How to establish an animal model of diabetes
Diabetes mellitus (DM) is a chronic metabolic disorder characterized by elevated blood glucose levels resulting from multiple disease factors. It is associated with familial inheritance, environmental factors, and autoimmunity, posing a significant public health concern due to its severe impact on human health. The complications of DM not only severely affect the quality of life of patients but also contribute significantly to disability and mortality. However, the specific pathogenesis of DM remains incompletely understood. Therefore, establishing appropriate animal models of diabetes is crucial to elucidate the pathogenesis of DM and its complications.
Currently, various methods are used to prepare DM animal models, including surgical pancreatic resection, chemical induction, spontaneous diabetes models, and transgenic animals. Among these, the most widely used method is the diabetes model induced by Streptozocin (STZ), which is suitable for long-term observation. STZ is a compound containing a nitrosourea moiety that selectively damages pancreatic islet β cells through the following mechanisms:
1. Direct destruction of pancreatic islet β cells: Primarily observed after the injection of high doses of STZ. Injection of STZ can lead to a decrease in the concentration of coenzyme I (NAD) in β cells, causing NAD-dependent energy and protein metabolism to cease, resulting in β cell death.
2. Induction of nitric oxide (NO) synthesis to damage pancreatic islet β cells.
3. Activation of autoimmune processes by STZ, further leading to β cell damage: Low-dose injection of STZ can destroy a small number of pancreatic islet β cells. The dead β cells can be engulfed by macrophages as antigens, resulting in the production of TH1 stimulatory factors. This dominance of TH1 cell lines leads to the production of IL-2 and IFN-γ locally in the islets, promoting the infiltration of inflammatory cells and activating the release of substances such as IL-1, TNF-α, IFN-γ, NO, and H2O2, causing cellular damage. The dead cells can serve as autoantigens, presented again to antigen-presenting cells for processing, leading to the release of cytokines that amplify the cellular damage effect, ultimately triggering DM.
Arcegen provides a highly successful modeling STZ (Cat#C331605): High purity ≥ 98% (HPLC), product performance comparable to imported products, supported by literature data, and priced at less than half of imported products.
1. Standard Operating Procedure (SOP) for constructing the STZ-induced diabetes model
1.1 Animal Preparation
It is preferable to use male animals as females may be influenced by hormonal factors affecting modeling efficiency. Research has shown that female animals have lower modeling rates and may exhibit higher mortality rates compared to males, especially in Type I diabetes.
For Type I diabetes, animals can generally be selected based on body weight. It is recommended to use rats weighing between 170-200g and mice weighing between 17-22g. After acclimation for 1-2 weeks, animals should be fasted and injected with STZ on an empty stomach, resulting in a relatively ideal modeling rate.
For Type II diabetes in rats (such as SD/Wistar), animals aged 4-5 weeks with a weight of 90-100g should be selected. They should be fed a high-fat, high-sugar diet for 4-6 weeks until their weight reaches approximately 240-280g. For mice (such as C57/ICR/Kunming mice), animals aged 4-5 weeks with a weight of 16-20g should be selected. They should be fed a high-fat, high-sugar diet for 4-6 weeks until their weight reaches approximately 30-35g. Newly acquired animals or those transitioning to a new environment should be fed a standard diet for adaptation for one week before selecting appropriate age/weight animals to switch to a high-fat, high-sugar diet for continued feeding.
In terms of modeling success rate, SD rats are recommended for rats, and C57 mice are recommended for mice.
1.2 Pre-modeling Feeding
Pre-modeling Feeding: For Type I diabetes models, which typically develop more rapidly, rats can usually begin modeling after a 2-week period of acclimation to standard diet feeding. For Type II diabetes models: Induction with a high-fat diet combined with low-dose STZ administration, pre-modeling feeding involves providing a high-fat, high-sugar diet to induce insulin resistance.
1.3 Preparation of Reagents
① High-fat, high-sugar diet
The high-fat, high-sugar diet is prepared by mixing the base rodent diet with sucrose, lard, and egg yolk in the following proportions by mass: lard 10%, sucrose 20%, egg yolk powder 10%, sodium cholate 0.5%, and base diet 59.5%.
② Preparation of 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 prepare Solution A. Weigh 2.94 g of sodium citrate (MW: 294.10) and dissolve it in 100 mL of double-distilled water to prepare 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, filter sterilize using a 0.22 μm membrane filter, and the resulting solution is the required sodium citrate buffer. It is recommended to prepare fresh as needed.
Weigh the STZ lyophilized powder and place it in a dry, sterile bottle covered with aluminum foil, keep it on ice, and dissolve it in pre-cooled sodium citrate buffer (1% w/v). Filter sterilize using a 0.22 μm membrane filter.
Note
1. After removing the STZ lyophilized powder from the -20°C freezer, allow it to thaw completely at room temperature in a dry and dark place for about 10 minutes (very important).
2. STZ is unstable and easily inactivated. After quickly weighing the required amount, any remaining reagent should be kept dry and protected from light. It is recommended to wrap it in dry aluminum foil (or tin foil).
3. During injection, if not proficient in the procedure, avoid completely dissolving all the STZ at once. It is advisable to dissolve STZ in batches based on proficiency level, for example, in groups of 10 or 15 mice per batch. Alternatively, pre-weigh the required amount of STZ according to the number of animals in each group and divide it into portions.
