The authors have no acknowledgments.

The patient gave written and oral informed consent to use medical data and the operative video for education and scientific purposes. This research was performed in compliance with all institutional, national and international guidelines for human welfare. Written informed consent was obtained from the patient for publication of this manuscript and any accompanying images.
1. Positioning
<ol>
	<li>Place the patient on a vacuum mattress in a supine French position (legs split). Lower the right arm alongside the body on an arm support and extend the left arm. Tilt the operating table 10-20&#176; in anti-Trendelenburg and 5-10&#176; to the left.</li>
</ol>
2. Robot docking
<ol>
	<li>After Verres needle insufflation on Palmers&#39; point, introduce four 8 mm robot trocars (R1-4) in a semi-curved line just above the umbilicus. The distance between the trocars is 6-7 cm: R1 in the right anterior axillary line, R2 in the right midclavicular line, R3 just right and above the umbilicus (camera) and R4 just medial in the left midclavicular line.</li>
	<li>Introduce two assistant 5 mm trocars 3-4 cm below to the left (vessel sealing device) and right of the umbilicus.</li>
</ol>
3. Mobilization
<ol>
	<li>Mobilize the hepatic flexure of the colon using robotic diathermia or laparoscopic sealing device.</li>
	<li>Introduce the liver retractor from the left and retract the liver from segment III and IV. This enables optimal exposure of the surgical site. Optionally, it could suspend the stomach.</li>
	<li>Identify the gastrocolic ligament and divide it with the vessel sealing device, hence opening the lesser sac.</li>
	<li>Continue the mobilization from lateral to medial until the hepatic flexor of the colon is freed.</li>
	<li>Perform Kocher&#39;s maneuver until the left renal vein is identified. Hereafter, dissect the right gastroepiploic vein free and ligate using a vessel sealer. Both steps are optional but improve exposure and control which may be useful in case of bleeding from the pancreatic head.</li>
	<li>Retract the pancreas and duodenum with the third robotic arm to entirely expose the abdominal aorta and inferior vena cava.</li>
	<li>Identify the right gastroepiploic vein and divide it with the laparoscopic sealing device and clips.</li>
	<li>Mobilize the pancreatic head using the cautery hook.</li>
</ol>
4. Intraoperative ultrasonography
<ol>
	<li>Introduce the ultrasonography probe and identify the trajectory of the pancreatic duct and the intra-pancreatic lesion.</li>
	<li>Demarcate the lesion with the cautery hook, with help of the ultrasonography probe.</li>
</ol>
5. Dissection
<ol>
	<li>Place a traction suture through the lesion.</li>
	<li>Lift the lesion up with the traction suture and enucleate the lesion circumferentially with the diathermic scissors.</li>
	<li>Cut a finger from a sterile surgical glove and introduce it into the abdominal cavity. Put the enucleated tissue in the finger and extract the specimen. The roof of the pancreatic duct is visible at the bottom of the enucleation site.</li>
	<li>Cut a finger from a sterile surgical glove and insert a dry sealant patch. Introduce the finger into the abdominal cavity. Position the sealant patch on the defect in the pancreatic parenchyma. Place two wet 10 x 10 cm gauzes on top and remove the gauzes after 3-5 minutes, the sealant patch remains on the pancreatic head. 
	NOTE: The sealant patch should not be made wet before positioning. Placement of a patch is optional; studies did not demonstrate its effectiveness in reducing the risk of pancreatic fistulas.</li>
</ol>
6. Drain placement
<ol>
	<li>Introduce a 18-20 French drain from the right side of the patient and advance it over the pancreatic head.</li>
	<li>Test produced drain fluids for amylase levels on the first and third postoperative day postoperatively to test for post-operative fistula. Consider placement of a stent in the pancreatic duct if the amylase level consistently exceeds 3 times the upper limit of the institutions normal serum amylase9.</li>
</ol>

