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 JoVE Neuroscience

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

1, 1, 1, 1, 1

1Department of Neurological Surgery, School of Medicine and Public Health, University of Wisconsin-Madison

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    Summary

    Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

    Date Published: 2/05/2011, Issue 48; doi: 10.3791/1978

    Cite this Article

    Uluç, K., Miranpuri, A., Kujoth, G. C., Aktüre, E., Başkaya, M. K. Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat. J. Vis. Exp. (48), e1978, doi:10.3791/1978 (2011).

    Abstract

    Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

    Protocol

    MCAO Rat Model

    Presurgical Preparations

    Aseptic technique should be used for all survival surgical procedures. Disinfect the surgical work surface with commercial disinfectant and prepare sterile surgical packs of instruments, drapes, gauze, swabs, sutures, and scalpel blades by autoclaving A surgical mask, hair bonnet and sterile gloves should be worn. A Germinator dry bead sterilizer is also used to resterilize surgical instruments between procedures if multiple rat surgeries will be done during one session. Prewarm a water-jacketed homeothermic blanket and place under an absorbent pad in order to prevent hypothermia of the rat during surgery.

    1. Place spontaneously hypertensive rat (or other rat strain of choice) into an induction chamber and induce anesthesia with 5% isoflurane (anesthesia machine should be set to 1.0 L/min O2 and 1.0 L/min N2O). Lower to 1-2% isoflurane to maintain anesthesia.
    2. Apply Artificial Tears ointment to both eyes.
    3. Shave the throat and left neck region beyond the prospective incision site using clippers (Oster A5 with #10 blade).
    4. Apply Betadine to a gauze pad and disinfect the skin starting from the center of the surgical region, spiraling outward. Rinse with sterile gauze pad containing 70% ethanol, moving pad in a similar pattern. Repeat both steps for a total of three cycles.
    5. Inject 0.2 mL of 0.5% bupivacaine subcutaneously along the prospective incision site.
    6. Place rat in sterile stockinette or cover with a sterile surgical drape.

    Transient occlusion of MCA

    1. Under an operating microscope, a ventral midline incision is performed and the superficial fascia is dissected.
    2. Beneath the superficial fascia, there is glandular tissue to the left and three muscles which form a triangle: the sternohyoid, which lies midline over the trachea; the digastric (easily identified with its shiny white tendinous portion); and lastly, the sternomastoid muscle.
    3. Careful sharp and blunt dissection is performed within the triangle to identify the carotid artery (exclusive blunt dissection may be preferred by some to minimize accidental tissue damage). The external, internal, and common carotid arteries (ECA, ICA, CCA) are exposed. The carotid artery is large and visibly pulses. The vagus nerve is seen coursing along the lateral aspect of both common and internal carotid arteries and is sharply dissected off the common and internal carotid arteries.
    4. Two ECA branches are then dissected sharply, the first branch heading medially and the second branch heading laterally. Both branches are cauterized and cut, which allows for greater ease in mobilizing the larger vessels.
    5. The ECA is now dissected more rostrally. The hyoid bone may be encountered and this will limit the extent of rostral dissection. The external carotid artery is tied off as distally as possible with a 6-0 silk suture.
    6. Another 6-0 silk suture is placed loosely around the ECA near the bifurcation with the ICA. Make sure not to occlude the vessel as the intraluminal suture to be used for the occlusion will be going through here at a later step.
    7. Microsurgical clips are placed on common and internal carotid arteries near the bifurcation.
    8. An initial partial arteriotomy is created between the two silk suture ties on the external carotid artery.
    9. Introduce a 2.2 -3.0 cm length of 4-0 monofilament nylon suture, with a kink 2.0 cm from its rounded, silicon-coated tip, into the ECA lumen down toward the CCA where the microsurgical clip is located. These sutures are commercially available (Doccol Corp., Redlands, CA, USA). Cut the remaining portion of the ECA (at the site of the partial arteriotomy) to free the stump and position the stump below the bifurcation of the ECA and ICA; this will more easily allow the intraluminal suture to slide into the ICA.
    10. Tighten the silk suture around the ECA stump to secure the intraluminal nylon suture and prevent bleeding, and then remove the microvascular clip from the ICA. Open the clip slowly before removing it to check for bleeding.
    11. Continue advancing the nylon suture from the ECA to ICA lumen to the middle cerebral artery (MCA). This length is typically 18-20 mm and is the reason for placing a kink in the suture prior to insertion. After a variable length of nylon suture is inserted, resistance may be felt. If this occurs with much of the nylon suture still present outside of the vessel, it indicates that the suture is likely entering the pterygopalatine artery (see note below). Pull back and curve the suture slightly to continue along the ICA, which will run more medially. Additionally, dissection of the origin of the PPA may be done to better visualize the path of the intraluminal filament. Continue to insert the nylon suture until resistance is felt at and after the 2 cm kinked position. At this point, the intraluminal suture has blocked the origin of the MCA. MCA occlusion can be confirmed by monitoring the reduction in regional cerebral blood flow using a laser Doppler flowmeter (see Materials table for one equipment source). Also note that the origin of the pterygopalatine artery off of the ICA can be directly tied off, if preferred, in order to avoid accidental intubation of this vessel with the intraluminal suture.
    12. Start the timer and record occlusion start time.
    13. Remove the microclip from the CCA.
    14. Close the incision (dermis, panniculus carnosus, subcutaneous tissue layers) quickly with 3-0 silk suture (a simple continuous pattern will facilitate reopening for reperfusion) and carefully place the rat in a recovery cage. Check that the cage floor around the nose and mouth are free of bedding material and monitor recovery from anesthesia.

