This work was supported by grants from the National Natural Science Foundation of China (No. 81702406) and the Natural Science Foundation of Guangdong Province of China (2016A030310207).

This protocol follows the guidelines of the human research ethics committee of Sun Yat-sen Memorial Hospital, Sun Yat-sen University. Written informed consent was obtained from the patients for performing this study.
1. Patient selection
<ol>
	<li>Select patients with a malignant tumor in the middle section of the liver, including patients with hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC).</li>
	<li>Ensure that these patients show no invasion of the portal or hepatic vein and no distant metastasis.</li>
</ol>
2. Patient preparation
NOTE: The patients were classified preoperatively as Child-Pugh A13.
<ol>
	<li>Administer antiviral drugs, such as entecavir or tenofovir (see Table of Materials), to the patients 1 week before the operation in case they are detected with hepatitis B. In our study,tThe ICG retention rate at 15 min was &#60;10%.</li>
	<li>Perform three-dimensional reconstruction in advance14. In our study, the future liver remnant (FLR) accounted for &#62;50% of the standard liver volume (SLV).</li>
</ol>
3. Surgical technique
<ol>
	<li>Preoperative preparation
	<ol>
		<li>Place the patient in a supine position, with the legs open and lying on the right side. Perform standard general anesthesia involving tracheal intubation and controlled ventilation. 
		NOTE: The dosage of anesthesia varies widely from patient to patient, determined mainly based on patient characteristics, age, and other medical conditions.</li>
		<li>Ensure that the surgeon stands on the right and the assistant on the left. Place the camera in the middle of the trocar sites and the camera holder in the middle.</li>
		<li>Place the trocar (see Table of Materials) as illustrated in Figure 1.</li>
		<li>Prepare fluorescent laparoscopic equipment and B-ultrasound with a laparoscopic probe (see Table of Materials).</li>
	</ol>
	</li>
	<li>Exploration phase
	<ol>
		<li>Perform abdominal cavity exploration from the epigastrium to the hypogastrium from left to right to reveal ascites, cirrhosis, and abdominal adhesions.</li>
		<li>Perform intraoperative ultrasound exploration of the liver. Evaluate the tumor size, location, relationship with surrounding blood vessels, and variations in the MHV and RHV. 
		NOTE: The tumor size is measured by intraoperative ultrasound. The relevant data are presented in Table 1.</li>
	</ol>
	</li>
	<li>Cholecystectomy and presetting of the hepatoduodenal ligament occlusion band
	<ol>
		<li>Anatomize Calot&#39;s triangle. Then, cut the cystic duct 5 mm from the common duct. Perform retrograde dissection of the gallbladder.</li>
		<li>Perform the hepatoduodenal ligament occlusion with a cotton string by using the Pringle maneuver15.</li>
		<li>Perform intermittent occlusion of the first porta hepatis by blocking for 15 min and opening for 5 min. Shorten the blocking time to 10 min for patients with obvious cirrhosis.</li>
	</ol>
	</li>
	<li>Resection of segments 4, 5, and 8
	<ol>
		<li>First, separate the liver parenchyma by resecting at 1 cm on the right side of the falciform ligament. Ligate and transect several branches of the segment 4 Glisson pedicle16 using clips.</li>
		<li>Second, separate the right anterior hepatic pedicle along the Laennec&#39;s capsule4. Ligate or occlude the right anterior hepatic pedicle.</li>
		<li>Administer a peripheral injection of 2 mL of 1:100 diluted ICG (see Table of Materials) for negative staining, to define the boundary between the right anterior section and the right posterior section (Figure 2).</li>
		<li>Dissect the liver parenchyma along the right fluorescent border. Transect the right anterior hepatic pedicle using a linear stapler after obtaining sufficient space.</li>
		<li>Separate the MHV at the root and transect it using a linear stapler.</li>
		<li>Under fluorescence tracing, reserve the trunk of the RHV, and transect the V5d and V8d branches of the RHV17. Finish resecting the specimens. 
