This work was supported by NIH R21 DC03969-01 (to JGB). We thank Dr. Fritz Lischka for capturing single taste bud cells, Dr. Tauf Huque for performing the RT-PCR shown in Figure 4B and Mr. D. Bayley and Ms. S. Alarcon for technical assistance. M. Yanina Pepino is a fellow supported by a NIDAT32 DA07313 Washington University School of Medicine, St. Louis, MO (Cottler, L.B. PI).

1. Introduction and General Considerations
The general procedure involves local anesthesia of a small (1 cm2) area of the dorsal surface of the anterior tongue, followed by removal of a number (6-8) of fungiform (taste) papillae from that anesthetized area using a curved spring micro-scissors (McPherson-Vannas type #SR 5603, Roboz, Rockville, MD). The removed fungiform papillae are approximately 0.25 mm3 each. There is very little non-papillae (taste) epithelial tissue removed along with each papilla. New papillae form in this area after 3 to 5 weeks and regain functioning taste buds (Spielman and Brand, unpublished observations). There is no noticeable effect on taste or flavor of food after removal of this small amount of taste material. From studies that have counted individual taste buds in the adult human tongue, it can be estimated that the human tongue contains approximately 5000 taste buds. Our procedure removes at most 20 to 25 of these taste buds (on average, ~ 3.5 to 4 taste buds per fungiform papilla in human)2, or about 0.4% of the total. In addition there is no pain associated with the surgical loss of this tissue. One feels, of course, slight pain at the injection site only, as the needle enters the tongue. After anesthesia has worn off, there is occasionally mild discomfort for less than a day, but this discomfort is much less than what one experiences after accidentally "biting" ones tongue.
Over the past several years we have developed an alternative procedure that does not include anesthesia. The technique for tissue removal is identical for both. The only difference in the procedure is that the surgeon is no longer confined to a 1 cm2 diameter area where the anesthesia was addressed. Rather s/he can choose any site on the dorsal surface of the tongue where prominent papillae appear. The total number of removed papillae are limited to 6-8 in both techniques, with or without anesthesia If performed properly, the discomfort of removing 6-8 fungiform taste papillae is considered less of an issue when compared with the pain associated with the administering of local anesthesia A general survey of individuals who underwent both procedures, i.e., with and without anesthesia, showed that an overwhelming majority of subjects would dispense without local anesthesia in all future biopsies. The surgeon who developed this technique (AIS) and several colleagues taught by the author who performed biopsies without anesthesia, have tried it on themselves and testify that the discomfort is temporary and is considered less than that of biting one's tongue. Finally, dozens of individuals who agreed to be biopsied were asked if they would be willing to try removal of one papilla without anesthesia and if they judge it to be too painful they would undergo anesthesia, have all agreed to continue with the removal of papillae without anesthesia.
The entire biopsy should not take longer than 5-10 minutes. If anesthesia is employed another 2-3 minutes is added. Biopsy is typically carried out in the morning hours. Before biopsy, volunteers are instructed to eat a light meal two hours before the scheduled start time. At biopsy, written informed consent is obtained. Exclusion criteria at the time of biopsy include those with serious chronic illness, whether controlled by medication or not (e.g., any gastrointestinal diseases, neurodegenerative diseases, cancer, blood dyscrasias, allergy to the local anesthetics, etc.) those with heart rate greater than 100 or less than 60; systolic blood pressure greater than 145 or less than 100; diastolic blood pressure greater than 90 or less than 60, and the opinion of the surgeon that the subject can participate in the study.
Finally, it is important to consider that the biopsy procedure should be carried out in the same facility where tissue will be processed for examination since tissue integrity is rapidly lost as we have often noted by the declining quality of mRNA. To minimize these problems the tongue tissue should be processed as quickly as possible.
2. Subject Preparation
<ol>
 <li>The subject is seated in a dental chair or a seat with a headrest.</li>
 <li>Check heart rate and blood pressure at the time of biopsy. Should the subject fall outside of the range of blood pressure, heart rate or oral competence, the surgeon recommends to the subject that he/she make immediate appointment to see a physician.</li>
 <li>Inspect the oral cavity for general tissue health, with attention to indicators of xerostomia, geographic tongue or any tongue lesions. Any subject showing signs of oral disease, including tongue lesions or xerostomia are excluded from the study. Instruct the subject to rinse the mouth with water to remove all debris from the oral cavity. If necessary the tongue is cleaned with a disposable tongue scrapper to remove plaque or debris, which could be carried into the tongue during injection of the anesthesia. The surgeon uses sterile gauze pads in a gloved hand to hold the anterior &frac14; of the tongue as it extends from the oral cavity.</li>
