No funding was received. JP and MM captured the photographs shown in Figure 1A. RB and MM captured the ones shown in Figure 3.

The following protocol will describe how to perform LAT in a clinical setting. The protocol is in accordance with the clinical practice and guidelines of the authors&#39; hospitals (Odense, N&#230;stved, Lleida, Bristol, and Preston). Written informed consent is obtained from the patient prior to the procedure. The main inclusion criteria for the procedure is recurrent pleural effusion of unknown cause, despite guideline-based work-up. Exclusion criteria include complete obliteration of the pleural space due to adhesions, skin infection at the site of entry, respiratory failure, cardiac instability, and uncorrectable coagulopathy.
1. Preparing for the procedure
<ol>
	<li>Examine the available image and perform thoracic ultrasound (US) to confirm the presence of pleural effusion and a safe distance (&#62;2 cm) from the parietal to visceral pleura at the expected entry site, preferably in the middle of the intercostal spaces 4 or 5 in the midaxillary line12.</li>
	<li>Mark the expected entry site with a designated pen.</li>
	<li>In case of small-volume effusions, induce an artificial pneumothorax by inserting a blunt needle (e.g., &#39;Boutin needle&#39;) or a small-bore chest tube under direct ultrasound guidance. After fluid removal, air is introduced via the needle or drain, and ultrasound confirms the disappearance of lung sliding8,12.
	<ol>
		<li>If in doubt, perform a lateral decubitus chest X-ray to confirm the presence and depth of the pneumothorax.</li>
	</ol>
	</li>
	<li>Place the patient in the lateral decubitus position on a pillow or roll, facing the operator, with the effusion side up. The pillow/roll spreads the contralateral ribs, making it easier to insert the trocar and cannula, and minimizing the patient&#39;s discomfort11.</li>
	<li>Prepare for the sterile procedure according to local standards:
	<ol>
		<li>For operator and any assistant: surgical hand scrub, sterile gown and gloves, mask, cap.</li>
		<li>For patient: cleanse the skin with an alcohol-based sterilizing solution, use sterile draping for coverage while leaving the expected entry site accessible.</li>
		<li>For tools: sterile table with a knife, trocar, cannula, endoscope, forceps, suction, and skin suture tools11(see Table of Materials).</li>
	</ol>
	</li>
	<li>Commence sedation with a benzodiazepine (e.g., midazolam 1 mg/mL) IV in incremental doses, combined with an opiate (e.g., fentanyl 25 microgram/mL) IV in incremental doses to control pain and cough. In select centers, LAT may be performed with thoracic nerve blocks or with remifentanil sedation12.</li>
	<li>Ensure sufficient additional sedation is available during the procedure in case of pain, cough, or agitation.</li>
</ol>
2. Patient preparation
<ol>
	<li>Anesthetize all layers step by step with 20 mL of lidocaine 1% (or mepivacaine 2%) plus adrenaline: the skin, subcutaneous tissue, intercostal muscle, parietal pleura, intercostal nerve, and rib periosteum (the caudal rim of the upper and cranial rim of the lower rib)7,11,18.</li>
	<li>End the local anesthesia by checking the correctness of the entry site through the aspiration of pleural fluid (or air if an artificial pneumothorax is present), and note the distance from the skin to the pleural space11,12.</li>
	<li>Have additional doses of local anesthesia available during the procedure in case of pain or agitation.</li>
	<li>Perform skin incision and insertion of trocar and cannula.
	<ol>
		<li>Make an approximately 10 mm incision in the skin and subcutaneous tissue parallel to the intercostal space to allow for the trocar and cannula to pass easily. 
		NOTE: A longer incision does not add maneuverability and needs to be closed with more stitches, which is associated with the risk of infections19.</li>
		<li>Ensure with the tip of the scalpel that adequate parietal pleural anesthesia has been achieved before trocar insertion.</li>
	</ol>
	</li>
	<li>Perform a careful blunt dissection of the various layers between the skin and the pleura with either a narrow-tip straight or curved forceps until the pleural cavity is entered, and a tract of sufficient width to allow the trocar and cannula to pass has been created. 
	NOTE: The release of fluid or air (the latter often heard as a &#39;hiss&#39;) confirms the location. Optional: Blunt dissection might not be necessary for semirigid procedures given the smaller diameter of the working channel.</li>
