Gli autori desiderano ringraziare la Divisione di Vector-Borne Diseases Branch Animal Resources, in particolare Andrea Peterson, Lisa Massoudi, Verna O&#39;Brien e John Liddell per la loro cura e manutenzione dei topi e conigli. Vorremmo anche ringraziare Amy Ullmann, Theresa Russell, e Barbara J. Johnson per il loro contributo verso questo manoscritto. Infine, vorremmo riconoscere Alissa Eckert presso l&#39;Ufficio del Direttore Associato per la Comunicazione presso il CDC per la produzione di illustrazioni grafiche e Judy Lavelle per dirigere tutti gli aspetti legali connessi con le riprese di questo manoscritto.

1. Raccolta emolinfa per preparazione del vetrino (Movie 1) <ol><li> Delicatamente rimuovere attivamente zecche alimentazione da un animale e posto in 3% di perossido di idrogeno topico per 5 minuti e poi in etanolo 70% per 10 minuti per sterilizzare superficie. </li><li> Con una penna pap disegnare un cerchio su un vetrino da microscopio silano rivestito e mettere il segno di spunta all&#39;interno del cerchio penna pap. <ol><li> Silano vetrini rivestiti vengono utilizzati per la migliore aderenza del emolinfa al vetrino da microscopio. </li></ol></li><li> Mostra il segno di spunta sotto un microscopio da dissezione (1X obiettivo, oculare 10X, ingrandimento 3,5 x). </li><li> Premere delicatamente sul dorso della zecca con una pinzetta per le gambe splay della zecca e immobilizzare il segno di spunta. </li></ol> Nota: Quando immobilizzare il segno di spunta non premere troppo forte perché questo può disturbare la midgut o forare il segno di spunta e contaminare l&#39;emolinfa. <ol start="5"><li> Amputare la gamba della zecca o gambe °e giunto distale con una punta sottile bisturi monouso. Per determinare l&#39;infettività via emolinfa solo 1 gamba deve essere amputata. Per la raccolta di emolinfa su un vetrino da più segmenti possono essere amputata. </li></ol> Nota: Non tagliare la gamba troppo vicino al corpo, poiché ciò potrebbe causare la contaminazione del midgut emolinfa. <ol start="6"><li> Dopo la tappa o le gambe sono tagliate continuare ad applicare delicatamente pressione dorso della zecca per la emolinfa di secernere fuori le gambe sul vetrino. Muovete il segno di spunta intorno sulla diapositiva per diffondere la emolinfa. </li></ol> 2. La rimozione della ghiandola salivare (Film 2 &amp; 3) <ol><li> Spot più 25 piscine microlitri di tampone fosfato (PBS) su un vetrino da microscopio e mettere un segno di spunta in una delle piscine PBS. </li><li> Mostra il segno di spunta sotto un microscopio da dissezione (1X obiettivo, oculare 10X, ingrandimento 3,5 x). </li><li> Stabilizzare la zecca con pinza a punta sottiles tenendo la base capitulum (apparato boccale) o la parte posteriore della zecca. </li><li> Inserire le pinze belle punte nella parte posteriore della zecca e tagliare la zecca dorso per esporre gli organi. Se desiderato il midgut può essere rimosso in questo momento e trasferito a un pool di PBS fresco su un vetrino da microscopio o una provetta da microcentrifuga contenente PBS. </li><li> Trova la coppia di ghiandole salivari (grappoli) situati bilateralmente lungo le gambe della zecca. Se le ghiandole salivari non sono visibili tra le macerie spunta spostare la parte principale segno di spunta, contenente ancora le ghiandole salivari, a un pool