This study did not receive any funding.

The Institutional Review Committee of the Istanbul Faculty of Medicine approved the study protocol. Before the start of the study, the patient was asked to sign an informed consent form outlining the purpose of use and publication of their data.
1. Preoperative procedures
<ol>
	<li>Perform the operations under general anesthesia. Prepare endoscopic and optical equipment in the operating room.</li>
	<li>Check the instruments before starting the procedure (Figure 1 and Table 1).</li>
</ol>
2. Surgical technique (Figure 2)
<ol>
	<li>Place the patient in the supine position under general anesthesia. Support the patient&#39;s head with a soft head support and bend it at a 10&#176; angle.</li>
	<li>Palpate the coronal suture. The Kocher point is just anterior to the coronal suture and approximately 2.5 cm to the right of the midline.</li>
	<li>Begin the operation by following routine asepsis and antiseptic rules.
	<ol>
		<li>Make a 5 cm curvilinear incision in the right frontal region, including Kocher&#39;s point, using a #20 blade.</li>
		<li>Ensure hemostasis with bipolar cautery and dissect the skin and subcutaneous tissue.</li>
		<li>Place the automated skin retractor.</li>
		<li>Scrape the periosteum using an Adson periosteal elevator.</li>
		<li>Drill a burr hole in the anterior region using a high-speed drill and perforator.</li>
		<li>Open the dura in a cruciform shape with a #11 blade and coagulate the underlying pia with bipolar cautery.</li>
		<li>Insert the endoscope sheath into the brain parenchyma perpendicular to the burr hole and remove the obturator while holding the endoscope sheath. After entering the lateral ventricle, cerebrospinal fluid is seen to exit the endoscope sheath. 
		NOTE: The examination of tumor markers such as alpha-feto-protein and B-Hcg in the cerebrospinal fluid (CSF) sample increases the accuracy of the histological examination.</li>
		<li>Insert the 6.1 mm wide endoscope into the endoscope sheath. Visualize the lateral ventricular cavity.
		<ol>
			<li>Identify the septal and thalamostriate veins in the lateral ventricle.</li>
			<li>Identify the choroid plexus. Follow the choroid plexus anteriorly up to the third ventricle going through the Foramen of Monro (FOM).</li>
		</ol>
		</li>
		<li>Pass the ventriculoscope through the FOM to access the third ventricle.</li>
		<li>Find the floor of the third ventricle, which is usually thin due to hydrocephalus.
		<ol>
			<li>Identify the mammillary bodies and infundibular recess in the third ventricle.</li>
			<li>Ensure that the hole at the base of the third ventricle is located at the most translucent point between the infundibular recess and the mammillary bodies.</li>
		</ol>
		</li>
		<li>Locate the basilar artery and puncture anterior to the arterial complex to prevent damage and hemorrhage during surgery.</li>
		<li>Use the tip of the 4F Fogarty catheter to puncture the base of the third ventricle. Then, repeatedly inflate and deflate the balloon of the catheter to widen the opening. See the jet flow of cerebrospinal fluid when the catheter balloon is deflated.</li>
		<li>If bleeding occurs during surgery, irrigate with copious amounts of warm fluid until all bleeding has visibly stopped and CSF has cleared. 
		NOTE: There are different opinions in the literature about the fluids used to manage bleeding. However, we use Ringer&#39;s lactate solution at normal body temperature in our clinic.</li>
		<li>Move the endoscope to the posterior region of the third ventricle where the pineal gland tumor is located. 
		NOTE: The entry point and angle of the endoscope play a pivotal role in accessing these two structures, which are positioned at different locations. This is essential to avoid damage to the fornix and critical vascular structures.</li>
		<li>Identify the massa intermedia and perform dissection to allow better visualization of the posterior third ventricle.</li>
		<li>Identify the tumor and examine the growth pattern. Carefully coagulate the surface of the tumor using bipolar cautery.</li>
		<li>After complete coagulation of the tumor surface, take a biopsy using biopsy forceps. Send the biopsy to the pathology unit for frozen analysis and resect the tumor according to the histopathological diagnosis.</li>
		<li>Control local hemorrhage with irrigation and bipolar cautery.</li>
		<li>Remove the Endoscopic System.</li>
		<li>Close the wound with sutures without a drain.</li>
	</ol>
	</li>
</ol>
3. Postoperative procedures
<ol>
	<li>The hospital stay is minimal. Mobilize the patient on the second day following surgery.</li>
	<li>Perform non-contrast cranial CT and CSF flow MRI to evaluate the patency of the third ventriculostomy and exclude the presence of bleeding.</li>
	<li>Discharge patients the following day if there are no complaints.</li>
	<li>Ask the patients to follow the pathology results and attend the outpatient clinic on the tenth day and at the end of the first month.</li>
</ol>