<ol>
	<li>Falconi, 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=ENETS+Consensus+Guidelines+Update+for+the+Management+of+Patients+with+Functional+Pancreatic+Neuroendocrine+Tumors+and+Non-Functional+Pancreatic+Neuroendocrine+Tumors.">ENETS Consensus Guidelines Update for the Management of Patients with Functional Pancreatic Neuroendocrine Tumors and Non-Functional Pancreatic Neuroendocrine Tumors.</a> Neuroendocrinology. 103 (2), 153-171 (2016).</li><li>Su, A. 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=Is+laparoscopic+approach+for+pancreatic+insulinomas+safe?+Results+of+a+systematic+review+and+meta-analysis.">Is laparoscopic approach for pancreatic insulinomas safe? Results of a systematic review and meta-analysis.</a> Journal of Surgical Research. 186 (1), 126-134 (2014).</li><li>Shi, 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=Pancreatic+enucleation+using+the+da+Vinci+robotic+surgical+system:+a+report+of+26+cases.">Pancreatic enucleation using the da Vinci robotic surgical system: a report of 26 cases.</a> The International Journal of Medical Robotics. 12 (4), 751-757 (2016).</li><li>Tian, 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=Propensity+score-matched+analysis+of+robotic+versus+open+surgical+enucleation+for+small+pancreatic+neuroendocrine+tumours.">Propensity score-matched analysis of robotic versus open surgical enucleation for small pancreatic neuroendocrine tumours.</a> British Journal of Surgery. 103 (10), 1358-1364 (2016).</li><li>Zhang, 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=Enucleation+of+pancreatic+lesions:+indications,+outcomes,+and+risk+factors+for+clinical+pancreatic+fistula.">Enucleation of pancreatic lesions: indications, outcomes, and risk factors for clinical pancreatic fistula.</a> Journal of Gastrointestinal Surgery. 17 (12), 2099-2104 (2013).</li><li>Liang, 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=Robotic+enucleation+for+pediatric+insulinoma+with+MEN1+syndrome:+a+case+report+and+literature+review.">Robotic enucleation for pediatric insulinoma with MEN1 syndrome: a case report and literature review.</a> BMC Surgery. 18 (1), 44 (2018).</li><li>Ore, A. S., Barrows, C. E., Solis-Velasco, M., Shaker, J., Moser, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robotic+enucleation+of+benign+pancreatic+tumors.">Robotic enucleation of benign pancreatic tumors.</a> Journal of Visualized Surgery. 3 (10), (2017).</li><li>Jin, J. B., 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=Robotic+Enucleation+for+Benign+or+Borderline+Tumours+of+the+Pancreas:+A+Retrospective+Analysis+and+Comparison+from+a+High-Volume+Centre+in+Asia.">Robotic Enucleation for Benign or Borderline Tumours of the Pancreas: A Retrospective Analysis and Comparison from a High-Volume Centre in Asia.</a> World Journal of Surgery. 40 (12), 3009-3020 (2016).</li><li>Bassi, C., 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+2016+update+of+the+International+Study+Group+(ISGPS)+definition+and+grading+of+postoperative+pancreatic+fistula:+11+Years+After.">The 2016 update of the International Study Group (ISGPS) definition and grading of postoperative pancreatic fistula: 11 Years After.</a> Surgery. 161 (3), 584-591 (2017).</li><li>Boone, B. 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=Assessment+of+quality+outcomes+for+robotic+pancreaticoduodenectomy:+identification+of+the+learning+curve.">Assessment of quality outcomes for robotic pancreaticoduodenectomy: identification of the learning curve.</a> JAMA Surgery. 150 (5), 416-422 (2015).</li><li>Zureikat, A. 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=250+robotic+pancreatic+resections:+safety+and+feasibility.">250 robotic pancreatic resections: safety and feasibility.</a> Annals of Surgery. 258 (4), 559-562 (2013).</li><li>Jilesen, A. P. 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=Postoperative+Outcomes+of+Enucleation+and+Standard+Resections+in+Patients+with+a+Pancreatic+Neuroendocrine+Tumor.">Postoperative Outcomes of Enucleation and Standard Resections in Patients with a Pancreatic Neuroendocrine Tumor.</a> World Journal of Surgery. 40 (3), 715-728 (2016).</li><li>Crippa, 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=Surgical+Management+of+Insulinomas:+Short-+and+Long-term+Outcomes+After+Enucleations+and+Pancreatic+Resections.">Surgical Management of Insulinomas: Short- and Long-term Outcomes After Enucleations and Pancreatic Resections.</a> JAMA Surgery. 147 (3), 261-266 (2012).</li><li>Li, 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=Laparoscopic+ultrasound+enhances+diagnosis+and+localization+of+insulinoma+in+pancreatic+head+and+neck+for+laparoscopic+surgery+with+satisfactory+postsurgical+outcomes.">Laparoscopic ultrasound enhances diagnosis and localization of insulinoma in pancreatic head and neck for laparoscopic surgery with satisfactory postsurgical outcomes.</a> Ultrasound in Medicine and Biology. 37 (7), 1017-1023 (2011).</li><li>Lu, W. 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=Enucleation+of+benign+or+borderline+pancreatic+head+tumors+adjacent+to+the+common+pancreatic+duct.">Enucleation of benign or borderline pancreatic head tumors adjacent to the common pancreatic duct.</a> Pancreas. 41 (2), 336-337 (2012).</li><li>Zhang, X. 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=Margin+status+and+long-term+prognosis+of+primary+pancreatic+neuroendocrine+tumor+after+curative+resection:+Results+from+the+US+Neuroendocrine+Tumor+Study+Group.">Margin status and long-term prognosis of primary pancreatic neuroendocrine tumor after curative resection: Results from the US Neuroendocrine Tumor Study Group.</a> Surgery. 165 (3), 548-556 (2019).</li><li>Gkegkes, I. D., Mamais, I. A., Iavazzo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robotics+in+general+surgery:+A+systematic+cost+assessment.">Robotics in general surgery: A systematic cost assessment.</a> Journal of Minimal Access Surgery. 13 (4), 243-255 (2017).</li></ol>