 <li>If anesthesia is used, a 1cc Tuberculin syringe with a 28 gauge x &frac12;' needle containing 0.25 mL lidocaine (2%) with epinephrine (1:100,000) is slowly injected into one site just below the dorsal surface of the anterior portion of the tongue. The site of anesthesia is distal to the collection site to avoid interference by the lidocaine with subsequent studies. For comparison, the amount of lidocaine injected (0.25 mL) is about one quarter of the amount normally given for nerve block in dental procedures (0.8 mL to 1 mL). After insertion of the needle but before injection of the anesthetic, aspiration ensures that no blood vessel is struck. This precaution is taken even though it is anatomically unlikely that a large vessel will be struck close to the dorsal surface. The only arteries on the dorsal surface, the dorsal lingual arteries are small blood vessels primarily located more posterior to the anterior third, where the tissue harvesting occurs. The larger lingual arteries and the lingual veins run on the ventral surface of the tongue, far removed from the dorsal area receiving anesthetic 3. The 1 cm anesthetized area appears to be slightly pale compared with the surrounding tissue. This is due to the swelling of the tissue as it compresses the small blood vessels. It is also due to the vasoconstrictive effect of the epinephrine. After about one minute, a probe is used to test the area for numbness. If the area is not numb, a second injection (0.1 to 0.2 mL) may be given.</li>
</ol>
3. Tongue Papillae Removal Technique
<ol>
 <li>The anterior quarter of the tongue of the subject is wrapped into sterile gauze (Figure 1) and held in the left hand of the surgeon (if performing the biopsy with the right hand). One corner of the gauze is left open to allow the surgeon to wipe the tongue free of saliva. The tongue must be held firmly, but, of course, not so as to cause unnecessary discomfort. The tip of the index finger of the left hand is positioned under the tongue directly under the site where the biopsy is performed. By pushing the index finger upward, the tongue becomes stretched over the tip of the finger making the papillae stick out for easy biopsy. </li>
 <li>Criteria for choosing papillae for biopsy: Taste papillae are easily distinguishable from the surrounding filliform papillae by their shape and size. Fungiform papillae are round and somewhat isolated from the surrounding. In their immediate vicinity there is a circular space devoid of filliform papillae (Figure 2A, blue arrows). This space is about 1 mm in diameter. Another distinguishing feature of the fungiform papillae is an extended capillary arborization visible on top of most fungiform papillae of young subjects. Although arborization makes locating fungiform papillae easier, they are not necessarily good predictors of the existence of taste buds in that papilla. As a rule one starts harvesting toward the front end and moves further away from the tip of the tongue to avoid any capillary bleeding from obscuring the site of the next tissue collection.</li>
 <li>Once a good papilla has been identified, a spring micro-scissors (McPherson-Vannas, curved, sharp cutting edge 5 mm, comb, tip width 0.2 mm overall length 3" (RS-5603, Roboz) is used to clip it off. To effectively use the micro surgical scissors one should not use the tip of the blade for cutting; rather, as far inside the blade as possible (Figure 1, enlarged image and Figure 3), close to the point of intersection with the opposite blade. Removal of the papilla is achieved with one cutting move of the scissors (Figure 1). The further inside the open blades the targeted papilla appears, the more effective is the cutting. Care should be taken that the overall position of the scissors must be parallel with the dorsal surface of the tongue. </li>
 <li>Sometimes the tongue becomes dry as the surgeon is trying to identify a papilla for removal. Once dry, the ability of the operator to clearly see which is a fungiform papilla becomes more challenging and makes sliding of the blades over the papilla very difficult. An overly dry tongue usually makes the metal blade stick to the tongue making the delicate surgical move less controllable. Letting the subject close his/her mouth and have saliva come in contact with the surface of the tongue helps the process. Overly wet tongue is not good either. Using the corner of the gauze that one uses to hold the tongue helps in this process. </li>
 <li>Any capillary bleeding that does occur is treated by blotting with sterile gauze. Bleeding usually stops within a few minutes and does not interfere with additional tissue harvesting. Because the papillae extend above the epithelial surface of the tongue, and only the upper 3/4 of a fungiform papilla is removed (Figure 2B), that part of the fungiform showing taste buds, little bleeding is expected. </li>
 <li>Using a small Dumont forceps (Pattern type), the removed papillae are placed in chilled basic Ringer buffer ( 130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na-Pyruvate, 20 mM HEPES-Na, pH 7.2; osmolality of ~ 305 milli osmols) for subsequent manipulations . It is imperative to NEVER pinch the papilla between the blades of a forceps. The tissue is very fragile and should be handled with extreme care.</li>
 <li>After removal of the papillae, the subject remains in the chair for ten to fifteen minutes. Each subject is told not to bite on their anesthetized tongue, but to keep their tongue at rest. Subjects should be visually monitored by the attending surgeon. Anesthesia is expected to wear off in approximately 30-60 minutes. Because the site of papillae removal may be slightly sore after the biopsy, they are instructed to be aware of spicy or hot (temperature) food, carbonated drinks or drinks that are either extremely cold or hot. A further indication is given to subjects that any slight reddish coloration of saliva is normal and will cease upon dissolution of the clot.</li>
 <li>Subjects are seen 30-40 days following removal of the papillae for evaluation of the site where the papillae were removed. By this time taste papillae should have completely regenerated.</li>
</ol>
5. Representative Results
Tissue collected can be used for immunohistochemistry, RT-PCR, in situ hybridization, calcium imaging, patch clamping and tissue culture. When performed correctly the appearance of the papillae are healthy looking, white, about 0.5-1 mm in diameter (Figure 2B).
