A method for optical imaging of living myocardial slices
Through the short-axis sectioning and optical mapping of cardiac in optogenetic transgenic mice, combined with fluorescent dye marking, the problem of high-throughput collection of electrocardiogram and calcium transient signals on live myocardial tablets was solved, and the function of cardiomyocyte population was realized Detailed study.
Patent Information
- Application Number
- CN201910349849.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2019-04-28
- Publication Date
- 2024-12-10
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
It is difficult for the prior art to perform non-injury high-throughput acquisition of electrocardiograms and calcium transient signals on live myocardial tablets to study the function of the cardiomyocyte population.
The hearts of optogenetic transgenic mice were used for live short-axis sectioning, and optical mapping was performed using an ultra-high score camera to detect the conduction characteristics of myocardial membrane potential and calcium signal, combining voltage sensitivity and calcium sensitivity fluorescent dye labeling, synchronously Record membrane potential and calcium signals.
It realizes synchronous recording of membrane potential and calcium signals on live myocardial tablets, obtains massive data sets, and evaluates regional myocardial tissue function and cell subpopulations function, which is suitable for high-throughput research.
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Figure CN110132914B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for optical imaging of a living myocardial slice, belonging to the technical field of optical imaging. Background Art
[0002] Cardiac basic research mainly uses traditional biological models from isolated cardiomyocytes to complex multicellular tissues, the latter including isolated hearts and intact tissue blocks (such as isolated myocardial strips and perfused myocardial blocks). However, the connection between myocardial cells and the extracellular matrix is destroyed during the enzymatic separation process of isolated cardiomyocytes, which seriously affects the electrophysiological function of cardiomyocytes; multicellular tissues have problems related to in vitro perfusion and tissue ischemia.
[0003] Optogenetics uses light to activate photosensitive ion channels and opsins to depolarize or hyperpolarize cells, and can control the "on" and "off" of excitable cells. This emerging technology is based on neuroscience and is now increasingly used in cardiac electrophysiology research. Blue light stimulation can cause electrical excitation of cardiomyocytes, restore electrical conduction of cardiomyocytes, achieve cardiac resynchronization, and even simulate arrhythmias; in vitro and in vivo experiments have confirmed the use of optogenetics to achieve photopacing of the heart; inhibit and manipulate rotors in cardiomyocyte monolayers, and clarify the role of cardiomyocytes and non-cardiomyocyte subpopulations in the heart.
[0004] Optical mapping has high temporal resolution (tens of microseconds) and spatial resolution (several micrometers), and can dynamically measure and track the electrical conduction, action potential, and calcium transients of the heart at multiple sites, with non-invasiveness, which is unmatched by traditional electrode technology. However, there is no combination of optogenetics, optical mapping, and living myocardial slices to achieve non-destructive high-throughput acquisition of ECG signals and calcium transient signals on living myocardial slices to study the function of the target cell population.
[0005] In view of this, it is necessary to study the method of optical imaging of living myocardial slices to address the shortcomings of existing technologies. Summary of the invention
[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for optical imaging of living myocardial slices. The present invention performs short-axis slicing of the heart of an optogenetic transgenic mouse and uses an ultra-high resolution camera for optical mapping to detect and analyze the conduction characteristics of myocardial slice membrane potential and calcium signals, opening up new research methods for cardiac physiology and pathology.
[0007] The technical solution of the present invention to solve the above technical problem is as follows: A method for optical imaging of a living myocardial slice comprises the following steps:
[0008] Step 1: Isolated Heart Perfusion
[0009] Step 1.1: Take the optogenetic transgenic adult mice and anesthetize them;
[0010] Step 1.2: Open the chest, quickly remove the heart, place it in Krebs solution at 37±1℃, remove the connective tissue, cannulate the aorta, fix it on the Langendroff system, and perfuse it with Krebs solution to obtain the perfused isolated heart;
[0011] Step 2: Loading calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye
[0012] Step 2.1: Mix the calcium-sensitive fluorescent dye Rhod-2AM and the surfactant Pluronic F127 at a ratio of 1:1, dilute in 1 ml of Krebs solution, and then slowly inject into the perfused isolated heart obtained in step 1.2, and then circulate in the Rhod-2AM dye for 20 minutes to obtain an isolated heart loaded with the calcium-sensitive fluorescent dye;
[0013] Step 2.2: Continue to dilute 30 μl of voltage-sensitive fluorescent dye RH237 in 1 ml of Krebs solution, and then slowly inject it into the isolated heart loaded with calcium-sensitive fluorescent dye obtained in step 2.1 to obtain an isolated heart loaded with calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye;
[0014] Step 2.3: The isolated heart loaded with the calcium-sensitive fluorescent dye and the voltage-sensitive fluorescent dye obtained in step 2.2 is perfused cyclically with a Krebs solution containing 10 μM Blebbistatin until the heart stops beating, thereby obtaining a heart after the heart stops beating;
[0015] Step 3: In vivo short-axis myocardial slice preparation
[0016] Step 3.1: placing the 2,3-butanedione monoxime solution in a refrigerator to freeze it into an ice-water mixture, taking out the heart after beating obtained in step 2.3 from the Langendorff system, and placing it in the 2,3-butanedione monoxime solution mixed with ice and water to obtain a heart treated with the ice 2,3-butanedione monoxime solution;
[0017] Step 3.2: embedding the heart treated with the ice 2,3-butanedione monoxime solution obtained in step 3.1 with low-temperature agar to obtain an embedded heart;
[0018] Step 3.3: stick the embedded heart obtained in step 3.2 onto the sample tray of the vibrating slicer, then place the tray in the sample bath, add ice 2,3-butanedione monoxime solution into the sample bath until the heart is completely immersed, and slice to obtain sliced myocardial slices;
[0019] Step 3.4: Place the sliced myocardium obtained in step 3.3 in an ice-cold 2,3-butanedione monoxime solution and incubate for 8 min-12 min;
[0020] Step 3.5: sequentially transfer to three portions of Krebs solution containing 10 μM Blebbistatin, each of which is incubated at room temperature for 8 min-12 min to obtain myocardial slices for optical mapping;
[0021] Step 4: Optical Mapping
[0022] Step 4.1: Place the myocardial slice for optical mapping obtained in step 3.5 in a slice bath containing 10 μM Blebbistatin-Krebs solution, equilibrate for 2 min-3 min, continuously fill with oxygen and carbon dioxide, and maintain the temperature at 37°C;
[0023] Step 4.2: Use a high-frequency camera on the optical mapping system to collect voltage signals and calcium signals after electrical stimulation and blue light stimulation, respectively, that is, obtain optical image data of the short-axis myocardial slice of the living body before and after electrical stimulation and blue light stimulation.
