A perfusion fluid and a normothermic blood mechanical perfusion system suitable for rat donor hearts

Abstract

The present invention relates to a perfusion solution, comprising blood and a modified Krebs-Henseleit solution, wherein the volume ratio of the two is 5:4; each liter of the modified Krebs-Henseleit solution contains 8-12 mmol / L glucose, 115-120 mmol / L NaCl, 4-5 mmol / L KCl, 23-27 mmol / L NaHCO 3 , 1-1.4mmol / L NaH 2 PO 4 、1.8-2.2mmol / L CaCl 2 and 0.5-0.52mmol / L MgCl 2 , the balance is water. The perfusion solution of the present invention can reduce the damage of the peristaltic pump to the red blood cells and reduce the adverse effects of hemolysis on the donor heart. The present invention also relates to a normal temperature blood mechanical perfusion system suitable for the donor heart of rats, which perfuses the perfusion solution into the donor heart to reduce ischemia-reperfusion injury and maintain a good physiological activity state during in vitro storage.

Description

Technical Field

[0001] The invention relates to the field of medical technology, in particular to a perfusion liquid and a normal temperature blood mechanical perfusion system suitable for a rat donor heart. Background Art

[0002] Heart transplantation is currently the most effective method for treating end-stage heart failure, but the shortage of donor hearts has largely limited the development of heart transplantation surgery at home and abroad. Currently, in clinical practice, donor hearts mainly come from brain-dead donors (Donation after brain death, DBD), while in the past five years, the use of hearts from donors after circulatory death (Donation after circulatory death, DCD) is considered an important strategy to expand the source of donor hearts.

[0003] During the heart transplantation process, the heart often needs to undergo a long and long-distance transportation from the donor to the recipient. During this process, the donor heart will continue to be ischemic and hypoxic. Therefore, it is the key to ensure the success of the transplantation operation to fully and effectively preserve the donor heart and reduce ischemia-reperfusion injury. Static cold storage (SCS) is a traditional method for preserving donor hearts. It reduces the metabolic level of the donor heart by low temperature so that it can tolerate ischemia and hypoxia for a certain period of time. However, compared with DBD hearts, DCD hearts need to undergo a long period of warm ischemia before being removed. During this period, the heart will deplete its energy reserves, produce a large number of toxic metabolites, and cause intracellular acidosis. This also means that the DCD heart lacks sufficient physiological reserves and is sensitive to ischemia and hypoxia during in vitro preservation, thus significantly reducing the quality of the donor heart and suffering greater ischemia-reperfusion injury during heart transplantation. Therefore, the traditional method of preserving donor hearts cannot meet the requirements of DCD hearts for preservation conditions. We urgently need a more perfect method for preserving DCD hearts to reduce their ischemia-reperfusion injury and improve the quality of donor hearts.

[0004] Mechanical perfusion is a method of donor heart preservation that has received widespread attention in the past decade. It can be simply divided into hypothermic machine perfusion (HMP) and normothermic machine perfusion (NMP). Compared with SCS, mechanical perfusion can continuously perfuse the isolated heart, continuously provide oxygen and metabolic substrates, reduce the ischemia and hypoxia time of the donor heart, and remove toxic metabolites in the microcirculation, thereby reducing ischemia-reperfusion injury. In addition, NMP is closer to physiological conditions. The donor heart is preserved at 35-37°C. During this process, the donor heart can restore metabolic activity, start the repair process, and even restore pulsation. Therefore, it is possible to objectively and comprehensively evaluate the function of the donor heart before transplantation. More importantly, during the process of mechanical perfusion preservation, the donor heart can maintain a low level or completely restore metabolic activity, which means that mechanical perfusion provides an excellent time window during which various myocardial protective drugs, biomolecules or stem cells can be used to repair the DCD heart, reduce its ischemia-reperfusion injury, improve the quality of the donor heart, and improve the survival rate of patients after transplantation. In addition, since mechanical perfusion is a method of preserving isolated organs, using drugs or stem cells to treat the donor heart during this process has a more "targeted therapy" effect without considering the impact on other tissues and organs or the impact of drugs on other organs.

[0005] Currently, more and more studies are beginning to be devoted to combining mechanical perfusion with drugs, stem cell therapy, etc., for the preservation and repair of DCD hearts. Therefore, before these new treatments are officially applied in the clinic, the mechanical perfusion model based on animal DCD hearts will be the core foundation of these studies.

[0006] However, as mentioned above, the DCD heart lacks sufficient physiological reserve and is sensitive to ischemia and hypoxia, and cannot tolerate ischemia-reperfusion injury. Since the donor heart will be kept at physiological temperature in the normothermic mechanical perfusion system, it requires more demanding perfusion conditions and organ protection. Therefore, the establishment of an animal DCD heart normothermic mechanical perfusion model is undoubtedly difficult. In addition, compared with animals such as pigs and monkeys, the rat heart is small in size and easily affected by perfusion pressure, gas emboli, etc., and the operation is difficult, which undoubtedly further increases the difficulty of model establishment. However, compared with the extremely high requirements for funds, laboratory conditions, etc. of large animals such as pigs and monkeys, using rats as research objects can greatly reduce research costs, can be implemented in general laboratories, and can establish different genetic modification or disease experimental models as needed, which is more suitable for early exploratory research. Therefore, it is very necessary to establish a normothermic mechanical perfusion model of rat donor hearts.

