Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Summary

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Abstract

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

Introduction

The liver is the major metabolic organ in mammals; it acts as a control tower for energy, hormones, and detoxification¹. Due to its importance, researchers have conducted many in vitro and in vivo experiments to elucidate the changes in the liver during inflammation. Intravital microscopy has been used to capture live images from the liver² . Indeed, various liver imaging methods have been introduced^2,3,4,5,6. However, in order to develop a more simplified surgical approach and achieve stabilization of the target organ and the immune cells, we designed a simple protocol that can guide researchers to minimize their effort for intravital imaging.

In this study, we introduced a stable and novel imaging chamber and surgical technique that could be used to minimize bleeding and possible infections. We confirmed that the liver remained intact for more than 2 h. With this method, we successfully identified morphologies of LCMs⁷ and neutrophils under septic condition. Since this method minimizes physical and biological confounding factors such as trembling and unexpected inflammation during surgical procedure, we anticipate that this method could be used to observe acute reactions of immune cells in the liver in response to reagents like LPS.

Protocol

All procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Yonsei University College of Medicine, Korea. LysM-green fluorescent protein (GFP; gfp/+) mice⁸ and CX3Cr1-GFP (gfp/+)⁹ mice at 8-12 weeks of age were used for intravital imaging. All surgical tools were autoclaved and disinfected with 70 % alcohol.

1. Custom-designed mouse liver imaging chamber with supporting tools

1. Mouse liver imaging chamber design
   1. Make a chamber of the following size for mouse liver imaging (Figure 1): inner chamber (120 mm length × 160 mm width × 30 mm height), outer chamber (100 mm length × 100 mm width × 24 mm height), bolts (Ф 3 mm × 30 mm height), nuts (Ф 4 mm), and wing-shaped nuts (Ф 4 mm).
   2. Measure the curing agent and elastomer base in 1:9 ratio to make a total of 120 g of silicone.
   3. Put the liquid silicone mixture in a 250 mL Erlenmeyer flask and mix it well. Then put the flask in a desiccator and remove air bubbles for about 1 h under vacuum through the pump.
   4. Pour the liquid silicone mix into a custom-designed mouse chamber as high as 10 mm height and let it cure for a couple of days (Figure 1A).
2. Preparation of cover slides and the liver fixing bed
   1. On the metal frame, attach the cover glass using silicone glue. Let it dry for 1 day (Figure 1B).
   2. In order to effectively hold the distilled water for the water immersion lens, a semipermanent water-blocking structure is essential. Make 1 mm rounding silicone glue and harden it overnight.
   3. Mix the curing agent and elastomer base and then pour it into a 6-well plate at a height of about 8-10 mm. Perform the hardening process as described in 1.1.2-1.1.4.

2. Preparation of LPS

1. Prepare sterile 1× phosphate-buffered saline (PBS) to dilute the LPS powder from Salmonella enterica serotype enteritidis. (1× PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄).
2. Pre-heat PBS in water bath at 37 °C, and open the LPS powder bottle (25 mg) carefully.
3. Using a pipet aid, pour the sterile PBS (5 mL) into the LPS powder bottle and gently stir or suspend the solution to prepare the stock solution.
   NOTE: After LPS powder is liquefied, harsh vortexing can cause bubbles. Avoid making bubbles to minimize loss when making aliquots in 1.5-mL tubes.
4. Store the stock LPS solution at −20 °C, and dilute it into a 1 mg/mL LPS solution for injection.
5. Inject the 1 mg/mL LPS solution (20 mg/kg) into the peritoneum of a non-anesthetized mouse using a 1-mL syringe. For the control animal, inject the same volume of PBS.
6. Wait for 2 h for sufficient inflammatory response.

3. Preparation of Texas-Red Dextran to stain blood vessels

1. Prepare two 15-mL conical tubes and 10 mL of sterile PBS.
2. Dilute Texas-Red Dextran powder (25 mg) in 3 mL of PBS and mix it well. Transfer the mixture to an empty 15-mL conical tube.
3. Transfer the remaining PBS (7 mL) to the 15-mL conical tube containing the dextran mixture (3 mL).
4. Connect a 0.45 µm syringe filter to the tip of a 10-mL syringe. The syringe filter should replace the needle.
5. Filter the dextran mixture (10 mL). Press the syringe evenly in order to avoid spilling.
6. Dispense 500 µL of 2.5 mg/mL dextran solution in the light-blocked 1.5-mL tube and store it at −20 °C. Once thawed, store it at 4°C.