		NOTE: ICG (25 mg ICG to 100 mL of sterile water) was diluted at a ratio of 1:100 for injection. Appropriate liver-cutting instruments, such as an ultrasonic scalpel, bipolar, cavitational ultrasonic surgical aspirator, etc., should be used, according to the surgeon&#39;s preference.</li>
	</ol>
	</li>
	<li>Resection of segments 5 and 8
	<ol>
		<li>Separate the right anterior hepatic pedicle along the Laennec&#39;s capsule.</li>
		<li>Perform a peripheral injection of 2 mL of 1:100 diluted ICG for negative staining to define the boundaries between the left, right anterior, and right posterior sections (Figure 3).</li>
		<li>First, process the left resection plane. Dissect the liver parenchyma along the left fluorescent border, preserve the trunk of the MHV, and transect the V5v and V8v branches of the MHV.</li>
		<li>Then, transect the right anterior hepatic pedicle using a linear stapler after obtaining sufficient space.</li>
		<li>Next, dissect the liver parenchyma along the right fluorescent border. Preserve the trunk of the RHV, and transect the V5d and V8d branches of the RHV. Finish resecting the specimen.</li>
	</ol>
	</li>
	<li>Hemostasis of residual liver after hepatectomy
	<ol>
		<li>Carefully check the residual liver and close the bleeding points one by one using bipolar electrocoagulation.</li>
	</ol>
	</li>
	<li>Specimen examination
	<ol>
		<li>After the surgery, visually confirm that the specimen margin is negative. Additionally, examine the specimens to confirm that the tumor capsule is complete and measure the distance from the tumor to the cutting edge.</li>
		<li>Immerse the specimens in formalin and send them to the pathology department. The operation is successful if the capsule is complete or the incision margin is &#62;1 cm.</li>
	</ol>
	</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+hepatectomy+for+centrally+located+malignant+liver+tumors:+A+systematic+review.">Central hepatectomy for centrally located malignant liver tumors: A systematic review.</a> World Journal of Hepatology. 6 (5), 347-357 (2014).</li><li>Takasaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glissonean+pedicle+transection+method+for+hepatic+resection:+a+new+concept+of+liver+segmentation.">Glissonean pedicle transection method for hepatic resection: a new concept of liver segmentation.</a> Journal of Hepato-Biliary-Pancreatic Surgery. 5 (3), 286-291 (1998).</li><li>Torzilli, 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=Ultrasound-guided+liver+resections+for+hepatocellular+carcinoma.">Ultrasound-guided liver resections for hepatocellular carcinoma.</a> Hepato-Gastroenterology. 49 (43), 21-27 (2002).</li><li>Takamoto, T., Makuuchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+surgery+for+primary+liver+cancer.">Precision surgery for primary liver cancer.</a> Cancer Biology &amp; Medicine. 16 (3), 475-485 (2019).</li><li>Makuuchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+treatment+for+HCC--special+reference+to+anatomical+resection.">Surgical treatment for HCC--special reference to anatomical resection.</a> International Journal of Surgery. 11, 47-49 (2013).</li><li>Aoki, 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=Image-guided+liver+mapping+using+fluorescence+navigation+system+with+indocyanine+green+for+anatomical+hepatic+resection.">Image-guided liver mapping using fluorescence navigation system with indocyanine green for anatomical hepatic resection.</a> World Journal of Surgery. 32 (8), 1763-1767 (2008).</li><li>Felli, 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=Laparoscopic+anatomical+liver+resection+for+malignancies+using+positive+or+negative+staining+technique+with+intraoperative+indocyanine+green-fluorescence+imaging.">Laparoscopic anatomical liver resection for malignancies using positive or negative staining technique with intraoperative indocyanine green-fluorescence imaging.</a> HPB. 23 (11), 1647-1655 (2021).</li><li>Ishizawa, T., Zuker, N. B., Kokudo, N., Gayet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+and+negative+staining+of+hepatic+segments+by+use+of+fluorescent+imaging+techniques+during+laparoscopic+hepatectomy.">Positive and negative staining of hepatic segments by use of fluorescent imaging techniques during laparoscopic hepatectomy.