[0024] The principle of the present invention is described as follows:
[0025] The present invention combines optogenetics, optical mapping and living myocardial slices, performs dual fluorescent dye labeling of calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye on living short-axis myocardial slices, and uses a high-frequency camera for optical mapping. The present invention uses living short-axis myocardial slices to avoid the damage to the electrophysiological function of myocardial cells by enzyme separation, and can obtain complex multicellular tissue structure and functional data. In combination with optogenetics, it can study different cardiac cell groups.
[0026] First, the photosensitive protein was transferred into the target myocardial cell population of mice using transgenic technology. Second, the hearts of adult mice were isolated, and voltage-sensitive and calcium-sensitive fluorescent dyes were used to label the myocardial cell membrane potential and intracellular calcium ions, respectively. Third, living short-axis myocardial slices were obtained by slicing. Fourth, blue light was used to stimulate the living short-axis myocardial slices, and the membrane potential and intracellular calcium signals of the living short-axis myocardial slices were synchronously recorded using a high-frequency camera.
[0027] Optogenetics is the use of gene manipulation technology to transfer light-sensitive genes (such as ChR2, eBR, NaHR3.0, Arch or OptoXR, etc.) into specific types of cells to express special ion channels or G protein-coupled receptors. Light-sensitive ion channels will be selective for the passage of cations or anions under light stimulation of different wavelengths, causing changes in the membrane potential on both sides of the cell membrane, achieving the purpose of selectively exciting or inhibiting cells. Optogenetics has two unique characteristics: high temporal and spatial resolution and cell type specificity.
[0028] Optical mapping is used to record cell transmembrane potential and intracellular calcium transients based on the use of voltage-sensitive fluorescent dyes and calcium-sensitive fluorescent dyes. The principle of fluorescent dyes as markers is that under the excitation of a light source of a certain wavelength, the fluorescent substance transitions to a high energy level, and then the energy level decays, releasing photons and energy, and the spectrum shifts to the long wavelength direction. This emitted light is recorded by the relevant detection device. Voltage-sensitive fluorescent dyes can sense changes in the local electric field. When the cell membrane potential changes, the cell membrane interacts with the dye molecules, and the dye molecules on the inside and outside of the cell membrane rearrange and rotate. When the membrane potential is low, there are more luminescent dye molecules, and when the membrane potential is high, the luminescent substance decreases, and the resulting brightness changes can be collected and recorded by the relevant recording device.
[0029] Living myocardial slices are fresh myocardial tissue sliced into thin slices, which retain the original multicellular structure and physiological characteristics of the heart, while its thin structure ensures sufficient oxygen and diffusion of metabolic substrates outside the body. Currently, living myocardial slices are used for culture or combined with immunohistochemical staining for microvascular and macrovascular network imaging.
[0030] The present invention combines optogenetics, optical mapping and living myocardial slices, can obtain massive data sets, can simultaneously record membrane potential (AP) and calcium transient (CaT), and obtain values such as duration, alternation time and conduction velocity of AP and / or CaT. Comprehensive analysis of the alternation time of AP and / or CaT is of great value in evaluating regional myocardial tissue function and studying cell subpopulations.
[0031] The method of the present invention also allows high-throughput studies: depending on the optical properties, camera resolution, slice size and position, images of multiple or all slices of a mouse heart can be acquired simultaneously rather than sequentially.
[0032] In step 2.1 of the present invention, Rhod-2 AM is a fluorescent calcium ion chelator with a chromophore similar to rhodamine, with an excitation wavelength of λex=552nm and an emission wavelength of λem=581nm. It has similar excitation and emission spectra to another fluorescent calcium ion chelator Fluo-3 and has no obvious migration when the calcium ion concentration changes. The probe does not emit fluorescence before binding to calcium ions, but when Ca is increased, 2+ The fluorescence intensity is greatly enhanced when the ion concentration is high. Rhod-2 AM has long excitation and emission wavelengths, so it is more suitable for experiments with cells / tissues with high levels of autofluorescence or for use with another short-wavelength fluorescent dye.
[0033] Pluronic F127, poloxamer (trade name: Pluronic), is a polyoxyethylene polyoxypropylene ether block copolymer, which is a new type of high molecular nonionic surfactant. In the present invention, Pluronic F127 is used together with Rhod-2 AM to promote the latter to enter cells.
[0034] In step 2 of the present invention, the calcium-sensitive fluorescent dye and the voltage-sensitive fluorescent dye are loaded, and the whole process needs to be operated in a dark environment.
[0035] In step 2.2 of the present invention, the voltage-sensitive fluorescent dye RH237 belongs to a styrene-based dye and is a fast-reaction potential probe used to monitor cell membrane potential. Its excitation wavelength is 540nm, its emission wavelength is 585nm, and its excitation spectrum is usually blue-shifted by 20nm and its emission spectrum is blue-shifted by 80nm.