	<li>Consider inserting a closing (&#34;mattress&#34;) suture (see Table of Materials), which is used later to seal the skin once the post-LAT chest drain is removed.</li>
	<li>Guided by the distance from the skin to the pleural space, insert the trocar and cannula with a cork-screw movement until a sudden release of resistance is felt. Then, remove the trocar and push the cannula forward to lie 1-3 cm within the pleural cavity11,12.</li>
</ol>
3. Inspection of the pleural cavity
<ol>
	<li>Ensure that the thoracoscope image is optimized for focus and color correction, and that the lens has been warmed or has demisting liquid applied.</li>
	<li>For semi-rigid LAT, insert the thoracoscope via the cannula and apply suction to remove the remaining fluid. For rigid LAT, insert a separate suction tube via the cannula to remove fluid before inserting the camera (Figure 1 and Figure 2).</li>
	<li>Inspect the pleural cavity systematically: the apex, mediastinal surface, diaphragm, and costal pleura back to the apex. As with other endoscopic techniques, experts use deliberate and minimal movements to achieve full inspection20,21. 
	NOTE: If using a rigid setup, initial inspection is best carried out using a 50-degree lens before switching to a 0-degree lens for intervention.</li>
	<li>Full visibility of the pleural space may be limited by fibrous pleuro-pulmonary adhesions12. Gently swipe away fine adhesions with the thoracoscope, but avoid those with a vascular supply.</li>
</ol>
4. Tissue biopsy
<ol>
	<li>Sample suspicious lesions on the parietal pleura with six to ten biopsies using appropriate forceps with a &#39;grip and strip&#39; approach (Figure 3A,B). Ensure that there is sufficient penetration of the parietal pleural membrane, as the invasion of subpleural fat can affect mesothelioma T classification8,12.
	<ol>
		<li>Take care to avoid the visceral pleura due to the risk of lung injury and subsequent persistent air leak.</li>
		<li>Exercise caution to avoid the intercostal neurovascular bundle due to the risk of bleeding and neuralgia. 
		NOTE: Safety tip: Feeling the rib with the forceps and obtaining biopsies from the parietal pleura &#39;on&#39; the rib is believed to reduce the risk of bleeding and damage to underlying vessels.</li>
		<li>For semirigid thoracoscope procedures, introduce the forceps via the endoscope working channel. Switching to narrow-band imaging may improve the visualization of areas with abnormal vascular patterns13,22.</li>
		<li>For rigid thoracoscope procedures, introduce over-scope forceps.</li>
	</ol>
	</li>
	<li>In the absence of clear malignant infiltration but with strong clinical suspicion of malignancy, perform random biopsies11.</li>
	<li>Send 1-2 biopsies for culture and tuberculosis analysis, especially in endemic areas, or in patients with risk factors23.</li>
	<li>If pleural infection is suspected during LAT, remove infectious debris (Figure 3C) and adhesions as far as possible12,24. Note that LAT is not an optimal procedure for the clearance of complex or widespread pleural infection9.</li>
</ol>
5. Termination and insertion of chest tube
<ol>
	<li>Remove the thoracoscope and cannula.</li>
	<li>Insert the post-LAT chest tube (typically 16-24 F, see Table of Materials) into the pleural space via the port tract.</li>
	<li>Close the incision with the &#34;mattress&#34; suture (as described in 2.6) or use another closing suture. 
	&#8203;NOTE: Talc pleurodesis and/or placement of an indwelling pleural catheter (IPC) can be optionally performed in combination with the LAT procedure and are now standard practice in many centers8. The description of talc pleurodesis or IPC procedures is beyond the scope of the current review.</li>
</ol>
6. Follow-up procedures
<ol>
	<li>Remove the chest tube when air and fluid production ceases, typically within 1-2 h. 
	NOTE: While a pre-removal chest X-ray or thoracic ultrasound is not mandatory, it may be helpful for assessing lung re-expansion.</li>
	<li>Close the incision with appropriate sutures or adhesive glue (see Table of Materials).</li>
	<li>Observe the patient for stability over 2-4 h. 
	NOTE: Although a post-removal chest X-ray or thoracic ultrasound is not mandatory, it can be helpful in assessing lung re-expansion. Patients who are well and stable after observation are discharged with a written action plan in case of serious symptoms25.</li>
	<li>Admit frail patients to the ward for further observation and treatment.</li>
</ol>

