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Question

How to Induce permanent diabetes by streptozotocin in

wistar rats?

We have injected STZ 55mg/kg body weight (i.p) in wistar rats. Initially

hyperglycemia was induced in animals (BG range between 500-600) but fasting

blood glucose levels came down gradually over period of 15 days. And after 15

days it is reduced to 127 from 522 in same animal.

Blood glucose was measured in 12h fasting condition and at the same time on

alternate days by using glucometer.

Calm conditions were maintained while handling animals.

Glucometer used was calibrated and strips were used of same code.

Please suggest how can we induce permanent hyperglycemia in animals.

TOPICS

Jun 26, 2012

POPULAR ANSWERS

Dennis Pillion · University of Alabama at Birmingham

We used high dose stz as well and occasionally animals would succumb

in the first 24 hours after injection. Apaparently beta cell destruction

caused increased insulin release and hypoglycemia initially, which was

followed by subsequent hyperglycemia. It proved advantageous to allow

animals access to water containing sucrose or glucose during the first

12-24 hours after stz adminstration.

Mar 21, 2013

Apurva Kumar · National Institute of Mental Health and Neuro Sciences

One simple rule-stz must be dissolved in cold citrate buffer JUST PRIOR

TO USE.

Jan 9, 2013

Deleted

You should perform your streptozotocine dose carefully because not only

dose dependent but also time dependent differences were reported in

this model.

In our group we usually inject 70 mg/Kg. After 4-6 days you can observe

an increase in blood glucose levels. My personal experience is that 6

weeks after toxin injection is enough to set a good model of type 1

diabetes, although longer period could be studied (3-4 months).

Diabetology

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CONTRIBUTORS (35) See all

Richard Mark Smith

Cardiff University

Anwar Qureshi

United Arab Emirates University

Mohammed Taha Al-Hariri

University of Dammam

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Pankaj PaliwalJiw aji University

8/5/2014 How to Induce permanent diabetes by streptozotocin in wistar rats?

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Take care because not all the rats injected will become diabetic.

Good luck!

Jun 26, 2012

Nireshni Chellan · South African Medical Research Council

Try multiple injections from 2 months.

Jun 26, 2012

Debrup Chakraborty · Henry Ford Health System

The concentration of STZ you have used is very low (55 mg/kg bw). You

can try with 150-200 mg/kg concentration of STZ (ip) and then see what

happens.

Jun 26, 2012

Vaibhavkumar Gawali · Medical University of Vienna

STZ (45 mg/kg, IV) can induce permenant hyperglycemia in rats, make

sure that you inject STZ immediately after dissolving it in citrate buffer,

pH 6.0

Jun 27, 2012

Dipali Bhoite · Haffkine Institute

Yes, I agree with Vaibhav's comment that 45mg/kg by IV can induce

permanent diabetes using freshly prepared citrate buffer.

Jun 27, 2012

Ayman Moawad Mahmoud · Manchester Metropolitan University

STZ (45 mg/kg ip) dissolved in COLD citrate buffer PH 4.5

Jun 27, 2012

Joana M Gaspar · New University of Lisbon

I always use 65mg/Kg,freshly dissolved in 10 mM sodium citrate buffer,

pH 4.5. At this dose and until 3 months the animals stay diabetic.

Good luck

Jun 29, 2012

Julien R Marshall · The University of the West Indies, Trinidad and

Tobago; Tbilisi State Medical University

STZ 50mg/kg in 10mM cold cıtrate buffer (4.5) İP in 10 week old sprague

dawley rats or younger with blood glucose in the range of 250-360mg/dl

by glucometer. but this must be done as soon as the buffer is prepared

STZ breaks done very quickly.

Jun 29, 2012

John Mellem · Durban University of Technology

I agree with Joana, I've just completed an STZ - rat model (male wistar

rats) using the same dose parameters that Joana spoke of, and the rats

were stable up to 8 weeks when the study was terminated.

Jul 4, 2012

Thamizhiniyan Venkatesan · Kookmin University

Prof. Julien Marshall, have mentioned that injection of STZ (50 mg)

8/5/2014 How to Induce permanent diabetes by streptozotocin in wistar rats?

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results in increased blood glucose level in the range of 250-360 mg/dl.

please mention that is it fasting or non-fasting blood glucose level.

Aug 3, 2012

Pankaj K. Bagul · Translational Health Science and Technology Institute

You can increase your dose up to 70-80 mg/kg however if you increase it

more you may get increased mortality. and in case if you are getting

normal blood glucose after few days or weeks try to give a small dose of

STZ again and see what happens.

