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 feeding for 1 week with free water and diet during the period.
1. Experimental preparation
<ol>
	<li>Assemble the equipment involved in microdialysis of blood sampling (see Table of Materials), as shown in Figure 1.</li>
	<li>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).</li>
	<li>Before use, filter the ACDs with a 0.22 &#181;m membrane filtration unit and remove bubbles ultrasonically. Keep ACDs at 37 &#176;C to reduce the stimulation for SD rats.</li>
</ol>
2. Patency inspection of microdialysis piping system
<ol>
	<li>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.</li>
	<li>Check the patency of the microdialysis piping system by perfusing ACDs18 into the piping system at a speed of 2 &#181;L/min. 
	&#8203;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.</li>
</ol>
3. Microdialysis probe implantation
<ol>
	<li>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 &#176;C using an animal temperature maintainer (see Table of Materials).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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. 
	&#8203;NOTE: The molecular cut-off by the blood microdialysis probe used in this study is &#62;20 kD. The collected blood samples contain substances with a molecular weight of less than 20 kD.</li>
</ol>
4. Microdialysis sampling
<ol>
	<li>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 &#181;L/min for 1 h.</li>
	<li>Collect microdialysis blood samples at a flow rate of 2 &#181;L/min and temporarily keep them in a 4 &#176;C fractional container. 
	&#8203;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 &#176;C for the following testing. When collecting samples, it is important always to observe whether the probe prolapse and/or leak out.</li>
</ol>
5. Post-sampling operation
<ol>
	<li>Anesthetize the SD rats with 2% isoflurane in an air-oxygen mixture at 0.6 L/min.</li>
	<li>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.</li>
	<li>Finally, euthanize the rats by inhalation of 5% isoflurane.</li>
	<li>Connect the probe to the pipeline and rinse overnight at a rate of 2 &#181;L/min with ultrapure water to completely wash out the residual salt in the pipe and probe.</li>
	<li>Remove the probe and soak it in ultrapure water. Store at 4 &#176;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.</li>
</ol>

