The embolic mouse MCAO data the Augusta group presented are excellent. How long did these mice survive? What is mortality if a long-term experiment is performed?
I also want to know what mortality the Iowa group had in their previous experiments.
Question: Can we use a thick and short blood clot to just block the MCA?
We sacrificed these embolic mouse models after 24, 48, and 72 hours. I had not kept these models for a longer time ( i.e., 28-30 days). However, it has a high mortality rate (50-70%) if used 10 mm clot size or more. It could minimize the mortality rate if the clot size is around 5-6 mm.
Question: Can we use a thick and short blood clot to just block the MCA?
Ans. For rats, it could use a short clot size of 10 mm. But I can't recommend more than 5 mm in mice.
Thank you for the information. I made the clot using a PE-50 tube and measured the diameter of the blood clot. It was around 0.25 mm. After washing, it became thin. The filament used in the mouse MCAO is 0.22 mm. If the blood clot is 5 mm, do you think it will block the blood flow to the PCA? Maybe we should do a pilot experiment to see how long they can survive after a 5 mm blood clot is injected.
# How did you measure the diameter of the clot around 0.25 mm? Have you used a device (slide clippers) or just used the scale?
# How did you wash the clot?
# During the washing of the clot, red cells around the clots will be washed off, so it becomes thinner than the usual diameter (as it was in PE-50 internal diameter). But it will be fine, and the clot will compact stretchable properties like rubber.
# Preparation of the clot is a crucial and tricky step. We can not expect all the clots will be good. It could be some will be bad, and some will be good clots.
# When the 5 mm clot is delivered using PE-10 or equivalent catheter/tubes, it will expand inside the proper place or ICA. The chance of mortality will be less, and the mice can survive longer.
Many of the questions you are asking were answered in the draft SOPs i sent last week. If you look at the drafts, you will see why/when/how we propose to make the clots. It tells you how to make them reproducibly, and how to wash them. And yes, we need to do a pilot stuidy to derive the proper length.
I should clarify my question- I understand that washing removes the red blood cells, but I don’t know why that is important. In human strokes there are likely red blood cells around the clots, no? Is it because the red blood cells are only loosely adhered and therefore introduce variability? Or some other reason?
Yes, i agree with that question. At our first SC meeting i asked Anil (Iowa) and he did not have an answer. Therefore, i think we should do a pilot study wiwth and without washing. Kahn (Augusta) opined that the clots move through the arterial tree much better if they are washed.
Yes, red blood cells loosely adhere to it, so after washing it off, it goes freely inside, and there is less chance of making a small ball or tag on the tips of the clots. It also has less chance of the rupture of the inside arterial wall.
I put the blood clot and a microscope calibration ruler under the microscope and measured its diameter using image software.
If the blood clot is not washed through the PE-10 tube, the neurological deficits will be transient in some animals. You also can make it thin by washing it.
We have used embolic clot model in Apoe deficient mice. 60 to 70% mortality by day 3 and 7. We have not followed these mice up to day 28. The clots contain human fibrinogen and are washed to make sure we are infusing stable clots. of course, these clots are not rich in RBCs.
The quality of blood clots: use a new syringe (no liquid inside) to take fresh blood and directly inject it into the tubes. If you mix the blood with others, the blood clots will be some good and some bad. If we use human fibrinogen, inject it into the animal intravenously before blood collection.
The mortality: The PCA supply should not be blocked by the blood clot in mice. Not an issue in rats. We used 3 mm blood clots (the blood clot was made using the PE-50) in mice for three mice last week. Two mice survived well and had infarcts in the cortex and striatum. One had an infarct in PCA and hippocampus (the blood clot was in ICA, I am thinking about whether we may use the filament to push the blood clot to the bifurcation of MCA and ACA if the LDF blood flow is not decreased).
I do not have any experience with the clot model, but I don't think we need to use it in both mice and rats. I suspect the rat model will be easier to accomplish, and rats are much more resilient with lower mortality. It would serve the purpose of testing the clot model and TNK with the surviving interventions toward the end of the trial.
