Preparation method of physical thrombus for animal model with cerebrovascular diseases

Abstract

The invention relates to the technical field of medical treatment, and particularly discloses a preparation method of physical thrombus for a cerebrovascular disease animal model, which comprises the following steps: S1, aseptically collecting fresh whole blood of an animal, and quickly transferring the whole blood into a precooled syringe assembly within 60 seconds after collection; s2, a piston of the injector is driven, so that the whole blood repeatedly passes through at least two needle heads with different inner diameters in a preset pulse type variable-speed mode in a constant-temperature environment (37 + / -0.5 DEG C), the whole blood passes through a needle head with a larger aperture during first-time injection, and the whole blood passes through a needle head with a smaller aperture during last-time injection; according to the present invention, the use of the exogenous thrombin and other biochemical reagents is fundamentally avoided, the interference of the exogenous thrombin and other biochemical reagents on the thrombus natural formation process and the final component is eliminated, and the physical shearing force on the blood when the blood flows through the needles of different specifications is controlled so as to directly activate the endogenous blood coagulation system in situ, such that the platelet aggregation and the fibrin network formation can be promoted.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a method for preparing physical thrombi for animal models of cerebrovascular diseases. Background Technology

[0002] In the study of cerebrovascular diseases, especially ischemic stroke (cerebral infarction) and hemorrhagic stroke (cerebral hemorrhage), constructing animal models that highly simulate human pathophysiological processes is crucial. Embolization using autologous or allogeneic thrombi is a key step in constructing cerebral infarction models, while preparing blood clots with specific characteristics is also significant for research on cerebral hemorrhage models. Currently, commonly used thrombus preparation techniques mainly rely on biochemical methods. Typical practices include: 1) directly adding exogenous thrombin to collected blood to induce the rapid conversion of fibrinogen into fibrin, thereby forming a clot; 2) using the Chandler extracorporeal arteriovenous loop method, simulating blood flow through continuous rotation of blood in a circular glass tube to promote natural coagulation; 3) injecting blood into a specific mold (such as a polyethylene tube) and then inducing solidification through chemical or physical methods (such as heating). These methods constitute the current mainstream basis for thrombus preparation.

[0003] However, while the aforementioned existing technologies relying on exogenous biochemical reagents (such as thrombin) are rapid, the resulting thrombi differ significantly from spontaneously generated thrombi in composition, structure, and formation kinetics. This may introduce non-physiological variables, affecting the accuracy of experimental results and the evaluation of drug efficacy (such as thrombolytic drugs). Secondly, physicochemical hybrid methods are often cumbersome, time-consuming, and have specific equipment requirements. Even slight fluctuations in preparation conditions can lead to large batch-to-batch variations in thrombus size, density, and mechanical properties, resulting in poor reproducibility. Furthermore, existing methods typically cannot flexibly and controllably prepare thrombi with different physical properties (such as dense versus loose), failing to accurately match the core material requirements of two drastically different disease models: cerebral infarction (requiring dense and stable emboli) and cerebral hemorrhage (involving loose hematomas). Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing physical thrombi for animal models of cerebrovascular diseases, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for preparing physical thrombi for animal models of cerebrovascular diseases, comprising the following steps:

[0007] Fresh whole blood from an animal is collected aseptically and rapidly transferred to a pre-cooled syringe assembly within 60 seconds of collection.

[0008] S2. Drive the piston of the syringe so that the whole blood is repeatedly passed through at least two needles with different inner diameters in a constant temperature environment (37±0.5℃) and a preset pulse speed change mode, wherein the first injection is through the larger diameter needle and the last injection is through the smaller diameter needle.

[0009] S3. During the injection process in step S2, the syringe barrel containing whole blood is subjected to a periodic axial rotation with a rotation frequency of 1-5Hz and an angle of 90-180 degrees.