1.4 Injection
Intraperitoneal or tail vein injection is performed on animals based on their fasting body weight. Compared to intraperitoneal injection, tail vein injection has higher drug utilization efficiency but is more challenging to perform. If not proficient in injection techniques, injections should alternate between two groups, and all injections should be completed within 30 minutes.
For Type I diabetes models:For mice, a single high dose is recommended at 100-200 mg/kg, or multiple low doses at 20-50 mg/kg injected continuously for five days.
For rats, a single dose is recommended at 40-70 mg/kg.
For Type II diabetes models:After 1-2 months of high-sugar, high-fat feeding, mice are recommended to receive a single dose at 70-120 mg/kg.
For rats, a single dose is recommended at 25-40 mg/kg.
Note: Pre-experimentation is necessary to determine the appropriate dosage as animal weight, fasting resistance, fasting time, injection technique, and feeding process may vary. Do not blindly follow literature dosages for experiments.
1.5 After Injection
After STZ injection, animals should be provided with sufficient water and food (basic living characteristics of diabetic rodents), and bedding should be changed daily to maintain dry cages. During housing, avoid exposure to direct sunlight and disinfect cages as frequently as possible.
Note: Following STZ induction, noticeable fluctuations in blood glucose levels occur in three phases: transient hyperglycemia (1-2 h), brief hypoglycemia (6-10 h), and sustained hyperglycemia (>72 h). Insulin and glucose supplementation must be appropriately administered.
1.6 Remediation for Substandard Models
For models that do not meet the desired standards, after stabilizing the animals, additional STZ can be administered (via intraperitoneal injection at a dosage of 10-20 mg/kg, selecting an appropriate dosage based on actual conditions), or wait until blood glucose levels return to normal before administering the regular dosage. However, achieving the desired effect often requires re-establishing the model under normal conditions.
2. Evaluation Indicators for STZ-Induced Diabetes Model
1. General Indicators:Polydipsia, polyphagia, polyuria symptoms, and weight loss.
2. Other Indicators:Fasting blood glucose levels, fasting serum insulin levels, serum insulin levels, insulin sensitivity, glucose tolerance, etc.
3. Serum Biochemical Indicators:Total cholesterol (T-Cho), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), C-reactive protein (CRP), blood urea nitrogen (BUN), alanine aminotransferase (ALT), etc.
4. Histopathological Sections:Pathological sections of pancreatic tissue.
3. Possible Reasons for Failure of STZ-Induced Diabetes Model
1. Poor Quality of STZ: STZ used for modeling should have a purity of not less than 98% (verified by HPLC analysis).
2. STZ Inactivation: STZ is susceptible to hydrolysis and should be stored in a dry environment to prevent moisture absorption. The powder form should not be left at room temperature for extended periods, and dissolved STZ is highly unstable with a half-life of 15 minutes at neutral pH. It is recommended to prepare STZ solutions immediately before use and preferably dissolve in acidic pH solutions, preferably in an ice bath.
3. Incorrect Intraperitoneal Injection: Failure to properly inject into the peritoneal cavity, resulting in injection into other organs such as the intestines.
For models that do not meet the desired standards, it is recommended to observe for an additional 3 days. If the model is still unsuccessful, re-administration of STZ is suggested.
(For more information on the use of STZ, please visit: https://www.Arcegen.com/products/detail/1002)
4. Analysis of High Mortality Rate in STZ-Induced Small/Big Rats
1. Low Body Weight of Rats:Rats with low body weight are more susceptible to adverse effects of STZ induction.
2. Inadequate Water Intake:Insufficient water intake can lead to dehydration and increase the risk of mortality.
3. Both Hyperglycemia and Hypoglycemia:Both high and low blood glucose levels can cause rat mortality. This can be mitigated by administering insulin or temporarily supplementing glucose:
Commonly, high blood glucose levels occur. Insulin supplementation involves administering intermediate-acting insulin such as NPH (Neutral Protamine Hagedorn) insulin at a dosage of 2-3 units per injection for 3-5 days, which typically reduces the mortality rate in rats.
For low blood glucose levels occurring after fasting, rats can be injected with 20% glucose intraperitoneally 4 hours after modeling to prevent mortality due to low blood sugar during injection.
4. Self-Mutilation:In situations of food deprivation and inadequate water supply, rats may resort to cannibalism and aggression towards each other. Therefore, it is essential to provide sufficient food and water, preferably from two different sources.
5. Infections: Diabetic rats have increased urine output, leading to damp bedding, which increases the risk of infection. Diabetic rats are more prone to infections compared to other rats, especially urinary tract infections and abdominal infections. Proper hygiene practices such as frequent changing of bedding and disinfection before and after invasive procedures such as intraperitoneal or subcutaneous injections and blood glucose testing are crucial. For example, after each blood glucose measurement, apply local treatment with tetracycline (or erythromycin ointment) to prevent infection.
5. Factors Influencing Diabetes Modeling
The factors influencing the establishment of diabetes disease models include the quality of STZ modeling reagents, animal conditions, and administration methods. Specifically, the reagent's main properties include purity, stability, and solubility. Animal conditions encompass genetic background, gender, body weight, rearing environment, and dietary structure. Administration methods include administration timing, intervals, and routes. Differentiated factors lead to differentiated modeling effects.
6. Guide for High Success Rate in STZ Modeling
For detailed instructions on the storage, dissolution, and aliquoting of STZ, please refer to https://www.Arcegen.com/products/detail/1002.
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