Making the clot is not difficult. The only thing is the catheter. We can find good catheters for rats, not mice because it is required to insert very deep. The modified PE-10 is used in mice, which may cause damage to the endothelial wall of ICA. We ligated the PPA and then just delivered the blood clot to CCA through ECA. Let the blood move the clot forward to the bifurcation of MCA and ACA. It still worked. The key is to figure out what size blood clot is selected (diameter and length). We are using PE-50 for making blood clots. To keep mice surviving well, a 2-3 mm blood clot is used for eMCAO. The rat model will be easier than the mouse model.
Yes, it is accessible in rats compared to the mice clot model. Initially, it can take time to learn these new techniques for the new surgeon, but later, it can be easy after practice in mice or rats.
The mortality rate depends upon the clot size. Therefore, a bigger clot size can cause higher mortality than a smaller clot size.
We usually make 5-6 of 12 inches PE-50 tubes clot at one time and keep them in the refrigerator for future use.
The PCA infarct of the model makes sense to me, as the infarcts were seen in a proportion of rabbits when the clots were delivered via the proximal segment of the ICA in our previous studies.
I had kept the PE-50 containg clot in the refrigerator around 1 month. Once it was washed with PBS, it can be used within 2-3 days. So I make many clot PE-59 tube in one time.
Dr. Khan, The data you presented last Friday are excellent. Did you do TTC to confirm the infarct size and location? The laser speckle images had blood flow reduced in the right hemisphere (from the frontal lobe to the occipital lobe). I want to know if the infarct was expanded to the territory of PCA.
You probably missed my first presentation, where I was shown clot delivery, CBF, and TTC staining after 24 hours of the clot delivery. The TTC staining has demonstrated the infarction of the right hemisphere.
In the last presentation, we optimized clot delivery with three different sizes of clots (3,4 and 5 mm) to show the dropped CBF just after the clot delivery.
Thank you for sharing your excellent data with us. In Dr. Zhang's study published in JCBF (Zhang Z et al. A mouse model of embolic focal cerebral ischemia. J Cereb Blood Flow Metab. 1997 Oct;17(10):1081-8): "Preliminary studies in 20 mice showed that animals (12 of 20) achieving a successful occlusion of the MCA exhibited a blanching of the ipsilateral skull and a about 70% reduction in rCBF immediately after injection of the clot."
Do you have of similar reproducibility data in your clot model?
Do you think measurement of MCA blood flow is necessary?
In your presentation, the clot seems located in the ICA segment connecting to the MCA. Do you think that the clot location might affect the injury size?
Do you have of similar reproducibility data in your clot model?
Ans. Yes, we have a similar reproducibility of the reduction of blood flow (60-70%), as I explained during the presentation last Friday.
Do you think measurement of MCA blood flow is necessary?
Ans. Yes. Measurement of CBF after clot delivery or filament model of MCAo will give you a clear picture to confirm whether you have successful surgery on those animals. So, I always measure CBF after MCAo surgery.
In your presentation, the clot seems located in the ICA segment connecting to the MCA. Do you think that the clot location might affect the injury size?
Ans. Yes, it can be.
I had shown TTC staining in my 1st clot model presentation after 24 hrs, where infarction was visible. However, in the last presentation, I just showed the sizes of the clot delivery and the reduction of CBF after that.
You may try it in your lab let's see if the result match with our lab.
Thank you for your informative answers. Had any mice shown no MCA blood flow reduction after injecting the clot in your previous studies or testing? Dr. Zhang's study showed that 12 among 20 mice had stroke, or 8 mice among 20 mice had no stroke after embolization.
If blood flow measurement is necessary, do you prefer mouse clot model or rat clot model. The rat skull bone is thicker so that the blood flow may not be directly monitored with a Doppler without thinning the bone area.
So far, I have never got such types of incidents in no MCA blood flow reduction after clot delivery. It could be possible due to missed clot delivery or a fragile/thinner clot has to be broken or lyse quickly inside the MCA regions or around.
Rat clot model: It is easier than mice clot. Easy to clot and deliver using PE-10. But there are some disadvantages that we cannot measure CBF due to thicker skull bone as I also explained in my 1st presentation or in the discussion.