[0010] S4. The thrombus mixture formed in steps S2-S3 is introduced into a microfluidic sorting chip, and physical thrombi with predetermined size and shape are screened out using the laminar flow principle.

[0011] Preferably, in step S1, the whole blood is autologous blood, and all operations from the completion of blood collection to the start of step S2 are completed within 3 minutes.

[0012] Preferably, in step S2, the pulse speed change mode is as follows: the push-out stage uses two speeds, one fast and one slow, and the pull-back stage uses a constant slow speed; wherein the initial speed of the push-out stage is not less than 8 cm / s and the final speed is not more than 2 cm / s.

[0013] Preferably, the at least two needles with different inner diameters include a 21G needle (with an inner diameter of about 0.51 mm) and a 30G needle (with an inner diameter of about 0.16 mm), and the total number of whole blood injections is 8 to 12, of which the number of injections through the smaller diameter needle is not less than two-thirds of the total number of injections.

[0014] Preferably, in step S2, the repeated passage operation is performed in a closed loop, which includes the syringe, needle and a replaceable elastic silicone tube, and the whole blood circulates within the loop without being exposed to the external environment.

[0015] Preferably, in step S3, the periodic rotation and the piston's pushing and pulling motion are synchronized with a specific phase difference, that is, the rotation begins in the middle of the piston's push-out stroke and resets at the beginning of the pull-back stroke.

[0016] Preferably, in step S4, the microfluidic sorting chip has a "Y"-shaped bifurcation channel. By adjusting the flow rate ratio of the buffer solutions on both sides, the thrombus is separated according to its mechanical stiffness, and harder thrombi used to simulate cerebral infarction or softer thrombus clumps used to simulate cerebral hemorrhage are collected.

[0017] Preferably, before starting step S2, the syringe assembly is placed in a temperature control module that is heated at a constant rate from 4°C to 37°C for 2 minutes.

[0018] A physical thrombus prepared according to any one of the above methods, the thrombus having a heterogeneous internal structure, wherein the hematocrit of the core portion is higher than that of the periphery, and the surface is covered with an activated platelet layer.

[0019] The use of a physical thrombus in the preparation of an embolic stroke model or a cerebral vascular microembolism model.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] (1) It fundamentally avoids the use of biochemical reagents such as exogenous thrombin, eliminating their interference with the natural formation process and final components of thrombi. By controlling the physical shear force experienced by blood when it flows through needles of different specifications, the intrinsic coagulation system can be directly and in situ activated, promoting platelet aggregation and fibrin network formation. Therefore, the structure, composition and mechanical properties of the prepared thrombus are closer to those of thrombi generated in the spontaneous pathophysiological process, significantly improving the authenticity and reliability of the animal model constructed in simulating the pathological state of human cerebral infarction or cerebral hemorrhage.

[0022] (2) The whole process only requires conventional syringe equipment, and there is no need to prepare and store easily inactivated biochemical reagents, which greatly reduces the technical threshold and experimental cost. By systematically adjusting the physical parameters such as the inner diameter of the needle, the number of injections and the speed, thrombi with specific differences in density, strength and morphology can be prepared stably and repeatedly, so as to flexibly meet the needs of different research directions from dense arterial embolism to loose bleeding clots. Attached Figure Description

[0023] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1:

[0026] Please see Figure 1 As shown, a method for preparing physical thrombi for animal models of cerebrovascular disease includes the following steps:

[0027] S1. Pre-flush syringe A, silicone tubing, and 21G needle with heparin sodium saline, then completely empty the tubing to ensure the inner wall of the instrument is moist but without obvious liquid residue; install the 21G needle on syringe B for blood collection.

[0028] After anesthetizing rats via intraperitoneal injection, the femoral artery was exposed and isolated. Blood was then rapidly collected via puncture using syringe B, yielding approximately 0.8 mL of fresh autologous whole blood. This process must be rapid, with the time from vascular puncture to blood collection controlled within 30 seconds.

