Les auteurs tiennent à remercier la Division de Vector-Borne Direction générale des ressources animales maladies, en particulier Andrea Peterson, Lisa Massoudi, Verna O&#39;Brien, et John Liddell pour leur entretien et de maintenance des souris et des lapins. Nous aimerions également remercier Amy Ullmann, Theresa Russell, et Barbara J. Johnson pour leurs contributions à l&#39;égard de ce manuscrit. Enfin, nous tenons à remercier Alissa Eckert dans le bureau du directeur adjoint pour la communication à la CDC pour la réalisation des illustrations graphiques et Judy Lavelle pour diriger tous les aspects juridiques liés à la prise de vue de ce manuscrit.

1. Collecte hémolymphe de préparation des lames (Film 1) <ol><li> Retirez délicatement activement tiques d&#39;alimentation d&#39;un animal et le lieu dans le peroxyde d&#39;hydrogène à 3% topique pour 5 minutes, puis dans l&#39;éthanol à 70% pendant 10 minutes à la surface de stériliser. </li><li> Avec un stylo pap dessiner un cercle sur une lame de microscope enduite de silane et de placer la tique dans le cercle stylo pap. <ol><li> Silane lames enduites sont utilisés pour une meilleure adhésion de l&#39;hémolymphe de la lame de microscope. </li></ol></li><li> Voir la tique sous une loupe binoculaire (1X objectif, oculaire 10x, un grossissement de 3.5X). </li><li> Poussez doucement sur le dos de la tique avec une pince à s&#39;évaser jambes de la tique et d&#39;immobiliser la tique. </li></ol> Remarque: Lorsque l&#39;immobilisation de la tique ne pas appuyer trop fort car cela peut perturber l&#39;intestin moyen ou de percer la tique et de contaminer l&#39;hémolymphe. <ol start="5"><li> Amputer la jambe de la tique ou les jambes à econjointe e distale avec une pointe fine scalpel jetable. Pour déterminer l&#39;infectiosité par l&#39;intermédiaire d&#39;hémolymphe seulement 1 jambe doit être amputée. Pour la collecte d&#39;hémolymphe sur une lame de plusieurs segments peuvent être amputé. </li></ol> Remarque: Ne pas couper la jambe trop près du corps, car cela peut provoquer une contamination intestin moyen de l&#39;hémolymphe. <ol start="6"><li> Après la ou les jambes sont coupées continuer à appliquer doucement la pression à dos de la tique pour l&#39;hémolymphe de sécréter sur les jambes sur la diapositive. Déplacez doucement la tique autour de la diapositive pour répandre l&#39;hémolymphe. </li></ol> 2. Ablation des glandes salivaires (Films 2 &amp; 3) <ol><li> Spot plusieurs piscines de 25 ul de tampon phosphate salin (PBS) sur une lame de microscope et placer une coche dans une des piscines de CPE. </li><li> Voir la tique sous une loupe binoculaire (1X objectif, oculaire 10x, un grossissement de 3.5X). </li><li> Stabiliser la tique avec pince à pointe amendes par la tenue du capitule base (pièces buccales) ou à l&#39;arrière de la tique. </li><li> Insérez les pinces fines pointes à l&#39;arrière de la tique et découper la tique est dos pour exposer les organes. Si vous le souhaitez l&#39;intestin moyen peut être retiré à ce moment et transféré à un pool de frais de PBS sur une lame de microscope ou à un microtube contenant du PBS. </li><li> Trouver la paire de glandes salivaires (raisin, comme les grappes) situés bilatéralement aux côtés des jambes de la tique. Si les glandes salivaires ne sont pas visibles parmi les débris tique déplacer la partie principale tique, contenant encore des glandes salivaires, d&#39;une piscine fraîche de PBS pour réduire la perturbation et la perte des glandes salivaires. </li></ol> Remarque: Plus tôt dans la tétée, les glandes salivaires sont plus difficiles à localiser, car ils ne sont pas aussi développé que par rapport à plus tard dans l&#39;alimentation. <ol start="6"><li> Retirer les glandes salivaires de la tique avec une pince fine pointe et les placer dans une piscine fraîche de PBS. </li><li> Wie à pointe fine pince transférer les glandes salivaires à l&#39;autre propre 25 PBS piscine pl, répétez cette étape de lavage 3-4 fois plus pour éliminer les micro-organismes externes et les débris tique. </li></ol> Remarque: Lavez les grappes des glandes salivaires en douceur afin de réduire la perte et la perturbation des glandes salivaires individuelles. <ol start="8"><li> Placez les glandes dans une piscine propre de PBS sur une lame de silane enduit ou dans un microtube contenant du PBS. </li></ol> 3. Le prélèvement de salive (Film 4) <ol><li> Retirez délicatement les tiques femelles adultes du lapin ou autre hôte à l&#39;aide de fines pinces à bout juste avant qu&#39;ils ne déposeront complètement gorgées de sang, des journées d&#39;environ 5-7 après fixation. </li><li> Respecter la tique près engorgée sur une extrémité d&#39;une lame de microscope avec du scotch. Le ruban doit être placé à environ ¾ de la hauteur dos de la tique vers la tête, laissant capitule base de la tique (pièces buccales) exposés.