Endoscopy from Kocher&#39;s Point Enabling Third Ventriculostomy and Pineal Biopsy

<ol>
	<li>Favero, G., Bonomini, F., Rezzani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pineal+gland+tumors:+A+review.">Pineal gland tumors: A review.</a> Cancers (Basel). 13 (7), 1547 (2021).</li><li>Nagasawa, D. 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=Pineal+germ+cell+tumors:+Two+cases+with+review+of+histopathologies+and+biomarkers).">Pineal germ cell tumors: Two cases with review of histopathologies and biomarkers).</a> J Clin Neurosci. 38, 23-31 (2017).</li><li>Tamrazi, B., Nelson, M., Bl&#252;ml, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pineal+region+masses+in+pediatric+patients.">Pineal region masses in pediatric patients.</a> Neuroimaging Clin N Am. 27 (1), 85-97 (2017).</li><li>Carr, C., O'Neill, B. E., Hochhalter, C. B., Strong, M. J., Ware, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+pineal+region+tumors:+A+review.">Biomarkers of pineal region tumors: A review.</a> Ochsner J. 19 (1), 26-31 (2019).</li><li>Iorio-Morin, 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=Histology-stratified+tumor+control+and+patient+survival+after+stereotactic+radiosurgery+for+pineal+region+tumors:+A+report+from+the+international+gamma+knife+research+foundation.">Histology-stratified tumor control and patient survival after stereotactic radiosurgery for pineal region tumors: A report from the international gamma knife research foundation.</a> World Neurosurg. 107, 974-982 (2017).</li><li>Liu, 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=Simultaneous+single-trajectory+endoscopic+biopsy+and+third+ventriculostomy+in+pediatric+pineal+region+tumors.">Simultaneous single-trajectory endoscopic biopsy and third ventriculostomy in pediatric pineal region tumors.</a> Acta Neurol Belg. 121 (6), 1535-1542 (2021).</li><li>Attri, 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=Endoscopic+third+ventriculostomy+and+simultaneous+tumor+biopsy+in+pineal+region+tumors+using+the+&amp;#34;single+burr+hole&amp;#34;+technique:+An+analysis+of+34+cases.">Endoscopic third ventriculostomy and simultaneous tumor biopsy in pineal region tumors using the &amp;#34;single burr hole&amp;#34; technique: An analysis of 34 cases.</a> Asian J Neurosurg. 15 (4), 976-982 (2020).</li><li>Abbassy, M., Aref, K., Farhoud, A., Hekal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outcome+of+single-trajectory+rigid+endoscopic+third+ventriculostomy+and+biopsy+in+the+management+algorithm+of+pineal+region+tumors:+A+case+series+and+review+of+the+literature.">Outcome of single-trajectory rigid endoscopic third ventriculostomy and biopsy in the management algorithm of pineal region tumors: A case series and review of the literature.</a> Childs Nerv Syst. 34 (7), 1335-1344 (2018).</li><li>Fukushima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+biopsy+of+intraventricular+tumors+with+the+use+of+a+ventriculofiberscope.">Endoscopic biopsy of intraventricular tumors with the use of a ventriculofiberscope.</a> Neurosurgery. 2 (2), 110-113 (1978).</li><li>Kawsar, K. A., Haque, M. R., Chowdhury, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoidance+and+management+of+perioperative+complications+of+endoscopic+third+ventriculostomy:+The+Dhaka+experience.">Avoidance and management of perioperative complications of endoscopic third ventriculostomy: The Dhaka experience.</a> J Neurosurg. 123 (6), 1414-1419 (2015).</li><li>Yang, 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=Endoscopic+third+ventriculostomy+and+biopsy+of+a+tectal+lesion+using+flexible+neuroendoscopy+and+urological+cup+forceps:+Illustrative+case.">Endoscopic third ventriculostomy and biopsy of a tectal lesion using flexible neuroendoscopy and urological cup forceps: Illustrative case.</a> J Neurosurg Case Lessons. 5 (16), (2023).</li><li>Revuelta Barbero, J. 