    Restoration of MCA blood flow (reperfusion)

    1. Shortly before the occlusion period should end, re-anesthetize the rat, disinfect the incision site with Betadine and 70% ethanol (3 cycles, as before) and reopen the incision by removing the closing sutures.
    2. Place a microclip on the CCA, as before.
    3. Withdraw the occluding suture partway from the ICA until the suture end is visible through the ICA. Do NOT fully remove the suture from the ICA/ECA!
    4. Place a microclip on the ICA above the end of the intraluminal suture.
    5. Completely remove the occluding suture and tightly tie off the ECA stump. Record end of occlusion (reperfusion start) time.
    6. Remove the microclip from the ICA.
    7. Remove the microclip from the CCA.
    8. Moisten the region with several drops of sterile saline and close the incision layers (dermis, panniculus carnosus, subcutaneous tissue) with 3-0 silk suture using a simple interrupted pattern.
    9. Administer 0.05 mg/kg buprenorphine (or other appropriate post-operative analgesic following your institutional guidelines) into the peritoneal space.
    10. Inject 5cc of prewarmed saline intraperitoneally. This will provide hydration during the recovery stage.
    11. Monitor recovery of rat from anesthesia.
    12. After the rat has recovered, test for behavioral indication of infarction by holding the rat by the tail and observing whether the rat can turn to both sides. Curling to one side only is expected. Simple scoring scales can be used to record initial post-infarct behavorial functioning (see Discussion).
    13. Additional injections of buprenorphine (same dosage as above) should be given every 6 to 8 hours for 24 hr of pain relief. Extend analgesia if animal is showing signs of discomfort.

    Representative Results

    Figure 1
    Figure 1. Occlusion of the middle cerebral artery by endovascular suture. A simplified diagram of the cranial circulatory system of the rat is shown with a silicon-coated intraluminal suture occluding the origin of the MCA. The OA and ST branches off of the left ECA have been ligated and a suture tie around the ECA stump holds the intraluminal suture in place. ACA, anterior cerebral artery; BA, basilar artery; CCA, common carotid artery; ECA, external carotid artery; ICA, internal carotid artery; MCA, middle cerebral artery; OA, occipital artery; PCA, posterior cerebral artery; PComA, posterior communicating artery; PPA, pterygopalatine artery; ST, superior thyroid artery. Figure adapted from Sasaki et al.4 and Lee3.

    Figure 2
    Figure 2. Representative magnetic resonance imaging of coronal sections of rat brain 24 hours after 1 hour transient MCAO. Brain edema accompanying focal ischemia is visualized in T2-weighted MRI images. The infarct zone appears hyperintense (bright) on T2 images. Similar infarct patterns can be seen with more advanced MRI techniques such as diffusion-weighted imaging and apparent diffusion coefficient mapping5.