[0036] In step 2.2 of the present invention, Krebs solution is a physiological nutrient solution used to maintain tissue activity. Krebs solution is prepared by itself, and the specific formula is: NaCl 119mM, NaHCO3 25mM, NaH2PO4 1.0mM, KCl 4.7mM, MgCl2 1.05mM, CaCl2 1.35mM and glucose 10mM, and the pH value is adjusted to 7.4. All reagents used are purchased from Sigma Company in the United States.
[0037] In step 2.3 of the present invention, after the heart stops beating, it is pink and has no visible beating.
[0038] In step 2.4 of the present invention, Blebbistatin is a cell permeable inhibitor that acts on non-myosin II ATPase inhibitors, does not inhibit myosin light chain kinase, inhibits the constriction of the cleavage furrow, and does not interfere with mitosis or the assembly of the contractile ring. In the present invention, it plays a role in inhibiting the excitation and contraction of myocardial tissue, but does not cause arrhythmia.
[0039] The above-mentioned Blebbistatin was purchased from Sigma Company of the United States, with a specification of 1 mg / vial.
[0040] In step 3.1 of the present invention, 2,3-butanedione monoxime (BDM) is a myocardial excitation and contraction inhibitor. The above 2,3-butanedione monoxime was purchased from Sigma Company in the United States, with a specification of 500g / bottle.
[0041] In step 3.3 of the present invention, the embedded heart is glued to the sample tray of the vibrating slicer with 502 glue.
[0042] Based on the above technical solution, the present invention can also be improved as follows.
[0043] Furthermore, in step 1.1, the optogenetic transgenic adult mice are composed of Pnmt-Cre transgenic mice and loxp system mice B6.Cg-Gt(ROSA)26Sor tm27.1(CAG-COP4H134R / tdTomato)Hze / J strain hybridization.
[0044] The above further beneficial effect is that the above optogenetic transgenic adult mice are Pnmt-Cre mice and loxp system mice B6.Cg-Gt (ROSA) 26Sor tm27.1(CAG-COP4H134R / tdTomato)Hze / Jstrain hybridization. The DNA between the loxP sites was cut by Cre recombinase, and the cut was reconnected under the action of DNA ligase. After recombination, the Pnmt promoter was connected to hChR2(H134R)-tdTomato, that is, Pnmt and hChR2(H134R)-tdTomato were expressed simultaneously.
[0045] The above-mentioned optogenetic transgenic adult mice were purchased from Oxford University.
[0046] Furthermore, the Pnmt-Cre transgenic mouse is a mouse in which Cre recombinase is inserted into the promoter of the Pnmt gene.
[0047] A further beneficial effect of the above is that Cre is encoded by the Cre gene of Escherichia coli phage P1 and is a 38kD protein consisting of 343 amino acids. It not only has catalytic activity, but also is similar to a restriction enzyme and can specifically recognize loxP sites.
[0048] Furthermore, the B6.Cg-Gt(ROSA)26Sor tm27.1(CAG-COP4H134R / tdTomato)Hze / J strain mice had CAG-loxP-STOP-loxP-hChR2(H134R)-tdTomato inserted at the (ROSA)26 position.
[0049] A further beneficial effect of the above is that the mouse has CAG-loxP-STOP-loxP-hChR2(H134R)-tdTomato inserted at the (ROSA)26 position, and the STOP element prevents CAG from driving the expression of hChR2(H134R)-tdTomato. However, when the (ROSA)26-LSL-hChR2(H134R)-tdTomato mouse is mated with a mouse expressing Cre recombinase throughout the body, the STOP element will be deleted in the cells expressing Cre, turning on the expression of hChR2(H134R) and tdTomato.
[0050] The loxP (locus of X-overP1) site is 34bp long, including two 13bp inverted repeat sequences and an 8bp spacer region. Among them, the inverted repeat sequence is the specific recognition site of the Cre recombinase, and the spacer region determines the direction of the loxP site. When the Cre recombinase encounters the loxP site, it will bind to the inverted repeat sequence region at both ends of the loxP site to form a dimer. This dimer combines with the dimer of other loxP sites to form a tetramer. Subsequently, the DNA between the loxP sites is cut off by the Cre recombinase, and the incision is reconnected under the action of DNA ligase.
[0051] Furthermore, in step 1.1, the anesthesia is performed by intraperitoneal injection of one of tribromoethanol, sodium pentobarbital, or a mixture of ketamine hydrochloride and Suminxin II in a volume ratio of 1:1; the anesthesia time is 10 minutes.
[0052] A further beneficial effect of the above method is that the mice are anesthetized in order to comply with animal experimental standards and facilitate the control of the mice.
[0053] Furthermore, the amount of tribromoethanol used is 0.5ml-0.8ml, and the mass percentage is 1.2%.
[0054] A further beneficial effect of the above is that the above dosage can anesthetize optogenetic transgenic adult mice.
[0055] Furthermore, in step 1.2, the perfusion rate is 3.5 ml / min-4.0 ml / min, and the perfusion time is 3 min.
[0056] A further beneficial effect of the above is that the above perfusion parameters can be used to completely drain the blood from optogenetic transgenic adult mice.
[0057] Furthermore, in step 2.1 and step 2.2, the injection time is 7 min-10 min.
[0058] Furthermore, in step 2.2, the voltage-sensitive fluorescent dye RH237 is prepared by dissolving RH237 solid in DMSO to prepare a solution with a concentration of 1.25 mg / ml.
[0059] The above further beneficial effect is: in step 2.2 of the present invention, the voltage-sensitive fluorescent dye RH237 belongs to a styrene-based fuel, which is a fast-reaction potential probe used to monitor cell membrane potential. Its excitation wavelength is 540nm, and its emission wavelength is 585nm. Usually, the excitation spectrum is blue-shifted by 20nm, and the emission spectrum is blue-shifted by 80nm. It is more suitable for storage when prepared into a stock solution to prevent RH237 from degradation.
[0060] Furthermore, in step 3.2, the temperature of the low-temperature agar is 37.5±5° C., and the concentration of the agar is 4%.