[0007] At present, only a few laboratories in the world have used the rat DCD heart normothermic mechanical perfusion model in their research, but all of them are based on the Langendorff system (an open ex vivo heart perfusion system with a compound electrolyte solution as the perfusion fluid). The Langendorff system uses a compound electrolyte solution as the perfusion fluid, which has a simple composition, lacks hemoglobin, and has poor oxygen carrying capacity. When the heart is perfused in vitro, it often requires a coronary artery flow rate several times higher than the physiological situation to meet the metabolic needs of the rat DCD heart, but it is also inevitable that the high flow rate will cause damage to the coronary endothelial cells. Moreover, the perfusion fluid lacks biological macromolecules with special functions in the blood, and cannot effectively meet the needs of maintaining a beating heart. In addition, since this normothermic mechanical perfusion model is an open perfusion system (the perfusion fluid does not return to the system after flowing through the donor heart), if drugs, biological molecules, etc. are added to the system and act on the donor heart through the perfusion fluid, they will face the problem of short action time or large amount required. OCS (Organ care system) is a heart normothermic mechanical perfusion system currently in clinical use. It is generally a closed-loop system consisting of an oxygenator, a heater, a centrifugal pump, and a perfusion pipeline. The perfusion fluid used is mainly composed of blood. Therefore, the rat DCD heart normothermic mechanical perfusion model established using the Langendorff system is far from clinical practice. Moreover, since the coronary artery resistance of the rat DCD heart increases and the coronary flow decreases after warm ischemia, and the oxygen supply capacity of the perfusion fluid is poor, the oxygen demand of the DCD heart cannot be fully met, and obvious myocardial infarction often occurs 1 hour after perfusion, and the measured cardiac function is likely not its true level. If an animal model based on the Langendorff system is used, it is likely that the protective effect of normothermic mechanical perfusion on the donor heart cannot be truly reflected, which will damage the authenticity and rigor of the research results.

[0008] So far, only one laboratory in Switzerland has conducted research based on the rat DCD heart normothermic blood mechanical perfusion model. They drew 4 ml of blood from another rat and mixed the obtained blood with Krebs-Henseleit solution (10 mmol / L glucose, 117 mmol / L NaCl, 5.9 mmol / L KCl, 25 mmol / L NaHCO3, 1.2 mmol / L NaH2PO4, 1 mmol / L CaCl2, 0.512 mmol / L MgCl2) in a 1:1 ratio to form a perfusion solution. This method has a relatively small blood ratio, low hemoglobin concentration, and weak oxygen supply capacity; the total priming volume is small, the peristaltic pump easily destroys red blood cells, and hemolysis is obvious and occurs early, so the donor heart can only be preserved for 1 hour; and after the blood is diluted, the osmotic pressure decreases, and different degrees of tissue edema will inevitably occur, and their model cannot evaluate the donor heart function in real time.

[0009] Therefore, the present invention provides a normal temperature blood mechanical perfusion system suitable for rat DCD donor hearts, with a closed-loop design, which can be used for donor heart preservation, reduce ischemia-reperfusion injury of donor hearts, and can evaluate the function of rat donor hearts in real time. Summary of the invention

[0010] In view of the technical problems existing in the prior art, one of the purposes of the present invention is to provide a perfusion fluid with a high oxygen supply capacity, capable of maintaining a long perfusion time, reducing the damage of the peristaltic pump to red blood cells, reducing the impact on the rat donor heart, and better meeting the needs of in vitro perfusion of the donor heart.

[0011] In view of the technical problems existing in the prior art, the second purpose of the present invention is to provide a room temperature blood mechanical perfusion system suitable for rat donor hearts, in which the silicone tube will not undergo difficult-to-recover deformation, the accuracy of the peristaltic pump flow control can be effectively maintained, and real-time evaluation of the donor heart function can be achieved.

[0012] In order to achieve the above object, the present invention adopts the following technical solution:

[0013] A perfusion fluid comprises blood and a modified Krebs-Henseleit solution, wherein the volume ratio of blood to the modified Krebs-Henseleit solution is 5:4; each liter of the modified Krebs-Henseleit solution contains 8-12 mmol / L glucose, 115-120 mmol / L NaCl, 4-5 mmol / L KCl, 23-27 mmol / L NaHCO3, 1-1.4 mmol / L NaH2PO4, 1.8-2.2 mmol / L CaCl2 and 0.5-0.52 mmol / L MgCl2, and the balance is water.

[0014] Furthermore, each liter of modified Krebs-Henseleit solution contains 10 mmol / L glucose, 117 mmol / L NaCl, 4.5 mmol / L KCl, 25 mmol / L NaHCO3, 1.2 mmol / L NaH2PO4, 2 mmol / L CaCl2 and 0.512 mmol / L MgCl2, with the remainder being water.

[0015] Furthermore, the perfusion solution also includes 25 g / L of mannitol.

[0016] Furthermore, the perfusion solution also includes 500 mg / L of methylprednisolone.

[0017] Furthermore, the perfusate also includes 160 IU / L of insulin.

[0018] A mechanical perfusion system for normal temperature blood of a donor heart of a rat comprises a controller, a blood reservoir, an oxygen cylinder, an oxygenator, a peristaltic pump, a water bath and a micro-infusion pump; the peristaltic pump comprises a silicone tube, a first extrusion device and a second extrusion device, the first extrusion device and the second extrusion device are respectively arranged beside the silicone tube, the oxygen cylinder is connected to the oxygenator for supplying oxygen to the oxygenator; two ends of the oxygenator are respectively connected to the donor heart and one end of the silicone tube, the other end of the silicone tube is respectively connected to the blood reservoir and one end of the micro-infusion pump, the blood reservoir stores perfusion liquid, the blood reservoir is arranged in a water bath, the water bath is used to store hot water, the other end of the micro-infusion pump is connected to a glucose solution, a sodium bicarbonate solution and an adrenaline solution; the controller is respectively connected to the peristaltic pump and the micro-infusion pump, the controller is used to control the first extrusion device and the second extrusion device to squeeze and release the silicone tube in turn, the direction of the first extrusion device squeezing the silicone tube is perpendicular to the direction of the second extrusion device squeezing the silicone tube.

[0019] Furthermore, a temperature control switch, a temperature measuring probe, a heater and a spiral stirrer are arranged in the water bath, and the temperature control switch is connected with the temperature measuring probe, the heater and the controller respectively.

[0020] Furthermore, the mass concentrations of the glucose solution and the sodium bicarbonate solution are 50% and 5% respectively; the injection volume of the adrenaline solution is 4.8×10 -5 mg / h / kg.

[0021] Furthermore, it also includes a balloon catheter and a pressure sensor; the balloon catheter is connected to the pressure sensor for real-time evaluation of the donor's heart function.