    Figure 3
    Figure 3. Representative coronal sections of rat brain stained with TTC 24 hours after 1 hour transient MCAO. TTC staining reveals white (unstained) infarcted regions of cerebral cortex and striatum in the same rat as that shown in Figure 2. Note that the general pattern of focal ischemic lesions is similar to that seen in the MRI T2-weighted images, although the final infarct area determined histologically may be slightly smaller than the T2 lesion.

    Discussion

    Rodent models of cerebral ischemia can be classified as global or focal and as reversible or irreversible. We use a reversible focal ischemia model in order to mimic the reperfusion injury that can occur following recanalization in human stroke patients. While the rat shares the advantage of similar cranial circulation to the human, the extensive intracranial collateral circulation due to the Circle of Willis and leptomeningeal anastomoses results in inconsistent infarct volumes6. We use the spontaneously hypertensive rat (SHR) strain because infarct size is greater in SHRs exposed to focal cerebral ischemia compared to normotensive rats7-10. Stroke induction in SHRs is also more consistent and may generate less mortality than in other commonly used strains11. In addition, physiological parameters should be monitored throughout the surgery, because fluctuations can add to the variability in infarct volumes; these parameters include blood pressure, arterial O2 and CO2 levels, hypothermia, and hyperglycemia, which can exacerbate ischemic injury2,12. Hypothermia is of particular concern because it can lessen the degree of neuronal ischemic injury, and therefore, placing the rat on a thermoregulatory heating pad during surgery is essential.

    We employ commercially prepared silicon-coated sutures in this procedure in an attempt to increase consistency in the infarct size13-15. Traditionally, intraluminal sutures used to occlude the MCA have been prepared by flame-rounding a 3-0 nylon suture tip or using a hot glue gun to coat the suture tip with a small glue bead16. These procedures produce sutures with varying tip diameters and thus, may require several attempts at occlusion before finding a suture that matches the vessel diameter of a given rat. Additional variations on the MCAO method described in this video include more distal occlusion, sparing the lenticulostriate branches in order to have a pure cortical infarct17, and simultaneous occlusion of the MCA and ipsilateral common carotid artery to reduce collateral blood flow6,9.

    The functional effects of stroke can be assessed using a variety of behavioral tests, in which deficits in specific tasks are reflected in a simple scoring scale. For example, Bederson et al.18 developed a neurological score grading rats on the extent of forelimb flexion in an elevated body position, asymmetric resistance to a lateral push, and open field circling behavior. Rats extending both forelimbs to the floor when elevated and exhibiting no other deficits were graded "Normal (0)", whereas rats with contralateral forelimb flexion only were classified with "Moderate deficits (1)". "Severe grade 2" rats displayed decreased resistance on the contralateral side to a lateral force and "Severe grade 3" rats additionally engaged in circling behaviors. Lower grade behavioral deficits were always observed in the next higher grade (e.g., grade 2 and 3 animals also displayed forelimb flexion) and these scores were predictive of differences in infarct size18. Thus, such a simple scoring system allows for a rapid, consistent semi-quantitative assessment of the functional deficits from stroke.

    Measurement of the extent of cerebral infarction is commonly accomplished by histological staining for tissue damage using TTC, cresyl violet (Nissl stain) or hematoxylin and eosin. Magnetic resonance imaging techniques, including diffusion-weighted MRI to visualize the infarct at early time points after occlusion, allow for the determination of an infarct volume in each individual rat without the need for harvesting tissue. This is especially valuable in studies aimed at determining the efficacy of a neuroprotective treatment following stroke; imaging the rat after MCAO but prior to treatment administration gives the investigator confidence that all experimental and control animals experienced similar strokes to start, reducing the confounding effects of variability in stroke induction.

    Disclosures

    The authors have no conflicting interests to disclose.
    This protocol was approved by the Institutional Animal Care and Use Committee at University of Wisconsin-Madison School of Medicine and Public Health and abides by the National Institutes of Health guidelines for the use of experimental animals.

    Acknowledgements

    We thank Beth Rauch and the University of Wisconsin Small Animal Imaging Facility for magnetic resonance imaging. The Department of Neurological Surgery at the University of Wisconsin-Madison provided funding for this work.