The authors have nothing to disclose.

There are six critical steps highlighted here: positioning and robot docking, mobilization, intraoperative ultrasonography, traction suture, dissection, and drain placement. Conversion to laparotomy should be performed in case of uncontrollable intraoperative bleeding or in case the tumor is not adequately located with ultrasound.
A liver retractor is useful to expose the surgical site. Intraoperative ultrasonography plays an important role in enabling a parenchyma-sparing enucleation. Preferably, a (interventional) radiologist should identify the lesion and especially its relationship with the pancreatic duct14. Applying a traction suture through the lesion eases the dissection, especially in a parenchyma-sparing resection.
Besides general complications of surgery, pancreatic fistula should be closely monitored after this procedure15. As shown here, the lesion had a close relationship with the pancreatic duct with only 1-2 mm distance between the insulinoma and the pancreatic duct. Because of this risk, a retro pancreatic drain was positioned and monitored at least during the first postoperative days9.
A microscopically margin-positive (R1) resection was obtained. Although microscopically margin-negative (R0) resection would be preferably, this is not deemed necessary, as this is not associated with improved long-term overall survival in pancreatic neuroendocrine tumors16.
Limitations of robotic surgery are the availability of the robotic system, the need for specific training, lack of tactile feedback and high costs17. The added degrees of freedom obtained by the robotic system may be useful for enucleation of insulinomas, especially in case of a close proximity to the pancreatic duct, as in this demonstrated case.
Robotic enucleation of insulinomas seems feasible; still, future prospective studies should confirm this suggestion. We believe that the described technique, with adequate ultrasonography guided localization of the lesion, could be a valuable alternative for open enucleation as stated in guidelines1. Further studies are needed to compare short- and long-term outcomes after robotic, open, and laparoscopic enucleation.