[0061] A further beneficial effect of the above method is that the heart is fixed and supported by low-temperature agar, which is convenient for slicing and does not affect the activity of myocardial tissue.
[0062] Furthermore, in step 3.3, the slicing speed is 0.05 mm / s, the frequency is 80 Hz, and the thickness is 300 μm / slice to 350 μm / slice.
[0063] A further beneficial effect of the above is that by using the above parameters, a myocardial tissue slice with uniform thickness and good activity can be obtained.
[0064] Furthermore, in step 4.1, the volume ratio of oxygen to carbon dioxide is 95:5.
[0065] A further beneficial effect of the above is that the pH value of the buffer solution is more stable by using oxygen and carbon dioxide in a volume ratio of 95:5.
[0066] The beneficial effects of the present invention are:
[0067] 1. The present invention provides a method for optical imaging of living myocardial slices, which integrates optogenetics, optical mapping and living myocardial slices, and can synchronously record myocardial cell membrane potential and calcium transients after blue light stimulation. Compared with the existing whole heart mapping method, it can conduct functional studies of different regions, different muscle layers or different cell groups of the heart.
[0068] 2. The present invention uses voltage-sensitive and calcium-sensitive fluorescent dyes to mark the myocardial cell membrane potential and calcium respectively on living myocardial slices, separates the two wavelengths of emitted light through a spectrometer, and uses a high-frequency camera to collect the membrane potential and intracellular calcium signals of the myocardial slices.
[0069] 3. The method of the present invention can obtain a large amount of data sets, can simultaneously record AP and CaT, and obtain values such as duration, alternation time and conduction velocity of AP and / or CaT. Comprehensive analysis of the alternation time of AP and / or CaT is of great value in evaluating regional myocardial tissue function and cell subpopulation function.
[0070] 4. The method of the present invention can be used for high-throughput research: depending on the optical properties, camera resolution, slice size and position, images of multiple or all slices of a mouse heart can be acquired simultaneously rather than sequentially.
[0071] 5. The method of the present invention is simple, easy to operate, has broad application prospects, and is suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS
[0072] Figure 1The present invention is a flow chart of the method for optical imaging of living myocardial slices.
[0073] Figure 2 The structure diagram of the optical mapping system in the method of optical imaging of living myocardial slices of the present invention is shown in the figure. In the figure, the components represented by the reference numerals are as follows: 1 is a sample bath; 2 is a living short-axis myocardial slice; 3 is blue light; 4 is LED light; 5 is a 635nm long-pass filter; 6 is a 585nm filter; 7 is a spectrometer; and 8 is a high-frequency camera.
[0074] Figure 3 This is the action potential signal of the living short-axis myocardial slice collected by the present invention.
[0075] Figure 4 This is the calcium transient signal of the living short-axis myocardial slice collected by the present invention.
[0076] Figure 5 This is a distribution diagram of the action potential activation time generated by electrical stimulation of the short-axis myocardial slice of the living body in the present invention.
[0077] Figure 6 This is the time distribution diagram of 50% repolarization of the electrical stimulation action potential in the present invention.
[0078] Figure 7 This is the time distribution diagram of 75% repolarization of the electrical stimulation action potential in the present invention.
[0079] Figure 8 This is a distribution diagram of the action potential activation time generated by blue light stimulation of the short-axis myocardial slice in the living body in the present invention.
[0080] Fig. 9 This is the time distribution diagram of 50% repolarization of action potential stimulated by blue light in the present invention.
[0081] Fig.10 This is the time distribution diagram of 75% repolarization of action potential stimulated by blue light in the present invention.
[0082] Fig.11 This is a distribution diagram of calcium transient time generated by electrical stimulation of a short-axis myocardial slice in a living body in the present invention.
[0083] Fig.12 This is a time distribution diagram of a 50% increase in calcium transients generated by electrical stimulation of a short-axis myocardial slice in a living body in the present invention.
[0084] Fig.13 This is a time distribution diagram of a 75% increase in calcium transients generated by electrical stimulation of a short-axis myocardial slice in a living body in the present invention.
[0085] Fig.14 This is a distribution diagram of calcium transient time generated by blue light stimulation of short-axis myocardial slices in living body in the present invention.
[0086] Fig.15 This is a time distribution diagram of a 50% increase in calcium transients generated by blue light stimulation of a short-axis myocardial slice in a living body in the present invention.
[0087] Fig.16 This is a time distribution diagram of a 75% increase in calcium transients generated by blue light stimulation of a short-axis myocardial slice in a living body in the present invention.
[0088] Fig.17 The method of the present invention can be used to analyze the characteristics and change gradients of AP and CaT in different regions of the short-axis myocardial slice of a living body (such as the left ventricular endocardium, the left ventricular endocardium, the interventricular septum, the right ventricle, etc.). In the accompanying drawings, the components represented by each reference numeral are listed as follows: 1 is the left ventricular endocardium, 2 is the left ventricular endocardium, 3 is the interventricular septum, and 4 is the right ventricle.
[0089] Fig.18 This is the AP map of the left ventricular epithelium.
[0090] Fig.19 This is the AP map of the left ventricular endocardium.
[0091] Fig. 20 This is the AP diagram of the ventricular septum.
[0092] Fig.21 This is the AP diagram of the right ventricle.
[0093] Fig. 22 This is the CaT map of the left ventricular endocardium.
[0094] Fig.23 This is the CaT map of the left ventricular endothelium.
[0095] Fig.24 This is the CaT map of the ventricular septum.
[0096] Fig.25 This is the CaT map of the right ventricle.
[0097] Fig.26 The data obtained by synchronously recording membrane potential and calcium transient signals using the present invention can be used to comprehensively analyze the excitation conduction diagram of the heart.
[0098] Fig. 27 The data obtained by synchronously recording membrane potential and calcium transient signals using the present invention can be used to comprehensively analyze the action potential diagram of the heart.