[0022] Furthermore, it also includes a display, which is connected to the pressure sensor.

[0023] In general, the present invention has the following advantages:

[0024] The present invention uses a blood-based perfusion fluid, which is combined with the normal temperature blood mechanical perfusion system of the present invention to realize a closed-loop perfusion system for real-time evaluation of the heart function of rat donors. In the perfusion fluid of the present invention, blood accounts for a large proportion, and the hemoglobin concentration is high, which improves the oxygen supply capacity of the perfusion fluid, and can keep the coronary flow of the rat donor heart within the physiological range during in vitro storage; the total amount of perfusion fluid is increased, the damage of the peristaltic pump to red blood cells is reduced, the adverse effects of hemolysis on the rat donor heart are reduced, and the perfusion time is extended; by increasing the calcium ion concentration in the Krebs-Henseleit solution, the free calcium ion concentration in the perfusion fluid is ensured to be closer to the physiological condition of the rat, and a good physiological activity state is maintained.

[0025] In addition, we added mannitol to the perfusion fluid to increase the osmotic pressure of the perfusion fluid and reduce edema in the donor heart; added methylprednisolone to reduce inflammatory response, stabilize cell membranes, and protect the donor heart; added insulin, continuously pumped adrenaline, supplemented glucose and sodium bicarbonate according to the results of blood gas analysis, and adjusted the acid-base balance to maintain the normal metabolism of the donor heart and stabilize its internal environment, which is more in line with the needs of extracorporeal perfusion of the donor heart. We placed the balloon catheter from the left atrium through the mitral valve into the left ventricle and connected it to a pressure sensor to achieve real-time evaluation of the donor heart function. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 . Flow chart of the operation flow and experimental design for implementing application example 4. (A) Operation flow; (B) Experimental design. The glucose concentration and pH of the perfusate are adjusted according to the results of blood gas analysis.

[0027] Figure 2 .Mechanical perfusion system of normothermic blood for donor hearts of rats with circulatory death. (A) Mechanical perfusion system of normothermic blood; (B) peristaltic pump; (C) oxygenator; (D) microinfusion pump; (E) water bath; (F) heater; (G) stirrer; (H) temperature control switch; (I) perfusion needle; (J) working state of the mechanical perfusion system of normothermic blood; (K) beating heart (arrow).

[0028] Figure 3 .Cardiac function during in vitro preservation of hearts from donors with circulatory death. (A) Typical development of carotid artery blood pressure after tracheal clamping in donor rats, line a: tracheal clamping; line b: circulatory death; (B) heart rate (HR); (C) developed pressure (DP); (D) maximum rate of rise of left ventricular pressure (dP / dt max ); (E) Maximum rate of decrease of left ventricular internal pressure (dP / d tmin ). Data are presented as median and interquartile range. 8 samples per group. *p<0.05vs.T0.

[0029] Figure 4 . Levels of ATP, GSH, SOD, and MDA in myocardial tissue of hearts from donors with circulatory death during in vitro storage. (A) Adenosine triphosphate (ATP); 5 samples in the P30 group, 6 samples in the P60 group, and 4 samples in the P90 group; (B) Reduced glutathione (GSH); 6 samples in each group; (C) Superoxide dismutase activity (SOD activity); 6 samples in each group; (D) Malondialdehyde (MDA); 6 samples in each of the P30 and P60 groups; 5 samples in the P90 group; Data are presented as medians and interquartile ranges.

[0030] Figure 5 .Expression of HNE, IL-6, and TNF-α in myocardial tissue during ex vivo storage of hearts from donors with circulatory death. (A) Representative immunohistochemical images of myocardial tissues in the P30, P60, and P90 groups, with a scale bar of 100 μm; (B) Average IOD of 4-hydroxynonenal (HNE), 6 samples in each group; (C) Average IOD of interleukin-6 (IL-6), 6 samples in each group; (D) Average IOD of tumor necrosis factor-α (TNF-α), 6 samples in each group of P30 and P90 groups; 5 samples in the P60 group. Data are presented as medians and interquartile ranges.

[0031] Figure 6 . Histopathological changes of hearts from donors with circulatory death during in vitro storage. (A) Representative hematoxylin-eosin staining images of myocardial tissues in the P30, P60, and P90 groups (×200 times, scale bar 200 μm); (B) Pathological scores, 6 samples in each group; (C) Representative hematoxylin-eosin staining images of myocardial tissues in the P30, P60, and P90 groups (×400 times, scale bar 100 μm); (D) Average cell area, 6 samples in each group; (E) Example of myocardial injury under hematoxylin-eosin staining Data are presented as medians and interquartile ranges.

[0032] Figure 7 .Myocardial apoptosis during in vitro storage of hearts from donors with circulatory death. (A) Representative images of TUNEL staining of myocardium during in vitro storage of hearts from donors with circulatory death; (B) Percentage of TUNEL-stained cells, 6 samples in each group. Data are presented as median and interquartile range.

[0033] Figure 8 . Evaluation of myocardial infarction during ex vivo storage of hearts from donors with circulatory death. The upper part shows representative images of myocardial TTC staining during ex vivo storage of hearts from donors with circulatory death; dark red: viable myocardial tissue; white: necrotic myocardial tissue. The lower part shows the quantification of infarct size in DCD hearts. Data are presented as median and interquartile range. Six samples per group.

[0034] Fig. 9 It is a schematic diagram of the first extrusion device and the second extrusion device of the peristaltic pump.

[0035] Fig.10 This is the working principle diagram of the peristaltic pump.

[0036] Description of reference numerals:

[0037] 1-donor heart, 2-oxygenator, 3-oxygen cylinder, 4-peristaltic pump, 41-first extrusion device, 42-second extrusion device, 43-silicone tube, 5-micro infusion pump, 6-water bath, 7-blood reservoir. DETAILED DESCRIPTION

[0038] The present invention will be described in further detail below.