Papillae collected for immunohistochemistry are processed based on existing and published protocols 4. A typical taste bud from a paraformaldehide-fixed taste papilla is shown in Figure 4A.
Biopsied papillae can also be enzymatically treated5 to achieve dissociation of taste bud cells. After the enzymatic dissociation, cells can be maintained for up to 4 hours in a humidified Petri dish at 4C. In this state they can be used for single cell PCR (Figure 4B), Ca-imaging, etc.
Cells show a variety of shapes, many being slender and bipolar (Figure 5A and 5B) as one would expect of a taste bud cell. When collecting cells or performing Ca-imaging, after about 20 minutes under a microscope, the cells begin to develop apoptotic blebs (Figure 6A and 6B, arrows) and many of them become round. Using a technique developed in our lab, cells were picked up individually using a patch-pipette, as shown in Figure 5A and 5B.
As mentioned above, papillae begin to regrow after 5 to 8 weeks. Figure 7A demonstrates the appearance of a tongue where 8 fungiform papillae were removed from left side a volunteer's tongue using no anesthesia. Notice 8 slight reddish spots, where the papillae were snipped off. No bleeding is observed after surgery. The present image was taken 10 minutes after the biopsy. Figure 7B is an image of the tongue of the same subject seen 40 days after the biopsy. Notice all papillae have regrown. To determine if the regenerated papillae are functional we asked our volunteer to agree to a second biopsy involving the exact same papillae. To correctly identify the appropriate papillae we were planning to re-harvest, we generated a grid (Figure 7C) that fits the 8 papillae that were removed in the first place and superimposed it over the tongue of the same volunteer after 40 days post initial biopsy. Aided by the contour of the tongue and the distance from the margin to the grid, once identified, we have removed a few of the same exact papillae and processed them for immunohistochemistry. Figure 4A is one of these papillae. Notice the outline of a taste bud (in yellow) and the immunopositive staining for Phospholipase C&beta;2 (PLC&beta;2), a type 2 taste cell transduction-associated enzyme. These data demonstrate that using the biopsy procedure as described herein, the fungiform papillae regenerate sufficiently by 40 days to have also in place likely functional taste buds.
<img src="/files/ftp_upload/2201/2201fig1.jpg" alt="Figure 1" /> 
 Figure 1. The anterior quarter of the tongue of the subject is wrapped into sterile gauze and held in the left hand of the surgeon. Tongue must be held firmly. The enlarged image on the right shows the proper position of the scissors and the position of the papilla in relation to the blade.
<img src="/files/ftp_upload/2201/2201fig2.jpg" alt="Figure 2" /> 
 Figure 2. (A) Fungiform papillae are shown with blue arrows. These are round structures somewhat isolated from its surrounding and in their immediate vicinity there is a circular space devoid of filliform papillae. (B) Fungiform taste papilla on the tip of the spring micro-scissors seconds after it has been removed.
<img src="/files/ftp_upload/2201/2201fig3.jpg" alt="Figure 3" /> 
 Figure 3. Self biopsy of a fungiform papilla using a mirror and no anesthesia. The inset illustrates the overall position of the scissors, which must be parallel with the dorsal surface of the tongue. If done properly and atraumatically, anesthesia is forgone by virtually all volunteers.
<img src="/files/ftp_upload/2201/2201fig4a.jpg" alt="Figure 4a" /> 
 Figure 4 A. Immunohistochemistry of a taste bud. A fungiform papilla visualized by Nomarski optics using antibodies to Phospholipase &beta;2 and tagged with Red Fluorescent Protein. The outline of the taste bud and a few labeled taste cells are shown in yellow. <img src="/files/ftp_upload/2201/2201fig4b.jpg" alt="Figure 4b" /> B. RT-PCR of a candidate sour taste receptor, Acid Sensing Ion Channel 1 (ASIC1). M= marker. Lanes 1 through 4 were single cell RT-PCR for ASIC1 from four different volunteers. After collection, fungiform papillae were enzymatically dissociated, cells individually picked up and expression of ASIC1 were done. Lanes 2 and 4 had normal expression of ASIC1.
<img src="/files/ftp_upload/2201/2201fig5.jpg" alt="Figure 5" /> 
 Figure 5. A. Image of single taste cells being picked up using a patch pipette. A large epithelial cell is visible in the lower left corner (5A). Two red blood cells are also visible above the patch pipette and behind the glass barrel. The taste cell is approximately 30-50 microns long and about 6 micron wide. For reference, a red blood cell is about 7-8 microns in diameter. B. shows another taste receptor cell surrounded by four epithelial cells. The taste cell looks crisp.
<img src="/files/ftp_upload/2201/2201fig6.jpg" alt="Figure 6" /> 
 Figure 6. A. Images of cells that started to develop apoptotic blebs (ghost like round blebs attached to the taste cell, arrows). B. Cells are no longer harvested when their appearance changes as shown here.
<img src="/files/ftp_upload/2201/2201fig7.jpg" alt="Figure 7" /> 
 Figure 7. A. Ten minutes after removal of 8 taste papillae using no anesthesia. The surgical sites are visible as slightly reddish spots. No bleeding is noticed. B. The same tongue 40 days after biopsy. Notice all papillae regenerated. To help identify the removed papillae as regrown, a grid was created with the site on the right side picture and shifted over the regenerated papillae on the left side image. C. The regenerated papillae appeared in the exact same spot. Harvesting papillae again from this group followed by immunohistochemistry demonstrated that the regenerated papillae contained functional taste cells (See Figure 4A).