[0029] S2. Immediately remove the 21G needle from syringe B and quickly inject the collected whole blood into the pre-cooled (stored at 4°C) syringe A. Connect the syringes in the following order to form a closed loop: Syringe A → 30G needle → silicone tubing → 21G needle (this needle serves as the loop interface; the needle tip is inserted into the other end of the silicone tubing). Ensure that the total time from blood collection to loop assembly does not exceed 2 minutes.

[0030] S3. Fix the assembled syringe A into the programmed temperature-controlled rotary device, start the device, and make the syringe heat up from 4℃ to 37.0℃ at a constant rate within 2 minutes, and maintain this temperature. Set the piston drive program to use pulse speed change mode. The ejection process is divided into two stages: the first 0.2 seconds are for rapid ejection (speed about 10 cm / s), and the next 0.3 seconds are for slow ejection (speed about 2 cm / s). The backflow process is carried out at a constant and slow rate (about 1 cm / s).

[0031] The syringe barrel reciprocates 90 degrees axially at a frequency of 3 Hz. The rotation is synchronized with the piston movement, and is set to begin rotating when the piston is pushed out to the middle of its stroke and to reset when the suction action begins.

[0032] The automatic program was started, and whole blood was circulated and injected within the loop. The first two cycles used a 21G needle tip as the outflow channel (simulating a large orifice), and the next eight cycles switched to a 30G needle tip as the outflow channel (simulating a small orifice). A total of 10 injection-absorption cycles were completed, taking approximately 2 minutes and 15 seconds in total.

[0033] S4. After the procedure is complete, disassemble the loop and gently inject all the contents of syringe A (including formed thrombus and residual blood) into 1 mL of normal saline. Mix slightly, and use a pipette to add the mixture to the inlet of the microfluidic sorting chip. Simultaneously, use two syringe pumps to inject normal saline as a buffer into the inlets of the two branch channels of the chip, adjusting the flow rate so that the flow rate ratio of the main channel to the branch channel collecting hard thrombus is 5:1. Under a microscope, it can be seen that harder, denser thrombus clumps tend to flow into the slower-flowing side channel. Collect the effluent from this channel.

[0034] The collected solution was filtered through a 200μm sieve, and the trapped thrombus was gently rinsed with physiological saline to obtain the target physical thrombus.

[0035] As can be seen from the above, the prepared thrombus is dark red, irregular in shape, and about 1.5-3.0 mm in length. Under an optical microscope, the thrombus structure is dense and does not easily disperse when gently squeezed, which meets the characteristics of dense thrombus required for the cerebral infarction model. When the thrombus is used in the rat middle cerebral artery embolism model, stable vascular occlusion can be achieved, and the cerebral ischemia model can be successfully constructed.

[0036] This embodiment successfully prepared a biomimetic, highly homogeneous, dense physical thrombus without using any anticoagulants or chemical inducers by controlling the time from blood collection to processing, employing physical stimulation that combines pulse speed variation with mechanical rotation, and finally using microfluidic sorting based on mechanical properties. The thrombus exhibits good repeatability and is suitable for detailed research on the mechanism of cerebral infarction.

[0037] Example 2

[0038] Please see Figure 1 As shown, a method for preparing physical thrombi for animal models of cerebrovascular disease includes the following steps:

[0039] S1. Anesthetize a New Zealand white rabbit and collect 5.0 ml of fresh autologous whole blood via puncture of the central auricular artery under sterile conditions.

[0040] S2. After blood collection, immediately (within 45 seconds) transfer the blood sample to a pre-cooled 2.5 ml syringe connected to a closed flexible silicone tubing loop via a three-way valve. The loop is initially fitted with an 18G needle (approximately 0.84 mm in inner diameter).

