clot stroke model

Submitted by bih001 on Wed, 03/29/2023 - 12:06
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Hello everyone,

During the site visit on 3/27/2023, we discussed with Pat and Jessica about making blood clot and the clot delivery tubing to be used.  PE50 tubing is commonly used to insert the clot delivery tubing into the MCA in the mouse and rat clot embolic stroke model. The PE50 tubing OD (0.99 mm) is too large and thus must be thinned down to 0.3-0.4 mm for the rat embolic model, as shown in the figure below. The thinning/extrusion technique via heating requires "significant" training to ensure a smooth extrusion.

As an alternative, a company called DOCCOL (www.dccol.com) produces a 0.36 mm OD tubing, as shown in the picture below. This microtubing may directly be used for the clot MCA embolus delivery purposes, eliminating the need for the "time consuming" thinning/extrusion technique. DOCOOL also sells a 0.22 mm OD tubing that may be used for the mouse clot delivery in the mouse embolic stroke model. 

Dr. Lyden asked a question about whether the DOCCOL 0.36 mm OD is too small to delivery a clot that is big enough to occlude the MCA. Our team will make different sizes of clots and then try to pass them through the Doccol catheter (s). We will report the results as soon as we have them. 

Meanwhile, please send us your questions, so we can answer them during our testing, e.g., making different types/sizes of clots.

Thank you!

Bingren

 


 

 

   Image removed.Image removed.

Submitted by huaxin.sheng on Thu, 03/30/2023 - 08:12

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What is the internal diameter of this catheter?  

Submitted by mkhanaugusta on Thu, 03/30/2023 - 09:42

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Augusta University has experience with human fibrinogen with arterial blood and washing clot. So, we can continue with it. 

Submitted by huaxin.sheng on Thu, 03/30/2023 - 10:01

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I checked the website and the ID of this Doccol catheter is 0.18 mm. 

                               OD (mm)            ID (mm)

PE10                        0.61                    0.28

PE 20                       1.09                    0.38

PE 50.                       0.965                 0.58

Alzet IT catheter.       0.36.                  0.18

Instech 2F PU.          0.69                   0.43

Doccol                      0.36                   0.18

PE50 blood clot       0.256 mm

Submitted by bih001 on Thu, 03/30/2023 - 10:22

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Could you please share your human fibrinogen with arterial blood and washing clot method, so we can try it?

The Alzet IT catheter is made with Polyurethane (PU). DOCCOL sells both Polyethlene (PE) and PU tubings.  The hardness of these two tubes may also need testing for the clot model. 

 

 

Order No.: 0007740

Catheter Material: Polyurethane (PU)

Specifications:

10 cm of 28G PU (0.36 mm OD; 0.18 mm ID) connected to 12.7 cm PU (0.84 mm OD; 0.36 mm ID) and ALZET connection (1.02 mm OD; 0.61 mm ID) with Teflon coated stylet wire. Total catheter length is 23.7 cm (9.33″). (Internal catheter volume is ~20 μl).

Optimum Animal Size:

Up to 300+ grams (cut distal end to desired length for proper placement)

Features & Benefits:

  • Designed for occipital insertion
  • Includes a Teflon coated stylet for easier placement
  • Protected tubing junction minimizes kinking at exit point from intrathecal space

 

Submitted by plyden on Fri, 03/31/2023 - 11:25

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All draft protocols are on the Google Drived where you can see, download, and comment. Please try out all the protocols and comment there or here or both. 

Will do,  

Also, we have tested the DOCCOL 0.36 mm microcatheter. I am happy to report that this DOCCOL 0.36 mm microcatheter works well to draw the washed clot made with PE50 tubing. Attached figure shows washed and non-washed clots, and DOCCOL catheter with the washed clot.

We have submitted a rat clot stroke model for UCSD IACUC review for about 3 weeks, and expect to get it approved in a few weeks.  We will test this DOCCOL catheter with the washed clot in the rat clot stroke model upon the IACUC approval. 

 

DOCCOL 0.36 mm microtubing with PE50 tubing clot

Submitted by lsansing on Fri, 03/31/2023 - 15:40

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We tried making clots from mouse.  Because we don't have femoral cut down on our approved IACUC protocol yet we used a submandibular venous blood sample.  We divided all volumes by 10 for mice.  Human fibrinogen 5uL (2mg/mL) and 10 cm PE-10 tubing.  The clot are incubating and we will post pictures when done.

