( D ) Volcano plot showing the 824 significantly differentially expressed genes from DESeq2 analysis. Top 30 differentially expressed genes are labelled. ( E ) Preranked GSEA results of EAU RPE showing significantly enriched gene sets from the Molecular Signatures Database Hallmark gene sets. ( F ) Heatmap of leading edge genes from inflammation-associated gene sets. All displayed genes are significantly differentially expressed between EAU and healthy RPE (p < 0.05, DESeq2 Wald Test). Inflammation-associated leading edge genes showed upregulation of chemokines, complement factors and MHC-II genes. Leading edge analysis of the Epithelial-to-Mesenchymal Transition (EMT) signature genes included extracellular matrix proteins and matrix metalloproteinases ( Mgp , Bgn , Pcolce , Serpine1 ), as well as typical transition markers ( Vim , Inhba ) and adhesion molecules ( Vcam1 , Itgav ). ( G ) Immunohistochemistry co-staining of RPE65 (yellow) and MHC-II (cyan) in EAU retina. Left, Hoechst staining of photoreceptor nuclei (blue); asterisk (*) highlights a cluster of infiltrating subretinal MHC-II hi immune cells. Dashed box denotes the RPE layer. Right top, RPE65 staining of RPE cells. Right middle, MHC-II staining predominantly at the basal surface of the RPE layer; asterisk indicating MHC-II hi immune cell located on apical side of RPE. Right bottom, merge of RPE65 and MHC-II staining with arrows indicating regions of strongest co-staining, suggestive of expression of MHC-II by the RPE during retinal inflammation. ( H ) Inset: an infiltrating (subretinal) CD45 + CD4 + T cell on the apical surface of the RPE layer. However, we did not identify overlap between MHC-II and RPE65 staining, suggesting this was not RPE-mediated. No evidence of colocalization of CD45 + CD4 + cells with RPE at the basal surface was found in analyzed sections. Scale bars = 50 μm.

Using a similar differential expression pipeline with DESeq2 and subsequent pre-ranked GSEA, we found 824 differentially expressed genes between EAU and healthy RPE (Fig.  5 D; Supplementary Dataset 4 ) and a similar enrichment of pro-inflammatory gene sets (Fig.  5 E; Supplementary Dataset 5 ). Leading edge analysis of the pro-inflammatory gene sets again revealed significant upregulation of a number of chemokines, complement and MHC-II genes (Fig.  5 F). As molecular evidence of breakdown of the outer blood-retinal barrier, we also found significant enrichment of the ‘Epithelial-to-Mesenchymal Transition’ (EMT) gene set in the RPE of eyes with active uveitis. Leading edge genes included mesenchymal markers ( Vim , Vcam1 , Spp1 , Inhba ), as well as proteases and extracellular matrix components ( Mgp , Bgn , Serpine1 , Pcolce ) (Fig.  5 F).

As with Müller glia, MHC Class II expression by RPE was validated at the protein level by IHC, with expression colocalizing with RPE65 predominantly on the basal side of the RPE layer during EAU (Fig.  5 G). We were able to identify CD45 + CD4 + T cells among infiltrating immune cells in the subretinal space (Fig.  5 H). However, unlike for Müller glia, we were unable to identify colocalization between such CD4 + T cells and MHC-II + RPE cells within the analysed sections, which would suggest this to be a relatively rare interaction.

Here, we present the first single cell transcriptomic characterization of immunization-induced experimental autoimmune uveitis in mice as a model for human posterior uveitis. Retinas with active grade 2 EAU at 21 days post-immunization were compared against healthy control by scRNA-seq profiling of retinal cell populations and immune infiltrate, revealing changes in a range of immune-related genes associated with breakdown of the blood-retinal barrier. Several manifestations of human posterior uveitis have been shown to be driven by a mixed Th1/Th17 immune response through cytokine and flow cytometric analyses 15 , 14 , 15 , 16 , 17 , 20 . The mouse EAU model of retinal inflammation has been shown to be Th1/Th17-driven 21 , 22 , thus recapitulating the inflammatory cytokine milieu in human disease and is suitable for exploring clinically relevant immune interactions.