Anesthetic Thoracoscopy for Diagnosis of Pleural Effusions

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The authors have no real or perceived disclosures to report.

This article provides a practical approach for performing LAT.
A single randomized trial compared rigid with semirigid LAT and found no differences concerning diagnostic yield or safety15. Evidence on how to optimize the critical steps is sparse. A group of one of the authors suggested eight critical steps for semirigid LAT and presented a competence checklist for learning LAT in a phantom based on their own experiences, narrative reviews8,<...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65734">Local Anesthetic Thoracoscopy for Undiagnosed Pleural Effusion</a>. The Authors section was updated from:
Uffe Bodtger1,2 
Jos&#233; M. Porcel3 
Rahul Bhatnagar4,5 
Mohammed Munavvar6,7 
Casper Jensen1 
Paul Frost Clementsen1,8 
Daniel Bech Rasmussen1,2 
1Respiratory Research Unit PLUZ, Department of Respiratory Medicine, Zealand University Hospital 
2Institute of Regional Health Research, University of Southern Denmark 
3Pleural Medicine Unit, Department of Internal Medicine, Hospital Universitari Arnau de Vilanova, IRBLleida 
4Respiratory Department, Southmead Hospital, North Bristol NHS Trust 
5Academic Respiratory Unit, University of Bristol 
6Lancashire Teaching Hospitals 
7University of Central Lancashire 
8Centre for HR and Education, Copenhagen Academy for Medical Education and Simulation
to:
Uffe Bodtger1,2 
Jos&#233; M. Porcel3 
Rahul Bhatnagar4,5 
Nick Maskell4,5 
Mohammed Munavvar6,7 
Casper Jensen1 
Paul Frost Clementsen1,8 
Daniel Bech Rasmussen1,2 
1Respiratory Research Unit PLUZ, Department of Respiratory Medicine, Zealand University Hospital 
2Institute of Regional Health Research, University of Southern Denmark 
3Pleural Medicine Unit, Department of Internal Medicine, Hospital Universitari Arnau de Vilanova, IRBLleida 
4Respiratory Department, Southmead Hospital, North Bristol NHS Trust 
5Academic Respiratory Unit, University of Bristol 
6Lancashire Teaching Hospitals 
7University of Central Lancashire 
8Centre for HR and Education, Copenhagen Academy for Medical Education and Simulation

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65734">Local Anesthetic Thoracoscopy for Undiagnosed Pleural Effusion</a>. The Authors section was updated.

Erratum: Local Anesthetic Thoracoscopy for Undiagnosed Pleural Effusion

The incidence and prevalence of pleural diseases are increasing worldwide, especially pleural effusions, which have more than 50 recognized causes1,2. Pleural malignancy is the leading cause of recurrent pleural effusion, mostly due to metastases from extrapleural lung, breast, or lymphoma malignancies3. Existing guidelines recommend pleural biopsies if the medical history, physical examination, radiology, and pleural tap for cytology, culture, and biochemistry fail to provide a diagnosis4. Pleural biopsies can be obtained either image-guided or under direct vision. Ultrasound (US)- or CT-guided percutaneous core biopsies allow sampling from most lesions in the costal part of the parietal pleura, with a diagnostic yield of 84% and 93%, respectively, in a recent systematic review5. US-guided pleural biopsy is superior in terms of a lower complication rate (4% vs. 7%), no irradiation, and availability as a bedside procedure5. Case reports show that lesions in the mediastinal pleura can be sampled using echoendoscopes6.
Thoracoscopy allows direct visual inspection and sampling of lesions in both the mediastinal, diaphragmatic, or costal pleura, making it the gold standard in the diagnosis of recurrent pleural effusion of an unknown cause4,7. Thoracoscopy is performed as either a local anesthetic thoracoscopy (LAT) or video-assisted thoracoscopic surgery (VATS)8. LAT (also known as medical thoracoscopy or pleuroscopy) is a single-port procedure, routinely performed by pulmonologists in the bronchoscopy suite, and it carries a diagnostic yield for malignancy of 93%9,10. LAT can be performed with either a semirigid or rigid endoscope, usually connected to a video-imaging source. A few publications discuss the benefits and disadvantages of these approaches8,11,12,13. Briefly, the semirigid thoracoscope resembles a bronchoscope, and biopsies are obtained using similar forceps. The rigid endoscope has a larger diameter, is cheaper, and allows for larger biopsies, although this does not readily translate into marked differences in diagnostic yield10,14,15. The safety of LAT is high, with a mortality rate of less than 0.5%, with this strongly related to pre-existing medical conditions3,4. Contraindications of LAT are fewer than for VATS and include complete obliteration of the pleural space due to adhesions, skin infection at the site of entry, respiratory failure, cardiac instability, and uncorrectable coagulopathy7,8,12. VATS is an in-hospital, 2 or 3-port procedure performed by thoracic surgeons, which must take place in a surgical theatre, requiring general anesthesia, intubation, single-lung ventilation, and post-operative admission. VATS has a diagnostic yield and complication rate similar to that of LAT but allows for more complex procedures to be undertaken10,16.
Guidelines recommend LAT as the first-choice thoracoscopy due to its high diagnostic yield, low risk, lower costs, and the possibility for daycase management, reserving VATS for selected cases4. The choice of a rigid or semirigid thoracoscope is usually determined by local preference. LAT is not a new procedure, as it was described as early as the mid-19th century, popularized by Jacobaeus in 1910, increasingly used to treat pleural tuberculosis until the 1950s, and &#34;rediscovered&#34; in the 1980s as an important tool to diagnose recurrent pleural effusions8,17.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chest tube</td>
			<td></td>
			<td></td>
			<td>16-24F</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td>narrow tip, straight or curved&#160;</td>
		</tr>
		<tr>
			<td>Indwelling pleural catheter</td>
			<td>Rocket Medical plc.</td>
			<td>R55400-16-MT</td>
			<td>or PleurX (from Becton Dickinson) or similar</td>
		</tr>
		<tr>
			<td>Local anaesthetics</td>
			<td></td>
			<td></td>
			<td>20 mL of lidocaine 1% (or mepivacaine 2%) &#177; adrenaline</td>
		</tr>
		<tr>
			<td>Non-absorbable suture</td>
			<td></td>
			<td></td>
			<td>Eg. Dafilon 2/0</td>
		</tr>
		<tr>
			<td>Rigid thoracoscope</td>
			<td>Karl Storz GmbH&#160;</td>
			<td>Hopkins-II</td>
			<td>with forceps 26072A and cannula+trocar 30120 NOL (or similar from eg. Richard Wolf GmbH)</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td>triangular</td>
		</tr>
		<tr>
			<td>Semirigid thoracoscope</td>
			<td>Olympus</td>
			<td>LTF-160</td>
			<td>with forceps FB-420K and cannula+trocar N1002130</td>
		</tr>
	</tbody>
</table>

