Name: dynamic continuous blood extraction from rat heart via noninvasive microdialysis technique
Uploaded: 2022-09-13T15:00:00.000+00:00
Duration: 4 min 52 s
Description: Watch this Scientific Journal Video about Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique at JoVE.com

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>

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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><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>&amp;nbsp;CMA Microdialysis AB</td><td>T55347</td><td></td></tr><tr><td>Catheter</td><td>&amp;nbsp;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>&amp;nbsp;CMA Microdialysis AB</td><td>3409501</td><td></td></tr><tr><td>Free movement tank for animals</td><td>&amp;nbsp;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>&amp;nbsp;CMA Microdialysis AB</td><td>7431100</td><td></td></tr><tr><td>Microdialysis collector</td><td>&amp;nbsp;CMA Microdialysis AB</td><td>CMA4004</td><td></td></tr><tr><td>Microdialysis in vitro stand</td><td>&amp;nbsp;CMA Microdialysis AB</td><td>CMA130</td><td></td></tr><tr><td>Microdialysis microinjection pump</td><td>&amp;nbsp;CMA Microdialysis AB</td><td>788130</td><td></td></tr><tr><td>Microdialysis syringe (1.0 mL)</td><td>&amp;nbsp;CMA Microdialysis AB</td><td>8309020</td><td></td></tr><tr><td>Microdialysis tubing adapter</td><td>&amp;nbsp;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&amp;nbsp; (SD) rats</td><td>Chengdu Dossy Experimental Animals Co., Ltd</td><td>SYXK(&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/64531/64531eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;)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>&amp;nbsp;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...

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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

Research

JoVE Journal

Medicine

2.2K Views.  Chengdu University of Traditional Chinese Medicine. Onze dynamische continue bloedafnametechniek van een rattenhart is erg belangrijk voor het analyseren van de bloedcomponenten en draagt bij aan het begrijpen van hart- en vaatziekten en hun gerelateerde ziekten. In vergelijking met traditionele bemonsteringsmethoden heeft deze techniek de voordelen van in vivo, real-time, dynamisch en is niet-invasief, zonder biologische micromoleculaire en titermonster voorbewerking voor een buitenlandse test. Deze techniek is niet alleen toepasbaar op hart-...

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

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. ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Neuroscience

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

Real-Time Dynamic Collection of Hippocampal Extracellular Fluid from Conscious Rats Using a Microdialysis System

A variety of central nervous system (CNS) diseases are associated with changes in the composition of hippocampal extracellular fluid (HECF). However, difficulty in obtaining HECF in real time from conscious rats has long restricted the evaluation of CNS disease progression and the effectiveness of ethnomedicine therapy. Encouragingly, a brain microdialysis technique can be used for continuous sampling with the advantages of dynamic observation, quantitative analysis, and a small sampling size. This allows the monitoring of changes in the extracellular fluid content for compounds from traditional herbs and their metabolites in the brain of living animals. The aim of this study was thus to accurately implant a cerebrospinal fluid microdialysis probe into the hippocampal region of Sprague Dawley (SD) rats with a three-dimensional brain stereotaxic apparatus, cutting off molecular weights greater than 20 kDa. The high-quality HECF was then obtained from conscious rats using a microdialysis sampling control system with an adjustable sampling rate from 2.87 nL/min - 2.98 mL/min. In conclusion, our protocol provides an efficient, rapid, and dynamic method to obtain HECF in awake rats with the help of microdialysis technology, which provides us with unlimited possibilities to further explore the pathogenesis of CNS-related diseases and evaluate drug efficacy.

The protocol here provides a detailed real-time dynamic sampling of extracellular fluid from the hippocampus of awake rats using a microdialysis system.

A variety of central nervous system (CNS) diseases are associated with changes in the composition of hippocampal extracellular fluid (HECF). However, ...

Microsurgical Skills of Establishing Permanent Jugular Vein Cannulation in Rats for Serial Blood Sampling of Orally Administered Drug

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, which could potentially affect results. The jugular vein cannulation (JVC) in rats is a widely used model for repeated blood collection but requires adequate training of surgery skills and animal care. This article details the microsurgical procedures for establishing and maintaining a permanent JVC rat model with specific focus on the placement and sealing of the jugular cannula. The importance of monitoring physiological (e.g., body weight, food, and water intake) and hematological parameters, was highlighted with results presented for 6 days post-surgery during the rat&#39;s recovery. The drug-plasma concentration-time profile of orally administered natural phenol ellagic acid was determined in the JVC rat model.

Detailed microsurgical techniques are demonstrated to establish a longer-term jugular vein cannulation rat model for sequential blood collection in the same animal. Physiological and hematological parameters have been monitored during the rat's recovery phase. This model has been applied to study pharmacokinetics of orally administered polyphenol without inducing animal stress.

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, ...

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

Author Spotlight: Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery 

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

Biology

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Here, we describe the preparation and technical details of murine heterotopic heart transplantation utilizing a circulatory death donor heart.

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

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