Our results reveal a major role for Müller glia in regulating inner BRB function through their production of chemokines and proinflammatory cytokines, as well as immune checkpoint ligand-receptor interactions with leukocytes during retinal inflammation. In addition, we supplemented the analysis with bulk RNA-seq of RPE from EAU to define gene expression changes in the main cellular constituent of the outer BRB. This identified a similar shift of the RPE toward a leukocyte-recruiting and antigen-presenting state during retinal inflammation. Using GSEA, we showed that inflammation-associated gene sets are upregulated in Müller glia and other retinal cells during EAU. Similar pathways were also found to be upregulated in the RPE. Notably, the ‘Response to Interferon Gamma’ gene set was significantly upregulated in both Müller glia and RPE, in keeping with previous observations in the Aire knockout mouse retina, which displayed a purely Th1-driven uveoretinitis caused by abolished central tolerance to retinal antigens 23 . Additionally, recent work has revealed an IFN-γ-responsive population of Müller glia in the retina which is poised to respond to injury in an optic nerve crush model 24 . Ligand-receptor interaction analysis showed that infiltrating T cells producing IFN-γ, TNF-α and lymphotoxins-α and -β are likely to be the main source of these proinflammatory cytokine signals to the Müller glia.

Interestingly, we also found that Müller glia moderately upregulate Cxcl1 , a chemokine that predominantly recruits neutrophils via CXCR1/CXCR2 receptors and is a target for upregulation by IL-17A. Indeed, a population of infiltrating neutrophils was present in the EAU retinas, whereas previously reported Aire -/- retinas did not appear to have a notable neutrophil infiltrate 23 . This suggests that Müller glia may alter gene expression broadly in response to IFN-γ, while IL-17A may subtly shape the chemokine expression profile during uveitis. This hypothesis is corroborated by previous work in a mouse model of central nervous system (CNS) neuroinflammation describing gliosis without blood–brain barrier breakdown, leukocyte infiltration or tissue damage in a mouse overexpressing IL-17A in astrocytes under the control of a GFAP promoter 25 .

Ligand-receptor interaction analysis indicates that Müller glia-produced chemokines, CXCL10, CXCL12 and CXCL16, are likely to be important in the recruitment of immune cells to the retina during inflammation. CXCL10 (also known as IP-10) is a chemokine that is upregulated in response to IFN-γ to target a variety of immune cells. Our data suggest the main cells targeted by CXCL10 are likely to be all T cell subsets, as well as NK cells and plasmacytoid dendritic cells (pDCs). CXCL10 levels have also been found to be increased in ocular fluids from patients with uveitis, suggesting these interactions parallel those in human disease 26 , 27 . CXCL12 expressed by Müller glia has an even broader range of target cells, with all infiltrating immune cells apart from naïve T cells being implicated. Its target receptor, CXCR4, has previously been shown to be important in leukocyte trafficking in both an ovalbumin-induced uveitis model and an adoptive transfer EAU model 28 , 29 . In both cases, the CXCR4 antagonist, AMD3100, attenuated leukocyte infiltration into the eye during disease induction, possibly due to the near pan-leukocyte targeting activity as seen in our data. Finally, the CXCL16-CXCR6 interaction was predicted to occur exclusively between Müller glia and T cells. CXCR6 has previously been reported as a marker for pathogenic effector T cell populations in experimental autoimmune encephalomyelitis (EAE) 30 . These effector T cell populations were preferentially recruited to the CNS where CXCL16 expression was found to be greater, thus a similar mechanism of T cell recruitment may exist in the retina.