[0041] S3. Place the entire device on the programmed temperature control module and uniformly raise the temperature from 4°C to 37°C within 90 seconds and maintain it. Then begin the thrombectomy procedure: drive the piston to expel blood in a pulse pattern, initially at a fast speed followed by a slower one, with an initial velocity of approximately 10 cm / s, decreasing to approximately 3 cm / s before the needle exit; then slowly aspirate at a uniform speed of approximately 1.5 cm / s. Simultaneously, a mechanical clamp drives the syringe barrel to perform a periodic axial rotation of 150 degrees at a frequency of 2 Hz, with the rotation synchronized with the piston's pushing and pulling motion (rotation during the ejection phase, resetting before aspiration).

[0042] After completing two cycles, quickly replace the needle at the end of the loop with a 23G needle (approximately 0.33mm inner diameter) via the three-way valve. Continue the injection cycle at a constant temperature of 37°C, adjusting the injection speed to 5-7 cm / s and the backflow speed to 1.5 cm / s, for a total of 6 cycles, with continuous syringe rotation. The entire mechanical molding process is completed in approximately 4 minutes.

[0043] S4. Inject all contents of the silicone tube loop into the microfluidic sorting chip. By adjusting the flow rate of the buffer solution on both sides of the chip's "Y"-shaped channel, a specific laminar flow field is created, separating the formed thrombus mixture according to its mechanical properties. The main thrombus clumps collected from the chip's other main outlet are softer, looser-structured, dark red thrombus clumps, with a size range of 500-1000 micrometers.

[0044] As can be seen from the above, the fibrin network inside the mass is relatively sparse and the red blood cell aggregation is obvious, which is consistent with the characteristics of hematoma after rupture of a blood vessel. This thrombus mass can be directly used to construct a rabbit lobar hemorrhage model, or after slight homogenization, it can be used to prepare a microembolism model to study secondary damage and treatment strategies after cerebral hemorrhage.

[0045] Example 3:

[0046] Please see Figure 1 As shown, a method for preparing physical thrombi for animal models of cerebrovascular disease includes the following steps:

[0047] S1. Anesthetize a C57BL / 6 mouse and collect 0.6 ml of fresh autologous whole blood via cardiac puncture;

[0048] S2. After blood collection, within 30 seconds, rapidly inject the blood into a 0.5 ml insulin syringe pre-placed in a 4°C ice box. This syringe is connected to a 5 cm long, 0.3 mm inner diameter microsilicone tube, with a 25G needle (approximately 0.26 mm inner diameter) attached to the end of the tube. Transfer the entire device to the temperature control unit, which linearly raises the temperature from 4°C to 37°C within 1.5 minutes and maintains a stable temperature.

[0049] S3. Initiate the thrombectomy procedure: A precise electronic plunger drives the piston, employing a progressively accelerating ejection mode, linearly increasing the speed from 3 cm / s to 6 cm / s, followed by a constant low-speed aspiration of 0.8 cm / s, constituting one cycle. During this process, the syringe, driven by a servo motor, performs a precise 90-degree axial oscillation at a frequency of 4 Hz. This oscillation is time-coupled with the piston movement and occurs during the latter half of each ejection. After completing 4 cycles, the needle at the connecting tube end is quickly replaced with a 31G needle (approximately 0.13 mm inner diameter) within a sterile operating chamber. Subsequent cycles are continued at a constant temperature of 37°C, with the ejection speed mode adjusted to a fluctuating pattern (periodically varying between 4-5 cm / s), and the aspiration speed maintained at 0.8 cm / s. A total of 10 cycles are performed, with continuous synchronous rotation. The entire mechanical thrombectomy operation is completed within 2 minutes and 15 seconds.

[0050] S4. The mixed products within the silicone tube are introduced into a custom-designed high-throughput microfluidic sorting chip. This chip features multi-level branched channels, which, through fluid control, efficiently separate and enrich thrombi according to size and rigidity. The final collected target product consists of a large number of uniformly sized (mainly distributed in the 50-150 micrometer range) microthrombi with a texture between dense and porous.