</li><li> Lorsque la bande se réunit le bord antérieur de la surface dorsale des tiques pipette 5 pi d&#39;une solution de pilocarpine 5% (dans le méthanol). Permettre à la bande pour absorber la pilocarpine sur le dos du cocher, sans permettre à la pilocarpine à venir en contact avec capitule base de la tique. </li><li> Monter un morceau de pâte à modeler non toxique sur la lame de microscope d&#39;environ un pouce de pièces buccales de la tique. </li><li> Utilisation de fines pinces à bout rompre la pointe d&#39;un tube capillaire tiré 28 au diamètre souhaité. </li><li> Voir capitule base de la tique sous une loupe binoculaire. </li><li> Doucement s&#39;adapter hypostome de la tique dans le tube capillaire permettant tiré les palpes maxillaires de résider à l&#39;extérieur du tube capillaire. </li><li> Appuyer sur l&#39;extrémité opposée du tube capillaire dans la pâte à modeler pour maintenir le tube capillaire en place. </li><li> Placez la tique monté saliver l&#39;intérieur d&#39;une chambre noire avec une humidité élevée (par exemple, une boîte à couvercle en polystyrène bordée de pap humide<html> serviettes er). Incliner la lame de sorte que les points de hypostome au fond du récipient, permettant gravité à l&#39;aide de prélèvement de salive. </li><li> Placez le récipient à la température ambiante. </li><li> Surveiller de près les tiques saliver pour la première heure, et de recueillir la salive comme il est généré par l&#39;expulsion hors des tubes capillaires avec un bulbe de pipette Pasteur. Après la première heure, vérifiez l&#39;accumulation toutes les heures pendant au moins 4 heures. Continuer à collecter la salive car il est généré. </li></ol> Remarque: l&#39;acquisition salive peut être arrêté une fois assez de salive sont recueillis pour l&#39;étude en cours d&#39;exécution. <ol start="12"><li> Si les tiques ne sont pas saliver ou si plus de salive est nécessaire, la salivation peut parfois être induite en utilisant le tube capillaire pour masser le hypostome. </li><li> Ajouter 0,1 volumes de cocktail inhibiteur de la protéase à la salive et les conserver à -80 ° C jusqu&#39;à ce que nécessaire. </li></ol> 4. Les résultats représentatifs e_content &quot;Film&gt; 1 montre comment tenir une nymphe I. scapularis et partiellement alimenté amputer les jambes pour recueillir hémolymphe sur une lame de microscope. Une fois la ou les jambes sont amputées un liquide clair est sécrétée (figure 1A et 1B). Si l&#39;intestin moyen est rompu l&#39;hémolymphe est trouble car il est sort de la jambe amputée (s) (figure 1C et 1D). L&#39;extraction des glandes salivaires après la nymphe a nourri pendant 48 ou 72 heures est démontrée dans les films 2 et 3. Après la tique est percé il ya généralement une grande quantité de débris (composé de la trachée, tubes de Malpighi, du sang, du tissu conjonctif, etc), pour empêcher la perte ou la perturbation des glandes salivaires de la tique se déplacer à un pool de frais de PBS. Les chiffres 2A et 2B montrent où les glandes salivaires sont situées après la nymphe a été coupée et la figure 2C montre enlevé grappes des glandes salivaires dans une mare de PBS. Le prélèvement de salive mis en place à partir de I. scapularis femelles adultes i<html> s montré dans le film 4 et la figure 3. Une coche saliver dans un tube capillaire est observé dans le film 5. Cette méthode de collecte de salive utilisée pour stimuler la salivation pilocarpine et peut produire plus de 20 ul de la salive par tique femelle adulte. <img alt="Figure 1." src="/files/ftp_upload/3894/3894fig1.jpg" /> Figure 1. Structures marquées d&#39;une nymphe I. scapularis. Film 1. Collection Ixodes scapularis hémolymphe. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie1.avi">Cliquez ici pour regarder un film</a> . <img alt="Figure 2." src="/files/ftp_upload/3894/3894fig2.jpg" /> Figure 2. Non contaminés (A &amp; B) et contaminés (C &amp; D) qui se dégage de la jambe hémolymphe de la nymphe. Movie 2. L&#39;extraction des glandes salivaires à partir d&#39;un 48 heures nourris I. nymphe scapularis.pload/3894/3894movie2.avi &quot;&gt; Cliquez ici pour regarder un film. L&#39;extraction Movie 3. La glande salivaire d&#39;un 72 heures nourris I. nymphe scapularis. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie3.avi">Cliquez ici pour regarder un film</a> . <img alt="Figure 3." src="/files/ftp_upload/3894/3894fig3.jpg" /> Figure 3. Ixodes scapularis glandes salivaires nymphe. (A &amp; B) Des exemples de glandes salivaires dans un 72 heures nourris nymphe, avant l&#39;extraction. (C) Suppression de cluster glande salivaire. Le prélèvement de salive Movie 4. Mettre en place d&#39;un adulte I. scapularis tique femelle. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie4.avi">Cliquez ici pour regarder un film</a> . <img alt="Figure 4." src="/files/ftp_upload/3894/3894fig4.jpg" /> Le prélèvement de salive Figure 4. De l&#39;adulte I. scapularis femelles tIcks. (A &amp; B) Cochez monté sur une glissière avec son hypostome à la fin tiré du tube capillaire avec la fin unpulled du tube capillaire détenu par la pâte à modeler. (C &amp; D) chambre humidifiée contenant adultes saliver I. scapularis tiques femelles. Film 5. Ixodes scapularis tique femelle saliver dans un tube capillaire. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie5.avi">Cliquez ici pour regarder un film</a> .