[0039] A perfusion fluid comprises blood and a modified Krebs-Henseleit solution, wherein the volume ratio of blood to the modified Krebs-Henseleit solution is 5:4; each liter of the modified Krebs-Henseleit solution contains 8-12 mmol / L glucose, 115-120 mmol / L NaCl, 4-5 mmol / L KCl, 23-27 mmol / L NaHCO3, 1-1.4 mmol / L NaH2PO4, 1.8-2.2 mmol / L CaCl2 and 0.5-0.52 mmol / L MgCl2, and the balance is water.

[0040] In the perfusion solution of the present invention, blood accounts for a relatively large proportion, and the hemoglobin concentration is relatively high, thereby improving the oxygen supply capacity of the perfusion solution and maintaining the coronary flow rate of the rat donor heart within the physiological range during the in vitro storage period; the total amount of the perfusion solution is increased, the damage of the peristaltic pump to red blood cells is reduced, the adverse effect of hemolysis on the rat donor heart is reduced, and the perfusion time is prolonged; and by increasing the calcium ion concentration in the Krebs-Henseleit solution, the free calcium ion concentration in the perfusion solution is ensured to be closer to the physiological condition of the rat, thereby maintaining a good physiological activity state.

[0041] In this embodiment, the blood in the perfusion solution is rat blood, and other animal blood or artificial blood may also be used.

[0042] Preferably, each liter of modified Krebs-Henseleit solution contains 10 mmol / L glucose, 117 mmol / L NaCl, 4.5 mmol / L KCl, 25 mmol / L NaHCO3, 1.2 mmol / L NaH2PO4, 2 mmol / LCaCl2 and 0.512 mmol / LMgCl2, with the remainder being water.

[0043] Preferably, the perfusion solution also includes 25 g / L of mannitol.

[0044] In the perfusion fluid of the prior art, the osmotic pressure decreases after the blood is diluted, and the donor heart that has undergone perfusion will inevitably have varying degrees of tissue edema, which makes it difficult to meet the needs of in vitro perfusion of the donor heart.

[0045] The invention adds mannitol into the perfusion solution, thereby increasing the osmotic pressure of the perfusion solution and reducing the edema of the donor heart, thus better meeting the requirements of in vitro perfusion of the donor heart.

[0046] Preferably, the perfusion solution also includes 500 mg / L of methylprednisolone.

[0047] The addition of methylprednisolone reduced the inflammatory response of the donor heart, stabilized the cell membrane, and played a role in protecting the donor heart.

[0048] Preferably, the perfusate also includes 160 IU / L of insulin.

[0049] Adding insulin is beneficial to maintaining the normal metabolism of the donor heart and stabilizing its internal environment.

[0050] like Figure 1 , Figure 2 , Fig. 9 , Fig.10 As shown, a normal temperature blood mechanical perfusion system suitable for a rat donor heart comprises a controller, a blood reservoir 7, an oxygen cylinder 3, an oxygenator 2, a peristaltic pump 4, a water bath 6 and a micro-infusion pump 5; the peristaltic pump 4 comprises a silicone tube 43, a first extrusion device 41 and a second extrusion device 42, the first extrusion device 41 and the second extrusion device 42 are respectively arranged beside the silicone tube 43, the oxygen cylinder 3 is connected to the oxygenator 2 to supply oxygen to the oxygenator 2; the two ends of the oxygenator 2 are respectively connected to the donor heart 1 and one end of the silicone tube 43, and the other end of the silicone tube 43 is separated into a first extrusion device 41 and a second extrusion device 42. The blood reservoir 7 is respectively connected to one end of the micro-infusion pump 5, and the blood reservoir 7 stores perfusion fluid. The blood reservoir 7 is arranged in a water bath 6, and the water bath 6 is used to store hot water. The other end of the micro-infusion pump 5 is connected to a glucose solution, a sodium bicarbonate solution and an adrenaline solution; the controller is respectively connected to the peristaltic pump 4 and the micro-infusion pump 5, and the controller is used to control the first extrusion device 41 and the second extrusion device 42 to extrude the silicone tube 43 in turn, and the direction in which the first extrusion device 41 extrudes the silicone tube 43 is perpendicular to the direction in which the second extrusion device 42 extrudes the silicone tube 43.

[0051] Specifically, the blood reservoir 7 is arranged in the water bath 6. By heating with hot water, the temperature of the perfusion liquid in the blood reservoir 7 is more uniform, and there will be no temperature inequality. The hot water stored in the water bath 6 allows the perfusion liquid stored in the blood reservoir 7 to be maintained at 35-37°C when it flows through the pipeline to the donor heart, which can keep the perfusion liquid and the donor heart in the best state, which is conducive to obtaining a better perfusion effect. The controller is connected to the peristaltic pump 4, and the perfusion pressure and flow rate of the perfusion liquid are adjusted by controlling the output power of the peristaltic pump 4. The micro-infusion pump 5 is respectively connected to the glucose solution, the sodium bicarbonate solution and the adrenaline solution, and is used to supplement the glucose solution, the sodium bicarbonate solution and the adrenaline solution into the perfusion liquid to maintain the glucose concentration of the perfusion liquid at 8-12mmol / L and the pH at 7.35-7.45.

[0052] Usually, the peristaltic pump 4 controls the flow rate by squeezing and releasing the silicone tube 43 with an extrusion device, so that the liquid flows in the silicone tube 43, and thus the resilience of the silicone tube 43 has a great influence on the flow control accuracy of the peristaltic pump. However, after being squeezed in one direction for a long time, the silicone tube 43 may not release stress in time, and the elastic material deformation cannot be restored in time, which may cause the silicone tube 43 to deform in a way that is difficult to recover, such as the silicone tube 43 becoming flat along the squeezing direction, and such deformation may cause the flow control accuracy of the peristaltic pump to decrease.

