Method Article

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

DOI:

10.3791/64531

⸱

September 13th, 2022

 ,  ,  ,  ,  , 
1School of Pharmacy, Chengdu University of Traditional Chinese Medicine, 2Research Institute of Integrated TCM & Western Medicine, Chengdu University of Traditional Chinese Medicine, 3School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine, 4Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine

In This Article

Summary

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The present protocol describes a simple and efficient method for the real-time and dynamic collection of rat heart blood using the microdialysis technique.

Abstract

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Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial infarction, arrhythmia, atherosclerosis, cardiogenic pulmonary edema, pulmonary embolism, and cerebral embolism. At the same time, it is urgent to break through the continuous heart blood sampling technique in live rats to evaluate the effectiveness of distinctive ethnic medicine therapy. In this study, a blood microdialysis probe was implanted in the right jugular vein of rats in a precise and noninvasive surgical procedure. Cardiac blood samples were then collected at a rate of 2.87 nL/min to 2.98 mL/min by connecting to an online microdialysis sample collection system. Even more momentously, the acquired blood samples can temporarily be stored in microdialysis containers at 4 °C. The microdialysis-based online continuous blood collection program from rat heart has greatly guaranteed the quality of blood samples, advancing and invigorating the scientific rationality of the research on systemic cardiovascular diseases and evaluating ethnomedicine therapy from the perspective of hematology.

Introduction

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With the acceleration of the pace of life and the increase of psychological pressure, cardiovascular diseases (CVDs) tend to occur in young, middle-aged, and elderly people1,2. The morbidity and mortality of CVDs are high, with the characteristics of acute onset, rapid progression, and a long course of the disease, which seriously affect the safety of patients3. The occurrence of CVDs may be closely related to the changes in some blood components, such as cholesterol, serum lipids, blood glucose, myocardial enzymes, and protein kinase K4,5,6. The patient's relevant situation can be managed most quickly by analyzing routine blood examination items. Hence, the quality of the blood samples determines the accuracy of the test results. However, conventional methods for blood collection have some inevitable drawbacks, which seriously affect the experimental results, such as large trauma area, small blood collection volume, high requirements for operators, inability to reflect drug changes in real-time, cumbersome blood sample pretreatment, large consumption of experimental animals, and failure to meet animal ethical requirements7,8,9. With continuous advances in medical technology, the quality of blood collection has also put forward higher requirements. Therefore, it is urgent to develop a new blood sampling technology to overcome the above shortcomings.

Microdialysis is an in vivo sampling technique based on dialysis principles10. Under non-equilibrium conditions, the compounds to be measured are diffused and perfused from the tissue along the concentration gradient into the microdialysis probe embedded in the tissue into the dialysate, which is continuously removed along with the dialysate, achieving the purpose of sampling from the living tissue11,12. Compared with traditional sampling methods, the microdialysis technique has splendid advantages in the following aspects13,14,15: continuous real-time tracking of the changes of various compounds in blood; sampling requires no tedious pre-processing and can truly represent the concentration of the target compound at the sampling site; probes can be implanted into different parts of the body to investigate the absorb, distribution, metabolism, excretion and toxicity of the target compounds; the acquired sample contains no biological macromolecules (>20 kD). Therefore, the higher quality blood samples ensure a better interpretation of CVDs and the mechanism treated by ethnic medicine.

Microdialysis sampling systems generally consist of micro-injection pumps, connecting tubes, animal-free movement tanks, microdialysis probes, and sample collectors16. As the most critical part of the device of the microdialysis system, common microdialysis probes comprise concentric probes, flexible probes, linear probes, and shunt probe17. Among these, flexible probes are soft and non-metallic probes, mainly used to collect samples from blood vessels and peripheral tissues such as heart, muscle, skin, and fat of awake and freely moving or anesthetized animals13. When in contact with blood vessels or tissues, the probe can be flexibly bent, thereby avoiding irreversible damage to the probe or sampling site. With the continuous development of probe technology, the application of microdialysis technology in various fields is also deepening. In this paper, the rat heart blood was dynamically and continuously acquired by the noninvasive microdialysis technology through the flexible probe designed for blood collection.