local anesthetic thoracoscopy for undiagnosed pleural effusion

Local anesthetic thoracoscopy (LAT) is a minimally invasive diagnostic procedure gaining recognition among chest physicians for managing undiagnosed pleural effusions. This single-port procedure is conducted with the patient under mild sedation and involves a contralateral decubitus position. It is performed in a sterile setting, typically a bronchoscopy suite or surgical theater, by a single operator with support from a procedure-focused nurse and a patient-focused nurse.
The procedure begins with a thoracic ultrasound to determine the optimal entry point, usually in the IV-V intercostal space along the midaxillary line. Lidocaine/mepivacaine, with or without adrenaline, is used to anesthetize the skin, thoracic wall layers, and parietal pleura. A designated trocar and cannula are inserted through a 10 mm incision, reaching the pleural cavity with gentle rotation. The thoracoscope is introduced through the cannula for systematic inspection of the pleural cavity from the apex to the diaphragm. Biopsies (typically six to ten) of suspicious parietal pleura lesions are obtained for histopathological evaluation and, when necessary, microbiological analysis. Biopsies of the visceral pleura are generally avoided due to the risk of bleeding or air leaks. Talc poudrage may be performed before inserting a chest tube or indwelling pleural catheter through the cannula. The skin incision is sutured, and intrapleural air is removed using a three-compartment or digital chest drainage system. The chest tube is removed once there is no airflow, and the lung has satisfactorily re-expanded. Patients are usually discharged after 2-4 h of observation and followed up on an outpatient basis. Successful LAT relies on careful patient selection, preparation, and management, as well as operator education, to ensure safety and a high diagnostic yield.

The described LAT technique using either the rigid or semirigid thoracoscopes (Figure 1 and Figure 2) enables the operator to perform conclusive biopsy sampling from the visceral pleural (Figure 3) with a diagnostic yield of 93% in a large systematic review on more than 5000 procedures (Table 1 and Supplementary File 1) (rigid: 93% [95% confidence interval 91%-95%]; semirigid: 93% [89%-97%])<sup cla...

Watch this Scientific Journal Video about Local Anesthetic Thoracoscopy for Undiagnosed Pleural Effusion at JoVE.com

Local Anesthetic Thoracoscopy for Undiagnosed Pleural Effusion

Local anesthetic thoracoscopy (LAT) is essential for diagnosing recurrent, undiagnosed pleural effusion when a guideline-based workup fails to provide a specific cause. LAT can be performed as a day-case procedure by chest physicians. Here, we present a step-by-step approach for a successful and safe procedure.

