RU2622983C1 - Method of intraoperative visualization of ischemic-reperfusion damage of myocardium - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: intraoperatively, indocyanine green (ICG) is intravenously injected and 10-30 minutes after administration, a fluorescent image is recorded with excitation radiation of 780-810 nm and registration radiation of 820-900 nm.

EFFECT: method makes it possible to detect areas of irreversible damage to the myocardium within the ischemic zone, to quantify the ischemic damage to the myocardium, does not require an examination of the obscuration of the operating room.

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Description

The invention relates to medicine, namely to cardiac surgery, and can be used to monitor the condition of the myocardium during surgical interventions on an open heart.

Intraoperative monitoring of the manifestations of reversible and irreversible ischemic-reperfusion injury (RPI) of the myocardium during heart surgery is important, both in extracorporeal circulation and cardioplegia, and on the working heart (off pump), to optimize the tactics of surgical intervention and cardiac anesthesiology support.

A known method of intraoperative visualization of myocardial ischemia, based on the registration of myocardial autofluorescence (Mayevsky A, Rogatsky GG Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies. Am. J. Physiol. Cell 292: C. 615-640, 2007 ) It is based on a change in the intracellular redox state of the respiratory coenzyme pyridine nucleotide (NAD), which in the cell can exist in oxidized (NAD +) and reduced (NADH) forms. In ischemia due to a violation of oxidation through the chain of mitochondrial electron transport, the normal balance between them is disturbed in favor of the restored form. Due to the fact that the reduced form, when excited by ultraviolet rays (320-380 nm), has intense fluorescence in the blue-green region of the spectrum (420-480 nm), it becomes possible to detect tissue under ischemic conditions. The implementation of the method consists in illuminating the surface of the heart with exciting radiation from a illuminator that generates radiation in the near ultraviolet region of 320-380 nm, constructing an image of the object in the light of autofluorescence in the region of 420-480 nm, recording it on television and evaluating the autofluorescence pattern available for viewing the surface of the heart. The method is closest to the claimed invention.

The known method has several disadvantages. Firstly, autofluorescence imaging is aimed at identifying early, reversible manifestations of ischemic damage, and does not allow to identify areas of irreversible damage to the myocardium within the ischemic zone. This is because most of the myocardium, which is in a state of irreversible IRP, dies through necrosis, while the restored NADH is destroyed by the action of activated enzymes (glycohydrolases) (Vivaldi MT, Kloner RA, Schoen FJ. Triphenyltetrazolium staining of irreversible ischemic injury following coronary artery occlusion in rats. Am J Pathol. 1985 Dec; 121 (3): 522-30). Secondly, such a visualization requires dimming the operating room in order to avoid the influence of external illumination on the autofluorescence signal.

The technical result achieved by the invention is to identify areas of irreversible damage to the myocardium within the ischemic zone, the possibility of research in an unclouded operating room, as well as a quantitative assessment of ischemic myocardial damage.

The indicated technical result is achieved in a method of intraoperative visualization of ischemic-reperfusion damage to the myocardium, including illumination of the heart surface with exciting radiation, image registration in the light of fluorescence and image analysis, in which Indocyanine green (IHC) is injected intravenously and 10 to 30 minutes after administration, fluorescent is recorded image with excitation radiation of 780-810 nm and registration radiation of 820-900 nm.

It turned out to be appropriate the introduction of ICG, previously associated with proteins.

The best result is achieved when using albumin as a protein.

To quantify ischemic damage to the myocardium, fluorescence signals are measured in areas of the image of the myocardium with and without ischemic damage and the index of ischemic reperfusion injury is calculated by the formula

IIIRP = F _and / F _n ,

where F _and is the signal from the myocardial site with ischemic damage, F _n is the signal from the myocardial site without ischemic damage.

The claimed method is based on experimentally discovered by the authors of the phenomenon of accumulation of ICG in the zone of irreversible myocardial IRP. The mechanism for visualizing an IRP using an ICZ consists in the appearance of a contrast in the glow of an ICZ between the IRP zone and the surrounding unaffected zone, which occurs as follows. After intravenous administration, the ICC immediately begins to bind to plasma proteins, including albumin, and is bound by the liver in bound form. An hour later, ICG is completely excreted by the liver and kidneys from the bloodstream in two phases. In the first, fast phase (20 minutes), the excretory organs are able to quickly, in 5-10 minutes, lower the concentration of ICG in the blood, and after 20 minutes in the plasma remains up to 4% of the initial concentration. The visualization of myocardial IRP zones is apparently associated with a violation of vascular permeability in the pathological zone, where it is delayed for a long time due to the low rate of removal of interstitial (intercellular) fluid from the body. If a patient with RTI (acute coronary syndrome, myocardial infarction), who restored blood flow through a heart attack-dependent coronary artery (thrombolysis or revascularization surgery), is injected with ICZ, then 20 minutes after the first phase of excretion of ICZ from the bloodstream, the IRP region begins to glow, and fluorescence in the unaffected zone, on the contrary, it falls, creating a positive contrast in the IRP region. Therefore, no earlier than 10 minutes later, one can see this contrast of the fluorescent luminescence of the ICC, which is clearly visible at the 20th minute and reaches its maximum by the 30th minute.