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=Combined+microsurgical-endoscopic+paramedian+supracerebellar-infratentorial+approach+for+resection+of+a+pineal+low-grade+glioma.">Combined microsurgical-endoscopic paramedian supracerebellar-infratentorial approach for resection of a pineal low-grade glioma.</a> Neurosurg Focus Video. 5 (1), 9 (2021).</li><li>Al-Saiari, 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=Simultaneous+biportal+endoscopic+management+of+pineal+region+tumors+in+patients+with+obstructive+hydrocephalus:+Technical+notes.">Simultaneous biportal endoscopic management of pineal region tumors in patients with obstructive hydrocephalus: Technical notes.</a> Chin Neurosurg J. 9 (1), 1 (2023).</li><li>Birski, 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=Endoscopic+versus+stereotactic+biopsies+of+intracranial+lesions+involving+the+ventricles.">Endoscopic versus stereotactic biopsies of intracranial lesions involving the ventricles.</a> Neurosurg Rev. 44 (3), 1721-1727 (2021).</li><li>Gurusamy, S. M., Yaakub, A., Wan Hitam, W. H., Ahmad Maulana, S. A., Wan Ibrahim, W. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pineal+gland+tumour+with+drop+metastases:+A+case+report.">Pineal gland tumour with drop metastases: A case report.</a> Cureus. 14 (10), e29855 (2022).</li><li>Panyaping, T., Tritanon, O., Wisetsathon, P., Chansakul, T., Pongpitcha, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+apparent+diffusion+coefficient+values+for+distinguishing+between+pineal+germ+cell+tumour+and+pineoblastoma.">Accuracy of apparent diffusion coefficient values for distinguishing between pineal germ cell tumour and pineoblastoma.</a> Clin Radiol. 78 (7), e494-e501 (2023).</li><li>Leone, 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=single+burr+hole+endoscopic+third+ventriculostomy+and+tumor+biopsy+for+pineal+lesions:+Feasibility,+safety,+and+benefits.">single burr hole endoscopic third ventriculostomy and tumor biopsy for pineal lesions: Feasibility, safety, and benefits.</a> World Neurosurg. 173, 5-11 (2023).</li><li>Morgenstern, P. 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=Pineal+region+tumors:+An+optimal+approach+for+simultaneous+endoscopic+third+ventriculostomy+and+biopsy.">Pineal region tumors: An optimal approach for simultaneous endoscopic third ventriculostomy and biopsy.</a> Neurosurg Focus. 30 (4), 3 (2011).</li><li>Zhang, Z., 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=Management+of+hydrocephalus+secondary+to+pineal+region+tumors.">Management of hydrocephalus secondary to pineal region tumors.</a> Clin Neurol Neurosurg. 115 (9), 1809-1813 (2013).</li><li>Xin, 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=Endoscopic-assisted+surgery+versus+microsurgery+for+pineal+region+tumors:+A+single-center+retrospective+study.">Endoscopic-assisted surgery versus microsurgery for pineal region tumors: A single-center retrospective study.</a> Neurosurg Rev. 44 (2), 1017-1022 (2021).</li><li>Choque-Velasquez, J., Resendiz-Nieves, J., Colasanti, R., Hernesniemi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+obstructive+hydrocephalus+associated+with+pineal+region+cysts+and+tumors+and+its+implication+in+long-term+outcome.">Management of obstructive hydrocephalus associated with pineal region cysts and tumors and its implication in long-term outcome.</a> World Neurosurg. 149, e913-e923 (2021).</li><li>Chibbaro, 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=Neuroendoscopic+management+of+posterior+third+ventricle+and+pineal+region+tumors:+Technique,+limitation,+and+possible+complication+avoidance.">Neuroendoscopic management of posterior third ventricle and pineal region tumors: Technique, limitation, and possible complication avoidance.</a> Neurosurg Rev. 35 (3), 331-338 (2012).</li><li>Ahmed, A. I., Zaben, M. J., Mathad, N. V., Sparrow, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+biopsy+and+third+ventriculostomy+for+the+management+of+pineal+region+tumors.">Endoscopic biopsy and third ventriculostomy for the management of pineal region tumors.</a> World Neurosurg. 83 (4), 543-547 (2015).</li></ol>