<ol>
	<li>Breslin, P. A., Spector, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+taste+perception.">Mammalian taste perception.</a> Curr Biol. 26 (4), R148-R155 (2008).</li><li>Miller, I., Doty, R. I., Dekker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+peripheral+taste+system.">Anatomy of the peripheral taste system.</a> Handbook of Olfaction and Gustation. , 521-547 (1995).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Figure+12.67.">Figure 12.67.</a> Gray's Anatomy: The Anatomical Basis of Medicine and Surgery. , (1995).</li><li>Huque, T., Cowart, B. J., Dankulich-Nagrudny, L., Pribitkin, E. A., Bayley, D. L., Spielman, A., Feldman, I., S, R., Mackler, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sour+ageusia+in+two+individuals+implicates+ion+channels+of+the+ASIC+and+PKD+families+in+human+sour+taste+perception+at+the+anterior+tongue.">Sour ageusia in two individuals implicates ion channels of the ASIC and PKD families in human sour taste perception at the anterior tongue.</a> PLoS One. 4 (10), e7347-e7347 (2009).</li><li>MacDonald, J. F., Mody, I., Salter, M. W., Spielman, A. I., Spielman, A. I., Brand, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamping+of+taste+cells+in+mouse.">Patch-clamping of taste cells in mouse.</a> Experimental Cell Biology of Taste and Olfaction. , 329-332 (1995).</li></ol>