Our results provide evidence that both Müller glia and RPE may be capable of antigen presentation via MHC Class II during retinal inflammation. Some controversy surrounds non-leukocyte MHC-II expression in the retina, as a previous study concluded that MHC-II expression was not attributable to retinal glial cells or RPE based on immunohistochemical co-staining for select markers 31 . However, our findings are consistent with previous in vitro studies of RPE cells and single cell transcriptomic analysis of Aire −/− retina suggesting MHC-II expression during inflammation 23 , 32 , 33 , 34 . Retinal glia 35 and RPE 33 , 36 have been shown to be able to stimulate T cells in culture following treatment with inflammatory cytokines, but in vivo evidence is scarce. MHC-II-mediated interactions between Müller glia/RPE and T cells may be rare, spatially restricted, transient or occur at specific phases of the disease course that make it challenging to find evidence of by IHC. We identified putative spatial colocalization of CD45 - MHC-II + Müller glia with CD4 + T cells in EAU retina, but functional antigen presentation is difficult to prove. Furthermore, as we and others have shown, MHC-II is expressed strongly and widely by infiltrating leukocytes, likely making these professional antigen presenting cells more important for T cell stimulation during retinal autoimmunity 37 . Thus, the functional consequences of in vivo MHC-II expression by Muller glia and RPE require further investigation. Even so, these findings may have significant implications for retinal gene therapy using adeno-associated viral (AAV) vectors since AAV transduction of RPE and Müller glia could lead to viral antigen presentation and clinically undesirable retinal immune cell infiltration 38 . Whether the retinal microglia are also capable of acting as antigen presenting cells remains uncertain, as it is currently difficult to distinguish infiltrating monocyte-derived macrophages from activated microglia without strict lineage tracing 39 , 40 .

In addition to this possible antigen-presenting role, our data also revealed that Müller glia may provide co-inhibitory signals to infiltrating lymphocytes through expression of the immune checkpoint ligands, PD-L1 ( Cd274 ), galectin-9 ( Lgals9 ), nectin-2 and nectin-3. PD-L1 has been studied as one of the mechanisms by which pigmented cells of the uvea are capable of suppressing T cell activation in vitro, and is upregulated in response to inflammatory cytokines 6 , 7 , 41 . Our observation of Müller glia-lymphocyte crosstalk via PD-L1 to PD-1 interaction provides a potential mechanistic explanation for the observation of spontaneous BRB breakdown and uveitis in patients receiving systemic PD-1 checkpoint inhibitors for cancer 42 . Galectin-9 is a member of a class of β-galactoside-binding proteins that often have pleiotropic effects with multiple binding partners 43 . For instance, the interaction of galectin-9 with TIM-3 functions as an inhibitory immune checkpoint with some beneficial effects in mouse models of autoimmune disease (including in the CNS), but in some cases the effect was not TIM-3-dependent, which highlights the complexity of galectin-9 signalling 44 , 45 . The role of galectin-9 in retinal inflammation remains poorly understood and potential benefit of targeted therapeutic intervention merits further investigation.

In order to capture an unbiased landscape of gene expression across retinal and immune cell populations, we analysed whole dissociated retinas. However, one of the limitations of this approach has been the capture of a relatively modest number of immune cells, an overrepresentation of rods, cones and bipolar cell, and underrepresentation of retinal endothelial cells, pericytes and perivascular macrophages which may coordinate their activities with Müller glia to regulate inner BRB function. Future work may focus on these rarer cell populations by enriching for non-neuronal populations prior to scRNA-seq. In addition, our data captures the transcriptional profile and cellular interactions during active retinal inflammation in EAU, while future work to characterize the temporal sequence of gene expression changes in BRB cells and infiltrating leukocytes as the disease onsets and subsides over the course of 6–8 weeks could provide further mechanistic insights.

Nonetheless, our data provide a wholistic molecular understanding of key local immune interactions during autoimmune retinal inflammation, which can help guide the development of targeted immunomodulatory interventions. While both Müller glia and RPE upregulate expression of chemokines that recruit immune cells to the retina and drive ocular inflammation, Müller glia also take part in numerous immune ligand-receptor interactions with the infiltrating leukocytes, which are likely to shape the severity and duration of tissue inflammation. Both Müller glia and RPE were found to upregulate MHC Class II, indicating a possible acquired antigen-presenting function during retinal inflammation. Furthermore, we observed an epithelial-to-mesenchymal transition signature in the inflamed RPE, which would contribute to outer BRB breakdown and correlate with clinical observations of pigment migration and secondary choroidal neovascularisation in posterior uveitis. Future work will aim to validate and characterize the functional importance of key BRB-leukocyte interactions at the protein level, with the aim of developing highly targeted therapeutics for uveitis and other neuroinflammatory diseases.

