How To Induce Permanent Diabetes by Streptozotocin in Wistar Rats

8/5/2014 How to Induce permanent diabetes by streptozotocin in wistar rats?
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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
QUESTION FOLLOWERS (60) See all

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Pankaj Paliwal
Jiwaji University
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 ctrate buffer (4.5) P 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)
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 -20C? 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 Universit 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
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
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
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
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
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
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
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
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
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 its 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
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 and 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
glucose efficiently and therefore similarly only transports STZ. As a
consequence of this it is also not quite true to say the anomer is more
toxic: it is this anomer that gets in to cells. It is thus also the
concentration of the anomer that is important in determining the toxicity
of a particular preparation. The commercially available preparations
have a defined composition, typically approximately 75-85% anomer, in
the solid compound.
However, as with all such compounds, the anomeric and 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 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 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 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
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.
I should add that the nutritional status of the animal affects the response.
This is obvious from the above as D-glucose will of course compete
with 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 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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Dec 10, 2013

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