[0053] The present invention provides two squeezing devices, a first squeezing device 41 and a second squeezing device 42, in the peristaltic pump 4. The first squeezing device 41 and the second squeezing device 42 are respectively arranged beside the silicone tube 43. Preferably, the first squeezing device 41 and the second squeezing device 42 squeeze the upper and lower sides and the left and right sides of the silicone tube 43 respectively. When the peristaltic pump 4 is working, the controller controls the first squeezing device 41 and the second squeezing device 42 to squeeze and release the upper and lower sides and the left and right sides of the silicone tube 43 in turn, so that the silicone tube 43 will not be squeezed in one direction for a long time, there will be no phenomenon of untimely stress release, and the silicone tube 43 will not be deformed and difficult to recover, and the flow control accuracy of the peristaltic pump can be effectively maintained.

[0054] Based on the prior art peristaltic pump that discloses an extrusion device for extruding the silicone tube 43, the applicant proposed the inventive concept of extruding different parts of the silicone tube 43 to solve the technical problem that the silicone tube 43 is difficult to recover its deformation due to long-term unidirectional extrusion. An extrusion device is expanded into a first extrusion device 41 and a second extrusion device 42, which are respectively located on different sides of the silicone tube 43, and are controlled by a controller to squeeze and release the different sides of the silicone tube 43 in turn. This is a structure that is easy for ordinary technicians in this field to think of and implement, and will not be repeated here.

[0055] A temperature control switch, a temperature measuring probe, a heater and a spiral stirrer are arranged in the water bath; the temperature control switch is connected with the temperature measuring probe, the heater and the controller respectively.

[0056] The spiral stirrer stirs the water in the water bath to keep the water temperature in the water bath uniform. The heater is used to heat the water in the water bath. The temperature probe is set in the hot water in the water bath to detect the hot water temperature in real time. The temperature control switch is used to control the heating action of the heater, thereby automatically adjusting the hot water temperature.

[0057] The mass concentrations of the glucose solution and the sodium bicarbonate solution are 50% and 5%, respectively.

[0058] It also includes a balloon catheter and a pressure sensor. The balloon catheter is connected to the pressure sensor for real-time evaluation of the donor's heart function.

[0059] The balloon catheter is placed from the left atrium of the donor heart through the mitral valve into the left ventricle, and the cardiac function of the donor heart can be evaluated in real time through the pressure sensor.

[0060] The device also includes a display connected to the pressure sensor.

[0061] The display can display the cardiac function image of the donor heart detected by the pressure sensor in real time, which helps the operator to understand the cardiac function status of the donor heart at any time.

[0062] Example 1

[0063] A perfusion solution includes blood, a modified Krebs-Henseleit solution, 25 g / L mannitol, 500 mg / L methylprednisolone, and 160 IU / L insulin, wherein the volume ratio of blood to the modified Krebs-Henseleit solution is 5:4; each liter of the modified Krebs-Henseleit solution contains 10 mmol / L glucose, 117 mmol / L NaCl, 4.5 mmol / L KCl, 25 mmol / L NaHCO3, 1.2 mmol / L NaH2PO4, 2 mmol / L CaCl2, and 0.512 mmol / L MgCl2, and the remainder is water.

[0064] Example 2

[0065] A perfusion solution includes blood, a modified Krebs-Henseleit solution, 25 g / L mannitol, 500 mg / L methylprednisolone, and 160 IU / L insulin, wherein the volume ratio of blood to the modified Krebs-Henseleit solution is 5:4; each liter of the modified Krebs-Henseleit solution contains 8 mmol / L glucose, 115 mmol / L NaCl, 4 mmol / L KCl, 23 mmol / L NaHCO3, 1 mmol / L NaH2PO4, 1.8 mmol / L CaCl2, and 0.5 mmol / L MgCl2, and the remainder is water.

[0066] Example 3

[0067] A perfusion solution comprises blood, a modified Krebs-Henseleit solution, 25 g / L mannitol, 500 mg / L methylprednisolone, and 160 IU / L insulin, wherein the volume ratio of blood to the modified Krebs-Henseleit solution is 5:4; each liter of the modified Krebs-Henseleit solution contains 12 mmol / L glucose, 120 mmol / L NaCl, 5 mmol / L KCl, 27 mmol / L NaHCO3, 1.4 mmol / L NaH2PO4, 2.2 mmol / L CaCl2, and 0.52 mmol / L MgCl2, and the remainder is water.

[0068] Application Example 4: DCD rat isolated heart normothermic blood mechanical perfusion model

[0069] In this example, a modified "DCD rat isolated heart normothermic blood mechanical perfusion model" was established. Figure 1 The perfusion system is a closed-loop perfusion system that uses a blood-based perfusion fluid and is composed of a perfusion pipe, a blood reservoir, an oxygenator, a peristaltic pump, a micro-infusion pump, and a water bath (water tank, temperature control switch, temperature probe, heater, and spiral stirrer). Figure 2 10 ml of blood was drawn from another rat, mixed with 8 ml of modified Krebs-Henseleit solution (10 mmol / L glucose, 117 mmol / L NaCl, 4.5 mmol / L KCl, 25 mmol / L NaHCO3, 1.2 mmol / L NaH2PO4, 2 mmol / L CaCl2, 0.512 mmol / L MgCl2), and methylprednisolone (500 mg / L), insulin (160 IU / L) and mannitol (25 g / L) were added to form the perfusion solution, and adrenaline solution (4.8 × 10 -5 mg / h / kg, pump speed is 0.1ml / h). The oxygen flow and perfusion flow are pre-set to 1:1 (perfusion flow is 1ml / min / 100g), the oxygen partial pressure is maintained at 150-250mmHg, the isolated heart temperature is maintained at 35-37℃, and 50% glucose solution and 5% sodium bicarbonate solution are supplemented according to the results of blood gas analysis to maintain the glucose concentration at 8-12mmol / L and pH at 7.35-7.45 to maintain the normal metabolism and homeostasis of the donor heart. The balloon catheter is placed from the left atrium through the mitral valve into the left ventricle and connected to a pressure sensor to achieve real-time evaluation of the donor heart function.

[0070] Experimental Animals: Male Sprague-Dawley rats.