    Materials

    Name Company Catalog Number Comments
    4-0 Nylon suture Doccol Corporation 2-3 mm silicon coating length, 0.39 mm tip diameter
    Spontaneously hypertensive rats Charles River Laboratories 290-300 g, male
    Gaymar T/Pump thermoregulatory pump and pads Gaymar Industries
    Vetroson V-10 bipolar electrosurgical unit Summit Hill Laboratories
    Germinator 500 glass bead sterilizer CellPoint Scientific
    Vasamedics Bpm2Laserflo blood perfusion monitor TSI Inc.

    References

    1. van Gijn, J. & Dennis, M. S. Issues and answers in stroke care. Lancet 352 Suppl 3, SIII23-27 (1998).
    2. Macrae, I. M. New models of focal cerebral ischaemia. British Journal of Clinical Pharmacology 34, 302 (1992).
    3. Lee, R. M. Morphology of cerebral arteries. Pharmacol Ther 66, 149-173 (1995).
    4. Sasaki, M., Honmou, O. & Kocsis, J. D. A rat middle cerebral artery occlusion model and intravenous cellular delivery. Methods Mol Biol 549, 187-195, doi:10.1007/978-1-60327-931-4_13 (2009).
    5. Bråtane, B. T., Bastan, B., Fisher, M., Bouley, J. & Henninger, N. Ischemic lesion volume determination on diffusion weighted images vs. apparent diffusion coefficient maps. Brain Res 1279, 182-188, doi:10.1016/j.brainres.2009.05.002 (2009).
    6. Chen, S. T., Hsu, C. Y., Hogan, E. L., Maricq, H. & Balentine, J. D. A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction. Stroke 17, 738-743 (1986).
    7. Dogan, A., Başkaya, M. K., Rao, V. L., Rao, A. M. & Dempsey, R. J. Intraluminal suture occlusion of the middle cerebral artery in Spontaneously Hypertensive rats. Neurol Res 20, 265-270 (1998).
    8. Ogata, J., Fujishima, M., Morotomi, Y. & Omae, T. Cerebral infarction following bilateral carotid artery ligation in normotensive and spontaneously hypertensive rats: a pathological study. Stroke 7, 54-60 (1976).
    9. Brint, S., Jacewicz, M., Kiessling, M., Tanabe, J. & Pulsinelli, W. Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries. J Cereb Blood Flow Metab 8, 474-485 (1988).
    10. Duverger, D. & MacKenzie, E. T. The quantification of cerebral infarction following focal ischemia in the rat: influence of strain, arterial pressure, blood glucose concentration, and age. J Cereb Blood Flow Metab 8, 449-461 (1988).
    11. Coyle, P. Different susceptibilities to cerebral infarction in spontaneously hypertensive (SHR) and normotensive Sprague-Dawley rats. Stroke 17, 520-525 (1986).
    12. Slivka, A. P. Hypertension and hyperglycemia in experimental stroke. Brain Res 562, 66-70 (1991).
    13. Bouley, J., Fisher, M. & Henninger, N. Comparison between coated vs. uncoated suture middle cerebral artery occlusion in the rat as assessed by perfusion/diffusion weighted imaging. Neurosci Lett 412, 185-190, doi:10.1016/j.neulet.2006.11.003 (2007).
    14. Schmid-Elsaesser, R., Zausinger, S., Hungerhuber, E., Baethmann, A. & Reulen, H. J. A critical reevaluation of the intraluminal thread model of focal cerebral ischemia: evidence of inadvertent premature reperfusion and subarachnoid hemorrhage in rats by laser-Doppler flowmetry. Stroke 29, 2162-2170 (1998).
    15. Shimamura, N., Matchett, G., Tsubokawa, T., Ohkuma, H. & Zhang, J. Comparison of silicon-coated nylon suture to plain nylon suture in the rat middle cerebral artery occlusion model. J Neurosci Methods 156, 161-165, doi:10.1016/j.jneumeth.2006.02.017 (2006).
    16. Longa, E. Z., Weinstein, P. R., Carlson, S. & Cummins, R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84-91 (1989).
    17. Shigeno, T., McCulloch, J., Graham, D. I., Mendelow, A. D. & Teasdale, G. M. Pure cortical ischemia versus striatal ischemia. Circulatory, metabolic, and neuropathologic consequences. Surgical neurology 24, 47-51 (1985).
    18. Bederson, J. B. et al. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17, 472-476 (1986).