Aug 3, 2012

Maria Leonor Silva · Cooperativa de Ensino Superior Egas Moniz

Make sure that STZ is freshly dissolved in 10 mM sodium citrate buffer

and administrated until 20 min after dissolved. My rats showed a

glycemia decrease on 12h fasting after STZ administration but high

levels on 8h fasting. Glycemia levels are very inconstant. Did you stored

STZ until need at -20ºC? Confirm that i.p is correctly administrated.

Good luck

Jan 7, 2013

Apurva Kumar · National Institute of Mental Health and Neuro Sciences

One simple rule-stz must be dissolved in cold citrate buffer JUST PRIOR

TO USE.

Jan 9, 2013

Edith Arany · The University of Western Ontario

The best route is IV. The other important point is the age of the animal...if

is young there is regeneration of beta cells.

Jan 23, 2013

Juweria Effendi · University of Karachi

I made diabetic models with 100 mg/kg STZ in 6 weeks wistar rats.

Make sure that the STZ is dissolved in cold citrate buffer at PH 4.5 and

inject it within a time frame of 5 mins of its dissolution. I used the

Intraperitonial route which was very comfortably managed.

Jan 29, 2013

Antonio Toniolo · Università degli Studi dell'Insubria

You can make a dose-finding exp. in your rats starting at 55mg/Kg, then,

1.5, 2, 3, 5.

You'll measure fasting glucose at 12 and 24 days. You'll find the dose

causing permanent hyperglycemia. Best wishes, Antonio

Jan 30, 2013

Mohamed Elseweidy · Zagazig University

I think that our ordinary dose of Streptozoticin to induce DM in rats may

be witin a range of 40-60 mg/kg body weight and more than this range

will be lethal and toxic. The response will appear within 3 days or little

more . Failure to acheive higher blood sugar level may be followed later

with reduced dose 20 or 25 mg/kg body weight,

Certain precaution for STZ storage ( kept in freezer) , color very faint

pink otherwise it will loose its potential.

Feb 4, 2013

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Sai Mangala · Sri Sathya Sai Institute of Higher Medical Sciences

Alloxan can also be used in place of STZ. But STZ gives you more stable

hyperglycemic values. Make sure the STZ is dissolved in cold citrate

buffer at 4.5 PH just prior to IP injection at a dose of 55 mg/kg body

weight. Weigh the quantity in appendorf tubes separately for individual

rats. Add same amount of buffer to all tubes to maintain uniformity of

volume injected.

Feb 5, 2013

Mohammed Taha Al-Hariri · University of Dammam

Simply you can dissolved the STZ in a distal water for more information

please read my paper

Thx

Feb 6, 2013

Salah Atteiah · King Abdulaziz University

I think the induction of diaibetes depends upon which type of diabetes

you in need. Is it a type I or type II. Regarding type II diabetes, the ideal

method used is the IP injectione of STZ ( 55-65mg/kg) 15 min after IP

injection of Nicotinamide ( 120mg/kg), in overnight fasted animals. Wait

for 21 days and measure the fasting blood sugar in at least 12h fasted

rats. Rats with fasted blood sugare over 130mg/dl can be considered

diabetic ( type II). This model is the most common one that can be used

for studying the effect of some new compounds or molecules with

expected antidiabetic activity. Some other models you can induce by

using STZ and High fat diet. Also, you can use Fractose feeding and

HFD or cholesterol to induce hyperglycemia and metabolic syndrom at

the same time. In my opinion, there is no need for complet dystruction of

the pancreas, to find little insulin to test the activity of this insulin in case

of insulin resistance.

Feb 7, 2013

Sharida Fakurazi · Putra University, Malaysia

We have also adopted similar technique described by Dr Salah Atteiah.

We have streptozotocin and nicotinamide on the first day, whilst,

reinjection the next day with Streptozotocin. Hyperglycemia developed

after 72 h. The level was maintained for the next 21 days. We also found

that Wistar rat is the best. Never use Sprague Dawley, as the level of

glucose could be inconsistent for the next 21 days. We are happy with

the model, as glucose is found high, insulin is still produced and beta

islet is still viable.

Feb 9, 2013

Salah Atteiah · King Abdulaziz University

Thank alot Dr Sharida for your additional information, but I have two

questions which are, 1) Are you using the same dose of STZ in the

secons injection? 2) For how many days in the control rats , diabetes can

continue in fasting rats ?