[0071] Experimental design:

[0072] (1) 26 male Sprague-Dawley rats (200-300 g, 8-12 weeks old) were used as DCD heart donor rats, and another 26 male Sprague-Dawley rats (300-400 g, 12-15 weeks old) were used as blood donor rats. All DCD hearts were preserved in vitro using a normothermic blood mechanical perfusion system designed by us. Cardiac function assessment and blood gas analysis were performed during in vitro preservation. At the end of perfusion, heart tissues were collected to evaluate tissue energy reserves, oxidative stress, apoptosis, inflammation, myocardial infarction, and histopathological changes.

[0073] (2) Experimental plan:

[0074] ① Eight DCD hearts will be kept in a normothermic blood mechanical perfusion system for 90 minutes, during which time cardiac function will be assessed;

[0075] ② Eighteen DCD hearts were preserved in a normothermic blood mechanical perfusion system for 30, 60 or 90 minutes (P30, P60 and P90 groups, 6 hearts in each group) and used for molecular biological analysis.

[0076] Establishing a normothermic blood mechanical perfusion system:

[0077] (1) The perfusion system consists of a peristaltic pump, an oxygenator, a 16# silicone tube, a blood reservoir, a micro-infusion pump and a water bath. The water bath contains water, a heater, a stirrer, a temperature control switch and a temperature probe. A humidified gas containing 95% O2 / 5% CO2 is used to continuously supply oxygen through the oxygenator. The oxygenator is wrapped in a thermal bag. The blood reservoir and the pipeline are partially immersed in the water of the water bath to heat the perfusion fluid. The removed DCD heart will be suspended in the blood reservoir through the perfusion needle, below the liquid level of the water bath to provide a warm environment. The water temperature of the water bath is set to 41-42°C to maintain the temperature of the isolated heart at 35-37°C.

[0078] (2) The perfusion system was primed with perfusion solution. The perfusion solution consisted of donor rat blood (10 mL) and modified Krebs-Henseleit solution (8 mL; containing 10 mmol / L glucose, 117 mmol / L NaCl, 4.5 mmol / L KCl, 25 mmol / L NaHCO3, 1.2 mmol / L NaH2PO4, 2 mmol / L CaCl2, and 0.512 mmol / L MgCl2), with mannitol (final concentration of 25 g / L), methylprednisolone sodium succinate (final concentration of 500 mg / L), and insulin (final concentration of 160 IU / L) added.

[0079] Table 1. Perfusion fluid composition

[0080]

[0081] Specific steps:

[0082] (1) Obtaining DCD rat hearts

[0083] ① Induce circulatory death in rats

[0084] Select 250-350g male Sprague-Dawley rats, induce anesthesia with 3% isoflurane inhalation, and maintain anesthesia with intraperitoneal injection of ketamine (75mg / kg) and xylazine (5mg / kg). Intubate the trachea, ventilator-assisted breathing (tidal volume 8-10ml / kg, respiratory rate 60 times / min), intubate the right common carotid artery, connect a pressure sensor, and monitor the rat's arterial blood pressure. Heparinize (2000IU / kg) through the right common carotid artery. After 15 minutes of heparinization, clamp the rat's trachea, and wait until the rat's arterial systolic pressure is less than 30mmHg or the arterial fluctuation disappears, and then count for 15 minutes.

[0085] ②Removing the donor heart

[0086] After 15 minutes, the rat's chest cavity was opened, and the ascending aorta, aortic arch, brachiocephalic trunk, left common carotid artery and left subclavian artery were freed. The aortic arch was clamped between the brachiocephalic trunk and the left common carotid artery and the left subclavian artery, and the inferior vena cava was cut 1 mm away from the right atrium. 20 ml of 4°C HTK cardioplegia solution was injected through the right common carotid artery of the rat, and the pressure was maintained at 60-80 mmHg, and crushed ice was placed in the chest cavity and on the surface of the heart. After the perfusion was completed, the aortic arch was cut off along the back of the third branch and the superior vena cava was cut off 1 mm away from the right atrium. The donor heart was lifted, the pulmonary artery and pulmonary vein were cut off, and the donor heart was removed. A 14G perfusion needle was inserted into the aorta of the donor heart, fixed with silk thread, and vented with 4°C modified Krebs-Henseleit solution.

[0087] (2) Prepare the perfusion solution

[0088] Another 300-400 g male Sprague-Dawley rat was selected and anesthesia was induced by inhalation of 3% isoflurane, and anesthesia was maintained by intraperitoneal injection of ketamine (75 mg / kg) and xylazine (5 mg / kg). The abdominal cavity was opened, the abdominal aorta was freed, the distal and proximal ends of the abdominal aorta were clamped respectively, the abdominal aorta was cut open near the distal aortic clamp, a 20G intravenous catheter was inserted, the proximal aortic clamp was loosened, and 10 ml of blood was drawn with a 20 ml syringe containing 0.2 ml (1250 U) heparin sodium injection. After mixing with 8 ml modified Krebs-Henseleit solution (10 mmol / L mmol / L glucose, 117 mmol / L NaCl, 4.5 mmol / L KCl, 25 mmol / L NaHCO3, 1.2 mmol / L NaH2PO4, 2 mmol / L CaCl2, 0.512 mmol / L MgCl2), methylprednisolone (500 mg / L), insulin (160 IU / L) and mannitol (25 g / L) were added to form the perfusion solution. Use perfusion solution to prefill the perfusion system, adjust the oxygen flow rate and perfusion flow rate to approximately 1:1 (perfusion flow rate is 1 ml / min / 100 g), and oxygenate for 15 min (PaO2 150-250 mmHg).

[0089] (3) In vitro preservation of rat DCD hearts

[0090] After the perfusion fluid was oxygenated for 15 minutes, the perfusion needle was connected to the perfusion tube and the perfusion began. The perfusion flow rate started at 2 ml / min and gradually increased to the target flow rate (1 ml / min / 100 g) within 10 minutes. After reaching the target flow rate, epinephrine (4.8 × 10 -5 mg / h / kg). During the perfusion, the isolated heart temperature was maintained at 35-37°C, the oxygen partial pressure of the perfusion fluid PaO2 was 150-250 mmHg, and 50wt.% glucose solution and 5wt.% sodium bicarbonate solution were supplemented according to the results of blood gas analysis to maintain the glucose concentration at 8-12mmol / L and the pH at 7.35-7.45. The perfusion period was divided into two stages: the stabilization period was 15 minutes after the start of perfusion, and the evaluation period (90 minutes) was 15 minutes later.

