Cerebral perfusion monitoring is very important for increasing accuracy and reducing variability in experimental stroke models. Laser Doppler technology can easily detect cerebral microvascular perfusion through the intact skull of mice and rats, particularly if you're using a deep penetration probe. The key problem here is to find a reliable system for securing the laser Doppler probe to the cranial coordinates during the surgical procedure.
In this video, we will show you our in-house developed system. We can be used for single site and multi-site cerebral hemodynamic monitoring. The rack stroke model shown in this video is obtained by inserting an inter luminal filament in the external carotid artery.
The filament is then advanced through the internal carotid artery in order to occlude the origin of the middle ceal artery producing a transient focal ceal ischemia. For more details on the procedure, please visit the link below. The cranial coordinates for single multi-site monitoring have been chosen after preliminary experiments with gelatin ink perfusion, which clearly delineated the hyper perfused area during middle cell artery occlusion for single-site monitoring.
Laser Doppler probe one is placed in the central territory supplied by the middle ceal artery for multi-site monitoring. Laser Doppler probe two is placed in the peripheral borders on territory between the middle and anterior ceal artery. To study the intercranial collateral flow, the positioning of laser doppler probes may be varied according to the experimental needs of each individual researcher.
The materials required are natural rubber, small plastic tubes, and the metal stylet. The prope holder can be customized to the size of the animal, the number and the size of the laser doppler probes, and the sable vascular territory, which needs to be monitored. Mark the positions of the probes and the brake mound.
The natural rubber according to the desired stereotaxic coordinates for middle ceal artery occlusion, the typical coordinates for the ischemic core are expected at Bgma minus one millimeters, five millimeters lateral to the midline. For a peripheral ischemic border zone territory, the expected coordinates can be set at bgma plus two millimeters, two millimeters lateral to the midline. Mark very clearly the position of the bgma with an X.This is the landmark for attaching the prop holder to the skull.
Insert the stylet into a small plastic tube. Its size needs to match the size of the probe. Insert the stylet into the natural rubber at the point where the probe has to be positioned.
Push the style into the rubber until the plastic tube has been inserted into the rubber too. Withdraw the style if multiple probes are needed. Repeat the same steps for other probe positions to ensure probes remain stable.
Rapid tape around the plastic tubes, single prope holders are obtained following the same procedure with one tube only. Chemical sterilization of several prope holders could be performed for future use. Gently shave the head of the rat, apply beta dime to a gauze pad and disinfect the skin.
The rat should be placed in the prom position on the operating table under ISO chlorine anesthesia administer lidocaine 2%five milligrams per kilo subcutaneously in the cranial area. Make a right paramedian skin incision for right ceal artery occlusion, and de dissect the subcutaneous tissue to reach the cranial fascia. Make a right paramedian incision in the cranial fascia and perform a blunt dissection.
To reach the skill bone, prepare a skill area which is large enough for the application of the probe holder. Please note there is no need for drilling or bone thinning, applying me broman to disinfect and dry the skull surface. Use a hair dryer set for cold air to accelerate drying the skull surface.
At this point, the cranial sutures and the bgma are clearly visible. Customize the probe holder by cutting the edges with sterile scissors. Apply a small quantity of surgical glue cyanoacrylate veterinary approved to the surface underneath the probe holder carefully avoiding the inferior opening of the plastic tubes.
Please note if a significant amount of glue remains between the optical surface of the probe and the skill, this may produce a low signal and may damage the probe. With multiple uses. Apply the probe holder to the skill surface carefully matching the BMA with the S land mine.
Apply gentle pressure over the prope holder. Use a hair dryer set for cold air to accelerate drying the surgical glue. Secure the prope holder by tying surgical thread around the prope holder and the head of the animal.
Be careful to position the surgical thread over the mandibula, avoiding the submandibular region and the neck. Fill the plastic tubes of the probe holder with an optical gel. For example, a common ultrasound gel.
This will increase the quality of the laser doppler signal. Place the props in the probe holder and verify the actual reading of the laser Doppler flow meter. Secure the probes tying them around the head of the animal.
Be careful to position the surgical thread over the mandibula, avoiding the submandibular region and the the multi-site system for CBR perfusion Monitoring is now complete Place the rats in the supplying position carefully avoiding tractional forces on the probes or prop holder. Start profusion monitoring during the surgical procedure for middle ceal artery occlusion when the endovascular filament reaches the origin of the middle ceal artery. A drop in ceal perfusion is visible during laser doppler monitoring.
If multi cy probes are used, different changes in ceal profusion may be seen according to their arterial territory. These representative perfusion tracings show how celebral hemodynamics can be studied using multi-site laser Doppler probes. A modest perfusion deficit occurs in probe two channel compared to probe one channel during both common carotid artery occlusion and middle ceal artery occlusion.
This hemodynamic pattern suggests functionally active inter cranial collaterals under ischemic conditions. Infarct volume is calculated on 19 consecutive coronal sections fixed in 4%perfor, MAGA, and stained with pre violet oh 0.1%corrected for interhemispheric asymmetries due to ceal edema and expressed in cubic millimeters. Immuno staining of molecular markers of neuronal loss and ischemic penumbra such as micro tubal associated protein two and heat shock protein 70 can be studied in relation to the multi-site hemodynamic monitoring of intercranial circulation.
The optimized system for sellable perfusion monitoring shown in this video could provide an easy and effective way to study the sellable hemodynamic changes in the experimental stroke field leading to the development of a new generation of celebral collateral therapeutics. Thank you for your attention and good luck with your experiments.