fresco di PBS per ridurre la rottura e la perdita delle ghiandole salivari. </li></ol> Nota: precedenti in una alimentazione, le ghiandole salivari sono più difficili da individuare perché non sono come sviluppato rispetto a seguito in alimentazione. <ol start="6"><li> Rimuovere le ghiandole salivari della zecca con una pinza a punta fine e posto in una piscina fresca di PBS. </li><li> With bella mancia pinza trasferire le ghiandole salivari a un altro pulito piscina di 25 microlitri di PBS, ripetere questo passaggio di lavaggio 3-4 volte di più per eliminare eventuali microrganismi esterni e detriti tick. </li></ol> Nota: Lavare i grappoli delle ghiandole salivari delicatamente per ridurre la perdita e la rottura delle singole ghiandole salivari. <ol start="8"><li> Mettere le ghiandole in una piscina pulita di PBS in una diapositiva silano rivestito o in una provetta da microcentrifuga contenente PBS. </li></ol> 3. Saliva di raccolta (Movie 4) <ol><li> Rimuovere delicatamente le zecche femmine adulte dal coniglio o di un altro host utilizzando una pinza a punta fine poco prima di cadere completamente off ingorgati, i giorni circa 5-7 post-attacco. </li><li> Rispettare il segno di spunta quasi engorged su un&#39;estremità di un vetrino da microscopio con lo scotch. Il nastro deve essere posizionato a circa ¾ della salita dorso della zecca verso la testa, lasciando capitulum base della zecca (apparato boccale) esposta.</li><li> Quando il nastro incontra il bordo anteriore della superficie dorsale pipetta zecche 5 pl di soluzione di pilocarpina 5% (in metanolo). Lasciare che il nastro stoppino della pilocarpina sul dorso di zecca, senza che il pilocarpina a venire in contatto con capitulum base della zecca. </li><li> Montare un pezzo di non-tossico plastilina sul vetrino di circa un pollice dal boccale della zecca. </li><li> Utilizzando belle pinze punta rompere la punta di un tubo capillare tirato 28 al diametro desiderato. </li><li> Visualizza capitulum base della zecca sotto un microscopio da dissezione. </li><li> Inserire delicatamente ipostoma della zecca nel tubo capillare tirato permettendo ai palpi mascellari di risiedere al di fuori del tubo capillare. </li><li> Premere l&#39;estremità opposta del tubo capillare nel plastilina per tenere il tubo capillare in posizione. </li><li> Mettere il segno di spunta montato la salivazione all&#39;interno di una camera oscura con umidità elevata (ad esempio, una scatola con coperchio di polistirolo rivestiti con pap bagnato<html> asciugamani er). Inclinare il vetrino in modo i punti ipostoma al fondo del contenitore, permettendo la gravità per aiutare nella saliva raccolta. </li><li> Posizionare il recipiente a temperatura ambiente. </li><li> Monitorare attentamente le zecche acquolina in bocca per la prima ora, e raccogliere la saliva in quanto è generato da espellendo fuori dei tubi capillari con una lampadina pipetta Pasteur. Dopo la prima ora, controllare accumulo ogni ora per almeno 4 ore. Continua a raccogliere la saliva come viene generato. </li></ol> Nota: l&#39;acquisizione di saliva può essere fermata una volta abbastanza saliva viene raccolto per lo studio in corso di esecuzione. <ol start="12"><li> Se le zecche non sono la salivazione o se più saliva è necessario, salivazione può occasionalmente essere indotta tramite