[0099] Fig.28 The data obtained by synchronously recording membrane potential and calcium transient signals using the present invention can be used to comprehensively analyze the calcium transient time course diagram of the heart.
[0100] Fig.29 The data obtained by synchronously recording the membrane potential and calcium transient signals using the present invention can be used to comprehensively analyze the delay diagram between the action potential and calcium transient of the heart.
[0101] Fig.30 For the control experiment of the present invention, the optical marking of the isolated heart. In the attached drawings, the components represented by the respective numbers are listed as follows: 1 is the atrium, 2 is the ventricle, and 3 is the apex.
[0102] Fig.31 is the membrane potential of the isolated heart.
[0103] Fig.32 Calcium transients in isolated hearts. DETAILED DESCRIPTION
[0104] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.
[0105] Example 1
[0106] The method for optical imaging of living myocardial slices in this embodiment is as follows: Figure 1 As shown, the following steps are included:
[0107] Step 1: Isolated Heart Perfusion
[0108] Step 1.1: Take an optogenetic transgenic adult mouse and anesthetize it.
[0109] The optogenetic transgenic adult mice were derived from Pnmt-Cre transgenic mice and B6.Cg-Gt(ROSA)26Sor tm27.1 (CAG-COP4H134R / tdTomato)Hze / J strain mice. The DNA between the loxP sites is cut by Cre recombinase, and the incision is reconnected under the action of DNA ligase. After recombination, the Pnmt promoter is connected to hChR2(H134R)-tdTomato, that is, Pnmt and hChR2(H134R)-tdTomato are expressed simultaneously. The above-mentioned optogenetic transgenic adult mice were purchased from Oxford University. The Pnmt-Cre transgenic mice are obtained by inserting Cre recombinase into the Pnmt gene promoter, and the B6.Cg-Gt(ROSA)26Sor tm27.1(CAG-COP4H134R / tdTomato)Hze / J strain mice had CAG-loxP-STOP-loxP-hChR2(H134R)-tdTomato inserted at the (ROSA)26 position.
[0110] The anesthesia was performed by intraperitoneal injection of tribromoethanol; the anesthesia time was 10 minutes.
[0111] Step 1.2: Open the chest, quickly remove the heart, place it in Krebs solution at 37±1°C, remove the connective tissue, cannulate the aorta, fix it on the Langendroff system, and perfuse it with Krebs solution at a rate of 3.5 ml / min for 3 minutes to obtain the perfused isolated heart.
[0112] Step 2: Loading calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye
[0113] Step 2.1: Mix the calcium-sensitive fluorescent dye Rhod-2AM and the surfactant Pluronic F127 in a ratio of 1:1, dilute in 1 ml of Krebs solution, and then slowly inject into the perfused isolated heart obtained in step 1.2 within 7 minutes. Then circulate in the Rhod-2 AM dye for 20 minutes to obtain an isolated heart loaded with the calcium-sensitive fluorescent dye.
[0114] Step 2.2: Continue to dilute 30 μl of voltage-sensitive fluorescent dye RH237 in 1 ml of Krebs solution. The voltage-sensitive fluorescent dye RH237 is prepared by dissolving RH237 solid in DMSO to a solution with a concentration of 1.25 mg / ml. Slowly inject the solution into the isolated heart loaded with calcium-sensitive fluorescent dye obtained in step 2.1 within 7 minutes to obtain an isolated heart loaded with calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye.
[0115] Step 2.3: The isolated heart loaded with calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye obtained in step 2.2 is perfused cyclically with Krebs solution containing 10 μM Blebbistatin until the heart stops beating. At this time, the heart is pink with no visible beating, thus obtaining an arrested heart.
[0116] Step 3: In vivo short-axis myocardial slice preparation
[0117] Keep away from light throughout the process.
[0118] Step 3.1: Freeze the 2,3-butanedione monoxime solution in a refrigerator to form an ice-water mixture, take out the arrested heart obtained in step 2.3 from the Langendorff system, and place it in the 2,3-butanedione monoxime solution mixed with ice and water to obtain a heart treated with the ice 2,3-butanedione monoxime solution.
[0119] Step 3.2: The heart treated with the ice 2,3-butanedione monoxime solution obtained in step 3.1 is embedded in low-temperature agar, wherein the temperature of the low-temperature agar is 37.5±5° C. and the concentration of the agar is 4%, to obtain an embedded heart.
[0120] Step 3.3: Glue the embedded heart obtained in step 3.2 to the sample tray of the vibrating slicer with 502 glue, then place the tray in the sample bath, add ice 2,3-butanedione monoxime solution to the sample bath until the heart is completely immersed, and slice at a speed of 0.05 mm / s, a frequency of 80 Hz, and a thickness of 300 μm / s to obtain sliced myocardial slices.
[0121] Step 3.4: Place the sliced myocardium obtained in step 3.3 in an ice-cold 2,3-butanedione monoxime solution and incubate for 8 minutes.
[0122] Step 3.5: Sequentially transfer to three portions of Krebs solution containing 10 μM Blebbistatin, each solution is incubated at room temperature for 8 min to obtain myocardial slices for optical mapping.
[0123] Step 4: Optical Mapping
[0124] like Figure 2 shown.
[0125] Step 4.1: Place the myocardial slice for optical mapping obtained in step 3.5 in a sample bath containing 10 μM Blebbistatin-Krebs solution, equilibrate for 2 minutes, continuously fill with oxygen and carbon dioxide in a volume ratio of 95:5, and maintain the temperature at 37°C.
[0126] Step 4.2: Use a high-frequency camera on the optical mapping system to collect voltage signals and calcium signals after electrical stimulation and blue light stimulation, respectively, that is, obtain optical image data of the short-axis myocardial slice of the living body before and after electrical stimulation and blue light stimulation.
