Streptozotocin Induced Diabetes Mellitus Animal Model

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.

 

So far, Streptozotocin (STZ) induced DM animal model is considered to be the most widely used in diabetes research, which is suitable for long-term observation. STZ is a DNA alkylating agent that enters cells exclusively via the GLUT2 glucose transport protein and specifically ablates the β-cell via 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.

 

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1. SOP--STZ induced DM animal 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 (170-200g) and mice (17-22g) 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

Conventional chow included 10% fat, 20% sucrose, 10% egg yolk powder, 0.5% sodium cholate

② STZ-sodium citrate buffer

50 mM Sodium citrate buffer (pH 4.5): prepare solution A (0.1 M citric acid monohydrate, FW=210.14) and solution B (0.1 M trisodium citrate, dihydrate, FW=294.12) with distilled water. Mix solution A and solution B in 1:1 or 1: 1.32 ratio. Adjust pH to 4.2-4.5. Sterilize with 0.22μm membrane filter. Use it right after it was ready.

Weigh appropriate amount of STZ into a sterile glass bottle and cover with aluminum foil. Dissolve the STZ with 50 mM sodium citrate buffer (pH 4.5) to a final concentration of 10 mg/mL or 20 mg/mL. Sterilize with 0.22μm membrane filter.

【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

After fasting animals for 6-12 h, administer STZ to the experimental animals through intraperitoneal injection or tail vein injection. Compared with intraperitoneal injection (i.p.), tail vein injection (i.v.) has higher drug utilization efficiency, but the operation is more difficult. Because STZ degrades within quickly after dissolving in the citrate buffer, the STZ solution should be prepared immediately before use and injected within 30 min of dissolution. The recommended dosage is as follows (For reference only):

T1DM: For mice, repeated low-dose of STZ (20-50 mg/kg, i.p., 5 consecutive days) or a single, high dose of STZ (100-200 mg/kg, i.p., once); For rat, a single dose of STZ (40-70 mg/kg, i.p., once).

T2DM: After fed with high-fat and high-sugar diet for 4-6 weeks, for mice, a single dose of STZ (70-120 mg/kg, i.p., once); For rat, a single dose of STZ (25-40 mg/kg, i.p., once).

【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

If the model fails the standard, STZ shall be injected after the animal is stable (i.p., 10-20 mg/kg STZ, the appropriate dose shall be chosen according to the actual situation), or the routine dose shall be injected after the blood glucose returns to normal. However, to achieve the desired effect, it is often necessary to wait animals to restore to the normal state and remould.

 

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 deliquesce and should be kept dry to avoid moisture. Do not keep the powder at room temperature for a long time. Dissolve STZ with acid pH value, preferably in an ice bath. The dissolved STZ is very unstable, so prepare the STZ solution immediately before use and inject within 30 min of dissolution.

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.

 

Ordering Information

Name	Catalog #
Streptozocin (Inquire)	60256ES

 

 

Citations & References (Incomplete statistics)

 

 

 

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)