Materials and methods

All animals were maintained by the Biomedical Science Division, University of Oxford, UK. Mice were housed in a 12-h light–dark cycle, with food and water available ad libitum. All animal procedures were approved by the Oxford University Animal Welfare and Ethical Review Board (AWERB) and the UK Home Office. All work was undertaken in accordance with the Association for Research in Vision and Ophthalmology (ARVO) guidelines for the humane use of laboratory animals in ophthalmic research. Results are reported in accordance with the ARRIVE guidelines. Immunisations were performed under inhalational anaesthesia with isoflurane, and imaging was performed under general anaesthesia by intraperitoneal injection of a mixture of 80 mg/kg ketamine and 10 mg/kg xylazine.

Induction, imaging and scoring of EAU

Induction of EAU was performed by immunising 6 to 10-week-old, female C57BL/6 J mice with 500 μg of IRBP1-20 peptide (GPTHLFQPSLVLDMAKVLLD; synthesised by Merck) emulsified in a 1:1 ratio with Complete Freund’s Adjuvant (CFA) containing 2.5 mg/ml heat-killed Mycobacterium tuberculosis (MTb). Mice received 100 μl of emulsion as two 50 μl injections, one in each hind flank. Mice were then injected with 1.5 μg Bordetella pertussis toxin as a 100 μl intraperitoneal injection. Littermate control mice were maintained untreated. At specified timepoints, mice retinas were imaged in vivo using scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT). The level of retinal inflammation was then graded based on a previously published protocol 13 by two independent scorers who were blinded to the treatment of the mouse.

Immunohistochemistry and confocal microscopy

Mice were euthanised and eyes enucleated and cleaned of excess tissue. Eyes were placed in 4% paraformaldehyde on ice for 20 min, followed by a brief rinse in PBS and then cryoprotected in a sucrose gradient (10%, 20%, 30%) at 4 °C. Eyes were then briefly incubated in optimal cutting temperature medium and frozen in moulds on dry ice. 16 μm sections were cut at − 20 °C with a cryotome and positioned onto Superfrost plus slides (VWR). Slides were washed in PBS, then blocked with 5% BSA, 5% serum of the secondary antibody host. Slides were then incubated with primary antibodies (full list of antibodies provided in Supplementary Table S4 ). overnight at 4 °C in a solution containing 1% BSA, 1% serum in PBS. Slides were washed with 0.05% Tween-20 in PBS, rinsed in PBS then, when necessary, stained with secondary antibodies under dark conditions for 2 h at room temperature. Slides were then briefly washed with 0.05% Tween-20 in PBS before counterstaining with Hoechst for 30 min in the dark. Coverslips were mounted with ProLong Diamond and sealed. Z-stack images of sections were captured on a LSM 710 confocal microscope (Zeiss) and data analysed in ImageJ.

Preparation of retinas for single-cell RNA-sequencing

After imaging at day 21 post-immunisation, mice were euthanised by cervical dislocation and eyes were enucleated. The retinas were carefully dissected out and placed into Hanks’ Balanced Salt Solution (HBSS) containing 10 mM HEPES on ice for transport. After all retinas were collected, they were dissociated using the Worthington Papain Dissociation system. Retinas were placed in a solution of 20 U/ml papain, 0.005% DNase I with 1 mM L-cysteine and 5 mM EDTA in Earle’s Balanced Salt Solution (EBSS), for 10 min at 37 °C with frequent, gentle agitation. Samples were then diluted by addition of 500 μl of EBSS to inactivate the papain and centrifuged at 300 ×  g for 5 min at room temperature. Pellets were resuspended in 525 μl of a solution containing 1 mg/ml ovomucoid and BSA and 100 U/ml DNase I in EBSS. The resulting suspension was carefully layered over 500 μl ovomucoid/BSA solution and centrifuged at 70 ×  g for 6 min. Supernatant was discarded and cells were resuspended in a solution of PBS containing 0.04% BSA.