[0127] Example 2
[0128] The method for optical imaging of living myocardial slices in this embodiment is as follows: Figure 1 As shown, the following steps are included:
[0129] Step 1: Isolated Heart Perfusion
[0130] Step 1.1: Take an optogenetic transgenic adult mouse and anesthetize it.
[0131] The optogenetic transgenic adult mice were derived from Pnmt-Cre transgenic mice and B6.Cg-Gt(ROSA)26Sor tm27.1 (CAG-COP4H134R / tdTomato)Hze / J strain mice. The DNA between the loxP sites is cut by Cre recombinase, and the incision is reconnected under the action of DNA ligase. After recombination, the Pnmt promoter is connected to hChR2(H134R)-tdTomato, that is, Pnmt and hChR2(H134R)-tdTomato are expressed simultaneously. The above-mentioned optogenetic transgenic adult mice were purchased from Oxford University. The Pnmt-Cre transgenic mice are obtained by inserting Cre recombinase into the Pnmt gene promoter, and the B6.Cg-Gt(ROSA)26Sor tm27.1(CAG-COP4H134R / tdTomato)Hze / J strain mice had CAG-loxP-STOP-loxP-hChR2(H134R)-tdTomato inserted at the (ROSA)26 position.
[0132] The anesthesia was performed by intraperitoneal injection of sodium pentobarbital; the anesthesia time was 10 minutes.
[0133] Step 1.2: Open the chest, quickly remove the heart, place it in Krebs solution at 37±1°C, remove the connective tissue, cannulate the aorta, fix it on the Langendroff system, and perfuse it with Krebs solution at a rate of 3.8 ml / min for 3 minutes to obtain the perfused isolated heart.
[0134] Step 2: Loading calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye
[0135] Step 2.1: Mix the calcium-sensitive fluorescent dye Rhod-2AM and the surfactant Pluronic F127 in a ratio of 1:1, dilute in 1 ml of Krebs solution, and then slowly inject into the perfused isolated heart obtained in step 1.2 within 8 minutes. Then circulate in the Rhod-2 AM dye for 20 minutes to obtain an isolated heart loaded with the calcium-sensitive fluorescent dye.
[0136] Step 2.2: Continue to dilute 30 μl of voltage-sensitive fluorescent dye RH237 in 1 ml of Krebs solution. The voltage-sensitive fluorescent dye RH237 is prepared by dissolving RH237 solid in DMSO to a solution with a concentration of 1.25 mg / ml. Slowly inject the solution into the isolated heart loaded with calcium-sensitive fluorescent dye obtained in step 2.1 within 8 minutes to obtain an isolated heart loaded with calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye.
[0137] Step 2.3: The isolated heart loaded with calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye obtained in step 2.2 is perfused cyclically with Krebs solution containing 10 μM Blebbistatin until the heart stops beating. At this time, the heart is pink with no visible beating, thus obtaining an arrested heart.
[0138] Step 3: In vivo short-axis myocardial slice preparation
[0139] Keep away from light throughout the process.
[0140] Step 3.1: Freeze the 2,3-butanedione monoxime solution in a refrigerator to form an ice-water mixture, take out the arrested heart obtained in step 2.3 from the Langendorff system, and place it in the 2,3-butanedione monoxime solution mixed with ice and water to obtain a heart treated with the ice 2,3-butanedione monoxime solution.
[0141] Step 3.2: The heart treated with the ice 2,3-butanedione monoxime solution obtained in step 3.1 is embedded in low-temperature agar, wherein the temperature of the low-temperature agar is 37.5±5° C. and the concentration of the agar is 4%, to obtain an embedded heart.
[0142] Step 3.3: Glue the embedded heart obtained in step 3.2 to the sample tray of the vibrating slicer with 502 glue, then place the tray in the sample bath, add ice 2,3-butanedione monoxime solution to the sample bath until the heart is completely immersed, and slice the heart at a speed of 0.05 mm / s, a frequency of 80 Hz, and a thickness of 320 μm / s to obtain sliced myocardial slices.
[0143] Step 3.4: Place the sliced myocardium obtained in step 3.3 in an ice-cold 2,3-butanedione monoxime solution and incubate for 10 minutes.
[0144] Step 3.5: Sequentially transfer to three portions of Krebs solution containing 10 μM Blebbistatin, each solution is incubated at room temperature for 10 min to obtain myocardial slices for optical mapping.
[0145] Step 4: Optical Mapping
[0146] like Figure 2 shown.
[0147] Step 4.1: Place the myocardial slice for optical mapping obtained in step 3.5 in a slice bath containing 10 μM Blebbistatin-Krebs solution, equilibrate for 2.5 minutes, continuously fill with oxygen and carbon dioxide in a volume ratio of 95:5, and maintain the temperature at 37°C.
[0148] Step 4.2: Use a high-frequency camera on the optical mapping system to collect voltage signals and calcium signals after electrical stimulation and blue light stimulation, respectively, that is, obtain optical image data of the short-axis myocardial slice of the living body before and after electrical stimulation and blue light stimulation.
[0149] Example 3
[0150] The method for optical imaging of living myocardial slices in this embodiment is as follows: Figure 1 As shown, the following steps are included:
[0151] Step 1: Isolated Heart Perfusion
[0152] Step 1.1: Take an optogenetic transgenic adult mouse and anesthetize it.
[0153] The optogenetic transgenic adult mice were derived from Pnmt-Cre transgenic mice and B6.Cg-Gt(ROSA)26Sor tm27.1 (CAG-COP4H134R / tdTomato)Hze / J strain mice. The DNA between the loxP sites is cut by Cre recombinase, and the incision is reconnected under the action of DNA ligase. After recombination, the Pnmt promoter is connected to hChR2(H134R)-tdTomato, that is, Pnmt and hChR2(H134R)-tdTomato are expressed simultaneously. The above-mentioned optogenetic transgenic adult mice were purchased from Oxford University. The Pnmt-Cre transgenic mice are obtained by inserting Cre recombinase into the Pnmt gene promoter, and the B6.Cg-Gt(ROSA)26Sor tm27.1(CAG-COP4H134R / tdTomato)Hze / J strain mice had CAG-loxP-STOP-loxP-hChR2(H134R)-tdTomato inserted at the (ROSA)26 position.