</a> Archives of Surgery. 147 (4), 393-394 (2012).</li><li>Procopio, 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=Ultrasound-guided+anatomical+liver+resection+using+a+compression+technique+combined+with+indocyanine+green+fluorescence+imaging.">Ultrasound-guided anatomical liver resection using a compression technique combined with indocyanine green fluorescence imaging.</a> HPB. 23 (2), 206-211 (2021).</li><li>Takemura, N., Ito, K., Inagaki, F., Mihara, F., Kokudo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Added+value+of+indocyanine+green+fluorescence+imaging+in+liver+surgery.">Added value of indocyanine green fluorescence imaging in liver surgery.</a> Hepatobiliary &amp; Pancreatic Diseases International. 21 (4), 310-317 (2022).</li><li>Maluccio, M., Covey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+understanding,+diagnosing,+and+treating+hepatocellular+carcinoma.">Recent progress in understanding, diagnosing, and treating hepatocellular carcinoma.</a> CA: A Cancer Journal for Clinicians. 62 (6), 394-399 (2012).</li><li>Lamade, W., 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+impact+of+3-dimensional+reconstructions+on+operation+planning+in+liver+surgery.">The impact of 3-dimensional reconstructions on operation planning in liver surgery.</a> Archives of Surgery. 135 (11), 1256-1261 (2000).</li><li>Pringle, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notes+on+the+arrest+of+hepatic+hemorrhage+due+to+trauma.">Notes on the arrest of hepatic hemorrhage due to trauma.</a> Annals of Surgery. 48 (4), 541-549 (1908).</li><li>Yamamoto, M., Ariizumi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glissonean+pedicle+approach+in+liver+surgery.">Glissonean pedicle approach in liver surgery.</a> Annals of Gastroenterological Surgery. 2 (2), 124-128 (2018).</li><li>Cho, 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=Relation+between+hepatic+and+portal+veins+in+the+right+paramedian+sector:+proposal+for+anatomical+reclassification+of+the+liver.">Relation between hepatic and portal veins in the right paramedian sector: proposal for anatomical reclassification of the liver.</a> World Journal of Surgery. 28 (1), 8-12 (2004).</li><li>Shindoh, 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=Complete+removal+of+the+tumor-bearing+portal+territory+decreases+local+tumor+recurrence+and+improves+disease-specific+survival+of+patients+with+hepatocellular+carcinoma.">Complete removal of the tumor-bearing portal territory decreases local tumor recurrence and improves disease-specific survival of patients with hepatocellular carcinoma.</a> Journal of Hepatology. 64 (3), 594-600 (2016).</li><li>Ishizawa, T., Saiura, A., Kokudo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+application+of+indocyanine+green-fluorescence+imaging+during+hepatectomy.">Clinical application of indocyanine green-fluorescence imaging during hepatectomy.</a> Hepatobiliary Surgery and Nutrition. 5 (4), 322-328 (2016).</li><li>Nishino, H., 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=What+is+a+precise+anatomic+resection+of+the+liver?+Proposal+of+a+new+evaluation+method+in+the+era+of+fluorescence+navigation+surgery.">What is a precise anatomic resection of the liver? Proposal of a new evaluation method in the era of fluorescence navigation surgery.</a> Journal of Hepato-Biliary-Pancreatic Sciences. 28 (6), 479-488 (2021).</li><li>Le Treut, Y. 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=The+technique+and+outcomes+of+central+hepatectomy+by+the+Glissonian+suprahilar+approach.">The technique and outcomes of central hepatectomy by the Glissonian suprahilar approach.</a> European Journal of Surgical Oncology. 45 (12), 2369-2374 (2019).</li><li>Rotellar, 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=Standardized+laparoscopic+central+hepatectomy+based+on+hilar+caudal+view+and+root+approach+of+the+right+hepatic+vein.">Standardized laparoscopic central hepatectomy based on hilar caudal view and root approach of the right hepatic vein.</a> Journal of Hepato-Biliary-Pancreatic Sciences. 27 (1), 7-8 (2019).</li><li>Muttillo, E. 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The authors have nothing to disclose.