    Comments

    34 Comments

    how do make a ²-3 mm silicon coating length, 0.39 mm tip diameter and how do put it on in the surure
    Reply

    Posted by: AnonymousMay 10, 2011, 6:24 PM

    We custom order 4-0 nylon sutures with 0.39 mm tip diameter and ²-3mm silicon coating length from Doccol Corporation ( http://www.doccol.com). Use their "My Special Design" option to customize coating length and tip diameter from their choices. You may need to try several sizes for your strain and size of rat. These values are what work best for us with SHRs in the ²80-300g range.
    Reply

    Posted by: AnonymousMay 10, 2011, 6:48 PM

    Could you please provide the information on the cauterizer used during the procedure
    Reply

    Posted by: AnonymousJune 22, 2011, 2:30 PM

    Here is the link to the manufacturer's site:

    http://www.summithilllaboratories.com/products_electro.htm
    Reply

    Posted by: AnonymousJune 22, 2011, 4:57 PM

    which software do you use and how to determine the volume of the infarction?
    kind regards
    Reply

    Posted by: AnonymousJuly 19, 2011, 6:14 PM

    We use the admittedly tedious method of drawing regions of interest in ImageJ software to measure infarct areas. We use the indirect measurement of infarct volume instead of directly measuring the infarct lesion to avoid the complications that arise from edema-induced changes in brain volume following stroke.

    The indirect formula is: ([contralateral hemisphere healthy area] minus [ipsilateral hemisphere healthy area]) times [slice thickness]; sum this across all of the sections to get infarct volume for the whole brain. We use this same approach whether it is on MRI or tissues stained with TTC or cresyl violet.
    Reply

    Posted by: AnonymousJuly 19, 2011, 6:32 PM

    very good for understanding.
    but how i save your video plz reply
    Reply

    Posted by: AnonymousJuly 22, 2011, 10:32 PM

    I notice in your video (which is excellent by the way) that you are using a very basic anaesthetic rig with just the vapouriser for isoflurane. Is this what you use to perform all of your MCA occlusions? If so, can you please provide a list of equipment I need to set up the exact same rig as yours?
    Thank you very much for your time and help.
    Reply

    Posted by: AnonymousOctober 16, 2011, 8:50 PM

    We use a setup with an isoflurane funnel-fill vaporizer (e.g., 911103), dual gas flowmeter, 45 inch bain non-rebreathing circuit (9²1410), and rodent nosecone (9²1431). We use a ² liter induction chamber (941444). Waste gases are vented into a fume hood for exhaust. If you do not have an exhaust hood available, then you will want a waste gas scavenging system, such as activated charcoal canisters. Several companies make appropriate equipment, (including SurgiVet and VetEquip, for example) and you can get a complete system mounted onto a tabletop bracket for a research setting. I have included VetEquip part numbers in parentheses for several items [I had to order replacements recently so I had them handy ;-) ] I would contact one of the companies and they can recommend a specific system if you are starting from scratch.
    Reply

    Posted by: AnonymousOctober 17, 2011, 3:59 PM

    Hi Gregory, thank you very much for providing that information!
    Reply

    Posted by: AnonymousOctober 17, 2011, 7:49 PM

    Gregory, is there an email I can contact you on? I have spoken to the Vetquip here in Australia and they have no idea what each of the individual components you have listed are!! Apparently the nomenclature is slightly different from country to country. Apologies in advance for the hassle.
    Reply

    Posted by: AnonymousOctober 17, 2011, 9:25 PM

    Hi Gregory,

    I have tried emailing you at the address you provided in the above post but it bounced? I have more questions re MCA occlusions. Could you possibly email me at michelle.chen@southernhealth.org.au?

