Mouse Models of Autoimmune Diabetes: The Nonobese Diabetic (NOD) Mouse

Abstract

There are now a number of different mouse models for type 1 diabetes. The best known is the nonobese diabetic (NOD) mouse which has a genetic susceptibility to autoimmune diabetes with some features that are similar to human type 1 diabetes. The mice also have a propensity to other autoimmune diatheses, including autoimmune thyroid disease and sialadenitis. In addition, it is well known that environmental factors affect the incidence of disease in these mice. While there are other rodent models, including numerous transgenic and knockout models, as well as those that express human proteins, none of these develop spontaneous diabetes over a period of time, when the natural history can be studied. We focus here on the unmanipulated NOD mouse and discuss features of the husbandry and investigation of the mice that allow for use of these long-studied mice in the pathogenesis of an autoimmune type of diabetes.

1. Introduction

In humans and mice, type 1 diabetes (T1D) is an autoimmune disease characterized by the infiltration of immune cells, which results in the destruction of insulin-producing pancreatic β cells. The nonobese diabetic (NOD) mouse strain was originally developed by Makino and colleagues during the selection of a cataractprone strain (cataract Shionogi mice) derived from the outbred Jcl: ICR line of mice [1, 2]. In the pancreatic islets, immune cell infiltration starts at about 5–6 weeks of age in females, with a delay in the male mice. Mononuclear cell infiltrates surround the islet at this point (peri-insulitis), followed by subsequent invasion of the islets and the onset of overt diabetes [3]. Typically, female NOD mice start to develop diabetes between 10 and 14 weeks of age, whereas diabetes in male mice occurs later [3], and they have a lower incidence of disease. For this reason, many investigators choose to study female mice. However, it should be noted that the age at which diabetes first develops varies between colonies in different research institutions. Although B cells, NK cells, dendritic cells, neutrophils, and macrophages can also be identified in the lesions, the destruction of insulin-producing β cells in islets is primarily dependent on autoreactive CD8+ T cells helped by CD4+ T cells.

The most important genetic factor contributing to T1D in NOD mice is the MHC. NOD mice have the haplotype H2^g7 comprising K^d, Aa^d, Ab^g7, E^null, and D^b; the MHC-I molecules H-2K^d and H-2D^b are found in other strains of mice, but it is the MHC-II molecule I-A^g7 (ortholog of HLA-DQ) which is unique, and the mice do not express I-E (ortholog of HLA-DR) because of a defective Eα locus [3]. I-A^g7 contributes significant susceptibility to developing diabetes [4]. In addition to the importance of the MHC to the progression of T1D, non-MHC genes are also found to contribute to the development of T1D. To date, at least fifty insulin-dependent diabetes (Idd) loci on at least 11 mouse chromosomes have been identified (Idd2-Idd12, Idd13a, Idd13b, and Idd14-Idd19) through crosses of NOD mice with various diabetes-resistant strains [4, 5]. These Idd loci contribute to the development of T1D at either initiation of insulitis or progression to overt diabetes or both. These loci act in a collective, recessive manner to determine development of insulitis as well as disease frequency and severity [6].

The environment is also an important factor, as it is very clear that NOD mice in different colonies have different incidences of diabetes [7], and the length of time over which this develops also varies, as discussed below.

A number of antigen-specific T cells have been identified and cloned from the NOD mouse, and T cell receptor (TCR) transgenic mice on the NOD genetic background have been generated (reviewed in ref. 4).

In addition to these various strains, there are also a number of immunodeficient derivative strains of NOD mice, which may be useful for studying individual components of the immune response. These include those which are globally immunodeficient that include NOD.scid mice and NOD.RAG2−/− mice as well as alpha-beta TCR-deficient, B cell-deficient, and more specialized models where individual immune subsets can be depleted. It should be noted that this chapter will provide an introductory overview of care of NOD mice and will not be a comprehensive account of NOD and derivative mice, which now number in the hundreds, many of which are available commercially.

2. Materials

2.1. Housing of Animals

1. Specific pathogen-free facilities.

2. Isolator or scantainer with appropriate cages.

3. Bedding.

4. Diet.

5. Personal protective equipment, including gloves, scrubs or overalls, hair net, face mask (if needed), and shoe covers.