Mouse clot model: A little bit tough only clot delivering method due to having not suitable tubing/microcatheter. Advantages: you can measure CBF by laser speckle and easy to handle it.
Thanks! Khan, Another issue might be about using blood clot fresh and consistent. I believe that Dr. Zhang' mouse study showing that 12 among 20 mice have stroke or 8 among 20 mice without stroke, may also because the blood clot autolysis after embolization. Dr. Zhang suggested to use the overnight clot within 4 h after PE10 tubing washing preparation.
It could be possible that a blood clot autolyzed in his case.
Fresh clots can be better than old clots. But I could not find any issue in our case, even in my old clot used after a week or two. But, of course, it depends upon how to prepare and store it.
In the large clot rabbit model with Justin Zivin, we settled on a "2-hit" model. We injected one clot, waited 5 min, and if the animal was looking normal, we injected a second clot. We had about an 80 to 90% "hit" rate. We did not use any transcranial monitoring at all. We had about a 50% rate of hemorrhagic transformation. to account for the 10 to 20% "no-hit" rate, we just increased the sample size in each experiment.
We measured the diameters of the blood clots made with PE50. The blood clots before washing were 0.391, 0.374, 0.380, and 0.385 mm. They were decreased to 0.227, 0.211, 0.218, and 0.222 mm after 15 times of washing.
clot model
Yes. We will do that.
Clot model
The embolic mouse MCAO data the Augusta group presented are excellent. How long did these mice survive? What is mortality if a long-term experiment is performed?
I also want to know what mortality the Iowa group had in their previous experiments.
Question: Can we use a thick and short blood clot to just block the MCA?
Clot Model
We sacrificed these embolic mouse models after 24, 48, and 72 hours. I had not kept these models for a longer time ( i.e., 28-30 days). However, it has a high mortality rate (50-70%) if used 10 mm clot size or more. It could minimize the mortality rate if the clot size is around 5-6 mm.
Question: Can we use a thick and short blood clot to just block the MCA?
Ans. For rats, it could use a short clot size of 10 mm. But I can't recommend more than 5 mm in mice.
clot model
Thank you for the information. I made the clot using a PE-50 tube and measured the diameter of the blood clot. It was around 0.25 mm. After washing, it became thin. The filament used in the mouse MCAO is 0.22 mm. If the blood clot is 5 mm, do you think it will block the blood flow to the PCA? Maybe we should do a pilot experiment to see how long they can survive after a 5 mm blood clot is injected.
I haven't used the clot…
I haven't used the clot model before- I'm curious, why are the clots washed?
clot model
# How did you measure the diameter of the clot around 0.25 mm? Have you used a device (slide clippers) or just used the scale?
# How did you wash the clot?
# During the washing of the clot, red cells around the clots will be washed off, so it becomes thinner than the usual diameter (as it was in PE-50 internal diameter). But it will be fine, and the clot will compact stretchable properties like rubber.
# Preparation of the clot is a crucial and tricky step. We can not expect all the clots will be good. It could be some will be bad, and some will be good clots.
# When the 5 mm clot is delivered using PE-10 or equivalent catheter/tubes, it will expand inside the proper place or ICA. The chance of mortality will be less, and the mice can survive longer.
# It is better to do a pilot study first.
Please read the documents i sent
Many of the questions you are asking were answered in the draft SOPs i sent last week. If you look at the drafts, you will see why/when/how we propose to make the clots. It tells you how to make them reproducibly, and how to wash them. And yes, we need to do a pilot stuidy to derive the proper length.
Link to files
I put the clot model dociments on my Google drive. See if you can access them:
https://drive.google.com/drive/folders/1XWlBEM5Ajz0fwWc4lrCVaG8uWUfkJjNJ?usp=sharing
I should clarify my question…
I should clarify my question- I understand that washing removes the red blood cells, but I don’t know why that is important. In human strokes there are likely red blood cells around the clots, no? Is it because the red blood cells are only loosely adhered and therefore introduce variability? Or some other reason?
thanks!
Washing clots
Yes, i agree with that question. At our first SC meeting i asked Anil (Iowa) and he did not have an answer. Therefore, i think we should do a pilot study wiwth and without washing. Kahn (Augusta) opined that the clots move through the arterial tree much better if they are washed.