Insulinoma is the most prevalent functioning pancreatic neuroendocrine tumor (F-PNET) with an annual incidence of 1-32/100,000 patients1. Pancreas-sparing surgery (i.e., enucleation) is mostly indicated for single lesions as pancreatic resections could be needed in multifocal or more extensive lesions1. General advantages of parenchymal-sparing enucleation over pancreatoduodenectomy or distal pancreatectomy include function preservation (both exocrine and endocrine), less blood loss, shorter operative time, and the absence of high risk anastomoses as required after pancreatoduodenectomy and central pancreatectomy.
A minimally invasive surgical approach aims to shorten the time to functional recovery with comparable oncologic outcomes1,2. Compared to open enucleation, robotic enucleation is associated with a shorter operative time and lower blood loss with a similar risk of postoperative pancreatic fistulas and major postoperative complications3,4. Compared to laparoscopic enucleation, robotic enucleation seems to be associated with less intraoperative blood loss, which could be related to the additional degrees of freedom during dissection that could lead to more accurate dissection5.
Three studies have so far addressed robotic enucleation of pancreatic neoplasms, one of which describes the technique to enucleate an insulinoma in the pediatric setting, the others describe techniques to enucleate benign pancreatic lesions6,7,8. In this study, we present a technique for robotic enucleation of an insulinoma originating from the pancreas. We fully acknowledge that many variations are possible to nearly every step. Accurate identification and meticulous dissection, especially with regards to the main pancreatic duct, are crucial.
This case shown here involves a 42-year old woman who presented with sweating, tremor and episodes of hypoglycemia. A fasting test confirmed endogenic insulin overproduction. CT and MRI were inconclusive; therefore, an endoscopic ultrasound of the pancreatic head was made. Endoscopic ultrasonography showed a non-bulging, hypoechoic lesion, which was fully embedded within the pancreatic head at 1-2 mm distance from the main pancreatic duct. The patient was consented for both a robotic pancreatoduodenectomy procedure and a robotic enucleation. Intraoperatively, the final decision was made to perform an enucleation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Arietta V70 Ultrasound</td>
			<td>Hitachi</td>
			<td>-</td>
			<td>The ultrasound system.</td>
		</tr>
		<tr>
			<td>Cobra Liver Retractor Diamond-Flex</td>
			<td>CareFusion</td>
			<td>89-6216</td>
			<td>Retracting the liver for optimal exposure of the surgical site.</td>
		</tr>
		<tr>
			<td>da Vinci Surgeon Console</td>
			<td>Intuitive Surgical</td>
			<td>SS999</td>
			<td>Used to control the surgical robot.</td>
		</tr>
		<tr>
			<td>da Vinci Vision Cart</td>
			<td>Intuitive Surgical</td>
			<td>VS999</td>
			<td>The vision cart houses advanced vision and energy technologies and provides communications across da Vinci system components.</td>
		</tr>
		<tr>
			<td>da Vinci Xi</td>
			<td>Intuitive Surgical</td>
			<td>K131861</td>
			<td>The surgical robot: &#39;patient side-cart&#39;.</td>
		</tr>
		<tr>
			<td>da Vinci Xi Endoscope with Camera, 8 mm, 30&#176;</td>
			<td>Intuitive Surgical</td>
			<td>470027</td>
			<td>The camera of the da Vinci robot.</td>
		</tr>
		<tr>
			<td>ENDOEYE Rigid Video Laparoscope, 10 mm, 30&#176;</td>
			<td>Olympus</td>
			<td>WA50042A</td>
			<td>To see within the intra-abdominal cavity.</td>
		</tr>
		<tr>
			<td>ENDOWRIST Fenestrated Bipolar Forceps</td>
			<td>Intuitive Surgical</td>
			<td>470205</td>
			<td>Used for dissection and coagulation.</td>
		</tr>
		<tr>
			<td>ENDOWRIST HOT SHEARS</td>
			<td>Intuitive Surgical</td>
			<td>470179</td>
			<td>Used for cutting and coagulation.</td>
		</tr>
		<tr>
			<td>ENDOWRIST Mega SutureCut Needle Driver</td>
			<td>Intuitive Surgical</td>
			<td>470309</td>
			<td>Used as a needle driver.</td>
		</tr>
		<tr>
			<td>ENDOWRIST Permanent Cautery Hook</td>
			<td>Intuitive Surgical</td>
			<td>470183</td>
			<td>Used for coagulation.</td>
		</tr>
		<tr>
			<td>ENDOWRIST PROGrasp Forceps</td>
			<td>Intuitive Surgical</td>
			<td>470093</td>
			<td>Used for dissection.</td>
		</tr>
		<tr>
			<td>LigaSure Dolphin Tip 37cm</td>
			<td>Medtronic</td>
			<td>LS1500</td>
			<td>Used for vessel sealing and dividing.</td>
		</tr>
		<tr>
			<td>Robotic ultrasonography transducer</td>
			<td>Hitachi</td>
			<td>L43K</td>
			<td>Used for intraoperative laparoscopic ultrasonography.</td>
		</tr>
		<tr>
			<td>TachoSil 4.8 cm x 4.8 cm</td>
			<td>Baxter Healthcare Corporation</td>
			<td>1144923</td>
			<td>Used for coagulation.</td>
		</tr>
	</tbody>
</table>