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Protocol

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The animal protocol has been approved by the Administrative Committee of Chengdu University of Traditional Chinese Medicine (Record number: 2021-11). Specified pathogen-free male Sprague Dawley (SD) rats (8-10 weeks, 260-300 g) were raised in independent ventilation cages, maintaining the laboratory environment at 22 °C and 65% relative humidity, and were used for the present study. The animals were obtained from a commercial source (see Table of Materials). All rats were habituated to adaptive feeding for 1 week with free water and diet during the period.

1. Experimental preparation

  1. Assemble the equipment involved in microdialysis of blood sampling (see Table of Materials), as shown in Figure 1.
  2. Prepare anti-coagulant citrate dextrose solution (ACDs), containing 3.50 mmol/L of citrate, 7.50 mmol/L of sodium citrate, and 13.60 mmol/L of glucose, as the perfusion fluid for microdialysis blood collection (see Table of Materials).
  3. Before use, filter the ACDs with a 0.22 µm membrane filtration unit and remove bubbles ultrasonically. Keep ACDs at 37 °C to reduce the stimulation for SD rats.

2. Patency inspection of microdialysis piping system

  1. Attach the inlet of the probe of the dialysis unit with the syringe needle, the tubing adapter, and the fep tubing (see Table of Materials).
    NOTE: The blue end of the microdialysis probe is for the fluid influx, and the transparent end is for the fluid outflow.
  2. Check the patency of the microdialysis piping system by perfusing ACDs18 into the piping system at a speed of 2 µL/min.
    ​NOTE: If ACDs flow from the sample collection site, the unimpeded microdialysis system can be used for further cardiac blood collection. If not, the blood probe must be checked for broken leaks or whether the pipe-pipe joint was sealed.

3. Microdialysis probe implantation

  1. Anesthetize the rats with 2% isoflurane in an air-oxygen mixture of 0.6 L/min, fasten the thoroughly unconscious rats on the operating table, and maintain the body temperature at 37 °C using an animal temperature maintainer (see Table of Materials).
  2. Remove the fur from the neck with an electric shaver and disinfect the surgical site with three alternating rounds of betadine and 70% alcohol. Inject bupivacaine (1.5 mg/kg) into the rat. Expose the right jugular vein by blunt dissection of soft tissue and perivascular fascia through a 1.5 cm incision along the midline of the neck.
    NOTE: All surgical instruments and tools used in the experiment should be sterilized in advance by autoclaving. Use sterile gloves and surgical drapes. The whole experimental operation should be carried out in a sterile environment. Follow local animal use guidelines for anesthesia and analgesia regimen.
  3. Make a detachable slipknot using a 4-0 surgical suture in the right jugular vein at the distal end of the heart to temporarily block blood flow, and make a 1.5 cm incision in the right jugular vein near the heart.
  4. Insert a needle-shaped catheter stylet (length 25 mm, diameter 0.7 mm, see Table of Materials) into the right jugular vein toward the proximal end of the rat heart, insert the blood microdialysis probe (total probe length 24 mm, membrane length 10 mm) in a catheter, and implant the probe with ophthalmic forceps along the oblique incision of the catheter stylet19.
  5. Remove the guided catheter stylet and fully immerse the semi-permeable membrane of the probe (membrane length 10 mm, membrane diameter 0.5 mm) in the right jugular vein. Unravel the detachable slipknot at the distal end of the heart to restore blood flow in the right jugular vein.
  6. Ligature the probe with the right jugular vein using 4-0 surgical sutures and subcutaneously thread the probe tail tube through the back of the neck. See Figure 2 for specific probe implantation steps.
    ​NOTE: The molecular cut-off by the blood microdialysis probe used in this study is >20 kD. The collected blood samples contain substances with a molecular weight of less than 20 kD.

4. Microdialysis sampling

  1. A week after the microdialysis probe implantation, rats recovering from surgical trauma undergo microdialysis sampling. Place the awake rat in a free-moving tank (see Table of Materials) and connect the probe to the microdialysis system. Equilibrate the probe dialysis membrane by irrigating ACDs with a rate of 2 µL/min for 1 h.
  2. Collect microdialysis blood samples at a flow rate of 2 µL/min and temporarily keep them in a 4 °C fractional container.
    ​NOTE: The obtained blood samples could be tested directly by centrifuging at 20,000 x g for 10 min at room temperature. Or it could be stored at -80 °C for the following testing. When collecting samples, it is important always to observe whether the probe prolapse and/or leak out.