<ol>
	<li>van Rensburg, W. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-mortem+evidence+of+a+diverse+distribution+pattern+of+atherosclerosis+in+the+South+African+population.">Post-mortem evidence of a diverse distribution pattern of atherosclerosis in the South African population.</a> Scientific Reports. 12 (1), 11366 (2022).</li><li>Katz, A. J., Chen, R. C., Usinger, D. S., Danus, S. M., Zullig, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+disease+prevention+and+management+of+pre-existent+cardiovascular+disease+in+a+cohort+of+prostate+cancer+survivors.">Cardiovascular disease prevention and management of pre-existent cardiovascular disease in a cohort of prostate cancer survivors.</a> Journal of Cancer Survivorship. , (2022).</li><li>R&#248;devand, L., Tesli, M., Andreassen, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+disease+risk+in+people+with+severe+mental+disorders:+an+update+and+call+for+action.">Cardiovascular disease risk in people with severe mental disorders: an update and call for action.</a> Current Opinion in Psychiatry. 35 (4), 277-284 (2022).</li><li>Izumi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+circulating+cathepsin+K+on+the+coronary+calcification+and+the+clinical+outcome+in+chronic+kidney+disease+patients.">Impact of circulating cathepsin K on the coronary calcification and the clinical outcome in chronic kidney disease patients.</a> Heart and Vessels. 31 (1), 6-14 (2016).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whey+protein+hydrolysate+alleviated+atherosclerosis+and+hepatic+steatosis+by+regulating+lipid+metabolism+in+apoE-/-+mice+fed+a+Western+diet.">Whey protein hydrolysate alleviated atherosclerosis and hepatic steatosis by regulating lipid metabolism in apoE-/- mice fed a Western diet.</a> Food Research International. 157, 111419 (2022).</li><li>Angelone, T., Rocca, C., Pasqua, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nesfatin-1+in+cardiovascular+orchestration:+From+bench+to+bedside.">Nesfatin-1 in cardiovascular orchestration: From bench to bedside.</a> Pharmacological Research. 156, 104766 (2020).</li><li>Bernardi, P. M., Barreto, F., Dalla Costa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+LC-MS/MS+method+for+evaluating+lung+penetration+of+tobramycin+in+rats+by+microdialysis.">Application of a LC-MS/MS method for evaluating lung penetration of tobramycin in rats by microdialysis.</a> Journal of Pharmaceutical and Biomedical Analysis. 134, 340-345 (2017).</li><li>Anderzhanova, E., Wotjak, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+microdialysis+and+its+applications+in+experimental+neurochemistry.">Brain microdialysis and its applications in experimental neurochemistry.</a> Cell and Tissue Research. 354 (1), 27-39 (2013).</li><li>Joukhadar, C., M&#252;ller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis:+current+applications+in+clinical+pharmacokinetic+studies+and+its+potential+role+in+the+future.">Microdialysis: current applications in clinical pharmacokinetic studies and its potential role in the future.</a> Clinical Pharmacokinetics. 44 (9), 895-913 (2005).</li><li>Stangler, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+and+microperfusion+electrodes+in+neurologic+disease+monitoring.">Microdialysis and microperfusion electrodes in neurologic disease monitoring.</a> Fluids and Barriers of the CNS. 18 (1), 52 (2021).</li><li>Young, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+microdialysis.">Cerebral microdialysis.</a> Critical Care Nursing Clinics of North America. 28 (1), 109-124 (2016).</li><li>O'Connell, M. T., Krejci, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+techniques+and+microdialysis-based+patient-near+diagnostics.">Microdialysis techniques and microdialysis-based patient-near diagnostics.</a> Analytical and Bioanalytical Chemistry. 414 (10), 3165-3175 (2022).</li><li>Hammarlund-Udenaes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+as+an+important+technique+in+systems+pharmacology-a+historical+and+methodological+review.">Microdialysis as an important technique in systems pharmacology-a historical and methodological review.</a> The AAPS Journal. 19 (5), 1294-1303 (2017).</li><li>Stahl, M., Bouw, R., Jackson, A., Pay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+microdialysis.">Human microdialysis.</a> Current Pharmaceutical Biotechnology. 3 (2), 165-178 (2002).</li><li>Pierce, C. F., Kwasnicki, A., Lakka, S. S., Engelhard, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+microdialysis+as+a+tool+for+assessing+the+delivery+of+chemotherapy+in+brain+tumor+patients.">Cerebral microdialysis as a tool for assessing the delivery of chemotherapy in brain tumor patients.</a> World Neurosurgery. 145, 187-196 (2021).</li><li>S&#248;rensen, M., Jacobsen, S., Petersen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+in+equine+research:+a+review+of+clinical+and+experimental+findings.">Microdialysis in equine research: a review of clinical and experimental findings.</a> Veterinary Journal. 197 (3), 553-559 (2013).</li><li>Dmitrieva, N., Rodr&#237;guez-Malaver, A. J., P&#233;rez, J., Hern&#225;ndez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+release+of+neurotransmitters+from+superficial+and+deep+layers+of+the+dorsal+horn+in+response+to+acute+noxious+stimulation+and+inflammation+of+the+rat+paw.">Differential release of neurotransmitters from superficial and deep layers of the dorsal horn in response to acute noxious stimulation and inflammation of the rat paw.</a> European Journal of Pain. 8 (3), 245-252 (2004).</li><li>Li, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+sampling+and+HPLC-MS/MS+quantification+of+sinomenine,+ligustrazine,+gabapentin,+paracetamol,+pregabalin+and+amitriptyline+in+rat+blood+and+brain+extracellular+fluid.">Microdialysis sampling and HPLC-MS/MS quantification of sinomenine, ligustrazine, gabapentin, paracetamol, pregabalin and amitriptyline in rat blood and brain extracellular fluid.