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+tick+oral+secretions+obtained+by+three+different+collection+methods.">Composition of tick oral secretions obtained by three different collection methods.</a> J. Med. Entomol. 10, 198-201 (1973).</li><li>Burgdorfer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolymph+test.+A+technique+for+detection+of+rickettsiae+in+ticks.">Hemolymph test. A technique for detection of rickettsiae in ticks.</a> Am. J. Trop. Med. Hyg. 19, 1010-1014 (1970).</li></ol>

Nous n&#39;avons rien à communiquer.

La collection de l &#39;hémolymphe tique, les glandes salivaires et la salive est important dans l&#39;étude des tiques transmission d&#39;agents pathogènes, la prévalence, la diffusion, la prolifération, et la persistance dans les deux la tique et l&#39;hôte 6,11-13,20,23,29. Il ya plusieurs façons de disséquer une tique 30,31. Toutefois, lors de la collecte des glandes salivaires, il est essentiel de disséquer la tique proprement les glandes salivaires ne sont pas rompues ou perdu dans l...

<table border="1"><tbody><tr><td> Réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Le peroxyde d&#39;hydrogène </td><td> Pêcheur </td><td> H312-500 </td></tr><tr><td> L&#39;éthanol </td><td> Acros </td><td> 61509-5000 </td></tr><tr><td> PBS </td><td> Bioproduits Boston </td><td> BM-2205 </td></tr><tr><td> Dumont fine pince (3C) </td><td> Pêcheur </td><td> NC9906085 </td></tr><tr><td> Silane lames de microscope traitées </td><td> Bioworld </td><td> 42763007-1 </td></tr><tr><td> Stylo Pap </td><td> Bioworld </td><td> 21750008-1 </td></tr><tr><td> Super Frost, plus des lames de microscope </td><td> Pêcheur </td><td> 12-550-18 </td></tr><tr><td> Pilocarpine </td><td> Sigma </td><td> P6503-5G </td></tr><tr><td> Cocktail d&#39;inhibiteurs de protéase </td><td> Sigma </td><td> P2714 </td></tr><tr><td> # 11 scalpel jetable </td><td> Plume </td><td> 2975 # 11 </td></tr><tr><td> Pâte à modeler non toxique </td><td&gt; Fisher </td><td> S17307 </td></tr><tr><td> Les tubes capillaires </td><td> Chase scientifiques verre, inc </td><td> 40A502 </td></tr></tbody></table>