[0154] The anesthesia was performed by intraperitoneal injection of a mixture of ketamine hydrochloride and Suminxin II in a volume ratio of 1:1; the anesthesia time was 10 minutes.
[0155] Step 1.2: Open the chest, quickly remove the heart, place it in Krebs solution at 37±1°C, remove the connective tissue, perform aortic cannulation, fix it on the Langendroff system, and perfuse it with Krebs solution at a rate of 4.0 ml / min for 3 minutes to obtain the perfused isolated heart.
[0156] Step 2: Loading calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye
[0157] Step 2.1: Mix the calcium-sensitive fluorescent dye Rhod-2 AM and the surfactant Pluronic F127 in a ratio of 1:1, dilute in 1 ml of Krebs solution, and then slowly inject into the perfused isolated heart obtained in step 1.2 within 10 minutes. Then circulate in the Rhod-2 AM dye for 20 minutes to obtain an isolated heart loaded with the calcium-sensitive fluorescent dye.
[0158] Step 2.2: Continue to dilute 30 μl of voltage-sensitive fluorescent dye RH237 in 1 ml of Krebs solution. The voltage-sensitive fluorescent dye RH237 is prepared by dissolving RH237 solid in DMSO to prepare a solution with a concentration of 1.25 mg / ml. Slowly inject it into the isolated heart loaded with calcium-sensitive fluorescent dye obtained in step 2.1 within 10 minutes to obtain an isolated heart loaded with calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye.
[0159] Step 2.3: The isolated heart loaded with calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye obtained in step 2.2 is perfused cyclically with Krebs solution containing 10 μM Blebbistatin until the heart stops beating. At this time, the heart is pink with no visible beating, thus obtaining an arrested heart.
[0160] Step 3: In vivo short-axis myocardial slice preparation
[0161] Keep away from light throughout the process.
[0162] Step 3.1: Freeze the 2,3-butanedione monoxime solution in a refrigerator to form an ice-water mixture, take out the arrested heart obtained in step 2.3 from the Langendorff system, and place it in the 2,3-butanedione monoxime solution mixed with ice and water to obtain a heart treated with the ice 2,3-butanedione monoxime solution.
[0163] Step 3.2: The heart treated with the ice 2,3-butanedione monoxime solution obtained in step 3.1 is embedded in low-temperature agar, wherein the temperature of the low-temperature agar is 37.5±5° C. and the concentration of the agar is 4%, to obtain an embedded heart.
[0164] Step 3.3: Glue the embedded heart obtained in step 3.2 to the sample tray of the vibrating slicer with 502 glue, then place the tray in the sample bath, add ice 2,3-butanedione monoxime solution to the sample bath until the heart is completely immersed, and slice at a speed of 0.05 mm / s, a frequency of 80 Hz, and a thickness of 350 μm / s to obtain sliced myocardial slices.
[0165] Step 3.4: Place the sliced myocardium obtained in step 3.3 in an ice-cold 2,3-butanedione monoxime solution and incubate for 12 minutes.
[0166] Step 3.5: Sequentially transfer to three portions of Krebs solution containing 10 μM Blebbistatin respectively, and incubate at room temperature for 12 min to obtain myocardial slices for optical mapping.
[0167] Step 4: Optical Mapping
[0168] like Figure 2 shown.
[0169] Step 4.1: Place the myocardial slice for optical mapping obtained in step 3.5 in a slice bath containing 10 μM Blebbistatin-Krebs solution, equilibrate for 3 minutes, continuously fill with oxygen and carbon dioxide in a volume ratio of 95:5, and maintain the temperature at 37°C.
[0170] Step 4.2: Use a high-frequency camera on the optical mapping system to collect voltage signals and calcium signals after electrical stimulation and blue light stimulation, respectively, that is, obtain optical image data of the short-axis myocardial slice of the living body before and after electrical stimulation and blue light stimulation.
[0171] Result analysis:
[0172] The method of Example 2 is used to obtain optical image data of the short-axis myocardial slice of the living body before and after electrical stimulation and blue light stimulation, such as Figure 3 and Figure 4 shown.
[0173] 1. Collect membrane potential signals of short-axis myocardial slices stimulated by electrical stimulation and light stimulation, and analyze and obtain Figure 5 , Figure 8 The distribution diagram of the action potential activation time of the entire myocardial slice is shown in the figure. Comparing the 50% and 75% time of the action potential repolarization under electrical stimulation and light stimulation is used to evaluate whether the cardiac electrical activity of the myocardial slice is abnormal. Figure 6 and Fig. 9 Compare, Figure 7 and Fig.10 Compare.
[0174] 2. Collect calcium transient signals of short-axis myocardial slices stimulated by electrical stimulation and blue light, and analyze and obtain the following Fig.11 , 14 The distribution diagram of calcium transient time of the whole myocardial slice is shown in the figure. The time when the calcium transient increases by 50% and 75% under electrical stimulation and blue light stimulation is compared to evaluate whether the calcium signal of the myocardial slice is abnormal. Fig.12 and Fig.15 Compare, Fig.13 and Fig.16 Compare.
[0175] 3. If Fig.17As shown, the characteristics and change gradients of AP and CaT in different areas of the in vivo short-axis myocardial slices (such as the left ventricular endocardium, left ventricular endocardium, interventricular septum, right ventricle, etc.) were analyzed.