Feb 9, 2013

Sharida Fakurazi · Putra University, Malaysia

I cant remember that of hand. It might be of the same dose.you still have

NAD to protect the islets. But but, let me double check next week and

come back to the question. 2 ) We do not fasted the rats.animals

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continued to be given food and water ad libitum. But we c an see that the

diabetic animals drink more water and urinate a lot more

Feb 9, 2013

Antonello Pileggi · University of Miami Miller School of Medicine

We use STZ freshly dissolved in citrate buffer to a dose of 60 mg/kg

given IV or IP, and repeat the treatment after 3 days. Some groups prefer

giving STZ after overnight fasting and giving access to food after STZ

injection. Try preparing small aliquots of STZ so that it is used soon after

reconstitution to prevent inactivation that will reduce efficacy. Keep

aliquots and buffer on ice at all times. Store your STZ stock at -20 C in a

vacuum bell.

Feb 11, 2013

Sharida Fakurazi · Putra University, Malaysia

Dr Salah Atteiah, I am coming back to your question which is 5 days ago.

This is how we induce diabetes. Animals were fasted over night for about

12h with free access to drinking water. Experimental type 2 diabetes was

induced with NAD at 150 mg/kg body weight, 15 mins later; it was

followed by i.p injection of STZ 65mg/kg b.wt. Induction procedure was

repeated 1 day later (Masiello et al., 1998). Animals are able to sustain

hyperglycemia for 21 days.

Feb 15, 2013

Younes Jahangiri Noudeh · Research Institute for Endocrine Sciences

We used Streptozotocin with dose of 65 mg/kg and it worked. Our Wistar

rats were diabetic until the end of our study with huge amounts of urine

(regardless of their high levels of blood sugar). By the way, you may

check the originality of your used material (Our material was from

Sigma).

Feb 15, 2013

Salah Atteiah · King Abdulaziz University

Dr Noudeh I think the using a dose of 65mg/kg will induce type I diabetes,

this type of diabetes is not so convenient to do screening for some drugs

with expected antidiabetic activity, or that increase glucose uttilization

through increasing insulin resptor sensitivity. So it needs partially working

pancreas especilly to test their capability for increasing insulin release

from pancreatic isles or not e.g. gliptins, sulphonylurea derivitives etc.....

Feb 15, 2013

Younes Jahangiri Noudeh · Research Institute for Endocrine Sciences

Dear Dr Salah,

Thank you for the comment. That's right. This dosage of STZ destroys

actually a high percentage, if not all, of pancreatic beta cells.

Feb 15, 2013

Sharida Fakurazi · Putra University, Malaysia

Dr Salah Atteiah, we checked for glucose, insulin and section the

pancreas to see the preservation of beta cells. At the moment, we are

pleased to say that the islets were preserved, insulin was secreted

Feb 15, 2013

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Chen Leo · University of Melbourne

Our method is pretty much in agreement with Dr Noudeh and Dr Pileggi.

However, we used a dose of 50mg/kg STZ (dissolved in 0.1M citrate

buffer), IV. These animals exhibit hyperglycaemia 1 week after injection

and lasted through the whole duration of our study(> 8 weeks post-STZ

injection).

Mar 14, 2013

John H Mcneill · University of British Columbia - Vancouver

I agree pretty much with the answers given. With STZ it depends not only

on the dose but also on the freshness of the solution and as stated

above the solution needs to be kept cold. Some people use i.p. and

others i.v. and the results appear to about the same. IN my lab it is also

somewhat dependent on who gives the injection. The more experienced

people tend to get diabetes at a lower dose than people just starting out.

For Type 1 diabetes we have settled on a dose of 60 mg/kg i.v. and have

almost 100% success in producing diabetes with this dose. These

animals can also live for a long time without insulin treatment although

growth is inhibited and cataracts appear within about 90 days.

Mar 21, 2013

Dennis Pillion · University of Alabama at Birmingham

We used high dose stz as well and occasionally animals would succumb

in the first 24 hours after injection. Apaparently beta cell destruction

caused increased insulin release and hypoglycemia initially, which was

followed by subsequent hyperglycemia. It proved advantageous to allow

animals access to water containing sucrose or glucose during the first

12-24 hours after stz adminstration.

Mar 21, 2013

Salah Atteiah · King Abdulaziz University

The problem depend upon that the fasting animals ( 12-14h) may

become normoglycemic despite of the hyperglycemia that was appeared

on them before fastening. So the problem still existed in both rats and

mice.

Mar 21, 2013

Archana Chavan · Parul Group of Institutes

I also experienced the same problem of reversal of DM (induced with

45mg/kg dose given i.p.) after nearly 20 days in some of the animals. I

need hyperglycemic condition at leaset for 8 weeks. So now I increased

my dose of STZ to 50 mg/kg, ip. I m also looking forward to answers to

this question from experts. from literature i learnet that STZ induced DM

is not reversible. but it reversed in some of my animals.