Yes, red blood cells loosely…
Yes, red blood cells loosely adhere to it, so after washing it off, it goes freely inside, and there is less chance of making a small ball or tag on the tips of the clots. It also has less chance of the rupture of the inside arterial wall.
blood clot
I put the blood clot and a microscope calibration ruler under the microscope and measured its diameter using image software.
If the blood clot is not washed through the PE-10 tube, the neurological deficits will be transient in some animals. You also can make it thin by washing it.
We have used embolic clot…
We have used embolic clot model in Apoe deficient mice. 60 to 70% mortality by day 3 and 7. We have not followed these mice up to day 28. The clots contain human fibrinogen and are washed to make sure we are infusing stable clots. of course, these clots are not rich in RBCs.
No experience with embolic clots in Rat.
Testuseruoi is Anil from…
Testuseruoi is Anil from Iowa.
Clot model
Thank you for the information.
ideas for blood clot model
The quality of blood clots: use a new syringe (no liquid inside) to take fresh blood and directly inject it into the tubes. If you mix the blood with others, the blood clots will be some good and some bad. If we use human fibrinogen, inject it into the animal intravenously before blood collection.
The mortality: The PCA supply should not be blocked by the blood clot in mice. Not an issue in rats. We used 3 mm blood clots (the blood clot was made using the PE-50) in mice for three mice last week. Two mice survived well and had infarcts in the cortex and striatum. One had an infarct in PCA and hippocampus (the blood clot was in ICA, I am thinking about whether we may use the filament to push the blood clot to the bifurcation of MCA and ACA if the LDF blood flow is not decreased).
I do not have any experience…
I do not have any experience with the clot model, but I don't think we need to use it in both mice and rats. I suspect the rat model will be easier to accomplish, and rats are much more resilient with lower mortality. It would serve the purpose of testing the clot model and TNK with the surviving interventions toward the end of the trial.
clot model
Making the clot is not difficult. The only thing is the catheter. We can find good catheters for rats, not mice because it is required to insert very deep. The modified PE-10 is used in mice, which may cause damage to the endothelial wall of ICA. We ligated the PPA and then just delivered the blood clot to CCA through ECA. Let the blood move the clot forward to the bifurcation of MCA and ACA. It still worked. The key is to figure out what size blood clot is selected (diameter and length). We are using PE-50 for making blood clots. To keep mice surviving well, a 2-3 mm blood clot is used for eMCAO. The rat model will be easier than the mouse model.
Clot model
Yes, it is accessible in rats compared to the mice clot model. Initially, it can take time to learn these new techniques for the new surgeon, but later, it can be easy after practice in mice or rats.
The mortality rate depends upon the clot size. Therefore, a bigger clot size can cause higher mortality than a smaller clot size.
We usually make 5-6 of 12 inches PE-50 tubes clot at one time and keep them in the refrigerator for future use.
clot model
The PCA infarct of the model makes sense to me, as the infarcts were seen in a proportion of rabbits when the clots were delivered via the proximal segment of the ICA in our previous studies.
clot model
How long (hours or days) can you keep/store the clot in the refrigerator for future use? We used fresh clots in our previous studies.
Clot model
I had kept the PE-50 containg clot in the refrigerator around 1 month. Once it was washed with PBS, it can be used within 2-3 days. So I make many clot PE-59 tube in one time.
No clot lysis
We did a clot lysis trial over the summer. TNK can lyse 24 and somewhat 48 hour clots, but that is all
Dr. Khan, The data you…
Dr. Khan, The data you presented last Friday are excellent. Did you do TTC to confirm the infarct size and location? The laser speckle images had blood flow reduced in the right hemisphere (from the frontal lobe to the occipital lobe). I want to know if the infarct was expanded to the territory of PCA.
Clot model and TTC staining
Hi Dr. Sheng,
You probably missed my first presentation, where I was shown clot delivery, CBF, and TTC staining after 24 hours of the clot delivery. The TTC staining has demonstrated the infarction of the right hemisphere.
