Diabetes mellitus (DM), a systemic chronic metabolic disease characterized by chronic elevated blood glucose caused by a combination of multiple disease factors, is a global health epidemic and related to family genetics, environmental factors, and autoimmunity. Even though DM known for millennia and great advances has been achieved in its diagnosis and management, there is currently no cure for the disease and its public health consequences are only growing. Therefore, it is particularly important to establish an appropriate animal model of DM and clarify the pathogenesis of DM and its complications. The commonly used DM animal models are surgical resection of pancreas, chemically induced diabetes, spontaneous diabetes animal model and transgenic animals, etc.
Currently, the diabetes disease model induced by Streptozotocin (STZ) is widely used, suitable for long-term observation. STZ is a nitrosourea compound that, once inside the body, can specifically destroy pancreatic β-cells through the following mechanisms:
(1) Injection of high-dose STZ may cause the concentration of intracellular coenzyme I (NAD) decreases in β-cells, and thus inhibit NAD dependent energy and protein metabolism, resulting in cell death;
(2) STZ increases nitric oxide (NO) production, which has been demonstrated to participate in β-cell damage;
(3) Administered at a low dose, STZ can trigger an autoimmune process leading to destruction of the β-cells of the pancreatic islets: dead β-cells, due to a low dose of STZ, can be phagocytized by macrophages as antigens to produce Th1 stimulating factor, making Th1-type lymphocytes dominant. Then Th1-type lymphocytes produces IL-2 and IFN- γ, causing the infiltration of inflammatory cells in the islet and the release of IL-1 and TNF- α, IFN- γ, NO and H2O2 to kill cells, eventually inducing DM.
Yeasen Biotech provides high success rate STZ (Cat#60256ES) for modeling: high purity ≥ 98% (HPLC).
1 Standard SOP for the construction of the STZ-induced diabetes model
1.1 Animal preparation
There is marked sexual dimorphism and males are more susceptible to develop diabetes. As estrogen interferes with STZ action, female animals are less sensitive to diabetogenic action of STZ than the male. Studies have shown that females have a poor modeling rate and a higher mortality rate than males, especially type I.
Type I diabetes(T1DM): Rats (170-200 g) and mice (17-22 g) are recommended. After one week of adaptive feeding, fast animals for 12 h followed by intraperitoneal injection of STZ, which can be easily operated and with high success rate.
Type II diabetes(T2DM): Rats (age 4-5 weeks, weigt 90-100 g, e.g SD or Wistar) and mice (age 4-5 weeks, weigth 16-20 g, e.g C57, ICR or Kunming) should be fed with high-fat and high-sugar diet for 4-6 weeks prior to STZ administration, and the body weight can be individually reach about 240-280g and 30-35g. SD is recommended for rats and C57 for mice.
1.2 Animal Feeding before STZ administration
T1DM: Allow all mice or rats free access to standard rodent chow diet and water 1-2 weeks of adaptive feeding prior to STZ treatment.
T2DM: Consumption of a high-fat and high-sugar diet to render insulin resistant prior to STZ treatment.
1.3 Reagent preparation
① High-fat and high-sugar diet
The high-fat and high-sugar diet is made by mixing basic rat feed with sucrose, refined pork fat, and egg yolk powder in a mass ratio: the proportions are 10% pork fat, 20% sucrose, 10% egg yolk powder, 0.5% sodium cholate, and 59.5% basic feed.
② STZ-sodium citrate buffer
Preparation of Solution A and Solution B: Weigh 2.1 g of citric acid (MW: 210.14) and dissolve it in 100 mL of double-distilled water to make Solution A; weigh 2.94 g of sodium citrate (MW: 294.10) and dissolve it in 100 mL of double-distilled water to make Solution B.
Preparation of Sodium Citrate Buffer: Mix solutions A and B in a certain ratio (1:1.32 or 1:1), adjust the pH to 4.2-4.5, and filter sterilize using a 0.22 μm filter membrane. This yields the required sodium citrate buffer; it is recommended to prepare and use it immediately.
Weigh the STZ lyophilized powder, place it in a dry, sterile bottle, wrap with aluminum foil, and put it on ice. Add pre-cooled sodium citrate buffer (1% w/v) to dissolve, and filter sterilize using a 0.22 μm filter membrane.
【Note】① After the STZ powder is taken out from the refrigerator, it shall be kept at room temperature and away from light for about 10 minutes until it’s completely thawed. ② After weighing, the bottle containing the sample of STZ must be covered with aluminum foil to protect it from light, as STZ is unstable. ③ Do not dissolve STZ at one time if you are not skilled in injection. It is recommended to dissolve STZ in groups according to the operation proficiency. Prepare STZ solution for one group at one time such as 10 or 15 rats /mice per group.