[0176] 4. If Figure 18-Figure 21 As shown in Figure 2, the membrane potential and calcium transient changes of different cardiac regions, such as the endocardium, ventricular septum, and epicardium of the left and right ventricles, were analyzed. Fig. 22 As shown, the CaT map of the left ventricular epithelium was analyzed. Fig.23 As shown in Figure 2, the CaT map of the left ventricular endocardium was analyzed. Fig.24 As shown, the CaT map of the ventricular septum was analyzed. Fig.25 As shown, the CaT map of the right ventricle was analyzed.
[0177] 5. If Fig.26 As shown, the data obtained by synchronously recording membrane potential and calcium transient signals in the present invention can be used to comprehensively analyze the excitation conduction of the heart.
[0178] 6. If Fig. 27 As shown, the data obtained by synchronously recording membrane potential and calcium transient signals in the present invention can be used to comprehensively analyze the action potential diagram of the heart.
[0179] 7. If Fig.28 As shown, the data obtained by synchronously recording membrane potential and calcium transient signals in the present invention can be used to comprehensively analyze the calcium transient time course diagram of the heart.
[0180] 8. If Fig.29 As shown, the data obtained by synchronously recording the membrane potential and calcium transient signals of the present invention can be used to comprehensively analyze the delay diagram between the action potential and calcium transient of the heart.
[0181] 9. If Fig.30 As shown in FIG. 1 , optical mapping records the membrane potential and calcium transients of the myocardial surface layer at different parts of the isolated heart. Fig.17 It can analyze the characteristics and change gradients of AP and CaT in different areas of the short-axis myocardial slices in vivo (such as the left ventricular endocardium, left ventricular endocardium, interventricular septum, right ventricle, etc.). 1. Atrium; 2. Ventricle; 3. Apex.
[0182] 10. Fig.31 As shown, compared with the present invention, optical mapping records the membrane potential of the surface layer of the left ventricular myocardium of an isolated heart, but cannot record the membrane potential of the myocardium endothelium.
[0183] 11. If Fig.32 As shown, compared with the present invention, optical mapping records calcium transients in the surface layer of the left ventricular myocardium of an isolated heart, but cannot record calcium signals in the myocardium.
[0184] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for optical imaging of living myocardial slices, characterized in that: The steps include: Step 1: Loading calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye Step 1.1: Mix the calcium-sensitive fluorescent dye Rhod-2 AM and the surfactant Pluronic F127 in a ratio of 1:1, dilute in 1 ml of Krebs solution, and then slowly inject into the perfused isolated heart. Then, circulate in the Rhod-2 AM dye for 20 minutes to obtain an isolated heart loaded with the calcium-sensitive fluorescent dye. Step 1.2: Continue to dilute 30 μl of voltage-sensitive fluorescent dye RH237 in 1 ml of Krebs solution, and then slowly inject it into the isolated heart loaded with calcium-sensitive fluorescent dye obtained in step 1.1 to obtain an isolated heart loaded with calcium-sensitive fluorescent dye and voltage-sensitive fluorescent dye; Step 1.3: The isolated heart loaded with the calcium-sensitive fluorescent dye and the voltage-sensitive fluorescent dye obtained in step 1.2 is perfused cyclically with a Krebs solution containing 10 μM Blebbistatin until the heart stops beating, thereby obtaining a heart after the heart stops beating; Step 2: In vivo short-axis myocardial slice preparation Step 2.1: placing the 2,3-butanedione monoxime solution in a refrigerator to freeze it into an ice-water mixture, taking out the heart after beating obtained in step 1.3 from the Langendorff system, and placing it in the 2,3-butanedione monoxime solution mixed with ice and water to obtain a heart treated with the ice 2,3-butanedione monoxime solution; Step 2.2: embedding the heart treated with the ice 2,3-butanedione monoxime solution obtained in step 2.1 with cold agar to obtain an embedded heart; Step 2.3: stick the embedded heart obtained in step 2.2 onto the sample tray of the vibrating slicer, then place the tray in the sample bath, add ice 2,3-butanedione monoxime solution into the sample bath until the heart is completely immersed, and slice to obtain sliced myocardial slices; Step 2.4: Place the sliced myocardium obtained in step 2.3 in an ice-cold 2,3-butanedione monoxime solution and incubate for 8 min-12 min; Step 2.5: sequentially transfer to three portions of Krebs solution containing 10 μM Blebbistatin, each of which is incubated at room temperature for 8 min-12 min to obtain myocardial slices for optical mapping; Step 3: Optical Mapping Step 3.1: Place the myocardial slice for optical mapping obtained in step 2.5 in a slice bath containing 10 μM Blebbistatin-Krebs solution, equilibrate for 2 min-3 min, continuously fill with oxygen and carbon dioxide, and maintain the temperature at 37°C; Step 3.2: Use a high-frequency camera on the optical mapping system to collect voltage signals and calcium signals after electrical stimulation and blue light stimulation, respectively, that is, obtain optical image data of the short-axis myocardial slice of the living body before and after electrical stimulation and blue light stimulation.
2. The method for optical imaging of living myocardial slices according to claim 1, characterized in that: In step 1.1 and step 1.2, the injection time is 7 min-10 min.
3. The method for optical imaging of living myocardial slices according to claim 1, characterized in that: In step 1.2, the voltage-sensitive fluorescent dye RH237 is prepared by dissolving RH237 solid in DMSO to prepare a solution with a concentration of 1.25 mg / ml.
4. The method for optical imaging of living myocardial slices according to claim 1, characterized in that: In step 2.2, the temperature of the low-temperature agar is 37.5±5° C., and the concentration of the agar is 4%.
5. The method for optical imaging of living myocardial slices according to claim 1, characterized in that: In step 2.3, the slicing speed is 0.05 mm / s, the frequency is 80 Hz, and the thickness is 300 μm / slice to 350 μm / slice.
6. The method for optical imaging of living myocardial slices according to claim 1, characterized in that: In step 3.1, the volume ratio of oxygen to carbon dioxide is 95:5.