This study was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korea government (MSIP; grant no. 2016R1A2B4008199 to Y-M. H.), and the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C1324).

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.&#160;All surgical tools were autoclaved and disinfected with 70 % alcohol.
1. Custom-designed mouse liver imaging chamber with supporting tools
<ol>
	...

<ol>
	<li>Girard, J. R., 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=Fuels,+hormones,+and+liver+metabolism+at+term+and+during+the+early+postnatal+period+in+the+rat.">Fuels, hormones, and liver metabolism at term and during the early postnatal period in the rat.</a> J Clin Invest. 52 (12), 3190-3200 (1973).</li><li>Marques, P. 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=Imaging+liver+biology+in+vivo+using+conventional+confocal+microscopy.">Imaging liver biology in vivo using conventional confocal microscopy.</a> Nat Protoc. 10 (2), 258-268 (2015).</li><li>Dasari, S., Weber, P., Makhloufi, C., Lopez, E., Forestier, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Microscopy+Imaging+of+the+Liver+following+Leishmania+Infection:+An+Assessment+of+Hepatic+Hemodynamics.">Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics.</a> J Vis Exp. (101), e52303 (2015).</li><li>Honda, 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=Intravital+imaging+of+neutrophil+recruitment+in+hepatic+ischemia-reperfusion+injury+in+mice.">Intravital imaging of neutrophil recruitment in hepatic ischemia-reperfusion injury in mice.</a> Transplantation. 95 (4), 551-558 (2013).</li><li>Jenne, C. N., Wong, C. H., Petri, B., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+spinning-disk+confocal+microscopy+for+the+intravital+analysis+of+platelet+dynamics+in+response+to+systemic+and+local+inflammation.">The use of spinning-disk confocal microscopy for the intravital analysis of platelet dynamics in response to systemic and local inflammation.</a> PLoS One. 6 (9), e25109 (2011).</li><li>Ritsma, 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=Intravital+microscopy+through+an+abdominal+imaging+window+reveals+a+pre-micrometastasis+stage+during+liver+metastasis.">Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis.</a> Sci Transl Med. 4 (158), 158ra145 (2012).</li><li>Sierro, F., 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=A+Liver+Capsular+Network+of+Monocyte-Derived+Macrophages+Restricts+Hepatic+Dissemination+of+Intraperitoneal+Bacteria+by+Neutrophil+Recruitment.">A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.</a> Immunity. 47 (2), 374-388 (2017).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96 (2), 719-726 (2000).</li><li>Jung, 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=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20 (11), 4106-4114 (2000).</li><li>Heymann, F., 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=Long+term+intravital+multiphoton+microscopy+imaging+of+immune+cells+in+healthy+and+diseased+liver+using+CXCR6.Gfp+reporter+mice.">Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.</a> J Vis Exp. (97), (2015).</li></ol>

The authors declare no competing financial interests.

The liver has been actively studied by many groups3,10, 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...

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 introduced2,3,4,5,6. 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.

<table border="0" cellpadding="0" cellspacing="0" width="1004">
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			<td height="61" style="height:61px;width:260px;">Lipopolysaccharides (LPS) from Salmonella enteria serotype enteritidis</td>
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two-photon intravital imaging of leukocytes during the immune response in lipopolysaccharide-treated mouse liver

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.

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...

Watch this Scientific Journal Video about Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver at JoVE.com

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

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.

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 ...