robotic enucleation of an intra-pancreatic insulinoma in the pancreatic head

Pancreatic parenchyma sparing surgery for insulinomas avoids the risk of endocrine and exocrine insufficiency, and potential high-risk anastomoses associated with pancreatic resection. Robotic surgery may be used as an alternative for open pancreatic enucleation without compromising dexterity and 3D-vision.
We present the case of a 42-year old woman who presented with sweating, tremor and episodes of hypoglycemia. A fasting test confirmed endogenic insulin overproduction. After inconclusive CT- and MRI imaging, endoscopic ultrasonography showed a hypoechoic lesion, which was fully within the pancreatic head. Although consent was obtained for pancreatoduodenectomy, robotic enucleation seemed feasible. After mobilization, intraoperative ultrasonography was used to identify the lesion and its relation with the pancreatic duct. Dissection was performed using a traction suture, hot shears and bipolar diathermia. A sealant patch was applied for hemostasis and a drain placed. The patient developed a grade B pancreatic fistula for which endoscopic sphincterotomy was performed; the surgical drain could be removed in the outpatient clinic after 20 days. Prospective studies should confirm the short- and long-term benefits of robotic enucleation of insulinomas.

Total operation time was 180 minutes with a blood loss of 5 mL. At the third postoperative day, drain amylase levels were still elevated. We therefore decided to attempt a stent placement in the pancreatic duct. During ERCP, this was technically not feasible, thus a pancreatic sphincterotomy was performed. This was classified as a grade B postoperative pancreatic fistula, due to the ERCP intervention9. The patient was discharged on postoperative day 7. After drain amylase had normalized, the drain could be removed in the outpatient clinic on postoperative day 20.
Histopathological examination revealed a grade 1 (mitotic index &#60;2/mm2 and Ki67 &#60;3%) well-differentiated neuroendocrine tumor measuring 1.5 cm with positive insulin staining (see Figure 1). Tumor cells were microscopically present at the resection margin (R1).
Comparable results from literature 
In general, a robotic docking time of 5-10 min has been described10 as well as an operative time of 206 &#177; 67 min, operative blood loss 43 ml (IQR 27-98)11 and a median tumor size of pancreatic neuroendocrine tumours of 16 mm (IQR 11-22)12. The expected postoperative hospital stay is 5 days (IQR 3-12)11, major morbidity rate 30%11, with a very low in hospital mortality rate (0%)12,13. The rate of clinically relevant pancreatic fistula rate is reported to be 30-40%11,12 and the delayed gastric emptying rate 0-26%12,13.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60290/60290fig1a.jpg" /> 
Figure 1: Specimen. <a href="https://www.jove.com/files/ftp_upload/60290/60290fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Robotic Enucleation of an Intra-Pancreatic Insulinoma in the Pancreatic Head at JoVE.com

Robotic Enucleation of an Intra-Pancreatic Insulinoma in the Pancreatic Head

Here, we present a robotic approach to enucleate an insulinoma in the pancreatic head.