Anatomical hepatectomy is based on portal territory, and its therapeutic effect on liver cancer is still being explored. The theoretical basis for this is that the spread of liver cancer along the portal vein is the main reason for its recurrence and metastasis18. In the 20th century, Makuuchi first proposed anatomical hepatectomy from the initial open portal vein puncture with methylene blue staining, revealing the landmark hepatic veins, which has advanced to the current preoperative analysis of...

Central hepatectomy is the main surgical method used to treat liver malignancies located in segments 4, 5, and 81,2. Standard central hepatectomy requires the complete resection of these segments, which necessitates exposing the sagittal portion, middle hepatic vein (MHV), and right hepatic vein (RHV)3. Segments 4, 5, and 8 belong to the category of central hepatectomy2.
With the advancement of precision liver surgery, anatomical hepatectomy has developed rapidly. However, the method to accurately determine the resection plane of hepatectomy is a key point and area of uncertainty. Generally, the Glisson pedicle is ligated to delimit the surface boundary according to the ischemic area4. For control of the deep plane, anatomical landmarks, including hepatic veins and portal branches, are often used under intraoperative ultrasound guidance during the transection5. Makuuchi&#39;s method shows the resection margin by injecting methylene blue into the portal vein under ultrasound guidance, exposing important hepatic veins at the liver resection plane after removing the specimen5,6,7.
Because hepatocytes specifically absorb indocyanine green (ICG), its fluorescence clearly shows the resection boundary. Anatomical hepatectomy guided by ICG fluorescence was first reported by Aoki in 20088. ICG fluorescent staining methods can be divided into positive and negative staining9. The negative staining technique is performed by intravenous ICG injection after clamping the segmental portal pedicle10. Then, the remnant liver displays fluorescence, but the portal territory does not. Compared with traditional methods, anatomical hepatectomy under fluorescence navigation can not only display the resection boundary on the surface of the liver, but also guide the deep resection plane. For hemihepatectomy and segmental hepatectomy, negative staining technology is recommended. To date, anatomical hepatectomy using fluorescence navigation has become a popular method for liver surgery11,12. This article describes a negative staining technique for fluorescent laparoscopic central hepatectomy. The patients were classified preoperatively by the Child-Pugh scoring system, and accordingly, patients with class A scores were selected13.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BK Flex Focus 800</td>
			<td>BK Medical</td>
			<td>8666-RF</td>
			<td>Intraoperative ultrasound</td>
		</tr>
		<tr>
			<td>Entecavir</td>
			<td>Bristol Myers Squibb</td>
			<td>H20052237</td>
			<td>Antiviral drugs</td>
		</tr>
		<tr>
			<td>Hakko Sonoguide PTC Needle</td>
			<td>Hakko-medical</td>
			<td>&#160;PTC-B 18G/20G</td>
			<td>Portal vein puncture</td>
		</tr>
		<tr>
			<td>Indocyanine green</td>
			<td>Dandong Yichuang Pharmaceutical</td>
			<td>0902007 GH102</td>
			<td>Fluorescent dye</td>
		</tr>
		<tr>
			<td>PINPOINT Endoscopic Fluorescence Imaging System</td>
			<td>Stryker</td>
			<td>PC9000</td>
			<td>Fluorescent laparoscope</td>
		</tr>
		<tr>
			<td>Tenofovir</td>
			<td>GILEAD</td>
			<td>H20180060</td>
			<td>Antiviral drugs</td>
		</tr>
		<tr>
			<td>Trocar</td>
			<td>Ethicon Endo-Surgery</td>
			<td>B5LT/B12LT</td>
			<td>Trocar</td>
		</tr>
	</tbody>
</table>

fluorescent laparoscopic central hepatectomy for liver cancer using indocyanine green negative staining

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ultrasound, important vascular structures, and surgeon experience. With the development of anatomical hepatectomy, visual surgery technology has gradually been applied to this type of surgery, particularly indocyanine green (ICG)-guided anatomical hepatectomy. As ICG can be specifically ingested by hepatocytes and used for fluorescence tracing, negative staining techniques have been applied according to different tumor positions. Under ICG fluorescent guidance, the surface boundary and deep resection plane can be more accurately displayed during liver resection. Thus, the tumor-bearing liver segment can be anatomically removed, which helps to avoid damage to important vessels and reduce ischemia or congestion of the remaining liver tissue. Finally, the incidence of postoperative biliary fistula and liver dysfunction is reduced; therefore, a better prognosis is obtained after the resection of liver cancer. Centrally located liver cancer is usually defined as a tumor located at segments 4, 5, or 8 that requires resection of the middle section of the liver. These are among the most difficult hepatectomies to perform because of the large surgical wounds and multiple vessel transections. Based on the specific tumor location, we formulated the required resection ranges by designing personalized fluorescent staining strategies. By completing anatomical resection based on the portal territory, this work aims to achieve the best therapeutic effect.