</a> Acta Pharmaceutica Sinica. 55 (9), 2198-2206 (2020).</li><li>Chauzy, A., Lamarche, I., Adier, C., Couet, W., Marchand, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+study+of+Aztreonam-Avibactam+distribution+in+peritoneal+fluid+and+muscle+of+rats+with+or+without+experimental+peritonitis.">Microdialysis study of Aztreonam-Avibactam distribution in peritoneal fluid and muscle of rats with or without experimental peritonitis.</a> Antimicrobial Agents and Chemotherapy. 62 (10), 01228 (2018).</li><li>Fang, X. X., Ardehali, H., Min, J. X., Wang, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+metabolic+landscape+of+iron+and+ferroptosis+in+cardiovascular+disease.">The molecular and metabolic landscape of iron and ferroptosis in cardiovascular disease.</a> Nature Reviews. Cardiology. , 1-17 (2022).</li><li>Samson, R., Ennezat, P. V., Le Jemtel, T. H., Oparil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+disease+risk+reduction+and+body+mass+index.">Cardiovascular disease risk reduction and body mass index.</a> Current Hypertension Reports. , (2022).</li><li>Kim, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=School+racial+segregation+and+long-term+cardiovascular+health+among+Black+adults+in+the+US:+A+quasi-experimental+study.">School racial segregation and long-term cardiovascular health among Black adults in the US: A quasi-experimental study.</a> PLoS Medicine. 19 (6), 1004031 (2022).</li><li>Qin, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+m6A+RNA+methylation+in+cardiovascular+disease+(Review).">Role of m6A RNA methylation in cardiovascular disease (Review).</a> International Journal of Molecular Medicine. 46 (6), 1958-1972 (2020).</li><li>Xu, C. M., Liu, C. J., Xiong, J. H., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+aspects+of+the+(pro)renin+receptor:+Function+and+significance.">Cardiovascular aspects of the (pro)renin receptor: Function and significance.</a> FASEB Journal. 36 (4), 22237 (2022).</li><li>Guvenc-Bayram, G., Yalcin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intermediary+role+of+the+central+cyclooxygenase+/+lipoxygenase+enzymes+in+intracerebroventricular+injected+nesfatin-1-evoked+cardiovascular+effects+in+rats.">The intermediary role of the central cyclooxygenase / lipoxygenase enzymes in intracerebroventricular injected nesfatin-1-evoked cardiovascular effects in rats.</a> Neuroscience Letters. 756, 135961 (2021).</li><li>Ahrens Kress, A. P., Zhang, Y. D., Kaiser-Vry, A. R., Sauer, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+blood+collection+techniques+in+mice+and+their+effects+on+welfare.">A comparison of blood collection techniques in mice and their effects on welfare.</a> Journal of the American Association for Laboratory Animal Science. 61 (3), 287-295 (2022).</li><li>Joshi, A., Patel, H., Joshi, A., Stagni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetic+applications+of+cutaneous+microdialysis:+Continuous+intermittent+vs+continuous-only+sampling.">Pharmacokinetic applications of cutaneous microdialysis: Continuous+intermittent vs continuous-only sampling.</a> Journal of Pharmacological and Toxicological Methods. 83, 16-20 (2017).</li><li>Reyes-Garc&#233;s, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+brain+sampling+using+a+microextraction+probe+reveals+metabolic+changes+in+rodents+after+deep+brain+stimulation.">In vivo brain sampling using a microextraction probe reveals metabolic changes in rodents after deep brain stimulation.</a> Analytical Chemistry. 91 (15), 9875-9884 (2019).</li><li>Kho, C. M., Enche Ab Rahim, S. K., Ahmad, Z. A., Abdullah, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+microdialysis+calibration+methods:+the+theory+and+current+related+efforts.">A review on microdialysis calibration methods: the theory and current related efforts.</a> Molecular Neurobiology. 54 (5), 3506-3527 (2017).</li><li>Zhuang, L. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+and+application+of+microdialysis+in+pharmacokinetic+studies.">Theory and application of microdialysis in pharmacokinetic studies.</a> Current Drug Metabolism. 16 (10), 919-931 (2015).</li><li>Zhang, Y. F., Huang, X. X., Zhu, L. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabonomics+research+strategy+based+on+microdialysis+technique.">Metabonomics research strategy based on microdialysis technique.</a> China Journal of Chinese Materia Medica. 45 (1), 214-220 (2020).</li><li>Carpenter, K. L., Young, A. M., Hutchinson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+monitoring+in+traumatic+brain+injury:+microdialysis.">Advanced monitoring in traumatic brain injury: microdialysis.</a> Current Opinion in Critical Care. 23 (2), 103-109 (2017).</li><li>Brunner, M., Langer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+versus+other+techniques+for+the+clinical+assessment+of+in+vivo+tissue+drug+distribution.">Microdialysis versus other techniques for the clinical assessment of in vivo tissue drug distribution.</a> The AAPS Journal. 8 (2), 263-271 (2006).</li><li>Tettey-Amlalo, R. N., Kanfer, I., Skinner, M. F., Benfeldt, E., Verbeeck, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+dermal+microdialysis+for+the+evaluation+of+bioequivalence+of+a+ketoprofen+topical+gel.">Application of dermal microdialysis for the evaluation of bioequivalence of a ketoprofen topical gel.</a> European Journal of Pharmaceutical Sciences. 36 (2-3), 219-225 (2009).</li><li>Dhanani, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+rates+of+combination+antibiotic+therapy+using+in+vitro+microdialysis+simulating+in+vivo+conditions.">Recovery rates of combination antibiotic therapy using in vitro microdialysis simulating in vivo conditions.</a> Journal of Pharmaceutical Analysis. 8 (6), 407-412 (2018).</li></ol>

