This work was supported by the National Natural Science Foundation of China (82104533), the China Postdoctoral Science Foundation (2020M683273), the Science &#38; Technology Department of Sichuan province (2021YJ0175) and the Key R&#38;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&#38;H TRADING PTE. LTD. (Singapore city, Singapore), for providing technical services for microdialysis techniques.

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 &#176;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 feed...

<ol>
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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...

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&#39;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 (&#62;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal anesthesia system</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>R500IE</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal temperature maintainer</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>69020</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood microdialysis probe</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>T55347</td>
			<td></td>
		</tr>
		<tr>
			<td>Catheter</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>T55347</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate</td>
			<td>Merck Chemical Technology (Shanghai) Co., Ltd</td>
			<td>251275</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>CP-5200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fep tubing</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>3409501</td>
			<td></td>
		</tr>
		<tr>
			<td>Free movement tank for animals</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>CMA120</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck Chemical Technology (Shanghai) Co., Ltd</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic forceps</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>F21020-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluran</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>R510-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>Beyotime Biotechnology Co., Ltd</td>
			<td>FS221</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis collection tube</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>7431100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis collector</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>CMA4004</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis in vitro stand</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>CMA130</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis microinjection pump</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>788130</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis syringe (1.0 mL)</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>8309020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis tubing adapter</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>3409500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microporous filter membrane</td>
			<td>Merck Millipore Ltd.</td>
			<td>R0DB36622</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-absorbable surgical sutures</td>
			<td>Shanghai Tianqing Biological Materials Co., Ltd</td>
			<td>S19004</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating table</td>
			<td>Yuyan Scientific Instrument Co., Ltd</td>
			<td>30153</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic forceps</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>F12016-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Merck Chemical Technology (Shanghai) Co., Ltd</td>
			<td>1613859</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley&#160; (SD) rats</td>
			<td>Chengdu Dossy Experimental Animals Co., Ltd</td>
			<td>SYXK(<img alt="Equation 1" src="/files/ftp_upload/64531/64531eq01.jpg" style="margin: auto;" />)2019-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>S14014-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Shanghai Bingyu Fluid technology Co., Ltd</td>
			<td>BY-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe needle</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>T55347</td>
			<td></td>
		</tr>
		<tr>
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dynamic continuous blood extraction from rat heart via noninvasive microdialysis technique

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 &#176;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.

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...

Watch this Scientific Journal Video about Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique at JoVE.com

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

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

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial ...

Our dynamic continuous blood collection technique from a rat heart is very important for analyzing the blood components, contributing to understanding cardiovascular disease and their related disease. Compared with traditional sampling methods, this technique has the advantages of in vivo, real-time, dynamic, and is non-invasive, with no biological micro moleculars and titre sample pre-process for a foreign test. This technique is not just applicable to cardiovascular diseases, but also should work for disease diagnosis and treatment by detecting blood biomarkers combined with high phase liquid chromatography and mass spectrometry.This method can be used to investigate cardiovascular diseases such as myocardial infarction, apnea and atherosclerosis. In addition, it also applies to cardiogenic pulmonary edema and pulmonary and cerebral embolism. After doing the experimental preparation, attach the inlet of the dialysis unit probe with the syringe needle, the tubing adapter, and the FEP tubing.Check the patency of the microdialysis piping system by perfusing anticoagulant citrate dextrose solution into the piping system. Make an incision to expose the right jugular vein by blunt dissection of soft tissue and perivascular fascia. Using a 4-0 surgical suture, make a detachable slip knot in the right jugular vein at the distal end of the heart to block the blood flow temporarily.Then make an incision in the right jugular vein near the heart. Insert a needle-shaped catheter stylet into the right jugular vein toward the proximal end of the rat heart. Insert the blood microdialysis probe in a catheter and implant the probe with ophthalmic forceps along the oblique incision of the catheter stylet.Remove the guided catheter stylet and fully immerse the semipermeable membrane of the probe in the right jugular vein. Unravel the detachable slip knot at the distal end of the heart to restore blood flow in the right jugular vein. Then use 4-0 surgical sutures to ligate the probe with the right jugular vein.Place the awake rat in a free-moving tank and connect the probe to the microdialysis system. Then irrigate the anticoagulant citrate dextrose solution at a rate of two microliters per minute for one hour to equilibrate the probe dialysis membrane. Collect microdialysis blood samples and temporarily keep them in a four degree Celsius fractional container.To reconfirm that the probe is in the right jugular vein, dissect the rat. Remove the microdialysis probe from the right jugular vein and put it into ultrapure water. Connect the probe to the pipeline and rinse overnight with ultrapure water to thoroughly wash out the residual salt in the pipe and probe.The normal blood samples appeared bright red while potential blood clots were observed in hypoxic samples. Samples obtained through the blood microdialysis technique were colorless, clear and transparent. To avoid the membrane's destruction and the probe of sliding or displacement, we should pay attention when we are placing and immersing the probe in the rat jugular vein.The blood samples obtained through the microdialysis technique can be used to analyze the biomarkers of different diseases and the blood distribution of drugs and rare metabolites.

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Research

JoVE Journal

Medicine

2.1K visualizações.  Chengdu University of Traditional Chinese Medicine. Nossa técnica dinâmica de coleta contínua de sangue de um coração de rato é muito importante para analisar os componentes sanguíneos, contribuindo para a compreensão da doença cardiovascular e sua doença relacionada. Em comparação com os métodos tradicionais de amostragem, esta técnica tem as vantagens de in vivo, em tempo real, dinâmica e não invasiva, sem micromoleculares biológicos e pré-processo de titulação de amostra para um teste estranho. Esta técnica não é apen...