    Thanking you kindly
    Reply

    Posted by: AnonymousMarch 14, 2012, 7:18 AM

    thanks for your excellent experiment.. but could you tell me kindly how can i get this video??
    Reply

    Posted by: AnonymousDecember 17, 2011, 11:34 AM

    This artical is really great .
    Reply

    Posted by: shen m.July 30, 2012, 8:30 PM

    thanke you for the video. Can you give me a reference please, where I can buy a microvascular clips, that you use. Thanke you!
    Reply

    Posted by: margarita B.October 3, 2012, 8:42 AM

    We use Mizuho Sugita AVM microclips (07-93² series; straight blades come in different lengths; we use 3mm or 4 mm) and clipholder. See the link for contact info to get a sales rep:

    http://www.mizuho.com/contact
    Reply

    Posted by: Gregory K.October 3, 2012, 7:24 PM

    Thank you for your share.My first question is that what is the aim for coagulation of ST artery and Occipital artery?Because the limitation ,we use 4 suture to ligation ST artery and Occipital artery,but bleeding is contineous ,not as your description in video,do you have some idea about the reason?
    Thanks for you help!
    Reply

    Posted by: janne l.November 27, 2012, 10:57 AM

    Good afternoon. I am not the author of the video, but I work with the same model. Perhaps The reason is that the blood pressure in these arteries is high and diamert of vessel is very small and your ligatures just come off? I gust can suggest using bipolar coagulation- it is very fast and easy-takes ²-5 seconds to one artery and prevents any bleeding.In Russia this device сost $ 1,000.
    Reply

    Posted by: margarita B.February 14, 2013, 8:41 AM

    Yes, we too use the bipolar. It is easy and effective and we don't experience any issues with bleeding of the ST and OA.
    Reply

    Posted by: Gregory K.February 14, 2013, 4:32 PM

    Good afternoon, colleagues.
    I've been working on this model and I am having the following problems: using rats weighing ²50-300 g and filaments Filament size 4-0, diameter 0.19 mm, length 30 mm; diameter with coating 0.39 + / - 0.0² mm; coating length 3 - 4 mm DOCCOL in rats is not formed area of stroke or it is extremely low. Tell me, what is it can be connected?
    Thanks for the reply.
    Margaret
    Reply

    Posted by: margarita B.February 14, 2013, 8:33 AM

    There is some variability inherent in the model. As such, you may find it necessary to perform a series of pilot experiments using sutures of different sizes. Doccol allows you to customize the silicon tip diameter and coating length. We use shorter coating length because it is easier to manipulate the suture during entry into the ECA and rerouting into the ICA. Longer coating would presumably give you a bigger margin of error in suture placement at the MCA origin while still occluding the vessel.
    Note that several other factors may come into play as well. Stroke size and reproducibility can vary with rat strain. While many people have had success with Sprague Dawley rats, the experience in our department has been that spontaneously hypertensive rats more consistently show stroke lesions. Even with SHRs, however, there can be some variability. Keeping the animals warm during surgery and recovery is also important. Hypothermia is neuroprotective. Room drafts can chill an animal post-ischemia enough to noticeably reduce infarct size (as a colleague of ours recently can testify).
    Good luck!
    Reply

    Posted by: Gregory K.February 14, 2013, 4:29 PM

    thank you very much for your reply.
    How the length of the silicone coating influences the variability of stroke?
    What length and diamert coverage you would be advised to rats weighing ²80-350 grams? line WISTAR
    Thank you!
    Reply

    Posted by: margarita B.February 26, 2013, 6:14 AM

    That is something that you will have to test yourself. We don't use Wistar rats (although SHRs are derived from Wistar Kyoto rats). We tried a few different silicon diameters and lengths in pilot experiments but didn't do enough numbers to be able to make definitive comparisons. Once we found that we were getting infarcts, we stuck with .39mm diameter, ²-3 mm length for our ²80-300 g SHRs. Our surgeons preferred the shorter coating length for a bit easier bending when advancing the suture from the ECA into the ICA but a longer coating (e.g. 4-5mm) may give a larger "margin of error" in terms of suture placement blocking the MCA.
    Reply

    Posted by: Gregory K.February 28, 2013, 5:46 PM

    Good afternoon. tell me please, what formula and application you use to calculate the volume of stroke using brain slices?
    Thank you!
    Reply

    Posted by: margarita B.April 4, 2013, 5:02 PM

    We use indirect lesion area measurement, summed across all sections analyzed to get an infarct volume. By indirect, I mean that we measure the healthy non-lesioned area and infer the lesioned area by subtraction. Please see comment number 6 above.