No, there is no clot model for mice. I am not sure anyone will want to do the model in mice. But either way, i would recomend we get the model sorted out in rats, and then decide if we want to also implement it in mice. Eager to hear thoughts of others. 

Submitted by huaxin.sheng on Sat, 04/01/2023 - 20:20

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For mice, we need to do it in a different way. Otherwise, the mortality is too high as the PCA is blocked.

The diameter of the mouse MCAO filament is 0.22 mm. We can use the PE-50 to make the blood clot and then wash the blood clot until its diameter is 0.22 mm. Cut the blood clot to 2-3 mm and inject it into the CCA. The blood itself will bring it to the bifurcation of ACA and MCA. PPA should be ligated before a blood clot is injected. We put the PE-10 in the ECA and keep the CCA open. Then slowly inject the blood clot into the CCA. The blood flow from the CCA pushes the blood clot forward. 

If the CCA is open when the blood clot is injected, the ICA diameter will be large and the blood clot can be quickly delivered to the site. In addition, it is not required to have the catheter placed into the distal ICA so the endothelial wall will not be damaged. 

As soon as the PCA is not blocked by the blood clot, the stroke mice will survive after surgery.

Submitted by bih001 on Tue, 04/04/2023 - 17:45

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We understand that different mouse and rat embolic model protocols have been used to achieve the research objectives. We would like to post the mouse and rat embolic stroke models used at UCSD for getting critiques, suggestions, and comments on the protocol.  

 

UCSD Mouse or Rat Embolic Model

Animals: Male Sprague Dawley rats weighing about 400 g or Male C57BL/6J mice weighing about 25 g.

Anesthesia:

Using a face mask, mice or rats will be anesthetized with 3.5% isoflurane for induction and 1.5% isoflurane for maintenance. Isoflurane is delivered with 70% N2O and 30% O2.

Rectal temperature will be maintained at 37° ± 1.0°C throughout the surgical procedure by means of a feedback-regulated heating system.

Preparation of the emboli:

All surgery-related steps will be performed aseptically.

Arterial blood from either common carotid artery or femoral artery of a donor mouse or rat will be withdrawn and retained in 10 cm (for mice) or 50 cm (for rats) of PE-50 tubing for 2 hours at room temperature and for 22 hours at 4°C.

Five centimeters of the PE-50 tubing containing clot will be cut and attached to a system built of two syringes interconnected by 40 cm PE-10 tube filled with saline.

The clot will be continuously moved back and forth from one end to the other end of the PE10 tubing for about 15 times.

The tip of the Doccol PI-218 microcatheter for mice (OD 0.218 mm, ID 0.175 mm, tubing length 150 mm) or Doccol PI-267 microcatheter for rats (OD 0.267 mm, ID 0.221 mm, tubing length 150 mm), is coated with a very thin layer of silicone.

Alternatively, we will make and use PE50-extruded clot delivery catheter in the following steps: (i) holding the PE-50 tubing (150 mm in length) by hand about 200 mm above the low - medium flame of a burner until the medial portion of the tubing becomes soft and flexible; (ii) move the soft tubing away from the flame and immediately stretch the tubing laterally until the medial portion of the tubing is elongated and its o.d. is reduced; (iii) measure the medial portion of the modified PE-50 tubing with a micrometer to the o.d. between 0.2-0.3 mm for mice and 0.3-0.4 mm for rats. Cut and discard the portion with the proper ODs; and (iv) sterilize the modified PE-50 tubing in Cidex OPA solution overnight, and then rinse with sterile saline at least three times before use. The entire length of the modified tubing should be at least 120 mm, including an about 22 mm long portion from the tip with 0.3–0.4 mm OD.

A single clot will be transferred to the corresponding Doccol or modified tubing for mice or rats.

Animal models for mice or rats:

All surgery-related steps will be performed aseptically.

Via a midline incision in the neck area, the right common carotid artery, the right external carotid artery (ECA), and the right internal carotid artery (ICA) will be isolated and carefully separated under a surgical microscope (e.g., Carl Zeiss, Inc., Thorn-wood, NY, USA).  