Single-cell RNA-seq library generation and processing

Library generation from dissociated retinas and initial data processing was performed by the Oxford Genomics Centre. scRNAseq transcriptome processing was performed using the Chromium 10X system involving GEM generation, post GEM-generation clean-up, cDNA amplification and DNA quantification. Chromium Single Cell Reagent Kits solution (10X SC RNA CITE-TSC, 10X SC RNA 5pr v2.0 Chemistry) was used to deliver a scalable microfluidic platform for digital scRNA-seq by profiling 500–10,000 individual cells per sample. A pool of ~ 3,500,000 10 × Barcodes were sampled separately to index each cell’s transcriptome. Libraries were generated and sequenced from the cDNAs and 10 × Barcodes were used to associate individual reads back to the individual partitions. The library was sequenced using the Illumina NovaSeq platform.

For initial data processing, Illumina’s bcl2fastq and cellranger mkfastq demultiplexes were used to convert the raw base call (BCL) files generated by Illumina sequencers into FASTQ files. Cellranger was used to perform alignment, filtering, barcode counting, and UMI counting from FASTQ files. This generated feature-barcode matrices for each sample, which was used for downstream analyses.

Analysis of single-cell RNA-seq data

Analysis was performed with the R programming language. The SoupX package 46 was used to correct raw feature-barcode matrices for ambient RNA contamination. Corrected matrices were then analysed using the Seurat package 47 . Commonly used QC metrics, such as UMI count, number of features, percentage mitochondrial RNA and percentage ribosomal RNA were used to filter out low quality cells. Samples were then normalised using the SCTransform function in Seurat, and integrated to remove batch effects and generate a single feature-barcode matrix.

The integrated matrix was then passed through standard dimensionality reduction and clustering pipelines in Seurat. Briefly, Principal Component Analysis (PCA) was used to determine dataset dimensionality, followed by shared nearest-neighbour graph construction and dimensionality reduction with the Uniform Manifold Approximation Projection (UMAP) method. Visualisations were performed using in-built functions in Seurat. Differential expression was performed by pseudobulking each identified cell type by sample and analysing with the DESeq2 package 48 . Ligand-receptor interaction analysis was performed with CellPhoneDB package in Python using the statistical analysis method 49 . DESeq2 and CellPhoneDB heatmaps were produced using the pheatmap package in R.

RPE isolation

RPE were isolated according to a previously established protocol 50 . Briefly, mice were euthanized and eyes were enucleated and cleaned of excess tissue. The cornea and lens were dissected out and eyecups were placed in a solution of HBSS containing 10 mM HEPES and 1 mg/mL hyaluronidase for 45 min at 37, 5% CO 2 . Eyecups were then placed in a solution of 10 mM HEPES in HBSS (with Ca/Mg), on ice for 30 min. Retinas were removed and the remaining eyecup was incubated in 0.25% trypsin–EDTA solution at 37C, 5% CO 2 for 45 min. RPE were isolated by gently shaking eyecups to dislodge RPE cells into a solution of 10 mM HEPES in HBSS (with Ca/Mg). Each sample contained RPE from both eyes from one mouse.

Bulk RNA-seq of RPE

RNA was extracted from samples using the Qiagen microRNeasy kit according to manufacturer’s protocol. Sequencing libraries were prepared using the NEBNext ® Ultra™ II Directional RNA Library Prep Kit for Illumina® according to the manufacturer’s protocol. During this process, the libraries were indexed using NEBNext® Multiplex Oligos for Illumina ® (Index Primers Set 4). The prepared libraries were quantified via a fluorometric method involving an Invitrogen Qubit dsDNA assay and qualified using electrophoretic separation on the Agilent BioAnalyzer 2100. This concentration and sizing information was used to calculate the molarity of each sample prior to pooling and sequencing of 150 bp paired-end reads using the Illumina NovaSeq 6000 platform. Sequencing reads were processed using the Nextflow (v22.04.5) 51 nf-core RNA-Seq pipline (v3.8) 52 . The quality of reads was assessed using FastQC where a mean Phred score > 30 was observed across the full length of reads across all samples. Reads were then pseudo aligned to the mouse reference genome (GRCm38) using Salmon (v1.5.2) 53 with > 65% of reads aligning across all samples. Transcript counts were converted to gene level counts using the tx2gene function from the R package tximport 54 . These were used for subsequent differential expression analyses of RPE in healthy vs uveitis with DESeq2. Tx2gene was also used to generate length normalised (transcript per million, TPM) counts for subsequent plotting.