The authors declare that they have no conflicts of interest.

The description of the ETV and biopsy for pineal tumors was first described in the 1970s. Historically, the fear of uncontrolled hemorrhage has always existed. However, owing to advances in endoscopic surgery techniques, bleeding control is not a major complication for experienced surgeons nowawadays9,10. According to several cases in the literature, endoscopic management has been found to be effective as an initial step for pineal region tumors presenting with h...

The pineal gland is a neuroendocrine gland located in the epithalamus that is responsible for regulating biological rhythms in vertebrates. Its anatomical boundaries include the posterior surface of the wall of the third ventricle, which forms the base of the gland, the splenium of the corpus callosum superiorly, and the thalamus surrounding both sides. It has a pinecone shape and extends posteriorly and inferiorly in the quadrigeminal cistern1,2. 
Pineal neoplasms are relatively rare tumors and are predominantly childhood malignancies, accounting for 3-11% of all childhood brain tumors compared with &lt;1% of adult brain tumors1,3,4. Age, sex, and ethnicity can modify the relative incidence of pineal neoplasms5. Pineal tumors are classified into three types: germ cell tumors, pineal parenchymal tumors, and tumors arising from neighboring anatomical regions. Germinomas are the most frequent pineal tumors, accounting for up to 50% of pineal tumors in Europe1,3.
Tumors of the pineal gland are pathologically diverse and their optimal management remains controversial6. Advances in neuroendoscopy have significantly contributed to treating pineal region tumors. Neuroendoscopic techniques are minimally invasive, effective, and safe in treating these tumors. With this technique, it is possible to treat hydrocephalus and obtain a biopsy simultaneously7. In most cases, simultaneous endoscopic third ventriculostomy (ETV) and tumor biopsy is the first-choice surgical procedure for pineal region tumors due to the effectiveness of radiotherapy and chemotherapy in most histopathological subtypes. Various techniques for simultaneous ETV and biopsy have been described, and there is currently no standard technique for performing this procedure8.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adson periosteal elevator</td>
			<td>Ruggles-Redmond</td>
			<td>RO263</td>
			<td>Semi-sharp, 5 mm, curved 6-3/8, length 164 mm</td>
		</tr>
		<tr>
			<td>Automatic skin retractors&#160;</td>
			<td>Integra</td>
			<td>3,72,245</td>
			<td>Heiss Automatic Skin Retractor Length - Overall (mm): 102 
			Length 1 - Tip/Jaw (mm): 8</td>
		</tr>
		<tr>
			<td>Balloon catheter&#160;</td>
			<td>Edwards Fogarty</td>
			<td>120804FP</td>
			<td>Length (cm): 80, Catheter size (F): 4,&#160; Inflated balloon diameter (mm): 9</td>
		</tr>
		<tr>
			<td>Biopsy Forceps</td>
			<td>Karl Storz LOTTA CLICKLINE Grasping Forceps</td>
			<td>28164 LE</td>
			<td>Rotating, dismantling, single action jaws, diameter 2.7 mm, working length 30 cm</td>
		</tr>
		<tr>
			<td>Bipolar coagulation electrode</td>
			<td>Karl Storz LOTTA</td>
			<td>28161 SF</td>
			<td>Diameter 1.3 mm, working length 30 cm</td>
		</tr>
		<tr>
			<td>Bisture</td>
			<td>Beybi</td>
			<td>24,02,502</td>
			<td>Beybi Bisture Tip. No: 20 and No: 11</td>
		</tr>
		<tr>
			<td>High-speed drill</td>
			<td>Medtronic Midas Rex MR8</td>
			<td>MR8&#8482; Electric Plus EM850</td>
			<td>Perforator tip used</td>
		</tr>
		<tr>
			<td>Obturator</td>
			<td>Karl Storz LOTTA</td>
			<td>28164 LLO</td>
			<td>Use with Operating Sheaths for ventricular puncture</td>
		</tr>
		<tr>
			<td>Operating sheath</td>
			<td>Karl Storz LOTTA</td>
			<td>28164 LSB</td>
			<td>Graduated, rotating, outer diameter 6.8 mm, working length 13 cm</td>
		</tr>
		<tr>
			<td>Ventriculoscope</td>
			<td>Karl Storz LOTTA Ventriculoscope with HOPKINS</td>
			<td>28164 LAB</td>
			<td>Wide angle telescope 30&#176;, angled eyepiece, outer diameter 6.1 mm, length 18 cm, working channel diameter 2.9 mm, irrigation/suction channel diameter 1.6 mm</td>
		</tr>
	</tbody>
</table>