A 6-0 silk suture will be loosely tied at the origin of the ECA and another suture will be ligated at the distal end of the ECA.

The right common carotid artery and ICA will be temporarily clamped using a microvascular clip (Codman & Shurtleff, Inc., Randolf, MA, U.S.A.).

The clot-loaded silicone-coated Doccol PI-218 microcatheter for mice or Doccol PI-267 microcatheter for rats will be introduced into the ECA lumen through a small ECA incision.

The suture around the origin of the ECA will be tightened around the intraluminal catheter to a degree that prevents bleeding.

The microvascular clip will be removed. An 8-9 mm length of catheter for mice and 18-19 mm length of catheter for rats will be gently advanced from the ECA into the lumen of the ICA. Previous studies show that the intraluminal catheters at these positions are about 1.5 mm from the origins of the mouse or rat MCA, respectively.

The embolus in the catheters will be injected with a pre-tested speed through the catheter.

For the pre-tested speed, before injection of the clot, the lab will need to test the injection speed (using a separate catheter and clot) that allows the clot to form a ball when it comes out from the tip of the catheter.    

To confirm the placement of the catheter in the correct position, the MCA blood flow will be monitored by laser Doppler flowmetry (LDF).

The LDF probe will be attached, via a small incision between the ear and eye, to the flat point/site of the temporal bone surface right about the MCA.  This flat point/site needs to be tested by moving the probe tip around the area until the maximal voltage is detected. For mice, this LDF attachment can directly or transcranially monitor the MCA blood flow. For rats, the bone in this area must be thinned via a hand-held drill to the level that the MCA blood flow can be monitored with a LDF probe. 

The MCA blood flow will be reduced significantly when the tip of the clot delivery catheter reaches or is close to the base/origin of MCA, or transiently increased initially when injecting the clot and then decreased when the clot blocks the MCA. The MCA blood flow is expressed as a percentage of pre-ischemic baseline values.

After injection of the embolus, the CCA remains to be clamped for 5 min, the clot delivery catheter will be slowly pulled out from the ECA, and then the ECA will be closed with a suture.

For mice, the original report of this model showed that mice (12 of 20) achieving a successful occlusion of the MCA exhibited a blanching of the ipsilateral skull and a ≥70% reduction in MCA blood flow immediately after injection of the clot. The remaining 8 mice, because of inadequate MCA occlusion, did not exhibit blanching of the ipsilateral skull, and MCA blood flow in these mice was reduced by 30% to 40% from pre-embolization levels, indicating inadequate MCAO (Zhang Z, Chopp M, Zhang RL, Goussev A. A mouse model of embolic focal cerebral ischemia. J Cereb Blood Flow Metab. 1997 Oct;17(10):1081-8).

For rats, the successful occlusion of the MCA is higher according to the report (Zhang RL, Chopp M, Zhang ZG, Jiang Q, Ewing JR. A rat model of focal embolic cerebral ischemia. Brain Res. 1997 Aug 22;766(1-2):83-92).

Submitted by huaxin.sheng on Wed, 04/05/2023 - 08:56

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I hope you have success when the blood clot is injected. Just be sure the blood clot size is smaller than the MCA diameter.  Will you be able to measure the diameter of the blood clot before injection? 

Submitted by mkhanaugusta on Wed, 04/05/2023 - 10:28

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                                                                                                                Clot preparation                                     M B Khan (Hess group: Augusta)  

                                                                                                                                                                                  March 2015                          

 

 

Stock solution (20 mg/ml):

  1. Weigh 20 mg human fibrinogen (Cat#341576; Millipore/sigma: Fibrinogen Human Plasma) and dissolved in 1 ml sterile 1x PBS.
  2. Aliquot 100 µl in a microcentrifuge tube and keep it at -80 C for future use.

Note: Stock aliquot should be used within 6 months, if possible. It may be inactive for a longer time.