Local anesthetic thoracoscopy (LAT) is a minimally invasive diagnostic procedure gaining recognition among chest physicians for managing undiagnosed ...

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472 Views.  Zealand University Hospital. Local anesthetic thoracoscopy (LAT) is essential for diagnosing recurrent, undiagnosed pleural effusion when a guideline-based workup fails to provide a specific cause. LAT can be performed as a day-case procedure by chest physicians. Here, we present a step-by-step approach for a successful and safe procedure.

Video: Local Anesthetic Thoracoscopy for Undiagnosed Pleural Effusion

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children. Recent investigations in animals have raised concerns that anesthesia exposure may lead to neuronal apoptosis, known as anesthesia-induced developmental neurotoxicity (AIDN). Furthermore, some clinical studies in children have suggested that anesthesia exposure may lead to neurodevelopmental deficits later in life. Nonetheless, an ideal animal model for preclinical study has yet to be developed. The neonatal piglet represents a valuable model for preclinical study, as they share a striking number of developmental similarities with humans.
The anatomy and physiology of piglets allow for implementation of rigorous human perioperative conditions in both survival and non-survival procedures. Femoral artery catheterization allows for close monitoring, thus enabling prompt correction of any deviation of the piglet's vital signs and chemistries. In addition, there are multiple developmental similarities between piglets and human neonates. The techniques required to use piglets for experimentation will require experience to master. A pediatric anesthesiologist is a critical member of the investigative team. We describe, in a general sense, the appropriate use of a piglet model for neurodevelopmental study.

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach

Anesthesia-induced developmental neurotoxicity (AIDN) research has focused on rodents, which are not broadly applicable to humans. Non-human primate models are more relevant, but are cost-prohibitive and difficult to use for experimentation. The piglet, in contrast, is a clinically relevant, practical animal model ideal for the study of anesthetic neurotoxicity.

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children.  Recent investigations in animals have raised concerns that ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

CO2-Lasertonsillotomy Under Local Anesthesia in Adults

Tonsil-related complaints are very common among the adult population. Tonsillectomy under general anesthesia is currently the most performed surgical treatment in adults for such complaints. Unfortunately, tonsillectomy is an invasive treatment associated with a high complication rate and a long recovery time. Complications and a long recovery time are mostly related to removing the vascular and densely innervated capsule of the tonsils. Recently, CO2-lasertonsillotomy under local anesthesia has been demonstrated to be a viable alternative treatment for tonsil-related disease with a significantly shorter and less painful recovery period. The milder side-effect profile of CO2-lasertonsillotomy is likely related to leaving the tonsil capsule intact. The aim of the current report is to present a concise protocol detailing the execution of CO2-lasertonsillotomy under local anesthesia. This intervention has been performed successfully in our hospital in more than 1,000 patients and has been found to be safe and to be associated with a steep learning curve.

CO2-Lasertonsillotomy Under Local Anesthesia in Adults

CO2-lasertonsillotomy under local anesthesia is an interesting alternative treatment method for tonsillectomy under general anesthesia for tonsil-related complaints in adults. This report presents a step-by-step protocol detailing the execution of CO2-lasertonsillotomy under local anesthesia.

Tonsil-related complaints are very common among the adult population. Tonsillectomy under general anesthesia is currently the most performed surgical ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...

Use of an Integrated Low-Flow Anesthetic Vaporizer, Ventilator, and Physiological Monitoring System for Rodents

Low-flow digital vaporizers commonly utilize a syringe pump to directly administer volatile anesthetics into a stream of carrier gas. Per animal welfare recommendations, animals are warmed and monitored during procedures requiring anesthesia. Common anesthesia and physiological monitoring equipment include gas tanks, anesthetic vaporizers and stands, warming controllers and pads, mechanical ventilators, and pulse oximeters. A computer is also necessary for data collection and to run equipment software. In smaller spaces or when performing field work, it can be challenging to configure all this equipment in limited space.
The goal of this protocol is to demonstrate best practices for use of a low-flow digital vaporizer using both compressed oxygen and room air, along with an integrated mechanical ventilator, pulse oximeter, and far infrared warming as an all-inclusive anesthesia and physiological monitoring suite ideal for rodents.

Here, we present a protocol to safely and effectively administer anesthetic gas to mice using a digital, low flow anesthesia system with integrated ventilator and physiological monitoring modules.

Low-flow digital vaporizers commonly utilize a syringe pump to directly administer volatile anesthetics into a stream of carrier gas. Per animal welfare ...