The authors have nothing to disclose.

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>
			<td>Ultrasonic cleaner</td>
			<td>Guangdong Goote Ultrasonic Co., Ltd</td>
			<td>KMH1-240W8101</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

dynamic-continuous-blood-extraction-from-rat-heart-via-noninvasive

Microsoft Bing

Research

JoVE Journal

Medicine

2.1K Views.  Chengdu University of Traditional Chinese Medicine. The present protocol describes a simple and efficient method for the real-time and dynamic collection of rat heart blood using the microdialysis technique.

Video: Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap &#8211; previously cast to match the average size of the rat heart &#8211; is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 &#181;L/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. ...

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the tail vein is thin so that it is sometimes hard to puncture. In addition, the tail vein has low blood flow and requires a long sampling time to get sufficient blood. The present report describes a simple blood sampling method, the vacuum blood collection method, which is usually used to obtain blood samples from patients, here used for non-anesthetized rats. The 22 G butterfly needle tip was inserted into one of the lateral tail veins approximately 2-3 cm from the tip of the tail at an angle of approximately 20&#176;, and blood was collected in the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube. The present experimental results show that the success rate was 95% in the experimental group and 90% in the beginner group. The success rate and puncture times were similar between two groups. The sampling duration was significantly shorter in the experimental group compared to beginner group. In conclusion, this vacuum blood collection method for sequential blood sampling from the tail vein of non- anesthetized rats is feasible and easy-to-learn, which might serve as a reliable alternative to other conventionally used blood sampling techniques for rats.

Here, we describe a simple tail vein blood sampling method in non-anesthetized rats using a vacuum extraction tube system. This method reduces the risk of direct exposure to blood and simplifies taking multiple samples from a single venipuncture.

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, such as gastric dilatation volvulus (GDV), mesenteric torsion, and colic, are observed in animals such as dogs and horses. An initial interruption of blood flow causes tissues to become ischemic. Although necessary to salvage viable tissue, subsequent reperfusion can induce further injury. The main mechanism responsible for IRI is free radical formation upon reperfusion and reintroduction of oxygen into damaged tissue, but there are many other components involved. The resulting local and systemic effects often impart a poor prognosis. 
Intestinal IRI has been the subject of extensive research over the past 50 years. An in vivo rodent model in which the base of the superior mesenteric artery (SMA) is temporarily ligated is currently the most common method used to study intestinal IRI. Here, we describe a model of intestinal IRI utilizing isoflurane anesthesia in 21% O2 medical air that yields reproducible injury, as demonstrated by consistent histopathology of the small intestines. Tissue injury was also assessed in the colon, liver, and kidneys.

We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine.

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, ...