1. Pathogenesis of Erectile Dysfunction
Erectile Dysfunction is due to the nitric oxide
(NO)-cyclic guanosine monophosphate (cGMP)-PDE5 pathway. NO synthase (NOS) generates
NO when phosphorylated by arginine as a neurotransmitter that wheels penile
smooth muscle relaxation, resulting in a normal penile erection after sexual
stimulation. Diabetes Mellitus can reduce the activity of NOS and the levels of
NO and cGMP in the corpus cavernosum of the penis, which may be one of the most
important mechanisms of Diabetes induced erectile dysfunction.
2. Diabetes Induction procedure without any
mortality in SD rats.
Step-1 : Preparation of 100 ml of 0.1 M sodium citrate buffer solution:
Use Autoclaved glassware only, to measure the
90 ml distilled water in a glass bottle, keep seperate10 ml same distilled water
to make up the sodium citrate buffer solution volume to 100 ml. Weigh
citric acid monohydrate 0.525g and Sodium Citrate Tribasic Dihydrate 0.74 g,
add 90 ml distilled water mixed it using a magnetic stirrer, measure the pH
4.5 adding the remaining 10 ml separated distilled water and make the volume 100ml,
and store the solution in Deep Freezer -80 Celsius for 10 minutes or in -20
Celsius for 15 minutes. Maintain the sodium citrate buffer solution in an icing bucket till the completion of the injection procedure without fail.
Step-2: Preparation
of Streptozotocin (STZ) solution in Sod citrate buffer:
Weigh only 120 mg of STZ, and add only in 12 ml of sod. citrate buffer
solution, and dissolve the Streptozotocin (Sigma Aldrich) completely in sod.
citrate buffer slowly using a vortex mixer. Filter it using a syringe filter, and
inject it within ten minutes into all 5 animals. Mix Streptozotocin only 10 minutes
before the intraperitoneal injection to the next 5 animal batches of animals. All
animals should be induced in batches of 5 animals only, if there is a delay of 10 minutes, then there will be a chance of degradation of Streptozotocin. If
Induction fails in some animals, try to re-induce by the same amount of STZ. One
more thing keep all animals 12 hrs fasting before Streptozotocin induction.
3.
Induction Diabetes confirmation by evaluating Blood Glucose Level
After the Streptozotocin injection, immediately add 5 % dextrose solution to a drinking
water bottle and maintain it for 24 hrs. The same day after 4hrs, check blood glucose
level using strips and a glucometer. Many animals will show blood glucose levels like non-induced animals’ blood glucose levels, and those animals will give a diabetic range of blood glucose levels the very next day after 24hrs. After 24 hrs. of STZ induction, there may be a change in a few animals that show blood glucose levels
below 200 mg/dl. Then, to these animals, you have to re-induced by using the same
dose level of Streptozotocin, one day after, keeping the animals on 12 hrs
fasting. On the 7th day check the blood glucose level above 300
mg/dl reading the animals you can confirm they as diabetic.
4.
Maintenance of Blood Glucose Level between 300-450mg/dl to induce Erectile
Dysfunction.
After diabetes confirmation, blood glucose levels above 500 mg/dl should be maintained between 300-450 mg/dl to induce erectile dysfunction. To maintain glucose levels use Human Actrapid Insulin (Novo nordisk India Private Ltd.) 1 unit for every 100 mg/dl blood glucose level. Maintenance of blood glucose levels between 300 to 450 mg is compulsory to induce erectile dysfunction, moreover, blood glucose levels above 500 mg/dl may be dangerous to animals and may cause often death in a long study (of more than 28 days).
ALLOXAN STREPTOZOTOCIN THEIR MECHANISM OF ACTION IN DIABETES INDUCTION
END OF DOCUMENT
Very informative blog. Here you have described all the methods in very easy way. I recommend this article to all preclinical scientist and biologist.
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