Pancreatic parenchyma sparing surgery for insulinomas avoids the risk of endocrine and exocrine insufficiency, and potential high-risk anastomoses ...

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7.8K Views.  University of Amsterdam. Here, we present a robotic approach to enucleate an insulinoma in the pancreatic head.

Video: Robotic Enucleation of an Intra-Pancreatic Insulinoma in the Pancreatic Head

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Robotic Lateral Pancreaticojejunostomy for Chronic Pancreatitis

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The recent rise of robotic and laparoscopic pancreatic surgery has found benefits such as reduced time to functional recovery. Few studies have reported on the feasibility, technique and outcome of robotic LPJ, especially including transection of the gastroduodenal artery. The present study describes the main steps for robotic LPJ in a patient with painful chronic pancreatitis with a dilated main pancreatic duct. The patient underwent robotic LPJ. The LPJ anastomosis is performed using a running suture technique in a longitudinal side-to-side manner. Routinely, the gastroduodenal artery is transected to drain the entire length of the main pancreatic duct. The patient is in French position; 7 trocars are placed (4 robotic, 2 laparoscopic assistants, 1 liver-retractor). After docking of the robot system, the omental bursa is opened, and the right gastroepiploic artery and vein are ligated at their base at the lower border of the pancreas. Intraoperative ultrasonography is performed in order to determine trajectory of the dilated main pancreatic duct which is opened for its entire length after the gastroduodenal artery has been suture ligated both cranially and caudally from the main pancreatic duct. A Roux limb is created, and a latero-lateral PJ is fashioned using several 3-0 barbed sutures. A stapled jejuno-jejunostomy is created at sufficient distance from the pancreatic anastomosis, aided by a 50 cm suture. The described technique for robotic LPJ is a complex but feasible operation for patients with treatment refractory CP and a dilated main pancreatic duct. Due to its complexity, implementation in high volume centers with extensive experience with CP surgery may improve outcomes.

Robotic lateral pancreaticojejunostomy (RLPJ) may be used in patients with painful, morphine dependent, chronic pancreatitis and a dilated main pancreatic duct. We describe a standardized and reproducible technique for RLPJ, which includes transection of the gastroduodenal artery.

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Robotic Taj Mahal Hepatectomy for Hilar Cholangiocarcinoma

Hilar cholangiocarcinoma is the most common malignant tumor of the biliary tract. Radical surgical resection is the only effective treatment option. In this study, a 32-year-old male patient with Bismuth Type IVa hilar cholangiocarcinoma underwent radical robotic resection of hepatic S4b, S5, and S1 (Taj Mahal hepatectomy) combined with regional lymphadenectomy, hilar bile duct reconstruction, and hepaticojejunostomy by the robotic surgical system. Postoperative pathological examination showed moderately-differentiated adenocarcinoma of the hilar bile duct. The surgical margins of the liver and bile ducts were negative. Recovery was smooth and the patient was discharged on the 17th postoperative day. The robotic surgical system and associated multiple instruments along with flexible and precise movements is suitable for the local hepatectomy around the porta hepatis, and delicate reconstruction of the hilar bile duct with a smaller diameter. This first clinical application study found that robotic Taj Mahal hepatectomy for hilar cholangiocarcinoma is safe and feasible and needs more experience for the evaluation of its long-term outcomes.

Robotic resection of hepatic S4b, S5, and S1 using the Taj Mahal procedure is feasible and safe for selected patients with hilar cholangiocarcinoma. The step-by-step details for this surgery are presented here.

Hilar cholangiocarcinoma is the most common malignant tumor of the biliary tract. Radical surgical resection is the only effective treatment option. In ...