No conflicts of interest declared.

One notable advantage to using human cells is their specificity. The procedure described here in detail provides tongue tissue for isolation of taste receptor cells from humans. These taste cells can be used for molecular studies, seeking to discover molecules important for transduction of taste information, and for calcium imaging studies that monitor taste cell activity to stimuli. Additionally, tongue tissue provided by this procedure can be analyzed with various techniques such as immunohistochemistry, in situ</e...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>McPherson-Vannas. Curved, sharp cutting edge 5mm. Comb, tip width 0.2 mm overall length 3"</td>
<td>&nbsp;</td>
<td>Roboz Surgical Instruments Co., Inc. Gaithersburg, MD 20898</td>
<td>RS-5603, Roboz</td>
<td>&nbsp;</td>
<tr>
<td>Small Dumont forceps</td>
<td>&nbsp;</td>
<td>Roboz Surgical Instruments Co.</td>
<td>RS-4915, Roboz</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

technique to collect fungiform (taste) papillae from human tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Watch this Scientific Journal Video about Technique to Collect Fungiform Taste Papillae from Human Tongue at JoVE.com

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

technique-to-collect-fungiform-taste-papillae-from-human-tongue

Research

JoVE Journal

Medicine

28.8K Views.  College of Dentistry, New York University. Conoscenza dei meccanismi molecolari alla base di trasduzione gustativa ha recentemente goduto di significativi passi avanti, soprattutto grazie all'utilizzo di modelli animali. Tuttavia, la grande diversità di sensibilità e specificità tra gusto garantisce mammiferi studi nel tessuto umano. Descriviamo una tecnica di biopsia per raccogliere cellule gustative viventi dalle papille sulla lingua umana.

Video: Technique to Collect Fungiform Taste Papillae from Human Tongue

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.

Object From Motion (OFM) and Time-constrained stereo protocols are sensitive tools to identify monocular and binocular dynamic visual function deficits, which are uniquely affected in optic neuritis patients. Furthermore, these tests may be used as quantitative noninvasive tools to assess the extent of myelination along visual pathways.

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and VII). Due to its autofluorescence, opaqueness, and thickness, it has not been found suitable for confocal microscopy. An alternative approach to the one presented here, which uses formalin-fixed sclera embedded in paraffin for immunohistochemistry, has technical challenges, especially when preheating the tissue for antigen retrieval. Since the sclera is relatively poor in both cells and vessels, the use of larger tissue samples was explored to help prevent overlooking cells and to understand their localization in relation to vessels and other anatomical sites. To allow for the analysis of larger tissue samples under the confocal microscope, a laminating technique was performed to create thin layers from the sclera. Following the analysis of results of CD31 blood vessels and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) positive cells, for which approval for scientific examination was obtained, the advantages and limitations of this method are discussed.

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Taste Exam: A Brief and Validated Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject&#39;s reported sensation and taste receptor genotype.

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...

Standardized Technique of Aortic Valve Re-implantation for Valve-sparing Aortic Root Replacement

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures prevents a number of cardiac surgeons from incorporating them into their practice. The aim of this protocol is to describe a simplified and user-friendly technique of an aortic valve-sparing root replacement (VSRR) procedure by re-implantation of the aortic valve. Proper selection of patients and limitations of the technique are discussed.
In 54 consecutive patients, normal appearing aortic valves were re-implanted in a commercially available polyester prosthesis with pre-shaped sinuses by a simplified and standardized technique. Placement of the first row of the proximal suture line, choice of the prosthesis size, and adjustment of the height of the commissures of the patient to the fixed height of the sinus portion of the prosthesis were slightly modified from the reference techniques with the aim of increasing its feasibility for use by other cardiac surgeons. Early mortality and morbidity as well as 5-year survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were collected in all patients.
Thirty-day mortality, re-sternotomy for bleeding, re-sternotomy for mediastinitis, and the incidence of stroke were very low, 1.8% for each (1 of 54). No patient required permanent pace-maker implantation. At 5 years, survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were 97.5%, 95.2%, and 91.6%, respectively.
Mid-term results of our standardized technique of re-implantation of the aortic valve for valve-sparing aortic root replacement are very good and compare with more complex techniques reported by experienced surgeons. By following the present protocol of the standardized re-implantation technique, a greater number of cardiac surgeons can perform this procedure with comparable good results.

Valve-sparing aortic root replacement has the advantage of preserving the patient&#39;s own aortic valve. The complexity of the reported techniques to date restricts their use to a limited number of cardiac surgeons. This protocol describes step-by-step a standardized technique reproducible by a greater number of cardiac surgeons.

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...