endoscopic third ventriculostomy and pineal biopsy from a single entry point

Pineal neoplasms have a significant impact on children although they are relatively uncommon. They account for approximately 3-11% of all childhood brain tumors, which is considerably higher than the &lt;1% seen in adult brain tumors. These tumors can be divided into three main categories: germ cell tumors, parenchymal pineal tumors, and tumors arising from related anatomical structures. Obtaining an accurate and minimally invasive tissue diagnosis is crucial for selecting the most appropriate treatment regimen for patients with pineal gland tumors. This is due to the diverse treatment options available and the potential risks associated with complete resection. In cases where patients present with acute obstructive hydrocephalus caused by a pineal gland tumor, immediate treatment of the hydrocephalus is necessary. The urgency stems from the potential complications of hydrocephalus, including increased intracranial pressure and neurological deficits. To address these challenges, a minimally invasive endoscopic approach provides a valuable opportunity. This technique allows clinicians to promptly relieve hydrocephalus and obtain a histological diagnosis simultaneously. This dual benefit enables a more comprehensive understanding of the tumor and assists in determining the most effective treatment strategy for the patient.

Preoperative magnetic resonance imaging (MRI) revealed a pineal tumor and triventricular hydrocephalus. Before the surgery, we ensured that our endoscope set, 4F Fogarty balloon catheter (see Figure 1), and all required materials (see Table 1) were checked. The size of the pineal tumor was initially measured at 30 mm x 15 mm x 20 mm. However, on postoperative MRI scans, it was observed that the tumor had increased in size to 35 mm x 52 mm x 45 mm, suggesting...

Author Spotlight: A Single-Entry Point Endoscopic Intraventricular Approach for Third Ventriculostomy and Pineal Biopsy

Pineal neoplasms frequently cause obstructive hydrocephalus and require histopathological diagnosis to decide the treatment regimen. They might be treated by surgical resection or chemoradiotherapy according to the pathological diagnosis. As a minimally invasive initial treatment, an endoscopic approach from Kocher's point enables both third ventriculostomy and biopsy.