In the last presentation, we optimized clot delivery with three different sizes of clots (3,4 and 5 mm) to show the dropped CBF just after the clot delivery.
reproducibility of the clot model
Hi Khan,
Thank you for sharing your excellent data with us. In Dr. Zhang's study published in JCBF (Zhang Z et al. A mouse model of embolic focal cerebral ischemia. J Cereb Blood Flow Metab. 1997 Oct;17(10):1081-8): "Preliminary studies in 20 mice showed that animals (12 of 20) achieving a successful occlusion of the MCA exhibited a blanching of the ipsilateral skull and a about 70% reduction in rCBF immediately after injection of the clot."
Do you have of similar reproducibility data in your clot model?
Do you think measurement of MCA blood flow is necessary?
In your presentation, the clot seems located in the ICA segment connecting to the MCA. Do you think that the clot location might affect the injury size?
Reproducibility of the clot model
Dr. Bengren,
Do you have of similar reproducibility data in your clot model?
Ans. Yes, we have a similar reproducibility of the reduction of blood flow (60-70%), as I explained during the presentation last Friday.
Do you think measurement of MCA blood flow is necessary?
Ans. Yes. Measurement of CBF after clot delivery or filament model of MCAo will give you a clear picture to confirm whether you have successful surgery on those animals. So, I always measure CBF after MCAo surgery.
In your presentation, the clot seems located in the ICA segment connecting to the MCA. Do you think that the clot location might affect the injury size?
Ans. Yes, it can be.
I had shown TTC staining in my 1st clot model presentation after 24 hrs, where infarction was visible. However, in the last presentation, I just showed the sizes of the clot delivery and the reduction of CBF after that.
You may try it in your lab let's see if the result match with our lab.
rat clot model
Hi Dr. Khan again,
Thank you for your informative answers. Had any mice shown no MCA blood flow reduction after injecting the clot in your previous studies or testing? Dr. Zhang's study showed that 12 among 20 mice had stroke, or 8 mice among 20 mice had no stroke after embolization.
If blood flow measurement is necessary, do you prefer mouse clot model or rat clot model. The rat skull bone is thicker so that the blood flow may not be directly monitored with a Doppler without thinning the bone area.
rat clot model
Hello Dr. Bingren,
So far, I have never got such types of incidents in no MCA blood flow reduction after clot delivery. It could be possible due to missed clot delivery or a fragile/thinner clot has to be broken or lyse quickly inside the MCA regions or around.
Rat clot model: It is easier than mice clot. Easy to clot and deliver using PE-10. But there are some disadvantages that we cannot measure CBF due to thicker skull bone as I also explained in my 1st presentation or in the discussion.
Mouse clot model: A little bit tough only clot delivering method due to having not suitable tubing/microcatheter. Advantages: you can measure CBF by laser speckle and easy to handle it.
So, I prefer mice.
clot model
Thanks! Khan, Another issue might be about using blood clot fresh and consistent. I believe that Dr. Zhang' mouse study showing that 12 among 20 mice have stroke or 8 among 20 mice without stroke, may also because the blood clot autolysis after embolization. Dr. Zhang suggested to use the overnight clot within 4 h after PE10 tubing washing preparation.
Clot model
Dr. Bingren,
It could be possible that a blood clot autolyzed in his case.
Fresh clots can be better than old clots. But I could not find any issue in our case, even in my old clot used after a week or two. But, of course, it depends upon how to prepare and store it.
2-hit model
In the large clot rabbit model with Justin Zivin, we settled on a "2-hit" model. We injected one clot, waited 5 min, and if the animal was looking normal, we injected a second clot. We had about an 80 to 90% "hit" rate. We did not use any transcranial monitoring at all. We had about a 50% rate of hemorrhagic transformation. to account for the 10 to 20% "no-hit" rate, we just increased the sample size in each experiment.
blood clot measurement
We measured the diameters of the blood clots made with PE50. The blood clots before washing were 0.391, 0.374, 0.380, and 0.385 mm. They were decreased to 0.227, 0.211, 0.218, and 0.222 mm after 15 times of washing.
clot model
Hi Huaxin, The diameters are great to know. The clot may be elastic, and thus may fit into a tubing ID that is smaller than the clot diameter.