This work was supported by the National Nature Science Foundation of China (81570399 and 81773735), the National Key Research and Development Program of China - Traditional Chinese Medicine Modernization Research project (2017YFC1702003), and Hei Long Jiang Outstanding Youth Science Fund (JC2017020).

All procedures involving animals and their care were approved by the Institutional Animal Care and Use Committee of Harbin Medical University.
NOTE: The tools and equipment required for the method are listed in the Table of Materials.
1. Preparation of materials and animals
<ol>
	<li>Order C57BL/6 male mice with weight between 20 g-25 g from a standard laboratory animal supplier.</li>
	<li>House the mice in a cycle of 12 h light: 12 h darkness at ...

<ol>
	<li>Baruch, 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=Aging-induced+type+I+interferon+response+at+the+choroid+plexus+negatively+affects+brain+function.">Aging-induced type I interferon response at the choroid plexus negatively affects brain function.</a> Science. 346 (6205), 89-93 (2014).</li><li>Zhang, 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=Positional+cloning+of+the+mouse+obese+gene+and+its+human+homologue.">Positional cloning of the mouse obese gene and its human homologue.</a> Nature. 372 (6505), 425-432 (1994).</li><li>Torres, M., 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=Parabiotic+model+for+differentiating+local+and+systemic+effects+of+continuous+and+intermittent+hypoxia.">Parabiotic model for differentiating local and systemic effects of continuous and intermittent hypoxia.</a> Journal of Applied Physiology (1985). 118 (1), 42-47 (2015).</li><li>Loffredo, F. S., 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=Growth+differentiation+factor+11+is+a+circulating+factor+that+reverses+age-related+cardiac+hypertrophy.">Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy.</a> Cell. 153 (4), 828-839 (2013).</li><li>Kamran, P., 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=Parabiosis+in+mice:+a+detailed+protocol.">Parabiosis in mice: a detailed protocol.</a> Journal of Visualized Experiments. (80), e50556 (2013).</li><li>Conboy, M. J., Conboy, I. M., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochronic+parabiosis:+historical+perspective+and+methodological+considerations+for+studies+of+aging+and+longevity.">Heterochronic parabiosis: historical perspective and methodological considerations for studies of aging and longevity.</a> Aging Cell. 12 (3), 525-530 (2013).</li><li>Eggel, A., Wyss-Coray, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revival+of+parabiosis+in+biomedical+research.">A revival of parabiosis in biomedical research.</a> Swiss Medical Weekly. 144, w13914 (2014).</li><li>Brack, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ageing+of+the+heart+reversed+by+youthful+systemic+factors!.">Ageing of the heart reversed by youthful systemic factors!.</a> EMBO Journal. 32 (16), 2189-2190 (2013).</li><li>Wu, J. M., 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=Circulating+cells+contribute+to+cardiomyocyte+regeneration+after+injury.">Circulating cells contribute to cardiomyocyte regeneration after injury.</a> Circulation Research. 116 (4), 633-641 (2015).