5. Post-sampling operation

  1. Anesthetize the SD rats with 2% isoflurane in an air-oxygen mixture at 0.6 L/min.
  2. Dissect to re-confirm that the probe is in the right jugular vein. Remove the microdialysis probe from the right jugular vein and put it into ultrapure water.
  3. Finally, euthanize the rats by inhalation of 5% isoflurane.
  4. Connect the probe to the pipeline and rinse overnight at a rate of 2 µL/min with ultrapure water to completely wash out the residual salt in the pipe and probe.
  5. Remove the probe and soak it in ultrapure water. Store at 4 °C to prevent the probe dialysis membrane from contracting.
    NOTE: If the volume of dialysis fluid is inconsistent with the volume of perfusion fluid, the probe may be blocked by coagulated blood. The probe can be placed in the pancreatic protein solution until the visible substance flakes off from the tip of the probe membrane.

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Results

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The present protocol allowed obtaining the cardiac blood from conscious rats according to sampling parameters set in the microdialysis equipment. Normal blood samples must be bright red, while animals with hypoxia, potential blood clots, or anemic disease may have dark purple or dark red. Samples obtained through the blood microdialysis technique are colorless, clear, and transparent, which can be used to analyze the serum markers of different diseases and the blood distribution of drugs and their metabolites by employin...

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Discussion

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CVDs are a common chronic disease in clinics with gradually increasing incidence in China, and the onset age tends to be younger, causing the concern and panic of most patients20,21. Being the leading cause of death in the world, CVDs can induce cerebral infarction and other high mortality diseases, seriously threatening the healthy life of patients22. CVDs, including ischemic heart disease, cardiomyopathy, atherosclerosis, high blood pres...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This work was supported by the National Natural Science Foundation of China (82104533), the China Postdoctoral Science Foundation (2020M683273), the Science & Technology Department of Sichuan province (2021YJ0175) and the Key R&D project of Sichuan Provincial Science and Technology Plan (2022YFS0438). Meanwhile, the authors would like to thank Mr. Yuncheng Hong, a senior equipment engineer at TRI-ANGELS D&H TRADING PTE. LTD. (Singapore city, Singapore), for providing technical services for microdialysis techniques.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Animal anesthesia systemRayward Life Technology Co., LtdR500IE
Animal temperature maintainerRayward Life Technology Co., Ltd69020
Blood microdialysis probe CMA Microdialysis ABT55347
Catheter CMA Microdialysis ABT55347
CitrateMerck Chemical Technology (Shanghai) Co., Ltd251275
Electric shaverRayward Life Technology Co., LtdCP-5200
Fep tubing CMA Microdialysis AB3409501
Free movement tank for animals CMA Microdialysis ABCMA120
GlucoseMerck Chemical Technology (Shanghai) Co., LtdG8270
Hemostatic forcepsRayward Life Technology Co., LtdF21020-16
IsofluranRayward Life Technology Co., LtdR510-22
Micro scissorsBeyotime Biotechnology Co., LtdFS221
Microdialysis collection tube CMA Microdialysis AB7431100
Microdialysis collector CMA Microdialysis ABCMA4004
Microdialysis in vitro stand CMA Microdialysis ABCMA130
Microdialysis microinjection pump CMA Microdialysis AB788130
Microdialysis syringe (1.0 mL) CMA Microdialysis AB8309020
Microdialysis tubing adapter CMA Microdialysis AB3409500
Microporous filter membraneMerck Millipore Ltd.R0DB36622
Non-absorbable surgical suturesShanghai Tianqing Biological Materials Co., LtdS19004
Operating tableYuyan Scientific Instrument Co., Ltd30153
Ophthalmic forcepsRayward Life Technology Co., LtdF12016-15
Sodium citrateMerck Chemical Technology (Shanghai) Co., Ltd1613859
Sprague Dawley  (SD) ratsChengdu Dossy Experimental Animals Co., LtdSYXK(figure-materials-1)2019-049
Surgical scissorsRayward Life Technology Co., LtdS14014-15
Surgical scissorsShanghai Bingyu Fluid technology Co., LtdBY-103
Syringe needle CMA Microdialysis ABT55347
Ultrasonic cleanerGuangdong Goote Ultrasonic Co., LtdKMH1-240W8101

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Tags

Blood MicrodialysisContinuous Blood SamplingRat HeartCardiovascular DiseaseJugular Vein CannulationNoninvasive TechniqueBlood BiomarkersMicrodialysis ProbeHematology AnalysisDisease Diagnosis

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