il tubo capillare per massaggiare il ipostoma. </li><li> Aggiungere 0,1 volumi di cocktail inibitore di proteasi per la saliva e conservare a -80 ° C fino a quando necessario. </li></ol> 4. Risultati rappresentativi e_content &quot;Movie&gt; 1 dimostra come tenere una ninfa parzialmente alimentato I. scapularis e amputare le gambe per raccogliere emolinfa su un vetrino da microscopio. Una volta che la gamba o gambe amputate un liquido chiaro viene secreto (figura 1A e 1B). Se il midgut si rompe la emolinfa appare torbido come è esce l&#39;arto (s) (figura 1C e 1D). L&#39;estrazione delle ghiandole salivari dopo la ninfa è stata l&#39;alimentazione per 48 o 72 ore è dimostrato nei film 2 e 3. Dopo il segno di spunta viene forato vi è generalmente un sacco di detriti (composto da trachea, Malpighi tubuli, sangue, tessuto connettivo, ecc), per prevenire la perdita o distruzione delle ghiandole salivari spostare il segno di spunta a un pool fresco di PBS. Le figure 2A e 2B mostrano dove si trovano le ghiandole salivari dopo che la ninfa è stato tagliato aperto e la figura 2C mostra rimosso cluster delle ghiandole salivari in una pozza di PBS. Saliva la raccolta istituito dalla I. femmine adulte scapularis i<html> s mostrato in film 4 e figura 3. Un segno di spunta la salivazione in un tubo capillare si osserva nel film 5. Questo metodo di raccolta saliva pilocarpina utilizzato per stimolare la salivazione e può produrre più di 20 pl di saliva per adulto tick femmina. <img alt="Figura 1." src="/files/ftp_upload/3894/3894fig1.jpg" /> Figura 1. Strutture etichetta di una ninfa scapularis I.. Movie 1. Raccolta emolinfa Ixodes scapularis. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie1.avi">Clicca qui per guardare film</a> . <img alt="Figura 2." src="/files/ftp_upload/3894/3894fig2.jpg" /> Figura 2. Incontaminata (A &amp; B) e contaminati (C &amp; D) emolinfa che trasuda dalla gamba della ninfa. Movie 2. L&#39;estrazione della ghiandola salivare da una 48 ore Fed I. ninfa scapularis.pload/3894/3894movie2.avi &quot;&gt; Clicca qui per guardare film. Movie 3. L&#39;estrazione della ghiandola salivare di 72 ore da un nutrito I. ninfa scapularis. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie3.avi">Clicca qui per guardare film</a> . <img alt="Figura 3." src="/files/ftp_upload/3894/3894fig3.jpg" /> Figura 3. Ixodes scapularis ninfa ghiandole salivari. (A &amp; B) Esempi di ghiandole salivari di una 72 ore alimentato ninfa, prima dell&#39;estrazione. (C) Rimosso cluster di ghiandola salivare. Saliva Movie 4. Di raccolta istituiti a da un adulto I. scapularis tick femminile. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie4.avi">Clicca qui per guardare film</a> . <img alt="Figura 4." src="/files/ftp_upload/3894/3894fig4.jpg" /> Figura 4. Prelievo della saliva da adulto I. scapularis t femminiliicks. (A e B) Spuntare montato su una slitta con ipostoma alla fine tirato del tubo capillare con l&#39;estremità unpulled del tubo capillare tenuto da plastilina. (C &amp; D) camera umidificata contenente la salivazione adulti I. scapularis zecche femminili. Movie 5. Scapularis zecche Ixodes femminile acquolina in bocca in un tubo capillare. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie5.avi">Clicca qui per guardare 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>

