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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 "glucose fluctuation method". 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.

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Protokół

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

1. Order C57BL/6 male mice with weight between 20 g-25 g from a standard laboratory animal supplier.
2. House the mice in a cycle of 12 h light: 12 h darkness at 24−26 °C with ad libitum access to water and food.

2. Parabiosis

1. Anesthetize the mice by intraperitoneal injection of 20 g/L 2,2,2-tribromoethanol at a concentration of 0.1 mL/10 g. Confirm proper anesthetization as indicated by muscle relaxation, slow and steady breathing, loss of skin stimulation reflex, and disappearance of corneal reflex.
2. Apply ophthalmic ointment with a Q-tip to prevent dry eyes.
3. Perform the parabiosis procedure as previously described⁵.
   1. Place the mice in the supine position. Thoroughly shave the left side of one mouse and the right side of the other mouse starting at approximately 1 cm above the elbow to 1 cm below the knee with an electric shaver. Use depilatory cream to totally clear the fur on the shaved skin.
   2. Wipe the shaved areas with iodophor. Place the mice on a heated pad covered by a sterile pad.
   3. Create longitudinal skin incisions starting from 0.5 cm above the elbow to 0.5 cm below the knee joint using a pair of sharp scissors on each animal's shaved side.
   4. Detach the skin from the subcutaneous fascia gently following the incision.
   5. Connect the olecranon and knee of the parabiont with a 3-0 suture.
   6. Suture the shaved skin with a continuous 5-0 suture.
4. Inject 0.5 mL of 0.9% NaCl subcutaneously to each mouse to prevent dehydration.
5. Inject tramadol (10 mg/25 g/day) intramuscularly to each mouse to relieve pain.

3. Validation of circulation chimerism

1. Glucose fluctuation method
   NOTE: Verify the successful construction of circulation chimerism between parabionts using the glucose fluctuation method (no fasting) on the 10th day after parabiosis surgery.
   1. Anesthetize the parabionts by intraperitoneal injection of 20 g/L 2,2,2-tribromoethanol to each mouse at a concentration of 0.1 mL/10 g.
   2. Fix the donor mice in a Venous visual-mouse tail fixator.
   3. Rub the caudal veins in the flank of one of the parabiont's tail with a cotton ball soaked in 70−75% alcohol to clean the tail and dilate the blood vessels.
   4. Hold a 1.0 mL syringe containing glucose in the right hand and keep the needle parallel to the vein (less than 15°).
   5. Insert the needle at a position approximately 2−4 cm from the tail tip.
   6. Inject 100 µL of glucose (1.2 g/kg) into the donor within 10 s.
      NOTE: The term donor refers to the parabiont that receives the glucose injection through the tail vein. The recipient is the other parabiont, which does not receive glucose directly.
   7. Clean the toes with iodophor. Cut off the toes of the donor and recipient mice with a scissor and collect a drop of blood at different time points after the injection of glucose (1 min, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, and 60 min). Remove the blood clot at each collection time point to avoid more damage.
      NOTE: The volume of collected blood was 10-25 ul for one test, and 90-225 ul in total.
   8. Drip the blood into the center of glucometer test strips for glucose level detection.
2. Use Evan's blue Counterstain as a positive control³.
   1. Inject 200 µL of 0.5% Evan's blue Counterstain intraperitoneally into the donor mouse.
   2. 2 h later, euthanize the parabionts by intraperitoneal injection of 20 g/L 2,2,2-tribromoethanol to each mouse at a concentration of 0.2 mL/10 g, and collect blood from both parabionts by cardiac puncture.
   3. Centrifuge the blood samples at 916 x g for 15 min.
   4. Collect serum from the supernatant.
   5. Dilute the serum with 0.9% NaCl at 1:50.
   6. Measure the absorbance of the diluted serum samples at 620 nm with a spectrophotometer.

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Wyniki

In six donor mice, blood glucose levels sharply increased to 26.5 µmol/L (173% increase) at an average of 1 min after the injection of 100 µL of glucose (1.2 g/kg) through the tail vein and then gradually decreased to 13.3 µmol/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 µmol/L). Based on the above results, the standard for circulation chimerism was set as follows: 1) a sharp increase in ...

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Dyskusje

Parabiosis refers to the surgical technique of connecting two living animals to establish a common vascular system by experimental means^6,7,8. 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...

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Materiały

Name	Company	Catalog Number	Comments
curved forceps	JZ surgical Instruments, China	J31340
fine scissors	JZ surgical Instruments, China	WA1030
needle forcep	JZ surgical Instruments, China	60017961
2.5 mL syringes	Agilent, USA	5182-9642
Tribromoethano	Sigma-Aldrich, USA	T48402-5G
penicillin	Solarbio, China	IP0150
tramadol	Yijishiye, China	YJT712520
glucometer	Roche Diabetes Care, Indiana	Accu-Chek Active test strips
Evan's blue Counterstain	Solarbio, China	G1810
depilatory cream	Nair, USA	LL9161
Warming Blanket (Heating pad)	Kent Scientific Corp, USA	TP-22G
Electrical shaver	Codos, China	CP-5000
3-0,5-0 surgical suture	Shanghai Medical Suture Needle Factory, China	SYZ 3-0#, SYZ 5-0#