Data availability

Code used for analysis is available on GitHub ( https://github.com/JoelQuinn ). Raw and processed single cell RNA-seq and bulk RNA-seq files have been deposited in the NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE241700 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241700 ).

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Acknowledgements

The project was funded by the Wellcome Trust (K.X., J.Q., C.A.D.), Oxford-MRC Doctoral Training Partnership (J.Q.), National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC) (A.S., M.E.M., J.L., R.E.M.) and the Medical Research Council (MRC) (C.P., S.J.D.). Figures 1 A and 5 A were created with BioRender.com.

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J.Q., S.J.D., R.C., R.E.M., C.A.D. and K.X. designed research; J.Q., A.S., M.E.M. and K.X. performed the research, J.Q., M.J-W., C.P., J.L. and C.A.D. analyzed the data; J.Q. A.S., C.P., M.J-W., M.E.M, J.L, S.J.D., R.C., R.E.M., C.A.D and K.X. wrote the main manuscript text. J.Q., A.S., C.A.D. and K.X. prepared Figs.  1 – 5 . J.Q., C.A.D and K.X. prepared supplementary Figs.  1 – 6 and supplementary tables 1 – 4 .

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Quinn, J., Salman, A., Paluch, C. et al. Single-cell transcriptomic analysis of retinal immune regulation and blood-retinal barrier function during experimental autoimmune uveitis. Sci Rep 14 , 20033 (2024). https://doi.org/10.1038/s41598-024-68401-y

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Pharmacological Modulation of Cognitive Test Solution in Mice of Two Genotypes

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Mice of two strains selected for successful solution of “object permanence” test and for lack of such solution demonstrated the differential reaction to injections of two drugs. The effects of injections of atomoxetine. which blocks the noradrenaline reuptake, and of ‘non-benzodiazepine” anxiolytic afobazol was different. The success of solutions increased in mice selected for this test “non-solution”: and decreased or was inefficient in mice, selected for successful solution of object permanence cognitive test.

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O. V. Perepelkina & I. I. Poletaeva

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Correspondence to I. I. Poletaeva .

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ETHICS APPROVAL AND CONSENT TO PARTICIPATE

All experiments were conducted in accordance of EC Directive 63-2010 on the use of animals in scientific research in the frame work of the research “Physiological and genetic bases of cognitive abilities in laboratory mouse.” Approved by Moscow State University Bioethical Committee 18.06.2014, No. 49.

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Perepelkina, O.V., Poletaeva, I.I. Pharmacological Modulation of Cognitive Test Solution in Mice of Two Genotypes. Dokl Biochem Biophys (2024). https://doi.org/10.1134/S1607672924701096

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Received : 10 May 2024

Revised : 20 June 2024

Accepted : 25 June 2024

Published : 28 August 2024

DOI : https://doi.org/10.1134/S1607672924701096

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Peroxiredoxin 6 is a natural radioprotector

Affiliations.

• 1 Institute of Cell Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, Pushchino, Moscow oblast, 142290, Russia. [email protected].
• 2 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, pr. Nauki 3, Pushchino, Moscow oblast, 142290, Russia.
• 3 Prokhorov Institute of General Physics, Russian Academy of Sciences, ul. Vavilova 38, Moscow, 119991, Russia.
• 4 Pushchino State Natural Science Institute, Pushchino, Moscow oblast, 142290, Russia.
• 5 Lobachevskii Nizhny Novgorod State University, Nizhny Novgorod, 603600, Russia.
• 6 Institute of Cell Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, Pushchino, Moscow oblast, 142290, Russia.
• PMID: 27193711
• DOI: 10.1134/S1607672916020095

After injection of 20 mg/kg peroxiredoxin 6 to male Kv:SHK mice 15 min before X-ray irradiation in the range of lethal doses (7-10 Gy), the mice remained alive for 30 days, whereas the mortality of the control animals was 100%. In the irradiated animals, peroxiredoxin 6 decreased the severity of radiation-induced leucopenia, granulocytopenia, and thrombocytopenia, increased the number of blood corpuscles, and prevented the mass death of epithelial cells and the destruction of the small intestine. Thus, peroxiredoxin 6 can be regarded as a prophylactic radioprotective agent.

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