From 2020 to 2022, six patients with HCC underwent fluorescent laparoscopic central hepatectomy. Among these, four patients underwent resection of segments 4, 5, and 8, and two patients underwent resection only of segments 5 and 8 (Table 1). None of the patients were converted to open surgery. Their ages ranged from 46 to 74 years. The tumor sizes ranged from 5 to 9 cm. The median operative time was 240 min, and the median intraoperative blood loss was 450 mL. The median postoperative hospital stay was 7...

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Fluorescent Laparoscopic Central Hepatectomy for Liver Cancer Using Indocyanine Green Negative Staining

The present protocol describes fluorescent negative staining in laparoscopic central hepatectomy. This technique can make hepatectomy more accurate and precise.

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ...

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631 Views.  Sun Yat-sen University. The present protocol describes fluorescent negative staining in laparoscopic central hepatectomy. This technique can make hepatectomy more accurate and precise. 

Video: Fluorescent Laparoscopic Central Hepatectomy for Liver Cancer Using Indocyanine Green Negative Staining

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare.

Here, we describe a minimally invasive laparoscopic technique for serial sampling of liver and mesenteric lymph nodes (MLN) in macaques that allows for increased sampling frequency, and reduces the potential for surgical complications when compared to performing a laparotomy.

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

The acute liver failure animal model developed in the current study presents a feasible alternative for the study of potential therapies. The current model employs the combined effect of physical and drug-induced hepatic injury and provides a suitable time window to study the potential of novel therapies.

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

Laparoscopic Radical Left Pancreatectomy for Pancreatic Cancer: Surgical Strategy and Technique Video

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological resection of left-sided pancreatic cancer. Several surgical techniques have been described in recent years, but few were specifically designed for minimally invasive approaches. This study describes and demonstrates a standardized and reproducible technique for an adequate oncological resection of pancreatic cancer: laparoscopic radical left pancreatectomy (LRLP).
A 61-year-old woman presented with an incidental finding of a 3 cm mass in the left pancreas suspect for malignancy. Imaging did not reveal distant metastases, central vascular involvement, or morbid obesity, hence the patient was suitable for LRLP. This study describes the main steps of LRLP for pancreatic cancer. First, the lesser sac is opened by transecting the gastrocolic ligament. The splenic flexure of the colon is mobilized and the inferior border of the pancreas including Gerota&#39;s fascia is dissected down to the inferior border of the spleen. The pancreas is tunneled and hung, including Gerota&#8217;s fascia with a vessel loop. At the pancreatic neck, a tunnel is created between the pancreas and the portal vein, likewise a vessel loop is passed. The pancreas is then transected using the graded compression technique with an endostapler. Both the splenic vein and artery are transected before completing the resection. The entire specimen is extracted in a retrieval bag via a small Pfannenstiel incision.
Duration of the surgery was 210 min with 250 mL blood loss. Pathology revealed a R0-resection (&#62;1 mm) of a well-to-moderately differentiated adenocarcinoma originating from an intraductal papillary mucinous neoplasm. A total of 15 tumor-negative lymph nodes were resected. This is a detailed description of LRLP for left-sided pancreatic cancer as is currently being used within the international, multicenter randomized DIPLOMA (Distal Pancreatectomy Minimally Invasive or Open for PDAC) trial.

Oncologically safe left pancreatectomy requires radical resection (R0), Gerota&#8217;s (perirenal) fascia resection, and adequate lymph node dissection. This study describes the technical details of laparoscopic radical left pancreatectomy (LRLP), used in the first international multicenter randomized trial comparing minimally invasive with open left pancreatectomy for pancreatic cancer, the DIPLOMA trial.

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

Indocyanine Green-Guided Intraoperative Imaging to Facilitate Video-Assisted Retroperitoneal Debridement for Treating Acute Necrotizing Pancreatitis

Video-assisted retroperitoneal debridement (VARD) is a feasible, minimally invasive necrosectomy method for treating severe acute necrotizing pancreatitis, if it does not resolve or is accompanied with infected necrosis in the retroperitoneum. As there are rarely any visually clear separating surface in white light image between necrotic debris and adjacent inflammatory normal tissues due to extensive retroperitoneal adhesions, VARD is accompanied with the risk of vascular injury, external pancreatico-cutaneous or enterocutaneous fistulae. In view of the above disadvantages, we apply real-time intraoperative near-infrared fluorescence imaging with indocyanine green (ICG) during VARD, which enables visualization of the well-perfused adjacent normal tissues. This modified technique (ICG-guided VARD) can provide a clear separating surface during debridement and reduce the risk of vascular or enteric injury. ICG-guided VARD may facilitate surgeons to perform safer debridement in treating severe acute necrotizing pancreatitis.