    Thanks.
    Reply

    Posted by: Gregory K.April 4, 2013, 5:50 PM

    Hi, pls is it important to shave the hair off the incision site, as i have watched other videos on jove with the hair not shaved. The Autoclips, bipolar coagulator all appear to be expensive, pls can you itemize a replacement for a low cost mcao produre. How best do you think a student can learn and perfect this procedure, as ethics can only approve less than 10 wistar rats for the pilot study.

    Thanks Alex
    Reply

    Posted by: Olusiji A.June 3, 2013, 8:36 AM

    Shaving the incision site is a part of proper aseptic surgical technique and will be desired by most institutional ACUCs I believe. To reduce expense, you can tie off vessels with suture in place of using a bipolar coagulator. Microvascular clips are expensive, unfortunately. There is no substitute for practice to attain proficiency. You may consider performing the procedure on both left and right sides with the first couple of rats to increase your opportunities at learning the technique with fewer rats.
    Reply

    Posted by: Gregory K.June 3, 2013, 4:30 PM

    Great Insight! Thanks
    Reply

    Posted by: Olusiji A.June 4, 2013, 4:59 AM

    Hello, please how many times can i expect to re-use a reusable silicon coated suture from doccol....Can 5 reusable silicon coated sutures be sufficient for reuse with 102 rats...Thanks
    Reply

    Posted by: Olusiji A.July 4, 2013, 11:26 AM

    The number of times you can reuse a "reuseable" suture will vary depending on it physical condition. We have most often gotten 2 or 3 uses per suture without complaint, occasionally more. Sutures that have been used too many times, however, can lose their silicon tip inside the vessel (!) or potentially increase the risk of hemorrhage if the silicon coating is no longer nice and smooth. I would say that there is very little chance that you'd get >20 uses per suture!
    Reply

    Posted by: Gregory K.August 19, 2013, 3:55 PM

    hello every body, i am experiencing a problem while inducing stroke through MCAO. i am using nylon silk with manually rounded tip by heat for blocking Middle cerebral artry. some times luckily i easily inserted this silk into the middle artry, while many times i failed because the silk fail to insert , facing resistance from pterygopalatine artery. i don't know what to do, i search a lot how i should locate the artry so that i should easily inserted without facing any problem. your kind help will be greatly appreciated. one thing there is no problem with the nylon silk and rounded tip, becoz i kept the tip diameter approximately low, so that it easily pass the Middle artry once it inserted.
    Reply

    Posted by: Fawad ali s.August 17, 2013, 4:14 AM

    You may consider dipping your suture in a heparin solution to reduce the likelihood of the suture sticking to the endovascular surfaces. We have done this in the past to try reduce the risk of hemorrhage when removing flame-round sutures (prior to using the silicon-coated sutures that we employ currently). This is just a guess, but an indirect benefit may also be that it might be a little easier to position within the artery and so avoid the PTA. Perhaps your best bet would be to extend the exposure during surgery high enough to visual the PTA branchpoint if this continues to be a problem.
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    Posted by: Gregory K.August 19, 2013, 4:03 PM

    Hello all, please i need a confirmation to the protocol from literatures i find online as to measuring infarct volume. Here are my questions:

    1. When sacrificing the animal (rat) for TTC staining, do we perform whole body perfusion fixation to fix the brain tissue, what i read from most articles is not that explanatory (Example :- After 24 h of reperfusion, the animal is euthanized using an overdose of Anesthesia followed by cervical dislocation and decapitated to remove the brain carefully.)

    2. To lower the extent of infarct variability between animals, i am considering or tying off the right CCA with a suture prior to occluding the MCA and only to remove the suture from CCA after reperfusion. Do you think doing will not significantly compromise the pathophysiological of the MCAO-reperfusion stroke model and if it will help reduces variability as i hope.

    Thanks a lot
    Olusiji Alex
    Reply

    Posted by: Olusiji A.April 10, 2014, 3:43 AM

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