</li><li>Kaiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Rejuvenation+factor'+in+blood+turns+back+the+clock+in+old+mice.">'Rejuvenation factor' in blood turns back the clock in old mice.</a> Science. 344 (6184), 570-571 (2014).</li><li>Rando, T. A., Finkel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+aging+and+rejuvenation--a+sense+of+humors?.">Cardiac aging and rejuvenation--a sense of humors?.</a> New England Journal of Medicine. 369 (6), 575-576 (2013).</li><li>Heidt, 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=Differential+contribution+of+monocytes+to+heart+macrophages+in+steady-state+and+after+myocardial+infarction.">Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.</a> Circulation Research. 115 (2), 284-295 (2014).</li><li>McPherron, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Through+thick+and+thin:+a+circulating+growth+factor+inhibits+age-related+cardiac+hypertrophy.">Through thick and thin: a circulating growth factor inhibits age-related cardiac hypertrophy.</a> Circulation Research. 113 (5), 487-491 (2013).</li><li>Villeda, S. 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=Young+blood+reverses+age-related+impairments+in+cognitive+function+and+synaptic+plasticity+in+mice.">Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice.</a> Nature Medicine. 20 (6), 659-663 (2014).</li><li>Katsimpardi, L., 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=Vascular+and+neurogenic+rejuvenation+of+the+aging+mouse+brain+by+young+systemic+factors.">Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors.</a> Science. 344 (6184), 630-634 (2014).</li><li>Starzl, T. E., 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=The+lost+chord:+microchimerism+and+allograft+survival.">The lost chord: microchimerism and allograft survival.</a> Immunology Today. 17 (12), 577-584 (1996).</li><li>Coleman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+historical+perspective+on+leptin.">A historical perspective on leptin.</a> Nature Medicine. 16 (10), 1097-1099 (2010).</li><li>Salpeter, S. J., 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=Systemic+regulation+of+the+age-related+decline+of+pancreatic+beta-cell+replication.">Systemic regulation of the age-related decline of pancreatic beta-cell replication.</a> Diabetes. 62 (8), 2843-2848 (2013).</li><li>Sheldon, R. D., 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=Gestational+exercise+protects+adult+male+offspring+from+high-fat+diet-induced+hepatic+steatosis.">Gestational exercise protects adult male offspring from high-fat diet-induced hepatic steatosis.</a> Journal of Hepatology. 64 (1), 171-178 (2016).</li><li>Martineau, M. G., 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=The+metabolic+profile+of+intrahepatic+cholestasis+of+pregnancy+is+associated+with+impaired+glucose+tolerance,+dyslipidemia,+and+increased+fetal+growth.">The metabolic profile of intrahepatic cholestasis of pregnancy is associated with impaired glucose tolerance, dyslipidemia, and increased fetal growth.</a> Diabetes Care. 38 (2), 243-248 (2015).</li><li>Wang, F., Liu, Y., Yuan, J., Yang, W., Mo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound+C+Protects+Mice+from+HFD-Induced+Obesity+and+Nonalcoholic+Fatty+Liver+Disease.">Compound C Protects Mice from HFD-Induced Obesity and Nonalcoholic Fatty Liver Disease.</a> International Journal of Endocrinology. 2019, 3206587 (2019).</li></ol>