 

Working solution (2 mg/ml):  900 µl blood+100 µl human fibrinogen or 450 µl blood+50 µl human fibrinogen

 Method:

  1. Bring 100 µl aliquot from – 80 C and keep it at room temperature till liquefying.
  2. Keep in a 1 ml syringe and withdraw arterial blood (900 µl or 450 µl) from the heart using with 1 ml syringe filled with human fibrinogen (100 µl or 50 µl) and mix with them (30-60 sec).
  3. Retained in 25-30 cm PE-50/PE-10 tubing overnight at room temperature.
  4.  Next day, keep the tubes in a 4 C refrigerator.
  5. Clot was taken out into sterile PBS or 0.9% saline and washed to remove most of the red blood cells.
  6. Clots: keep into clean sterile PBS at room temperature for retraction.
  7. Clots again washed with PBS
  8. Cut into 5 cm long fibrin-rich core pieces.
  9. A single clot of 9±0.5 mm (8 mm) was transferred to a modified PE-10 tube/catheter filled with PBS.
  10. Adjust the length of the clots according to need /delivery. It is better to use between 2-5 mm for mice and 8-10 mm for rats to avoid a higher mortality rate of the animals.

 

We use PE50 clot. The clot is highly sticky and elastic. The diameter will change when very slowly injecting out of the microcatheter (ID 0.18), so that clot will form a small ball to occlude MCA base.  Also, select curved a "curved" clot. 

Yes, we customize it by myself. It just pooled the tip little bit (~10 mm) and cut the end of the tips diagonally. It will be insert easily in the ICA.

You can also use custom-made commercially. That can be saved your time.

Submitted by mkhanaugusta on Wed, 04/05/2023 - 12:36

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The delivery clot tube can be passed through PPA (Pterygopalatine) and push the syringe thumb rest, the clot will be delivered smoothly with flow and it will be passed/crossed the PCA (Posterior cerebral artery) junction. The clot will be reached in MCA region via PcomA ( Posterior communicating artery).

It may or may not stay the clot at the junction of PCA if the thumb rest pressure is very slow. Usually, it cant be! So maintain the thumb rest pressure during delivery of the clot.

Submitted by plyden on Wed, 04/12/2023 - 21:46

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Rather than pass the catheter so far up, i prefer to ligate the PPA first. However, Jessica prefers the  method Khan is saying: push the catheter up past the PPA. 

Submitted by huaxin.sheng on Thu, 04/13/2023 - 07:50

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Did anyone check the endothelial wall of ICA to see if the catheter caused wall damage in some mice, which may affect blood flow during recovery?

The catheter can cause ICA endothelial wall damage if the thickness of the catheter diameter is more than the ICA diameter. So gently push the catheter tips inside the ICA.  

I also prefer to ligate the PPA first, or it can be used with small micro-clips, commercially available for microvessels surgery. Sometimes either filament or clot is stuck onto the junction of ICA and PPA. It also depends on the surgeon's skills. Some surgeons do not ligate the PPA. They lift it slightly and insert the filament.

 

Submitted by bih001 on Thu, 04/13/2023 - 10:48

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The more close to the MCA base that clot delivery tube is inserted into ICA, the more consistent/reproducible we may get. However, if all testing sites use the same ICA insertion distance, we may have more animals per group, which may overcome the potential variation issues.    

Submitted by huaxin.sheng on Fri, 04/14/2023 - 09:14

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We prefer to add human fibrinogen into the syringe before blood collection and inject the blood into the tubing immediately after blood collection. 

This is not a uniform clot made due to improper mixing of blood and human fibrinogen. So, these steps are critical to preparing the clot.

Submitted by mkhanaugusta on Fri, 04/14/2023 - 09:37

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First, we take human fibrinogen (50 ul) in a syringe (insulin injection syringe 1 mL) and then collect blood around 450 ul, mixing using ups and down to the syringe a couple of times and injecting the blood in the PE-50 tube as I explained in our sites protocol. Improper mixing can make a nonuniform clot. 

We withdraw blood from femoral artery or CCA directly into a PE50 tubing and leave the PE50 tubing at 37C for 2 h and 4C for overnight. Has anyone compared the clot formation between the blood withdrawal to a syringe then to a PE50 tubing, and directly into PE50 tubing?    

Submitted by mkhanaugusta on Fri, 04/14/2023 - 13:00

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We did not compare. What is the reason for reaching it? 

We withdrew from the heart using a pre-filled fibrinogen (50 ul) syringe, mixed it quickly with blood (450 ul), transferred it into PE-50 (25-30 cm), kept it at room temperature overnight, and then stored it at 4 C for future use.