Dear Fawad

First of all you have to check the filament size and the diameter of filament tip. generally if you used the SD rats that weight about 300 g and the age is about the 8-9 weeks then the length of nylon filament that undergo from carotid bifurcation is about 18 mm and the tip diameter should be 365-375 micrometer. If you insert more length then anterior cerebral artery might also be occluded to produce the large infarct. So take care in the probe preparation. Nowadays commercial probe are available and you can try by them which might be good notion to overcome your problem.

I hope you are good technician not to damage the vagus nerve during surgery which leads to the death of animal as well.

Good luck

Thankx Bhakta GAIRE for your timely support. Yes i am using SD rats (weight approximately 250 g, and 4 weeks of age). I am aware of the tip diameter so i kept the tip approximately in range so that it enter the internal Carotid easily. Next thing is length size, its true that i uses a bet larger length of nylon (>3 cm, b/w 3-3.3 cm) and i inserted the whole except up to 0.5 mm for knotting with external artry. My professor say mostly that my skills are not very good but i take care of the vagus nerve (laterally the CCA) and i carefully maintain the space between the two.

Now in your opinion where does the problem exist. The length or ...?

regards

fawad

I used 2.2 cm nylon filament for 8 weeks old SD rats, of winch I insert 18 mm from the carotid bifurcation and 4 mm remains the outer part. In your case, you are using the very younger rats and also inserted the filament by 25-28 mm. So I think your filament passes not towards the MCA but towards the ACA. Another think you should keep the body temperature around 37 degree during the operation and operation time should be below 20 min. Use the sterile device during surgery and try to reduce the inflammation. you can use etoh everytime before surgery to sterile the surgical tools. First try by using shorter filament try once. i suggest you to use elder rats for better success of the result. moreover the MCA is almost 10 times smaller than that of internal carotid artery so tip diameter should not be based on ICA. I wonder that you are saying there is no bleeding. There might be intracerebral hemorrhage so you please check the rat brain by slicing as well.

You can discuss with your professor about my suggestion as well.

Good luck

Dear Fawad, I usually buy comemrcially available filaments as they have improved significantly success of MCAo experiments (i.e., mortality is now almost zero!).

Nevertheless, if you prefer home made filements, you should try to use 0.37 mm tips for rats weighing approx 300g (not less then 270g!), inserting them approx 17-18 mm from CCA bifurcation (as Dr Bhakta also suggests). Another useful strategy to reduce mortality is to cover the tip of the filament with silicon, givin it a soft texture that mor unlikely will damage rat vessels.

Hope this is helpful

GL!

2day i perform 3 surgery keeping in view all the above modifications. i kept the length size 2.3 cm, inserting approximately 18-20 mm. But still no fruits all died. i swear i am very confused. i spent my whole time with rats. Before they died,, the rats produce a characteristic sound (cracking like) and then take a long breath and die. Their movements are also very sluggish when they awaken particularly the hind lamb. i tried my level best not to harm vagus.

My professor does not allow silicon coated suture, accordingly all my seniors did the same and with good results.

Dear Fawad,

I perform MCAO by your schem, but with control of local cerebral blood flow. I think you should use rats not less then 300 g. Also suture should be insertet not deeper then 18 mm (for 300 g rat). It is very important thing - to use Doppler flowmetry to control local cerebral blood flow. It is nececcary, otherwise- you are doing blind experiment.

What may be orther reason of rat's deth - the quality of rats. I had such experiance - we had very vulnerable rats, which died nejavascript:xt day after surgery (I used occlusin 90 min and reperfusion). When we have got other delivery of animals - all became ok.

You also can try to change your experimental protocol - use not 24 hours after permanent MCAO, but 6-7 hours. Rats will still be anesthetized and alive during that time. If you will make slices and stain them with TTC you will see infarction. If you use some neuroprotective agents within 60 mun after MCAO - you also may see the effect.

Thanks Elena Tukhovskaya for responding.

This is almost true, that rat with bigger size survive with greater percentage, But my Professor believe that rat with size less than 250g produce good infarction, so i strictly followed my prof.

I did collect samples after 6-12 hours ( when rats are died), but surprisingly no variations in the expression level of apoptotic proteins/or related proteinS like Collagen response mediator protein (CRMP2) from westren blots results.

You are welcome! But do you use flowmetry? Because if you use little anymals, you could control the length of occlusion suture by blood flow reduction (occlusion of MCA). If you dont have flowmeter, it is realy nececcary to acquire it. You will get more stable and uniform results.

thankx alot Elena Tukhovskaya !

Dear Fawad,

The weight 250 gr is not suitable for MCAO.The best results are derived from the weight 280_320.Also there are some probale reasons for rat death.One of the most impotant reason  is cardiac asthma which includes hard breathing accompained some times with pink disharge from the noise which is due to vagous damage.The solution is very simple.You can use lidocaine25% over the carotid sheet and that is enough.The second reason can be SAH.

Good luck

Dear fawad

Lidocaine 0.25% is correct.

Good luck

an MCAO lasting for 24 hours?still hoping for any better outcome than death?decrease the duration of ischemia if it does not affect your study. If you are not interested in reperfusion, any duration of ischemia would suffice. If i were you, i would not go beyond 6 hours

During the first 24-48 h of ischemia there is a huge intracranial pressure due to vasogenic edema (if you have acces to MRI images you will se a displacement of the middline of the brain. In very large infarctions edema may be fatal and rats die within 24-48 h, depending on severity.

When you are performing a transitory mcao, try to reduce alittel bit the duration of the occlusion before reperfusion (60 min as reference is fine). When you practice a permanent oclussion, try to use a slightly smaller filament.

When you reduce a little bit the size of your lessions, edema becomes serious but no fatal, and your survival rates will increase.

Furthermore, during the first 78 h animals have problems to get acces to water and food, try to change food pellets for a paste, prepared with pellet powder and water, and leave a bit on the bottom of the cages. In severe cases you may use i.p. or s.c. injections of saline to keep the animals hydrated.

I tend to agree with the previous post. Permanent MCAO via the intraluminal filament technique is well known to have a high mortality rate, irrespective of the filament size or coating. It is highy likley the lesions are very large (as you describe) and the rats have high intracranial pressure that is leading to the high mortality. I do not believe this can be solved by changing the filament, you must reduce the severity of the model or the survival time. I would advise you to sit with your supervisor and discuss this limiation in order to determine how best to achieve the aims of your work.

In this model in rat, a marked hyperthermia occurs and can kill the animals. Some researchers put the rat in a cool place to reduce this mortality.

I know this answer is super late, Because now I am going on with the MCAO project, would you mind which vessels that you occluded to make permanent MCAO ? 

I did MCAO permanent in Wistar rats  male, 250 gram. If the rats died, it was majority because of the bleeding and too much anastetia agents. Otherwise the rats survived.

I hope I still get a response