saliva, salivary gland, and hemolymph collection from ixodes scapularis ticks

Les tiques se trouvent dans le monde entier et affligent les humains avec de nombreuses maladies transmises par les tiques. Les tiques sont des vecteurs d&#39;agents pathogènes qui causent la maladie de Lyme et fièvre récurrente à poux (Borrelia spp.), Rocky fièvre pourprée des montagnes (Rickettsia rickettsii), l&#39;ehrlichiose (Ehrlichia chaffeensis et E. equi), l&#39;anaplasmose (Anaplasma phagocytophilum), l&#39;encéphalite (tic- virus de l&#39;encéphalite transmise par), de la babésiose (Babesia spp.), de la fièvre à tiques du Colorado (Coltivirus), et la tularémie (Francisella tularensis) 1-8. Pour être correctement transmis à l&#39;hôte de ces agents infectieux différentielle réguler l&#39;expression génique, interagir avec des protéines de tiques, et migrent à travers la tique 3,9-13. Par exemple, l&#39;agent de la maladie de Lyme, Borrelia burgdorferi, s&#39;adapte à travers l&#39;expression différentielle des gènes aux stades de fête et de la famine de cycle enzootique de la tique 14,15. En outre, comme une tique Ixodes consomme unerepas de sang Borrelia reproduire et migrer de l&#39;intestin moyen dans le hémocèle, où ils se rendent aux glandes salivaires et sont transmis à l&#39;hôte avec la salive expulsées 9,16-19. Comme une tique se nourrit de l&#39;hôte répond généralement avec une forte réponse immunitaire innée et hémostatique 11,13,20-22. En dépit de ces réponses de l&#39;hôte, I. scapularis peut nourrir pendant plusieurs jours en raison salive de la tique contient des protéines qui sont immunomodulateur, des agents lytiques, les anticoagulants, et fibrinolysins pour aider la tique nourrir 3,11,20,21,23. Les activités immunomodulatrices possédés par la salive de tique ou extrait des glandes salivaires (SGE) de faciliter la transmission, la prolifération et la diffusion de nombreux agents pathogènes transmis par les tiques 3,20,24-27. Pour mieux comprendre comment tiques agents infectieux provoquer une maladie, il est essentiel de disséquer les tiques se nourrissent activement et de recueillir la salive des tiques. Ce protocole vidéo montre les techniques de dissection pourla collection de l &#39;hémolymphe et la suppression des glandes salivaires de nourrir activement I. nymphes scapularis après 48 et 72 heures le placement de la souris après. Nous démontrons également le prélèvement de salive d&#39;un adulte femelle I. tique scapularis.

Watch this Scientific Journal Video about Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks at JoVE.com

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

La collection de l &#39;hémolymphe tique infectée, des glandes salivaires et la salive est important d&#39;étudier comment les tiques agents pathogènes causent la maladie. Dans ce protocole, nous montrer comment recueillir les glandes salivaires et l&#39;hémolymphe de se nourrir<em&gt; Ixodes scapularis</em&gt; Nymphes. Nous démontrons également le prélèvement de salive de la femelle<em&gt; I. scapularis</em&gt; Les adultes.

La salive, les glandes salivaires, et Collection hémolymphe de tiques Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

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Research

JoVE Journal

Immunology and Infection

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

29.2K vues.  Centers for Disease Control and Prevention. La collection de l 'hémolymphe tique infectée, des glandes salivaires et la salive est important d'étudier comment les tiques agents pathogènes causent la maladie. Dans ce protocole, nous montrer comment recueillir les glandes salivaires et l'hémolymphe de se nourrir Ixodes scapularis Nymphes. Nous démontrons également le prélèvement de salive de la femelle I. scapularis Les adultes.

Vidéo: Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

ARN interférence dans les tiques

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Recherche

Immunologie et infection

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Hybridation<em&gt; In situ</em&gt; Des glandes salivaires, des ovaires et des embryons de moustiques vecteurs

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Alimentation des tiques sur les animaux pour la transmission et Xénodiagnostic dans la recherche la maladie de Lyme

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

Élevage<em&gt; Ixodes scapularis,</em&gt; La tique à pattes noires: alimentation sur des souris étapes Immature

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Une méthode pour la mesure de la fonction des glandes salivaires chez les souris

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

Préparation du murin des glandes salivaires sous-maxillaires pour la microscopie intravitale verticale

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

Isolement des cellules épithéliales salivaires des glandes salivaires humaines pour la croissance in vitro comme Salispheres ou Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

Purification des préparations extracellulaires de vésicule à haut rendement loin du virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

Divulgation de la collection d’hémolymphe et inoculation des blastospores de Métarhizium dans les tiques de Rhipicephalus MICROPLUS vers les études de pathologie des invertébrés

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

Imagerie en temps réel de la régénération de la glande sous-mandibulaire de la souris

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

Pleins feux sur l’auteur : Étude des interactions macrophage-cellules épithéliales dans la régénération des glandes salivaires après une blessure 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Interrogation des interactions cellule-cellule dans la glande salivaire par imagerie ex vivo de cellules vivantes

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...