May 4, 2013

Fred Levine · Sanford-Burnham Medical Research Institute

It is likely that the effective dose of streptozotocin was insufficient. It is an

extremely unstable drug. We take it down to the animals as a powder and

put it in solution seconds before administration. If that doesn't work well,

then go up on the dose. Finally, you can try switching to alloxan, which

we find to be a bit more stable than streptozotocin.

May 7, 2013

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Patricia Gail Wilson · U.S. Department of Veterans Affairs

I agree that STZ is an unstable drug. Is there any way you can preweigh

it out into tubes and then dissolve it in buffer in the animal room just prior

to injection? I also would suggest that you once dissolved in the buffer

you use it up quickly. You can preweight aliquots that will only allow you

to inject a few animals at once for example. That's what we do. Plus keep

in mind that STZ should be kept cold and protected from light. I believe

once dissolved in buffer that STZ is only good for a few minutes.

Jun 4, 2013

Anwar Qureshi · United Arab Emirates University

well, 60 md/dl STZ dissolved in freshly prepeared Citrate buffer PH 4.5

willl induced Permanent diabeted (Type 1) in wistar rats. It work very well

in our lab.

Jun 10, 2013

Jon Gunnarsson Mabley · University of Brighton

I agree with the answers here but we injected the STZ (80 mg/kg) i.v. into

the tail vein of rats and that gave us a permanent diabetes. We never

found the i.p. route to be that effective in inducing diabetes with STZ in

rats.

Jul 13, 2013

Edith Arany · The University of Western Ontario

Do not forget that beta cells can regenerate after STZ treatment and this

also depends on the age of the animal

Jul 16, 2013

Rajaram Krishnasamy · Anna University, Chennai

Alloxan (120mg/kg b.w.) can be used for the permanent diabetes

Jul 17, 2013

Indumati Sharma · University of Mysore

Even my rats have become hypoglycemic..........does the fasting duration

(to check FBG) effect the FBG? My rats were almost 20h fasted before I

checked its FBG. Could this have effected my results?

Jul 19, 2013

Richard Mark Smith · Cardiff University

I have just posted this answer elsewhere on RG. Sorry it is long but I

think it may be of interest here. With regard to permanence of diabetes:

it is simply a dose effect relating to log kill of beta cells. Permanent

diabetes can be achieved reliably if the right dose is established and all

the variables outlined below are controlled.

STZ has two intracellular effects accounting for it’s cellular toxicity: DNA

alkylation and NAD+/NADH depletion by a PARP dependent mechanism,

the latter being suggested to be most important in beta cell toxicity. This

matters as it demonstrates that STZ will be toxic to any cell within which it

achieves adequate intracellular concentrations.

STZ is taken up into cells by GLUT2, differential expression of GLUT2 by

different tissues explaining much (but not all) of the differential tissue

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toxicity and the well described strain/species differences. It is a very toxic

compound so effective use requires a concentration to be found that

results in a toxic concentration in beta cells but a non-toxic concentration

in the other tissues that it will enter. In our early use of STZ (20 years

ago!) we performed autopsies on animals that died and found liver and

kidney to be particularly affected, which is not surprising now that we

know about GLUT2 mediated uptake. If the dose is too high widespread

toxicity will be seen. Unfortunately the safe and effective window is quite

narrow. For mice we titrate new batches in 20mg/kg steps. I have

attached data from one such experiment. The formatting has scrambled

a bit but the X-axis is fasting glucose at day 3 post injection. The other

important data is that 40% mortality was seen in the 180mg/kg group and

no mortality was seen in any other group. You will see that the dose

window is quite small.

This is all fairly basic stuff. What is crucially important is that, just like

glucose, STZ exists in α and β anomeric forms. This is not the same as

the D/L enantiomers where the whole molecule is a mirror image, but

defines the position of a single hydroxyl group: often referred to as up or

down! This is important for a couple of reasons. GLUT2 only transports α

glucose efficiently and therefore similarly only transports α STZ. As a

consequence of this it is also not quite true to say the α anomer is more

toxic: it is this anomer that gets in to cells. It is thus also the

concentration of the α anomer that is important in determining the toxicity

of a particular preparation. The commercially available preparations

have a defined composition, typically approximately 75-85% α anomer, in

the solid compound.

However, as with all such compounds, the α anomeric and β anomeric

forms will equilibrate in solution, so as soon as STZ is dissolved

equilibration begins. Much of the degradation of STZ is in fact this

equilibration with loss of the α anomer with resulting loss of efficacy. True

degradation is slow. Early work describes an effective half life of about 19

minutes, giving some idea of the rate at which equilibration proceeds.

