Protocol
Species-Wistar albino rats
No of rats-15
Body weight of rats-300-380gm
Gender -M
STZ- freshly prepared in citrate buffer, pH-4.2
Fasting-6hrs, water ad libitum
Glucose feeding -15%glucose in drinking water after 30 minutes of STZ induction
Observations
After 18 hrs
Its noticed that there was only less amount of water intake (drinking water was added with 15% glucose )
15% glucose in drinking water replaced with normal water
After 72 hrs
poly urea
no of deaths-1
After 96 hours
poly urea
no of deaths-13
On Sixth day
Blood glucose level of (not fasting) rat which is alive-481 mg/dl
Alex,
40mg/kg STZ is considered a low dose and you should have not encountered the problem you did.
But then at such a low dose, there is absolutely no need for you to give rats 15% glucose (which is a rather high concentration) instead of water.
How long did you continue glucose instead of water?
My guess is that you killed your rats by giving them way too much glucose and no water. Repeat the experiment without glucose and your rats will be fine. At the STZ dose of your choice, they will not develop raging hyperglycemia.
Dear Alam,
15% glucose was mixed with drinking water for 18 hrs after STZ induction
Can you be sure your injection technique has not punctured the internal organs? This is a very strange occurrence in so many animals. As Tausif has stated, you are well with the safe range for single bolus injection.
Alex,
Rather than answering your personal message, I'll include details here so others can also benefit or add from their experience to help you.
Depending on the dose of STZ, one can cause some damage to ß-cells to all the way up to virtually total destruction of all ß-cells.
It seems that you are not interested in total destruction of ß-cells. What is the target of your experiment?
I prefer to use 1x10-4N HCl to dissolve STZ and immediately inject it after dissolution (usually under 2 min).
Since I am interested in total destruction of ß-cells, we use ~80mg/kg STZ.
In smaller rats, it is easy to locate and inject STZ through the tail vein. You have to restrain or anesthetize your rats. You are using quite large rats and although, their tails veins will be proportionately larger and easier to find but if you are inexperience, it may be difficult to see. In larger rats (~ >150gm), another convenient route of administration in male rats is penile vein.
If you choose to use peritoneal injections, make sure that you are delivering STZ in peritoneal cavity and not in any of the internal organs. You will have to enlist help of someone to firmly hold the rat, if not anesthetized, and lift the belly skin away from the body, judge the thickness of skin and depth where the needle must reach so as to not end up in any organs. I suppose, this is easier to show than to describe.
In the final analysis, at 40mg/kg STZ, there is no reason why your rats should die if everything is done carefully. You can fast large rats overnight (access to water only), treat them with STZ, no food (access to water) for additional 1-2hr, followed by feeding them ad libitum. No need to replace water with glucose.
Best,
Dear All,
Happy that we could get healthy diabetic rats without mortality
Revised protocol for the pilot study
Species-Wistar albino rats
No of rats-3
STZ dose -40mg/kg I.P in citrate buffer (pH-4.5)
Body weight (g)-365, 330 and 340
Fasting blood glucose levels after 72hrs of STZ induction-252, 250 and 389 mg/dl respectively
Notes:
Changes from the protocol mentioned in question
-Rats were given food, and water ad libitum before STZ administration and after
-30 mns After STZ administration 15% glucose in drinking water was given.
We kept rats for 6hrs fasting on day time - it might have effected ( or)
Fasting itself might have effected irrespective of light or dark cycle.
Regards
Easy solution- divide the dose and inject about an hour apart. I hade done this several times and survival rate is 100%. After about 3 days you can observe the signs of diabetes and cloudy serum.
II am in agreement wth Tausif. You do not need add glucose after the experiment. Rats may be old and perhaps obese for this type of experiment, but in your question I do not find the purposed metabolic design.
Hi for all
Really I have No idea but I highly agree with Tausif Alam
Regards
Hi all
STZ (40 mg/kg, I.P) is low dosage for induction of diabetes. Sometime after fasting all of STZ should be absorbed to beta cells. After diabetic condition glucose is risk for animal. No need for giving it, indeed insulin is choice for first day after diabetes induction.
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