[0051] As can be seen from the above, these microthrombi exhibit a significant gradient distribution of cellular components and have an activated platelet-fibrin complex surface. This batch of microthrombus suspension is suitable for establishing a mouse cerebral microcirculation embolism model. It can be perfused via the internal carotid artery to simulate common clinical sporadic microembolic events and can be used to evaluate the efficacy of antiplatelet drugs or study the pathophysiological changes of the ischemic penumbra.

[0052] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing physical thrombi for animal models of cerebrovascular diseases, characterized in that, Includes the following steps: S1. Aseptically collect fresh whole blood from the animal and rapidly transfer the whole blood into a pre-cooled syringe assembly within 60 seconds of collection; S2. Drive the piston of the syringe so that the whole blood is repeatedly passed through at least two needles with different inner diameters in a constant temperature environment (37±0.5℃) and a preset pulse speed change mode, wherein the first injection is through the larger diameter needle and the last injection is through the smaller diameter needle. S3. During the injection process in step S2, the syringe barrel containing whole blood is subjected to a periodic axial rotation with a rotation frequency of 1-5Hz and an angle of 90-180 degrees. S4. The thrombus mixture formed in steps S2-S3 is introduced into a microfluidic sorting chip, and physical thrombi with predetermined size and shape are screened out using the laminar flow principle.

2. The method for preparing a physical thrombus for an animal model of cerebrovascular disease according to claim 1, characterized in that: In step S1, the whole blood is autologous blood, and all operations from the completion of blood collection to the start of step S2 are completed within 3 minutes.

3. The method for preparing a physical thrombus for an animal model of cerebrovascular disease according to claim 1, characterized in that: In step S2, the pulse speed change mode is as follows: the push-out stage uses two speeds, one fast and one slow, and the pull-back stage uses a constant slow speed; wherein the initial speed of the push-out stage is not less than 8 cm / s and the final speed is not more than 2 cm / s.

4. The method for preparing a physical thrombus for an animal model of cerebrovascular disease according to claim 1, characterized in that: The at least two different inner diameter needles include 21G needles (inner diameter of about 0.51 mm) and 30G needles (inner diameter of about 0.16 mm), and the total number of whole blood injections is 8 to 12, of which at least two-thirds of the injections are through the smaller diameter needle.

5. The method for preparing a physical thrombus for an animal model of cerebrovascular disease according to claim 1, characterized in that: In step S2, the repeated passage operation is performed in a closed loop, which includes the syringe, needle and a replaceable elastic silicone tube, and the whole blood circulates within the loop without being exposed to the external environment.

6. The method for preparing a physical thrombus for an animal model of cerebrovascular disease according to claim 1, characterized in that: In step S3, the periodic rotation and the piston's pushing and pulling motion are synchronized with a specific phase difference, that is, the rotation begins in the middle of the piston's push-out stroke and resets at the beginning of the pull-back stroke.

7. The method for preparing a physical thrombus for an animal model of cerebrovascular disease according to claim 1, characterized in that: In step S4, the microfluidic sorting chip has a "Y"-shaped bifurcation channel. By adjusting the flow rate ratio of the buffer solutions on both sides, the thrombus is separated according to its mechanical stiffness, and harder thrombi used to simulate cerebral infarction or softer thrombus clumps used to simulate cerebral hemorrhage are collected.

8. The method for preparing a physical thrombus for an animal model of cerebrovascular disease according to claim 1, characterized in that: Before starting step S2, the syringe assembly is placed in a temperature control module that is heated at a constant rate from 4°C to 37°C for 2 minutes.

9. A physical thrombus prepared by the method according to any one of claims 1-8, characterized in that, The thrombus has a heterogeneous internal structure, with a higher hematocrit in the core and a surface covered with an activated platelet layer.

10. The use of a physical thrombus according to claim 9 in the preparation of an embolic stroke model or a cerebral vascular microembolism model.

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