Indocyanin green (cardiogrin) is used in medicine to solve various problems. It is used as an absorption dye that allows for intravital studies, and as a fluorescent substance emitting in the infrared (IR) region of the spectrum. ICF IR fluorescence is widely used in ophthalmology to visualize the state of the ocular fundus vessels (Pasechnikova N.V., Gut Yu., Gut I. Basic principles and clinical use of green angiography indocyanine in the diagnosis of ocular fundus pathology. Ophthalmological Journal No. 2. 2008. С . 63-67). Visualization in the light of ICG fluorescence is also used in general surgery for detecting tumors, determining sentinel lymph nodes, lymphography, and imaging of the bile ducts (Alander J.T. et al. A Review of Indocyanine Green Fluorescent Imaging in Surgery. International Journal of Biomedical Imaging Volume 2012, Article ID 940585, 26 pages doi: 10.1155 / 2012/940585). In cardiac surgery, ICGs are used during an operation for the purpose of angiography to check for obstruction of blood flow (Yamamoto, M. et al. Assessing intraoperative blood flow in cardiovascular surgery. Surg Today. 2011 Nov; 41 (11): 1467-74).

However, the authors did not find information about the use of the ICH to detect irreversible IRP in accessible sources of information.

Visualization of the zone of ischemic damage in the light of near infrared fluorescence using a fluorescent dye, the preparation of ICZ, provides the opportunity to conduct research in an unclouded operating room.

Preliminary binding of ICG to proteins increases the selectivity of fluorophore accumulation in the zone of irreversible myocardial damage.

The binding of ICG to albumin protein allows you to get the most contrasting picture of the fluorescence of the IRP zone.

The determination of the index of ischemic-reperfusion injury (IIRP) according to the formula NNRP = F _and / F _n increases the convenience and reliability of assessing myocardial IRP.

The invention is illustrated by drawings, which represent:

in FIG. 1 is a block diagram of a device for fluorescence imaging in the near infrared region of the spectrum on which the studies were conducted;

in FIG. 2 - photographs of the rat’s heart in ordinary light - the left column, and in the light of IR fluorescence - the right column; A, B - photographs of the working heart at the time of the beginning of the intravenous administration of the ICZ solution B, D - photographs of a working heart 20 minutes after restoration of blood flow; D, E - photographs of the surface of the extracted rat heart 30 minutes after the end of ischemia; G, H - photographs of the IRP zone in the heart section: G - after histochemical staining of the section with 2,3,5-triphenyltetrazolium chloride (TTC), 3 - view of the same section in the light of IR fluorescence.

The method is carried out, for example, as follows.

During a heart operation in extracorporeal circulation 10-30 minutes before the study, an ICZ solution (0.2-2 mg / ml) in a volume of 2-5 ml (0.2-0.5 mg / kg) is administered intravenously . It is also possible to use a mixture of ICZ with albumin. When using 20% human albumin as a solvent, the optimal concentration is 2.5 mg / ml. Then carry out a television registration in the light of infrared fluorescence when excited by radiation in the region of 780-810 nm and registration in the region of 820-900 nm. For registration, for example, a digital television camera developed at the Russian Science Center in Seoul (Kang Uk, Papayan G.V. Fluorescent endoscope system having improved image detection module. US Patent No. US 7635330. 12.22.2009) can be used. It provides the possibility of television visualization and measurement using a computer of the brightness of individual structures of the object simultaneously in the visible region and near infrared region of the spectrum in the range of 820-900 nm. The complex built on its basis (Fig. 1), in addition to the television camera 1, also includes a semiconductor laser 2 with a wavelength of 808 nm. It provides excitation of infrared fluorescence. Laser radiation is directed to the surface of the myocardium 3 through a fiber light guide 4. Image capture and analysis is carried out using computer 5, equipped with a special program that allows you to display the same object in two adjacent frames - black and white and color. In a black-and-white frame, the image of the object is formed in the light of infrared fluorescence due to the use of laser lighting, in a color frame it is presented in the usual form due to its illumination with an operating lamp. A conventional image is used to anatomically identify areas of increased IR fluorescence. Using the program in an interactive mode, the signals are measured in areas of the myocardium image with ischemic reperfusion damage and without damage, and the index of ischemic reperfusion damage is determined from them.