[0091] (4) Cardiac function assessment

[0092] After the stabilization period, the balloon catheter was placed from the left atrium through the mitral valve into the left ventricle, and the other end was connected to the pressure sensor and bioinformatics analyzer. At the beginning of the evaluation period (T0) and every 30 minutes thereafter (T30, T60, T90), 0.15 ml of normal saline was injected into the balloon to evaluate the DCD heart function. Heart function indicators included developed pressure (DP, the difference between systolic and diastolic pressure), heart rate (HR), maximum rate of rise of left ventricular pressure (dP / dt max ) and the maximum rate of decrease of left ventricular pressure (dP / dt min )

[0093] (5) Blood gas analysis

[0094] At T0, T30, T60 and T90, part of the perfusate was taken for blood gas analysis.

[0095] (6) Specimen collection

[0096] After the experiment, the DCD hearts were removed and serially sectioned along the long axis of the left ventricle (four left ventricular slices, 1-2 mm thick). The first apical slice was used for adenosine triphosphate (ATP) level determination, the second for superoxide dismutase (SOD) activity, reduced glutathione (GSH) and malondialdehyde (MDA) levels, the third for histological and immunohistochemical analysis, and the fourth for the assessment of myocardial infarction size.

[0097] (7) Molecular biology experiments

[0098] ① Tissue energy reserve

[0099] Myocardial tissue was weighed and diluted with boiled double distilled water at a ratio of 1:9 to prepare tissue homogenate, which was then centrifuged and the supernatant was collected. Tissue ATP levels were determined using the phosphomolybdic acid colorimetric method (A095-1-1, Nanjing Jiancheng). An automatic microplate reader (CLARIOstar, BMG, Germany) was used for analysis.

[0100] ② Oxidative stress

[0101] Myocardial tissue was weighed and diluted with physiological saline 1:9 to prepare tissue homogenate, which was then centrifuged and the supernatant was collected. The SOD activity and MDA and GSH contents in the culture supernatant were determined by 5,5'-dithiobis-(2-nitrobenzoic acid) colorimetry, thiobarbituric acid reaction method and water-soluble tetrazolium-1 method (A001-3-2, A003-1-2, A006-2-1, Nanjing Jiancheng), respectively. The analysis was performed using an automatic microplate reader. The expression of 4-hydroxynonenal (HNE) was determined by immunohistochemical analysis.

[0102] ③Inflammatory response

[0103] The expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was determined by immunohistochemical analysis.

[0104] ④Immunohistochemical analysis

[0105] Myocardial tissue sections were fixed with 4% paraformaldehyde solution, embedded in paraffin, and 4 μm thick sections were prepared. Immunoreactivity was evaluated for IL-6 (1:500, Abcam, ab9324, USA), TNF-α (1:200, Abcam, ab109322, USA), and HNE (1:1000, Abcam, ab46545, USA). Antigen-antibody reactions were observed by diaminobenzidine reaction and observed under an optical microscope. Four non-overlapping areas were randomly selected for analysis in each section. Images were analyzed using ImageJ software. The expression of IL-6, TNF-α, and HNE was measured using integrated optical density (IOD).

[0106] ⑤ Histological analysis

[0107] The paraffin blocks were additionally sliced ​​4 μm thick and stained with hematoxylin-eosin. Four non-overlapping areas were randomly selected for each section under light microscopy, and the samples were evaluated using pathological scores. The pathological scores included grades 0-4: grade 0: none, grade 1: minimal (focal myocardial cell damage), grade 2: mild (occasionally myocardial fiber disarray with multifocal myocardial fiber degeneration and inflammatory response), grade 3: moderate (diffuse inflammation and / or extensive myocardial fiber degeneration with wavy lesions and detached nuclei), and 4: severe (diffuse inflammatory process with myocardial necrosis: cell nuclei are shrunken and cells are severely damaged). In addition, cell edema was evaluated by calculating the average area of ​​each cell using ImageJ.

[0108] ⑥ Apoptosis

[0109] The above paraffin blocks were also used to prepare 4 μm thick sections for detecting cardiomyocyte apoptosis by terminal deoxyribonucleic acid transferase-mediated nick end labeling (TUNEL) technique.

[0110] ⑦ Assessment of myocardial infarction area

[0111] Ventricular sections were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 30 minutes to distinguish viable from non-viable myocardium. The percentage of infarct area was evaluated using ImageJ.

[0112] ⑧Statistical analysis

[0113] Results are presented as median and interquartile range. Kruskal-Wallis test and Dunn's multiple comparison test were used for comparison between groups. Cardiac function results were analyzed using Friedman test, and Dunn's multiple comparison test was used to compare values ​​at different time points. p < 0.05 was considered statistically significant. GraphPad Prism 8.3 software (GraphPadSofware, Inc., San Diego, CA, USA) was used for statistical analysis.

[0114] Experimental Results

[0115] After tracheal clamping induced asphyxia, the blood pressure of the donor rats dropped rapidly ( Figure 3 ). All DCD hearts recovered sinus rhythm within 5 minutes after the start of perfusion.

[0116] (1) Heart function

[0117] During the evaluation period, the HR of the DCD heart gradually increased in the first 30 minutes and then gradually decreased in the following 60 minutes, but the differences at each time point were not statistically significant ( Figure 3 B) In addition, the DP and dP / dt of the DCD heart max It started to increase rapidly from T0 and remained relatively stable from T30 to T90. The DP of the DCD heart at T60 and T90 was significantly different from that at T0 ( Figure 3 C) dP / dt of DCD heart at T60 max Significantly higher than T0( Figure 3 D) dP / dt of DCD heart min The values ​​at T30 and T60 were significantly lower than those at T0 ( Figure 3 E).