The overall goal of this procedure is to observe the morphology and functionality of leukocytes inside the liver during septic shock. The main advantage of this technique is that you can obtain direct videos of leukocytes inside the liver. Prior to surgery, use 70%ethanol to disinfect the surgery table and all instruments.Then set up the tools for liver surgery and adjust the heating plate to 37 degrees Celsius. After anesthetizing LPS or PBS injected mice according to the text protocol, apply a small amount of eye ointment to prevent the retina from drying out. Use a razor and hair removal cream to remove the hair around the abdomen.Then use wet gauze to clear the abdomen. While carrying out a retro-orbital injection using an insulin syringe, inject 200 microliters of Texas red dextran solution. Use a marker to mark the right subcostal area from the xyphoid process to the end of the rib.Then apply povidone iodine to the mouse's abdomen to prevent secondary infection. Use forceps and scissors to cut the epidermis. Next, using micro scissors and forceps open the abdominal cavity and carefully expose the liver.Use cotton swabs to roll out the left lateral lobe. Then pour 500 microliters of sterile PBS on the abdominal cavity to prevent the liver from drying out. Place the mouse in the right decubitis position in a custom designed chamber and apply a small amount of tissue glue to the silicon bed.Then use cotton swabs to carefully attach the liver. Liver tissue is extremely friable. Never use sharp or heavy tools when exposing and holding the liver.A few seconds later, wet the liver again with 500 microliters of sterile PBS to prevent it from drying out. Then place the metal frame on the liver and use nuts to firmly fix it. Always place supporting nuts before placing the metal frame.This will help reduce the pressure of the metal frame. To carry out intravital imaging of the liver, run the Zen software to turn on the laser. Set the wavelength to 880 nanometers and set the green and red channels.Then adjust the laser power according to fluorescence intensity. Place the chamber on the imaging stage and position the 20 power water immersion objective lens close to the cover glass. Fix the silicone rubber heater under the mouse body to maintain its body temperature.Next draw up 500 microliters of distilled water on the metal frame. And then position the objective lens close to the liver. Adjust the focus and determine the depth and time of the imaging area.For long term imaging, inject half a dose of the previous prepared Zoletil and Rompun anesthetic mixture for each hour during imaging for ethical reasons. Maintain the body temperature at 37 degrees Celsius and always keep an eye to the mouse since anesthetization times differ for every mouse. To analyze the data open Velocity and import the raw data from the library.Then remove the noise from the image and take a snapshot. Finally fit the edited items to 100%and export the files as JPEG or TIF files for snapshot images and AVI files for movies. Intravital imaging analysis of control mice showed that a small number of neutrophils were circulating in the blood vessel.Whereas the number of neutrophils was significantly increased in LPS treated mice. In LPS condition a lot of neutrophils were circulating inside the bloodstream but they did not migrate outside the vessel. As seen in these movies, compared to control mice, neutrophils in the LPS treated mice were more firmly adhered to the luminal surface of the vessel which made neutrophils more frequently detected in the vessel.Apart from the motility of neutrophils, liver capsular macrophages, or LCMs, in CX3CR1-GFP mice were observed on the surface of liver tissue rather than deep within the tissue. In addition, LCMs showed little movement or activation in the liver even in LPS treated mice. Once mastered this technique can be done in 30 minutes if it is performed properly.While attempting this procedure, it's important to remember to set the incision line from xiphoid process to the end of the right subcostal abdomen. Following this procedure, other methods like treatment injection can be performed in order to answer additional questions like how the liver recovers from damage and infection. After its development this technique paved the way for researchers in the field of medicine to explore the leukocyte's reaction during sepsis and test potential drugs.After watching this video you should have a good understand of how to expose a liver with minimal abdomen incision. Don't forget that working with lipopolysaccharide can be extremely hazardous and precautions such as sterilization and hand washing should always be taken while performing this procedure.

two-photon-intravital-imaging-leukocytes-during-immune-response

Research

JoVE Journal

Immunology and Infection

10.0K vistas.  Yonsei University College of Medicine. The overall goal of this procedure is to observe the morphology and functionality of leukocytes inside the liver during septic shock. The main advantage of this technique is that you can obtain direct videos of leukocytes inside the liver. Prior to surgery, use 70%ethanol to disinfect the surgery table and all instruments.Then set up the tools for liver surgery and adjust the heating plate to 37 degrees Celsius. After anesthetizing LPS or PBS injected mice according to the text protoco...