The de la Garza-Rodea paper is of course crucial in this regard and

describes this chemistry pretty definitively. What this publication does not

take into account is the wider aspects of the biology. From memory I think

they used the same does of STZ for immediate administration and

delayed administration. Given the above these preparations will

inevitably and predictably have different efficacy/toxicity. At equilibrium

there is a slight excess of β anomer. The rate at which equilibrium is

achieved is of course affected by temperature and the buffer used. So

aqueous and citrate buffer, pH4-7, solution at room temperature or kept

on ice and any time point for injection from 10 minutes to 3 hours are all

possible. What will differ is the percentage of the initial α anomer that is

still present in solution and therefore the toxicity of the particular prep at

that particular time. STZ equilibrates particularly quickly in aqueous

solutions at room temperature, much less quickly in citrate buffer at pH 4-

4.5 on ice. The problem with the 2-3 hour equilibrated protocol is that

this preparation will have approximately half the concentration of ‘active’

α anomer compared to fresh STZ. Hence the lower incidence of diabetes

in many peoples hands and the suggestion that multiple doses are

needed. Diabetes can be consistently and reliably induced with a single

dose if all the above parameters are considered when deciding on the

protocol for your particular experiments. Given your logistic constraints I

can see that the equilibrated protocol might work best, but do make sure

that you work out the correct dose.

I mention this as it is crucially important to recognise these differences as

the dose of STZ used by one person may not be right in your model with

your logistic constraints.

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I should add that the nutritional status of the animal affects the response.

This is obvious from the above as α D-glucose will of course compete

with α STZ for uptake and the amount of glucose ‘available’ will therefore

impact on beta cell toxicity. Administration of carbohydrate post injection

has been advocated to counter the hypoglycaemia that can be seen with

insulin release from targeted beta cells. This may be true (although we

have not found this to be a big problem with correct doses and may

relate to overdosing with hepatic toxicity preventing also counter

regulatory mechanisms). This post dose carbohydrate may be more

important in preventing ongoing toxicity. This was important in our

experiments as we were transplanting islets so needed to avoid toxicity to

the transplanted tissue. We found transplanted beta cells to be fine at 3

days post injection of STZ. What matters is that this nutritional variable is

controlled also in your protocol. I would suggest that the best way is to

fast animals overnight as discussed elsewhere on Research Gate at

length. This may be a problem for the multiple dose regimens. I am not a

fan of post STZ glucose administration as I have not found it to be

necessary and it introduces another variable with regard to beta cell

killing to be controlled. But this is a personal view.

So in summary it is all about understanding the biology and chemistry

and as a result getting the dose of the α anomer right for your model.

Hope this helps

Dec 9, 2013

Richard Mark Smith · Cardiff University

One further thought: I would disagree with one of the earlier posts and

suggest that it is not possible to induce type 2 diabetes with STZ. Type 2

DM is a syndrome with a central metabolic disorder that is far more

complex than hyperglycaemia and hyperglycaemia induced insulin

resistance. All STZ can reproduce is varying degrees of hyperglycaemia

due purely to beta cell deficiency/failure. Careful choice of species/strain

with or without dietary manipulation may reproduce SOME aspects of

T2DM. As posted elsewhere I would encourage the use of the term STZ

induced diabetes. Whatever the protocol, whatever the degree of beta

cell loss, STZ alone induces 'pure' beta cell loss. The regimens that

cause partial beta cell loss best reproduce early stages of T1DM, in

reality pre-diagnosis stages. They do not reproduce T2DM!

Dec 9, 2013

John H Mcneill · University of British Columbia - Vancouver

I agree with Richard Smith's comments. Type 1 diabetes induced by STZ

is a dose related process. The higher the dose the greater the number

of beta cells killed. It is also related to the experience of the individuals in

giving the STZ. I find that inexperienced people doing the injections are

more likely to end up with a higher number of rats that do not become

hyperglycemic as compared to people with experience. After nearly 30

years of using STZ we routinely use 60 mg/kg of STZ and I have been

fortunate in having technicians who have been with me that long. I agree

also that STZ does not produce Type 2 diabetes but rather an animal

with limited viable beta cells and, depending on the dose, perhaps

hyperglycemia. Such animals witl respond to insulin releasing drugs

since there is still insulin to release. They will also respond to insulin

enhancing drugs but they do not have the characteristics or a Type 2

diabetic animal such as the diabetic ZDF strain of rats or several mouse

strains.

STZ dose graph.docx

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