[0118] (2) Energy reserve

[0119] Three ATP values ​​below 0 were excluded (2 in the P90 group and 1 in the P30 group). No significant differences were found in the ATP content of cardiac tissues between the P30, P60, and P90 groups ( Figure 4 ).

[0120] (3) Oxidative stress

[0121] The GSH level in the P30 group was lower than that in the P60 and P90 groups, but the difference was not statistically significant. However, there was no statistically significant difference in the SOD activity, MDA and HNE levels of the DCD hearts among the P30, P60 and P90 groups ( Figure 4 ).

[0122] (4) Inflammatory response

[0123] There was no significant difference in IL-6 and TNF-α levels in the DCD hearts of the P30, P60, and P90 groups ( Figure 5 ).

[0124] (5) Histopathology

[0125] A small amount of focal myocardial cell damage, myocardial fiber disorder and hemorrhage were observed in the DCD hearts of the P30, P60 and P90 groups, but no obvious acute inflammation and myocardial necrosis were observed. The pathological score was 1 point (interquartile range, 1-1.25) in the P30 group, 2 points (interquartile range, 1-2) in the P60 group, and 1 point (interquartile range, 1-2) in the P90 group. In addition, there was no significant difference in the pathological score and the average area of ​​myocardial cells (an indicator of cell edema) among the three groups ( Figure 6 ).

[0126] (6) Apoptosis

[0127] The apoptosis of myocardial cells in the DCD hearts of the P30, P60 and P90 groups was not obvious, and there was no significant difference among the three groups ( Figure 7 ).

[0128] (7) Myocardial infarction

[0129] There was no obvious myocardial infarction in the DCD hearts of the P30, P60, and P90 groups, and there was no significant difference among the three groups ( Figure 8 ).

[0130] In summary, through the modified "DCD rat isolated heart normothermic blood mechanical perfusion model" we established, the DCD rat heart can be preserved in vitro for at least 90 minutes, and the DCD heart can be in a stable state during this period. The DCD hearts recovered sinus rhythm within 5 minutes after the start of perfusion. The function of the DCD heart gradually recovered within 30 minutes after the start of perfusion and remained at a stable level for the next 60 minutes. During the in vitro preservation, there were no significant changes in the oxidative stress, apoptosis, tissue energy status, histopathological changes, cell edema and inflammatory response of the DCD heart. In addition, the histopathological changes of the DCD heart were mild, with no signs of acute inflammation and necrosis, and there was no obvious myocardial infarction in the DCD heart during in vitro preservation.

[0131] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A perfusion solution suitable for rat donor hearts, characterized in that: It is composed of blood and modified Krebs-Henseleit solution in a volume ratio of 5:4; The modified Krebs-Henseleit solution contains 8-12 mmol / L glucose, 115-120 mmol / L NaCl, 4-5 mmol / L KCl, 23-27 mmol / L NaHCO3, 1-1.4 mmol / L NaH2PO4, 1.8-2.2 mmol / L CaCl2 and 0.5-0.52 mmol / L MgCl2 per liter of the modified Krebs-Henseleit solution, and the balance is water.

2. A perfusion solution suitable for rat donor hearts according to claim 1, characterized in that: The modified Krebs-Henseleit solution contains 10 mmol / L glucose, 117 mmol / L NaCl, 4.5 mmol / L KCl, 25 mmol / L NaHCO3, 1.2 mmol / L NaH2PO4, 2 mmol / L CaCl2, 0.512 mmol / L MgCl2 per liter of the modified Krebs-Henseleit solution, and the balance is water.

3. A perfusion solution suitable for rat donor heart according to claim 1, characterized in that: Also included was 25 g / L of mannitol.

4. A perfusion solution suitable for rat donor heart according to claim 1, characterized in that: Also included is 500 mg / L of methylprednisolone.

5. A perfusion solution suitable for rat donor heart according to claim 1, characterized in that: It also includes 160 IU / L of insulin.

6. A perfusion solution suitable for rat donor hearts according to claim 1, characterized in that: It comprises a controller, a blood reservoir, an oxygen cylinder, an oxygenator, a peristaltic pump, a water bath and a micro-infusion pump; the peristaltic pump comprises a silicone tube, a first extrusion device and a second extrusion device, the first extrusion device and the second extrusion device are respectively arranged beside the silicone tube, the oxygen cylinder is connected to the oxygenator for supplying oxygen to the oxygenator; the two ends of the oxygenator are respectively connected to the donor heart and one end of the silicone tube, the other end of the silicone tube is respectively connected to the blood reservoir and one end of the micro-infusion pump, the blood reservoir stores perfusion fluid, the blood reservoir is arranged in a water bath, the water bath is used to store hot water, the other end of the micro-infusion pump is connected to a glucose solution, a sodium bicarbonate solution and an adrenaline solution; the controller is respectively connected to the peristaltic pump and the micro-infusion pump, the controller is used to control the first extrusion device and the second extrusion device to squeeze and release the silicone tube in turn, the direction in which the first extrusion device squeezes the silicone tube is perpendicular to the direction in which the second extrusion device squeezes the silicone tube.

7. A perfusion solution suitable for rat donor hearts according to claim 6, characterized in that: A temperature control switch, a temperature measuring probe, a heater and a spiral stirrer are arranged in the water bath; the temperature control switch is connected with the temperature measuring probe, the heater and the controller respectively.

8. A perfusion solution suitable for rat donor hearts according to claim 6, characterized in that: The concentrations of the glucose solution and the sodium bicarbonate solution are 50wt.% and 5wt.% respectively; the injection amount of the adrenaline solution is 4.8×10-5mg / h / kg.

9. A perfusion solution suitable for rat donor hearts according to claim 6, characterized in that: It also includes a balloon catheter and a pressure sensor; the balloon catheter is connected to the pressure sensor for real-time evaluation of the donor's heart function.

10. A perfusion solution suitable for rat donor hearts according to claim 6, characterized in that: The device also includes a display connected to the pressure sensor.

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