This protocol presents Indocyanine Green-guided video-assisted retroperitoneal debridement (ICG-guided VARD) for treating severe acute necrotizing pancreatitis.

Video-assisted retroperitoneal debridement (VARD) is a feasible, minimally invasive necrosectomy method for treating severe acute necrotizing ...

Robotic Left Hepatectomy using Indocyanine Green Fluorescence Imaging for an Intrahepatic Complex Biliary Cyst

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. Surgery is the cornerstone treatment for patients with BC. While total BC excision and Roux-Y hepaticojejunostomy is the treatment method of the choice in patients with extrahepatic BC (i.e., Todani I-IV), patients with intrahepatic BC (i.e., Todani V) benefit the most from a surgical liver resection. In recent years, minimally invasive liver surgery (MILS) including robotic MILS has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. Robotic major MILS is still considered technically demanding and a detailed description of the technical approach during robotic major MILS has only been limitedly discussed in the literature. The current article describes the main steps for a robotic left hepatectomy in a patient with a large BC Todani Type V. The patient is in French position with 5 trocars placed (4 robotic, 1 laparoscopic assistant). After mobilizing the left hemiliver, the left and right hepatic artery are dissected carefully followed by a cholecystectomy. Intraoperative ultrasound is performed to confirm localization and margins of the BC. The Left hepatic artery and left portal vein are isolated, clipped, and divided. Indocyanine green (ICG) fluorescence imaging is used regularly during the entire procedure to visualize and confirm biliary tract anatomy and the BC. Parenchymal transection is performed with robotic cautery hook for the superficial part and robotic cautery spatula, bipolar cautery, and vessel sealer for the deeper parenchyma. The postoperative course was uncomplicated. A robotic left hepatectomy is technically demanding, yet a feasible and safe procedure. ICG-fluorescence imaging aids in delineating the BC and bile duct anatomy. Further, comparative studies are needed to confirm clinical benefits of robotic MILS for benign and malignant indications.

Robotic liver surgery has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. However, robotic left hepatectomy is still technically demanding. We describe our surgical technique of a robotic left hepatectomy using indocyanine green fluorescence imaging for a large biliary cyst.

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. ...

Laparoscopic Pancreatoduodenectomy for Pancreatic Cancer Using In-Situ No-Touch Isolation Technique

Laparoscopic pancreatoduodenectomy (LPD) is a standard radical operation for pancreatic head malignant tumors by now. Due to the complex laparoscopic resection and reconstruction techniques, it is difficult to perform LPD for patients with locally advanced pancreatic head cancer after neoadjuvant therapy. Our team initiates LPD using the in-situ No-Touch isolation technique. The innovation and optimization of this modified No-Touch isolation technique emphasize exploring the distal section of superior mesenteric vein&#160;(SMV) and the left side of the superior mesenteric artery (SMA) prior to evaluating the resectability by subcolonic mesenteric approach, which is an ideal exploring approach. After that, we use the median-anterior, and left-posterior of SMA approaches to cut off the blood flow of the pancreatic head to make the tumor isolated intact, then move and dissect the tumor. It is a process fitting the surgical principle of tumor-free. This article aims to demonstrate the feasibility and safety of performing LPD using the in-situ No-Touch isolation technique, which might elevate the R0 resection rate. It is an oncological ideal operation process.

Laparoscopic Pancreatoduodenectomy for Pancreatic Cancer Using In-Situ No-Touch Isolation Technique

No-Touch isolation procedures might prevent the dissemination of cancer cells from the primary tumor. However, these techniques are not widely accepted in laparoscopic pancreatoduodenectomy (LPD) by now. We herein present in-situ No-Touch isolation LPD with partial resection and reconstruction of the superior mesenteric vein (SMV) for pancreatic cancer after neoadjuvant therapy.

Laparoscopic pancreatoduodenectomy (LPD) is a standard radical operation for pancreatic head malignant tumors by now. Due to the complex laparoscopic ...