The authors have nothing to disclose.

Parabiosis refers to the surgical technique of connecting two living animals to establish a common vascular system by experimental means6,7,8. The advantage of this model is that the substances produced by a single individual can act on both animals at the same time. Thus, the parabiosis model can be used to explore the role of a substance or factor in a related disease, producing many meaningful and innovative conclusions. In v...

Parabiosis is a modeling method in which two living organisms are joined together surgically and develop as a single physiological system with a shared circulatory system1. Such models have been widely used for studying physiology owing to the advantage that the substances produced by a single individual can act on both animals at the same time via the shared circulatory system. Since the mid-1800s when parabiotic experiments were pioneered by Paul Bert2, the methods for constructing parabiotic models have become standardized. However, a straightforward and convenient method for verifying the successful establishment of blood chimerism has been missing. It has been reported that cross-circulation can be successfully assessed by intraperitoneally injecting 0.5% Evans blue dye in one of the parabionts followed by measurement of the absorbance of Evans blue in the blood of both parabionts with a microplate reader3. Another method requires a specific mouse breed that contains CD45.1+- and CD45.2+-labeled monocytes in each parabiont. Cell cytometry is then used to determine blood chimerism by measuring the frequency of the two markers in monocytes from spleen or blood4. However, these methods are often lethal or cumbersome to the animals, and a safe and simple method for quick and reliable verification of parabiotic models is highly desirable. In this study, we established a new method for this purpose, which was validated in a mouse model of parabiosis. Glucose concentration in blood samples drawn from a tail vein is measured using a glucometer, and the pattern of changes of glucose level in donor and recipient mice is considered an indication of circulation chimerism. We named this method the &#34;glucose fluctuation method&#34;. The application of this validation method is not limited to mice but could be extended to diverse pathological models except for those with serious dysregulation of glucose metabolism. The procedure is simple, timesaving, and safe.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>curved forceps</td>
			<td>JZ surgical Instruments, China</td>
			<td>J31340</td>
			<td></td>
		</tr>
		<tr>
			<td>fine scissors</td>
			<td>JZ surgical Instruments, China</td>
			<td>WA1030</td>
			<td></td>
		</tr>
		<tr>
			<td>needle forcep</td>
			<td>JZ surgical Instruments, China</td>
			<td>60017961</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 mL syringes</td>
			<td>Agilent, USA</td>
			<td>5182-9642</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethano</td>
			<td>Sigma-Aldrich, USA</td>
			<td>T48402-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin</td>
			<td>Solarbio, China</td>
			<td>IP0150</td>
			<td></td>
		</tr>
		<tr>
			<td>tramadol</td>
			<td>Yijishiye, China</td>
			<td>YJT712520</td>
			<td></td>
		</tr>
		<tr>
			<td>glucometer</td>
			<td>Roche Diabetes Care, Indiana</td>
			<td>Accu-Chek Active test strips</td>
			<td></td>
		</tr>
		<tr>
			<td>Evan&#39;s blue Counterstain</td>
			<td>Solarbio, China</td>
			<td>G1810</td>
			<td></td>
		</tr>
		<tr>
			<td>depilatory cream</td>
			<td>Nair, USA</td>
			<td>LL9161</td>
			<td></td>
		</tr>
		<tr>
			<td>Warming Blanket (Heating pad)</td>
			<td>Kent Scientific Corp, USA</td>
			<td>TP-22G</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical shaver</td>
			<td>Codos, China</td>
			<td>CP-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0,5-0 
			surgical suture</td>
			<td>Shanghai Medical Suture Needle Factory, China</td>
			<td>SYZ 3-0#, SYZ 5-0#</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting establishment of shared blood supply in parabiotic mice by caudal vein glucose injection

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for detecting the successful establishment of blood chimerism in parabionts by a caudal vein injection of glucose. Parabiotic mice were constructed. Glucose was injected into the donor mouse through the tail vein, and the fluctuation of blood glucose level was measured in both mice using a blood glucometer at different time points. Our results showed that after glucose injection, the blood glucose level in donor mice increased sharply after 1 min and decreased slowly thereafter. Meanwhile, the blood glucose level of the recipient mice peaked 15 min after injection. Similar results were obtained with Evans blue, used as a positive control for glucose. The synchronous fluctuation of blood glucose levels indicates that blood flow between the two mice was established successfully.

In six donor mice, blood glucose levels sharply increased to 26.5 &#181;mol/L (173% increase) at an average of 1 min after the injection of 100 &#181;L of glucose (1.2 g/kg) through the tail vein and then gradually decreased to 13.3 &#181;mol/L at 60 min. In recipient mice, blood glucose slowly increased after injection and reached the first peak level at 15 min (47% increase, 12.2 &#181;mol/L). Based on the above results, the standard for circulation chimerism was set as follows: 1) a sh...

Watch this Scientific Journal Video about Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection at JoVE.com

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for ...