1.4 STZ injection
Administer the injection intraperitoneally or via the tail vein based on the animal's fasting weight. Compared to intraperitoneal injection, tail vein injection has a higher drug utilization efficiency but is more technically challenging. If the injection technique is not proficient, alternate between two groups for the injection, and the injection should be completed within 30 minutes.
T1DM: For mice, a single high dose is recommended at 100-200 mg/kg, and a multiple low doses at 20-50 mg/kg for five consecutive days; for rats, the dose is suggested to be 40-70 mg/kg, administered in a single injection.
T2DM:After feeding with a high-sugar and high-fat diet for 1-2 months, for mice, a dose of 70-120 mg/kg is recommended, administered in a single injection; for rats, the suggested dose is 25-40 mg/kg, administered in a single injection.
【Note】It is recommended to conduct pre-experiment to determine the appropriate dosage of STZ, due to different weight, drug tolerance, fasting time, injection method and feeding process of experimental animals. Do not blindly carry out the experiment directly according to the dosage in literatures.
1.5 Post-injection
After the injection of STZ, allow the animals free to water and food. Change the padding every day. Keep the cage clean and dry. Avoid strong sunlight. Disinfect as often as possible.
【Note】After STZ injection, animals’ blood glucose level will show three phases: transient hyperglycemia (1-2 h), transient hypoglycemia (6-10 h), and continuous hyperglycemia (>72 h). Insulin and glucose must be properly provided.
1.6 Remedy of animal modeling
For models that do not meet the criteria, STZ can be administered additionally once the animal has stabilized (with an intraperitoneal injection at a dose of 10-20 mg/kg, choosing an appropriate dose based on the actual situation), or wait until blood glucose levels return to normal and then inject at the routine dose. However, to achieve the desired effect, it is often necessary to restart the modeling process after returning to a normal state.
2 Evaluation of STZ induced DM animal models
① General characteristics: loss of body weight, polydypsia, polyphagia and polyuria.
② Fasting blood glucose (FGB), serum insulin level (FINS), oral glucose tolerance (OGT), fasting serum insulin (FSI) and insulin sensitivity.
③ Serum biochemical indexes: T-Cho、TG、HDL-C、LDL-C、CR、BUN、Alt, etc.
④ Pathology of pancreas: H&E staining.
3 Possible reasons for the failure of STZ induced DM model
① Poor quality of STZ. The purity of STZ for modeling shall not be less than 98% (HPLC detection).
② STZ degradation. STZ is easy to damp solution, should be kept dry, to avoid moisture. The powder avoids long room temperature placement, and the dissolved STZ is very unstable, with a half-life of neutral pH of 15 min, and should be used in place. Please dissolve the STZ with an acidic pH value, preferably in an ice bath.
③ STZ solution was injected into intestines or other organs.
If the model fails the DM standard, it is recommended to observe for another 3 days. If still fails, repeat the injection procedure.
4 The causes of high mortality of animals induced by STZ
① Animals are underweight.
② Insufficient supply of drinking water.
③ Hyperglycemia or hypoglycemia, usually hyperglycemia. This can be alleviated by insulin injection or temporary glucose supplementation.
Insulin supplement method: for example, if Novolin N or NPH (neutral protamine zinc insulin) is given for 2-3 units each time, the general mortality of rats will be lower after 3-5 days.
Glucose supplement method: Intraperitoneal injection of 20% glucose 4 hours after STZ injection can avoid the death of rats due to low blood glucose causing by fasting.
④ Experimental animals kill each other due to lack of food and water supply.
⑤ Infection. DM animals are more prone to infection than others, especially urinary tract infection and abdominal infection, due to polyuria. It is necessary to disinfect before and after invasive operations such as intraperitoneal injection, subcutaneous injection and blood collecting. For example, tetracycline (or aureomycin eye ointment) can be applied to the wound after blood glucose measurement each time to prevent infection.
5 Factors affecting diabetes modeling
Factors affecting the diabetes disease model include the quality of the STZ modeling reagent, the condition of the animals, and the method of administration. The main performance characteristics of the reagent include purity, stability, and solubility. The condition of the animals mainly includes genetic background, sex, gender, weight, breeding environment, and dietary structure. Methods of administration include timing of administration, intervals between doses, and routes of administration. Differentiated factors bring about differentiated modeling effects.
6 Product recommendation
|
Product name |
Cat# |
Specification |
|
Streptozocin (STZ) |
60256ES60 |
100 mg |
|
60256ES76 |
500 mg |
|
|
60256ES80 |
1 g |
|
|
Citric Acid, Monohydrate |
60347ES25 |
25 g |
|
Citric Acid Trisodium Salt, Dihydrate |
60348ES25 |
25 g |
|
Solvent For Streptozocin STZ |
60750ES76 |
500 mL |
7 Use the literature related to this product
[1] Xi Z, et al. Dual-modified nanoparticles overcome sequential absorption barriers for oral insulin delivery. J Control Release. 2022 Feb;342:1-13. (PMID: 34864116, IF:7.727)