The overall goal of this procedure is to deliver an Eloqua of inject safely and accurately to a desired region of mirroring myocardium under ultrasound guidance. This is accomplished by first manipulating the transducer orientation so that it is aligned parallel to the axis of the delivery needle. Next, the recumbent and anesthetized mouse is appropriately placed on the animal platform to facilitate targeted injection.
Then the optimal acoustic window is defined for accurate delivery of inject eight through minor adjustments to the animal platform and displaying a wide field of view. Finally, under ultrasound guidance, the needle tip is carefully advanced into the chest wall and myocardium and inject. Eight is safely and effectively delivered ultimately.
Cross-sectional microscopy of the mirroring myocardium is used to show the successful delivery of a tissue marker to the desired myocardial target. Visual demonstration of this method is critical. As the integrated rail system animal platform injection syringe control and transducer clamp must all be spatially aligned correctly for accurate delivery of inject eight.
This method can help answer key questions in the field of regenerative biology, such as those investigating therapeutic strategies for recovering myocardial performance. In embarked hearts Generally individual new to this method, we are struck most of proficiency on target. Injection carefully attention to detail in setting up needle alignment and the optimal ultrasound the winter will ensure success.
We first had the idea of this method when trying to device some method for the original of flux genes in the adult mouth heart After performing echocardiography, to assess baseline cardiac anatomy, place an empty syringe with the sheath needle with the bevel oriented upwards in the syringe clamp and secure the ultrasound transducer probe in the scan head clamp. Loosen the scan head clamp ball lock joint, and manipulate the transducer orientation so that it is aligned parallel to the axis of the needle. Fix the scan head position by tightening the scan head clamp ball lock joint.
Next liberally apply ultrasound gel to the transducer head. Carefully unhea the needle and use the needle mount controls to advance the needle directly under the transducer and within the ultrasound gel. For visualization.
Use the needle mount control to make minor adjustments so that the needle can be clearly seen along its length on the ultrasound image. Then use the scan height control to move the transducer superiorly from the animal platform to allow placement of the anesthetized mouse onto the animal platform, remove the syringe that was used for alignment from the syringe clamp and carefully discard it. Load the new needle and syringe with the inject date to the final target volume, allowing for dead space.
In the syringe tip, place the syringe into the syringe clamp without adjusting its x-axis alignment and use the injection control to fully retract the syringe. Turn on the integrated warmer of the heating platform and set it to 37 degrees Celsius. Place the animal platform 180 degrees from the usual imaging orientation with the anesthesia hose clamp and head of the animal closest to the operator, which allows the heart to be ipsilateral to the syringe clamp and needle.
After preparing the mouse for echocardiography and anesthetizing it in an induction chamber with 2%ISO fluorine, use hair removal cream to remove the chest hair and apply lubricating gel to the eyes. Place the anesthetized mouse supine atop the heated animal platform and attach a nose cone to deliver one to 3%isof fluorine. Gently insert a rectal probe and apply electrode gel to the four paws before taping them to the ECG electrodes.
Once the mouse is secure on the animal platform, lower the transducer onto the depleted chest to obtain the optimal acoustic window for injection. In the short axis imaging plane, rotate the animal platform 20 to 30 degrees clockwise for a clear ultrasound of both the heart and the needle tip as it approaches the chest. Apply plenty of ultrasound gel over the left side of the chest and optimize the acoustic window by using the echocardiography controls.
To set a wide field of view, use the animal platform adjustment controls to pan back and forth from the apex to the base of the heart. To target the desired injection location in the left ventricular myocardium, refer to the text protocol for view recommendations. Next, starting with the syringe in the fully retracted syringe clamp, turn the injection control clockwise to slowly advance the syringe towards the animal's chest.
Then with appropriate sedation confirmed by a tail pinch, advance the needle through the chest wall of the mouse and into the myocardium. Carefully observing the position of the beveled needle tip at all times. When the entire beveled needle tip is within the target myocardium, stop advancing the needle and slowly push on the syringe plunger to deliver the inject.
A transient echo bright appearance to the injected myocardial region may be evident after successful injection. Once the inject has been administered promptly rotate the injection control knob counterclockwise to withdraw the needle before discarding it. Keep the animal under anesthesia for several minutes of echocardiographic observation to confirm preserved ventricular function and no post-procedural implications.
After intra myocardial injection, place the mouse in a cage on its own and allow it to recover from anesthesia under observation, when it has regained sternal recumbent, place it in a heated cage with water and food. This figure shows an example of a successful injection with blue dye infiltrating the myocardium at the level of the mid papillary muscle. These panels illustrate multiple injection targets of red fluorescent microbeads in the anterior and posterior left ventricular walls to visualize the delivery of adenovirus to myocardium by ultrasound guided injection and adenovirus was injected in which the cardiomyocyte specific troponin T promoter drives expression of mammalian codon optimized Cree.
The domain of CRE mediad recombination was determined using ROSA 26 MTMG mice in which CRE recombination turns off red fluorescent protein and turns on green fluorescent protein. As shown here, a single 50 microliter injection regionally activated GFP expression within seven days Once mastered this technique can be done in 10 minutes if it is performed properly Following this procedure. Other methods like tissue section microscopy can be performed in order to answer additional questions like the extent and effect of inject eight delivery.
After watching this video, you should have a good understanding of how to safely and accurately inject into a targeted region of rine.Myocardium. Don't forget that working with adeno virus can be generous and precautions such as preparation and a valve safety cabinet should always be taken.