4. Surgical process

1. Disinfect the surgery table and all instruments using 70% alcohol prior to the surgery. Then, set the tools for the liver surgery and adjust the heating plate to 37 °C (Figure 2A, B).
2. For anesthesia, intraperitoneally inject the mice with 30 mg/kg of tiletamine/zolazepam  and 10 mg/kg of xylazine. These solutions can be mixed and diluted in PBS for simultaneous injection. Confirm anesthetization by gently pressing the toe of the mouse.  Proceed next step when there is no reflex.
3. Apply small amount of eye ointment to prevent the dryness of mouse's retina.
4. Remove body hair around the abdomen area using hair removal cream (Figure 2C).
5. Inject 200 µL of Texas-Red Dextran solution (concentration of 2.5 mg/mL) via the tail vein or retro-orbital injection using an insulin syringe (Figure 2D).
6. Mark the right subcostal area with a marker. The line should be drawn from the xiphoid process to the end of the rib. Cut the epidermis with forceps and scissors (Figure 2E, F).
7. Open the abdominal cavity using micro-scissors and forceps, and expose the liver carefully. The left lateral lobe should be rolled out using cotton swabs (Figure 2G, H).
8. Pour 500 µL of sterile PBS on the abdominal cavity to prevent dryness of the liver.
9. Place the mouse in the right-decubitus position in a custom-designed chamber, and apply a small amount of tissue glue to the silicon bed. Then, carefully attach the liver using cotton swabs (Figure 2I).
   NOTE: Because the liver tissue is extremely friable, do not mechanically pull out the liver. Gently roll it out with cotton swabs.
10. A few seconds later, wet the liver again with 500 µL of sterile PBS to avoid drying out of the liver. Place the metal frame on the liver and fix it firmly with nuts (Figure 2J).

5. Intravital imaging of the liver with two-photon microscopy

1. Run ZEN software (Carl Zeiss) to turn on the laser, set the wavelength to 880 nm, and set green, and red channels. Adjust laser power according to fluorescence intensity (Figure 3A). Emission filter information is as follows: Green/Alexa488, 500-550 nm and Red/Alexa 555, 575-610 nm.
2. Place the chamber on the imaging stage and place the 20X water-immersion objective lens close to the cover glass.
3. Fix the silicone rubber heater under the mouse body to maintain its body temperature (Figure 3B).
4. Drop 500 µL of distilled water on the metal frame, and then pull the objective lens close to the liver (Figure 3C).
5. Adjust the focus and determine the depth and time of the imaging area.
6. Conduct imaging.
7. For long-term imaging, inject half a dose of tiletamine/zolazepam and xylazine mixture  each hour during imaging. Maintaining the body temperature of the mice at 37 °C is essential during this stage. Always keep on eye to the mouse, since anesthetization time differs from every mouse.
   NOTE: This method is not suitable for recovery after surgery and imaging. We ethically sacrifice the mouse immediately after surgery.
8. Check the pedal reflexes for every 30 min for stable anesthetization.

6. Data analysis

1. Open the data analysis software Volocity and then import the raw data in the library into the software.
2. Remove noise from the image to suit the situation and take a snapshot.
   NOTE: To obtain smooth images, use the 'Fine' or 'Median' filter. Furthermore, use the 'Contrast enhancement' feature to make dark or blurry images clearer.
3. Fit the edited items to 100% and export as JPEG or TIFF files for snapshot images and AVI files for movies.

Results

A simple and very stable imaging chamber was devised. The bottom of chamber was covered with silicone, which prevented slipping of the mouse body and organs. Additionally, because silicon has the proper amount of elasticity, it could prevent expected damage induced by the pressure of the cover slide (Figure 1A, C). Second, tissue-safe glue (Vet bond) was applied in order to fix the extracted liver on the circle-shaped silicone bed. This metho...

Discussion

The liver has been actively studied by many groups^3,10, due to importance of its function. For instance, Heymann et al. suggested a delicate method using agarose. Although this method was found to be highly precise, the agarose setting takes time. However, with our methodology, all procedures could be carried out within 30 min, minimizing other confounding factors. Second, stabilizing the liver is extremely important because the heartbeat can disturb the video. T...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Custom designed chamber	Live Cell Instruments		Custom-designed size
Metal cover slide	Live Cell Instruments		Custom-designed size
Cover glass	Electron Microscopy Sciences	#72204-03
Sylgard 184 Silicone elastomer kit	Dow Corning
Erlenmeyer flask	DURAN	21 216 36
Cell culture plate (Flat type)	HYUNDAI Micro Co.	H31006
Desiccator	ADARSH	55204
High Q BOND SE 5 mL	BJM LAB		To make semi-permanent dam
Flexible Silicone Rubber Heaters	Omega	SRFR/SRFG SERIES, M1250/0800
DC power supply	Toyotech	DP30-03A
Phosphate-buffered saline			Home-made
Lipopolysaccharides (LPS) from Salmonella enteria serotype enteritidis	Sigma-Aldrich	L6011
Texas Red Dextran	Thermo Fisher Scientific	D1830
15 mL High-clarity polypropylene conical tube	FALCON	14-959-49B
0.45 μm filter (Minisart Syringe Filter)	Sartorius	16555k
1.5ml Micro Tube, Amber	Axygen	MCT-150-X	For light-blocking
1 mL syringe	Korea Vaccine	KV-S01
3 mL syringe	Korea Vaccine	KV-S03
10 mL syringe	Korea Vaccine	KV-S10
0.3 mL insulin syringe	Becton Dickinson	324900
Hair removal cream 100 mL	Body natur
Cotton swab	ABDI
Zoletil 50 5 mL	Virbac
Rompun 10 mL	Bayer
Heating plate	Live Cell Instruments	PH-S-10	Custom-designed size
DC controller	Live Cell Instruments	CU-301
Microdessection Scissors	Harvard Apparatus	72-5418
Scissors	Roboz	RS-5882
Forceps	Roboz	RS-5137
Vet bond	3M	1469SB
ZEN software (Black Edition)	Carl Zeiss		Program for LSM 7 MP
LSM 7 MP	Carl Zeiss
Volocity	PerkinElmer		Analysis program