Our protocol provides an improved verification method for circulation chimerism and a method for successful parabiosis model setup for biomedical research. Compared with existing methods for the detection of blood chimerism in parabiosis that are often lethal or cumbersome, our protocol is safe and simple, allowing a quick and reliable verification. Although this method is specific to the detection of blood chimerism in parabiosis models, it can be applied to any biomedical studies involving parabiotic procedures.The most challenging step is glucose injection. Therefore be sure to practice the tail vein injection prior to performing an experimental procedure, or use a venous visual mouse tail fixator. After confirming a lack of response to pedal reflex, place two 20 to 25-gram anesthetized C57 black 6 male mice in the supine position, and use an electric shaver to thoroughly shave the left side of one mouse and the right side of the second mouse from approximately one centimeter above the elbow to one centimeter below the knee.Remove any remaining hair with depilatory cream before making longitudinal skin incisions in the cleared skin of each animal, starting from 0.5 centimeters above the elbow to 0.5 centimeters below the knee joint, and gently detach the skin from the subcutaneous fascia. Connect the olecranon and knee of the parabiont with 3-0 suture, and suture the shaved skin with a continuous 5-0 suture. Then subcutaneously inject 0.5 milliliters of 0.9%sodium chloride to prevent dehydration, and intramuscularly inject 10 milligrams of tramadol per 25 grams of mouse to relieve pain, into each mouse per day for three days.To verify the successful construction of circulation between chimerism between parabionts by glucose fluctuation, on the 10th day after parabiosis surgery, secure the donor mice in a venous visual mouse tail fixator, and use a 70 to 75%ethanol-soaked cotton ball to rub the caudal veins in the flank of one parabiont tail. Insert a one-milliliter syringe needle into the caudal vein two to four centimeters from the tail tip and as parallel to the vein as possible, and inject one to two grams per kilogram of glucose in 100 microliters of saline solution into the donor on a period of 10 seconds. Next use scissors to remove the toes from the donor and recipient animals to allow the collection of a drop of blood at various time points of interest after the injection.Then add a drop of each blood sample into the center of a glucometer test strip for glucose level detection. To verify the successful construction of circulation by Evans blue dye absorbance, inject 200 microliters of 0.5%Evans blue counterstain intraperitoneally into the donor mouse, and collect blood from both parabionts by cardiac puncture after two hours. Separate the serum by centrifugation, and transfer the serum into a conical tube for dilution at a one to 50 ratio in 0.9%sodium chloride.Then measure the absorbance of the diluted serum samples at 620 nanometers on a spectrophotometer. In this representative analysis of the blood glucose levels in six donor mice, the blood glucose sharply increased to 26.5 micromolar per liter at an average of one minute after the injection of 100 microliters of glucose through the tail vein. Blood glucose levels then gradually decreased to 13.3 microliters per liter at 60 minutes.In recipient mice, the blood glucose slowly increased after the injection, reaching the first peak level at 15 minutes. The concentration of Evans blue dye in the serum of parabionts also indicated a successful construction of circulation chimerism, and subcutaneous vascular junctions between the parabionts could be clearly observed. In parabionts that did not establish blood chimerism 15 days after parabiosis surgery, the recipient mice did not exhibit an increased blood glucose level within 60 minutes after glucose injection into the donor animal.The blood concentration of Evans blue in the two recipients was also not elevated, demonstrating that the glucose fluctuation method is as sensitive as the Evans blue method. Moreover the blood insulin level was markedly decreased one hour after glucose injection because of the quickly increased glucose levels and recovered to normal levels by three hours, indicating that the effects of the injected glucose on insulin metabolism are restorable. Take care to make sure that the needle is inserted directly into the tail vein.After confirming the blood chimerism of the parabionts by this method, physiopathological changes can be studied in live parabiotic animals.

detecting-establishment-shared-blood-supply-parabiotic-mice-caudal

Research

JoVE Journal

Biology

8.1K visualizações.  Harbin Medical University. Nosso protocolo fornece um método de verificação aprimorado para o chimerismo de circulação e um método para uma configuração bem-sucedida do modelo de parabiose para pesquisa biomédica. Comparado com os métodos existentes para a detecção de chimerismo sanguíneo na parabiose que muitas vezes são letais ou complicados, nosso protocolo é seguro e simples, permitindo uma verificação rápida e confiável. Embora este método seja específico para a detecção do chimerismo sangu?...