Endoscopic Third Ventriculostomy and Pineal Biopsy from a Single Entry Point

Pineal neoplasms have a significant impact on children although they are relatively uncommon. They account for approximately 3-11% of all childhood brain ...

endoscopic-third-ventriculostomy-pineal-biopsy-from-single-entry

Research

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Medicine

465 Views.  Istanbul University Faculty of Medicine. Pineal neoplasms frequently cause obstructive hydrocephalus and require histopathological diagnosis to decide the treatment regimen. They might be treated by surgical resection or chemoradiotherapy according to the pathological diagnosis. As a minimally invasive initial treatment, an endoscopic approach from Kocher's point enables both third ventriculostomy and biopsy. 

Video: Author Spotlight: A Single-Entry Point Endoscopic Intraventricular Approach for Third Ventriculostomy and Pineal Biopsy

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

Endaural Endoscopic Atticoantrotomy (Retrograde Mastoidectomy) using a Constant Suction Bone-drilling Technique

Endoscopic middle ear surgery is a widely employed minimally invasive surgical technique to address middle ear and mastoid pathology. Bone drilling is the main technical challenge of endoscopic middle ear surgery. The accompanying video describes the detailed protocol of a constant-suction bone-drilling technique and the procedure of endaural exclusive endoscopic atticoantrotomy (retrograde mastoidectomy) using this technique. The main components of this bone-drilling technique include a soft and flexible suction tube, which is placed into the tympanic cavity to provide constant suction, and a soft sleeve, which is wrapped around the drill shaft to prevent the high-speed rotating shaft from damaging the lens of the endoscope. With these simple modifications, the traditional otological electrodrill can be used for drilling a tiny endaural incision in endoscopic middle ear surgery. Based on this bone-drilling technique, endaural endoscopic atticoantrotomy (retrograde mastoidectomy) can be successfully established for the removal of various amounts of bone, depending on the extent of the lesion. The short-term postoperative outcome seems promising.

Endaural Endoscopic Atticoantrotomy Retrograde Mastoidectomy using a Constant Suction Bone-drilling Technique

In this paper, we present the protocol and short-term outcomes of endaural exclusive endoscopic atticoantrotomy using a constant suction bone-drilling technique.

Endoscopic middle ear surgery is a widely employed minimally invasive surgical technique to address middle ear and mastoid pathology. Bone drilling is the ...

Endoscopic Ultrasound-Guided Biliary Drainage: Endoscopic Ultrasound-Guided Hepaticogastrostomy in Malignant Biliary Obstruction

Patients with unresectable malignant biliary obstruction often require biliary drainage to decompress the biliary system. Endoscopic Retrograde Cholangiopancreatography (ERCP) is the primary biliary drainage method whenever possible. Percutaneous Transhepatic Biliary Drainage (PTBD) is used as a salvage method if ERCP fails. Endoscopic Ultrasound-Guided Biliary Drainage (EUS-BD) provides a feasible alternative biliary drainage method where one of the methods is EUS guided Hepaticogastrostomy (EUS-HGS). Here we describe the EUS-HGS technique in a case of unresectable malignant hilar biliary obstruction to achieve biliary drainage.
Presented here is the case of a 71-year-old female with painless jaundice and weight loss for 2 weeks. Computed Tomography (CT) imaging showed a 4 x 5 cm hilar tumor with lymphadenopathy and liver metastasis. EUS fine needle biopsy (FNB) of the lesion was consistent with cholangiocarcinoma. Her bilirubin levels were 212 &#181;mol/L (&lt;15) during presentation.
A linear echoendoscope was used to locate the left dilated intrahepatic ducts (IHD) of the liver. The segment 3 dilated IHD was identified and punctured using a 19 G needle. Contrast was used to opacify the IHDs under fluoroscopic guidance. The IHD was cannulated using a 0.025-inch guidewire. This was followed by the dilation of the fistula tract using a 6 Fr electrocautery dilator along with a 4 mm biliary balloon dilator. A partially covered metallic stent of 10 cm in length was deployed under fluoroscopic guidance. The distal part opens in the IHD and the proximal part was deployed within the working channel of the echoendoscope that subsequently released into the stomach. The patient was discharged three days after the procedure. Follow up performed in the second and fourth weeks showed that the bilirubin levels were 30 &#181;mol/L and 14 &#181;mol/L, respectively. This indicates that EUS-HGS is a safe method for biliary drainage in unresectable malignant biliary obstruction.

