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09:56 min
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February 14th, 2021
DOI :
February 14th, 2021
•0:05
Introduction
1:34
Dissection of the Left Common Carotid
4:09
Cannulation of the Left Common Carotid Artery
5:11
Placement of the Telemetry Device Body and ECG Leads
7:29
Results: Analysis of BRS Using the Sequence Method on Simultaneous Telemetric ECG and BP Recordings
8:58
Conclusion
필기록
Baroreflex sensitivity describes the relationship between changes in arterial blood pressure and reflex changes in heart rate. When blood pressure increases, heart rate decreases and vice versa. The combined telemetric electrocardiogram and blood pressure measurements in conscious mice allow the determination of baroreflex sensitivity.
The main advantage of this technique over older methods for determining baroreflex sensitivity is that it measures the spontaneous reflex. The mice are awake and do not need to be restrained. No invasive procedures such as injecting vasoactive drugs to induce blood pressure changes are required.
The baroreflex can be used for risk stratification of patients with cardiac disease such as arterial hypertension, myocardial infarction, and heart failure. Pathological changes of the baroreflex sensitivity are associated with increased risk for cardiac mortality. It is therefore an important prognostic marker for cardiovascular disease.
The microsurgical details such as puncturing and cannulating the carotid artery are the most challenging steps of the protocol. Care must be taken to ensure that there's sufficient tension on the sutures to temporarily stop blood flow and to slightly lift the artery for cannulation. Begin by using a trimmer to shave the surgical area of an anesthetized mouse from below the chin towards the transversal pectoral muscles.
After positioning the mouse in a supine position, apply eye ointment to protect the animal's eyes during anesthesia. Apply depilatory cream to the previously shaved surgical area. After three to four minutes, remove hair and depilatory cream using a cotton pad and warm water, making sure that the skin is clean and free of any residual hair or cream so that the wound will not be contaminated during the operation.
Make a one to 1.5 centimeter straight midline incision through the skin of the neck starting immediately below the chin. Create a subcutaneous space at both sides of the incision by separating the skin from the underlying connective tissue with blunt dissection scissors, being careful not to pinch the skin too strongly with the forceps, which can cause necrosis and lead to impaired wound healing after surgery. Separate the parotid and submandibular glands using cotton tip applicators to expose the musculature overlying the trachea.
Retract the left salivary gland with curved dissection forceps to identify the left carotid artery located laterally to the trachea. Carefully dissect the carotid artery from adjacent tissue using curved forceps, being very careful not to injure the vagal nerve that is running along the vessel. Continue blunt dissection to expose the left carotid artery to about 10 millimeters in length and fully separate it from the vascular fascia and the vagus nerve.
Pass a non-absorbable five by zero silk suture underneath the isolated portion of the carotid artery while slightly lifting the blood vessel with curved forceps to reduce friction between the sutures and the carotid artery. Place the suture cranially just proximally to the bifurcation of the carotid artery. Then form a knot and tie it to permanently ligate the vessel.
Fix both ends of the cranial occlusion suture to the surgery table with surgical tape. Pass a second occlusion suture underneath the carotid artery and place it caudally at approximately five millimeter distance to the cranial suture, tying a loose knot. Fix both suture ends with surgical tape.
Position a third suture between the cranial and caudal occlusion suture to keep the catheter in place while cannulating the artery and make a loose knot. Gently pull the caudal occlusion suture and fix it with tension to temporarily stop blood flow and to slightly lift the artery. Carefully penetrate the artery proximal to the cranial occlusion suture with the bent needle.
While slightly elevating the carotid artery with the bent needle, grip the catheter with vessel cannulation forceps, introduce it into the small puncture, and let it slide slowly into the vessel. Simultaneously, gently pull back the bent needle. When the catheter reaches the caudal occlusion suture, slightly tighten the secure suture to keep the catheter in place.
Loosen the caudal occlusion suture so that the catheter can be further moved until its tip is positioned in the aortic arch. Once positioned properly, secure the catheter with all three sutures and cut the ends as short as possible without pulling the knots too tight. Form a subcutaneous tunnel from the neck directed towards the left flank of the animal and form a small pouch using small blunt dissecting scissors.
Irrigate the tunnel with a one milliliter syringe filled with warm sterile 0.9%sodium chloride solution and introduce approximately 300 microliters of the solution into the pouch. Carefully lift the skin with blunt forceps and introduce the transmitter device body into the pouch, being careful not to pull the blood pressure catheter out of the carotid artery. Form a thin tunnel to the right pectoral muscle with blunt dissecting scissors, and place the negative lead into the tunnel using blunt forceps.
Fix the terminal end of the lead with a stitch to the pectoral muscle using six by zero absorbable suture material. Form a loop in the positive lead, position its tip at the left caudal rib region, and secure its position using a six by zero absorbable suture. Close the skin with single knots using five by zero non-absorbable suture material.
Additionally, apply a small amount of tissue adhesive on every knot to keep the animal from biting the suture and prevent dehiscence. Apply 10%povidone iodine hydrogel to the wound to prevent wound infection during the recovery phase. For preemptive pain relief, inject carprofen in 0.9%sodium chloride subcutaneously while the mouse is still under anesthesia.
Position one-half of the cage on a heating platform set to 36 degrees Celsius for 12 hours after surgery and transfer the mouse to the warm area so that the animal has the option of staying on the warm area or moving to the cooler part of the cage when awake. Following data acquisition, inspect blood pressure and ECG traces and screen for a three-hour sequence of low activity. To perform baroreflex sensitivity or BRS analysis, open the BRS analysis panel.
Manually inspect every sequence displayed in the panel and exclude ectopic beats, sinus pauses, arrhythmic events, or noisy data to successfully exclude them from the analysis. This protocol can be used for precise BRS analysis and a broad range of ECG or BP-derived parameters like ECG intervals, BP variability, and arrhythmia detections. A healthy mouse that has sufficiently recovered from surgery shows a physiological increase of activity, heart rate, and BP during the dark phase.
The sequence method was used to determine BRS where systolic blood pressure and RR intervals were examined on a beat-to-beat basis during short sequences of three or more beats with a spontaneous rise or fall in systolic blood pressure. To evaluate the correlation between RR and systolic blood pressure, both parameters were plotted against each other and the slope of the linear regression line was calculated for each sequence. The total number of sequences per 1, 000 beats and average gain of spontaneous BRS reflected by the slopes of the linear regression lines calculated from the RR by systolic blood pressure relation are parameters of interest in the analysis.
During phases of higher activity, signal quality might decrease and induce noise. Setting two low values for the minimum correlation coefficient also results in false detections of sequences that do not reflect baroreflex activity, but result from arrhythmic beads. For the interpretation of baroreceptor sensitivity data, it is important to consider the functional levels involved in the baroreceptor reflex.
At the neuronal level, afferent, central, or efferent components of the reflex may be altered. At the cardiovascular level, reduced or increased responsiveness of the sinus node may cause changes in baroreceptor sensitivity. With this data, one cannot only determine baroreflex sensitivity and the function of the cardiac sinoatrial node, but also a wide range of ECG and blood pressure-derived parameters.
These include, for example, systolic and diastolic blood pressure, ECG intervals, heart rate variability, or cicada rhythms.
The baroreflex is a heart-rate regulation mechanism by the autonomic nervous system in response to blood-pressure changes. We describe a surgical technique to implant telemetry transmitters for continuous and simultaneous measurement of electrocardiogram and blood pressure in mice. This can determine spontaneous baroreflex sensitivity, an important prognostic marker for cardiovascular disease.
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