The method is illustrated by an example of experimental studies on an in-vivo model of rat myocardial ischemia-reperfusion. The essence of the model of local ischemic-reperfusion damage to rat myocardium is to perform thoracotomy on an anesthetized rat and 30 minutes ligation of the anterior descending branch of the left coronary artery.

In conditions of mechanical ventilation through the tracheostomy (respiratory rate

- 60 / min, tidal volume - 3 ml / 100 g body weight) and maintaining a constant body temperature (37.0 ± 0.5 ° C), using a thermostatically controlled operating table, left-sided thoracotomy was performed in the fourth intercostal space. Using an atraumatic needle, a polypropylene ligature (6-0) was placed under the left coronary artery (LCA) between the anatomical landmarks, which were the medial edge of the left atrial left ear and the pulmonary cone on the right. Reversible 30-minute myocardial ischemia was formed using an occluder [Himori N, Matsuura A. A simple technique for occlusion and reperfusion of coronary artery in conscious rats. Am J Physiol. 1989 Jun; 256 (6 Pt 2): H1719-25]. After 25 minutes of ischemia, a 10-minute infusion of the ICZ solution in a volume of 1 ml (0.25 mg / ml ICZ) or ICZ + albumin in a volume of 1 ml (0.25 mg / ml ICZ + albumin 20% 0.1 ml) was carried out, that is, the introduction began 5 minutes before the end of ischemia. In-vivo television monitoring and video recording of the myocardial state was carried out using the device of FIG. one.

Then the heart was dissected, myocardium was photographed from the outer surface of the heart and in the slice, followed by measuring the intensity of the ICG glow in the area of ischemic reperfusion damage and in the surrounding intact tissue with the calculation of the IIIRP.

Photos of the working heart (Fig. 2A, B, C, D) were obtained intraoperatively (in vivo) through the wound window in the fourth intercostal space during the formation of local ischemic-reperfusion damage to the rat myocardium. Post-mortem images of the extracted rat heart (Fig. 2D, E, G, Z) were obtained 30 minutes after the end of the ischemia.

Figs. 2A, B show the moment of the beginning of the intravenous administration of the ICZ solution when an occluder was created on the left coronary artery, creating myocardial ischemia. The occluder prevented the passage of the fluorophore only into the ischemic myocardium, which is confirmed by a defect in the filling of the fluorescent dye and the lack of fluorescent luminescence in the ischemic zone, while there was a luminescence (Fig. 2B) in the rest of the myocardium, in which the blood flow was saved and where the fluorescent dye entered. ICC was administered continuously for 10 minutes, while the occluder was removed at the 5th minute of its intravenous administration and the fluorophore with blood flow passed through the infarct-dependent coronary artery and stained the ischemic zone, which had not been illuminated before, therefore, the remaining 5 minutes of intravenous administration of ICZ fluorescence observed on the entire surface of the heart.

20 minutes after the restoration of blood flow, a persistent intense glow of the IRP zone is visible (Fig. 2B, D), where the accumulation of ICG occurred, with a low intensity of the fluorescent glow of the surrounding tissues.

In FIG. 2E shows the fluorescent glow of the epicardium in the area of the myocardial IRP, while in normal shooting this glow is not fixed (Fig. 2D).

A heart section was histochemically stained with 2,3,5-triphenyltetrazolium chloride (TTC). The RPI area in FIG. 2G corresponds to a white, unpainted red-brick color, myocardial tissue, and in FIG. 2Z - luminescence zone of the ICZ accumulated during the reperfusion. The contours and size of the unpainted area of the IRP in FIG. 2G correspond to the contours and sizes of the glow zone in FIG. 2Z, which indicates the accumulation of ICG in the area of irreversibly damaged myocardium. The maximum IIRP value in this zone was 6.3.

Using the claimed method allows to identify areas of irreversible damage to the myocardium within the ischemic zone, to quantify ischemic damage to the myocardium, and does not require dimming of the operating room during the study.

Claims (6)

1. A method of intraoperative visualization of ischemic-reperfusion damage to the myocardium, including illumination of the surface of the heart with exciting radiation, image registration in the light of fluorescence and image analysis, characterized in that the patient is injected with green indocyanine and 10-30 minutes after the injection, a fluorescence image is recorded upon excitation radiation 780-810 nm and emission registration 820-900 nm. 2. The method according to p. 1, characterized in that the injected indocyanine green, previously associated with proteins. 3. The method according to p. 2, characterized in that albumin is used as the protein. 4. The method according to PP. 1-3, characterized in that they measure the fluorescence signals in areas of the image of the myocardium with and without ischemic damage and calculate the index of ischemic reperfusion damage according to the formula IIIRP = F _and / F _n , where F _and is the signal from the myocardial site with ischemic damage, F _n is the signal from the myocardial site without ischemic damage.

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