Endoscopic Ultrasound-Guided Biliary Drainage (EUS-BD) is an alternative method of biliary decompression in malignant biliary obstruction. Here we describe the technique of EUS guided-Hepaticogastrostomy (EUS-HGS) in a case of unresectable malignant hilar biliary obstruction.

Patients with unresectable malignant biliary obstruction often require biliary drainage to decompress the biliary system. Endoscopic Retrograde ...

Endoscopic Cholesteatoma Surgery

Implementation of endoscopes in cholesteatoma surgery resulted in considerable changes in the management of cholesteatoma in the last two decades. Compared to the microscopic approach with an excellent but straight-line view and limited illumination, the introduction of endoscopes provides a wide-angled panoramic view. Moreover, angled lenses allow the surgeon to visualize the middle ear and its hidden recesses through a transcanal, minimally-invasive approach. The endoscope enables the surgeon to remove limited cholesteatoma of the middle ear and its recesses using an exclusive endoscopic technique by taking advantage of these benefits. This reduces the rate of residual disease and sparing external incisions and excessive temporal bone drilling as in a transmastoid approach. Since transcanal endoscopic access is mainly a one-handed technique, it implies the need for specific procedures and technical refinements. This article describes a step-by-step guide as a surgical manual for endoscopic removal of epitympanic cholesteatoma. Different techniques for cholesteatoma dissection and bone removal for epitympanectomy, including curettage and powered instruments such as drills and ultrasonic devices with their outcomes, are discussed. This may offer ear surgeons insight into technical refinements and the latest technological developments and open the horizon for different techniques.

The present protocol describes a step-by-step guide for the complete endoscopic removal of epitympanic cholesteatoma with different techniques for cholesteatoma dissection and bone removal for epitympanectomy.

Implementation of endoscopes in cholesteatoma surgery resulted in considerable changes in the management of cholesteatoma in the last two decades. ...

Ultrasound in Evaluating Diaphragm Dysfunction and Monitoring Treatment

Measuring Diaphragm Thickness and Function Using Point-of-Care Ultrasound

The diaphragm is the main component of the respiratory muscle pump. Diaphragm dysfunction can cause dyspnea and exercise intolerance, and predisposes affected individuals to respiratory failure. In mechanically ventilated patients, the diaphragm is susceptible to atrophy and dysfunction through disuse and other mechanisms. This contributes to failure to wean and poor long-term clinical outcomes. Point-of-care ultrasound provides a valid and reproducible method for evaluating diaphragm thickness and contractile activity (thickening fraction during inspiration) that can be readily employed by clinicians and researchers alike. This article presents best practices for measuring diaphragm thickness and quantifying diaphragm thickening during tidal breathing or maximal inspiration. Once mastered, this technique can be used to diagnose and prognosticate diaphragm dysfunction, and guide and monitor response to treatment over time in both healthy individuals and acute or chronically ill patients.

Author Spotlight: Unraveling the Impact of Mechanical Ventilation on Diaphragm Function and Patient Outcomes 

Diaphragm thickness and function can be assessed in healthy individuals and critically ill patients using point-of-care ultrasound. This technique offers an accurate, reproducible, feasible, and well-tolerated method for evaluating diaphragm structure and function.

The diaphragm is the main component of the respiratory muscle pump. Diaphragm dysfunction can cause dyspnea and exercise intolerance, and predisposes ...