2.2. Monitoring Onset of Diabetes

1. Diastix urine test strips.

2. Glucometer and test strips.

3. Scalpel or surgical scissors.

4. Anesthetic spray, if needed.

3. Methods

3.1. Housing of Animals

The incidence of diabetes in NOD mice varies between different institutions and laboratories. It has been suggested that environmental factors are as important as genetic susceptibilities in shaping the risk of T1D development [4]. Many environmental factors in NOD mice have been implicated in altering diabetes susceptibility, including exposure to dietary factors such as wheat [8, 9] and gluten [9], exposure to infectious organisms [10–12], and changes to the gut microbiota [4]. The incidence of diabetes is highest when NOD mice are maintained in a relatively germ-free environment; however, this dramatically decreases when mice are exposed to dirty environments and/or infections [3]. Exposure to pinworms [13], Salmonella typhimurium [11], mouse hepatitis virus (MHV) [14], and Schistosoma mansoni [10] has all been associated with abrogation of diabetes development. Accordingly, consideration should be given to routine monitoring of colonies for health monitoring of breeding, and experimental animals should be undertaken according to Federation for Laboratory Animal Science Associations (FELASA) or similar recommendations [15].

Appropriate housing and dietary requirements need to be carefully considered within the host institution and animal housing facilities:

1. NOD mice are generally bred and maintained in specific pathogen-free (SPF) facilities that are carefully monitored for the presence of mouse pathogens. Thus, breeding pairs and small colonies can be kept in micro-isolators, although, more commonly, mice of this type are housed in individually ventilated cages or scantainers.

2. It is good practice to rotate breeders, and because of the propensity of the mice to develop diabetes, it is advisable to restrict the breeding to 2–3 litters, as the females will not be able to care for the litters if they develop diabetes (which can occur any time after the age of 10–14 weeks as above).

3. A new colony can be established with 2–4 breeding pairs. NOD mice are generally very good breeders, regularly producing litter sizes of eight mice or more. The colony may be maintained by brother-sister mating.

4. Limiting the number of staff members to access to the animal facilities may be useful, and all individuals involved in the care and use of the mice should be educated as to the needs for housing and monitoring experimental and potentially diabetic animals. Dependent on local institutional rules, researchers and caretakers should wear protective equipment, including hair coverings, gloves, scrubs, shoe covers or overalls, and masks, to prevent the spread of pathogens to the mice and exposure to allergens to humans.

5. Food, water, bedding, cages, and other materials that mice will contact should be sterilized or disinfected. Water should be acidified to pH 2.5–3.0 to control Pseudomonas species contamination.

6. Mice should be assessed for diabetes regularly, depending on the experimental conditions and strain (see Note 1). NOD female mice may develop diabetes at 10–14 weeks old. Wet bedding should be changed immediately.

7. Sentinel cages can be set up within each isolator and scantainer to monitor for infection. Fecal pellets may be collected and screened by PCR for infection by pinworm, for example.

3.2. Monitoring for Diabetes

Regular monitoring of mice for diabetes is important to maintain colony incidence and highlight any potential contamination that may interfere with studies. Monitoring the welfare of the mice is also critical to prevent undue stress to diabetic animals:

1. Animals are tested daily to weekly, depending upon experimental conditions (see Note 1) by urinalysis using Diastix test strips, in the first instance. A small drop of urine is placed on the test strip.

2. Following manufacturer’s directions, the test strip is assessed for any color change and compared to the scale provided.

3. If there is no color change, the mouse is likely to be nondiabetic.

4. If glycosuria is noted by a color change on the urine test, indicating positive urinalysis, the mouse should be retested 24 h later. If a positive urinalysis is confirmed, then blood glucose is measured.

5. Positive diabetes diagnosis is determined by blood glucose measurement:

   1. Spray tail with anesthetic spray if needed.
   2. Using a clean scalpel, the tip of the mouse tail is cut.
   3. A small drop of blood is placed on the test strip mounted in a glucometer; set up according to manufacturer’s directions (see Note 2).
   4. Diabetes is generally diagnosed with a blood glucose over 13.9 mmol/L (250 mg/dL).

6. Mouse gender and age are recorded to establish an incidence curve (Fig. 1) and should be monitored regularly to assess maintenance of a high incidence of diabetes. A decrease in the incidence of diabetes may be an early warning of infection.

7. Diabetic mice can be used for further experiments or culled by approved methods (see Note 3).

Fig. 1.

Incidence of spontaneous diabetes in NOD mice. NOD mice were observed over a period of 35 weeks and tested weekly for glycosuria with diabetes confirmed by a blood glucose of >13.9 mmol/l (250 mg/dl)