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La raccolta di emolinfa tick, ghiandole salivari, e la saliva è importante nello studio delle zecche trasmissione dei patogeni, la prevalenza, la diffusione, la proliferazione, e la persistenza sia il segno di spunta e l&#39;host 6,11-13,20,23,29. Ci sono diversi modi per sezionare un segno di spunta 30,31. Tuttavia, quando la raccolta delle ghiandole salivari è fondamentale per analizzare correttamente il segno di spunta in modo che le ghiandole salivari non sono rotti o persi nei resti della ze...

<table border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Il perossido di idrogeno </td><td> Pescatore </td><td> H312-500 </td></tr><tr><td> Etanolo </td><td> Acros </td><td> 61509-5000 </td></tr><tr><td> PBS </td><td> Boston Bioproducts </td><td> BM-2205 </td></tr><tr><td> Dumont fine pinza (3C) </td><td> Pescatore </td><td> NC9906085 </td></tr><tr><td> Silano vetrini da microscopio trattati </td><td> Bioworld </td><td> 42763007-1 </td></tr><tr><td> Pap penna </td><td> Bioworld </td><td> 21750008-1 </td></tr><tr><td> Super gelo più vetrini da microscopio </td><td> Pescatore </td><td> 12-550-18 </td></tr><tr><td> Pilocarpine </td><td> Sigma </td><td> P6503-5G </td></tr><tr><td> Cocktail di inibitori della proteasi </td><td> Sigma </td><td> P2714 </td></tr><tr><td> # 11 monouso bisturi </td><td> Piuma </td><td> 2975 # 11 </td></tr><tr><td> Modellare l&#39;argilla non tossico </td><td&gt; Fisher </td><td> S17307 </td></tr><tr><td> Tubi capillari </td><td> Chase scientifica Glass, inc </td><td> 40A502 </td></tr></tbody></table>

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

Le zecche sono presenti in tutto il mondo e di affliggere gli esseri umani con molte malattie trasmesse da zecche. Le zecche sono vettori di agenti patogeni che causano la malattia di Lyme e zecche febbre ricorrente (Borrelia spp.), Febbre delle Montagne Rocciose (Rickettsia rickettsii), ehrlichiosi (Ehrlichia chaffeensis e E. equi), anaplasmosi (Anaplasma phagocytophilum), encefalite (tick- virus dell&#39;encefalite Borne), babesiosi (Babesia spp.), febbre tick Colorado (Coltivirus), e la tularemia (Francisella tularensis) 1-8. Per essere correttamente trasmessi nell&#39;ospite questi agenti infettivi differenziale regolare l&#39;espressione genica, interagiscono con le proteine ​​zecca, e migrano attraverso il segno di spunta 3,9-13. Ad esempio, l&#39;agente della malattia di Lyme, Borrelia burgdorferi, si adatta attraverso l&#39;espressione genica differenziale alle fasi festa e la carestia del ciclo di leucosi della zecca 14,15. Inoltre, come una zecca Ixodes consumafarina di sangue Borrelia replicarsi e migrare dal midgut nel hemocoel, dove si spostano per le ghiandole salivari e vengono trasmessi nell&#39;ospite con la saliva espulsi 9,16-19. Come un segno di spunta alimenta l&#39;host risponde in genere con una forte risposta immunitaria innata emostatico e 11,13,20-22. Nonostante queste risposte di accoglienza, I. scapularis può alimentare per diversi giorni, perché la saliva tick contiene proteine ​​che sono immunomodulatori, agenti litici, anticoagulanti e fibrinolysins per aiutare il segno di spunta di alimentazione 3,11,20,21,23. Le attività immunomodulanti possedute dal saliva tick o estratti di ghiandola salivare (SGE) facilitare la trasmissione, la proliferazione e la diffusione di numerose zecche patogeni 3,20,24-27. Per capire meglio come zecche agenti infettivi causare la malattia è indispensabile analizzare attivamente l&#39;alimentazione zecche e raccogliere la saliva tick. Questo protocollo video mostra tecniche di dissezione perla raccolta di emolinfa e la rimozione delle ghiandole salivari da attivamente alimentazione I. ninfe scapularis dopo 48 e 72 ore dopo il posizionamento del mouse. Dimostriamo anche il prelievo della saliva da una femmina adulta I. scapularis tick.

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 raccolta di emolinfa zecca infetta, ghiandole salivari, e la saliva è importante studiare come zecche patogeni causare la malattia. In questo protocollo si dimostra come raccogliere emolinfa ghiandole salivari e di nutrirsi<em&gt; Ixodes scapularis</em&gt; Ninfe. Dimostriamo anche il prelievo della saliva da femmina<em&gt; I. scapularis</em&gt; Adulti.

Saliva, le ghiandole salivari, e Collection emolinfa dalle zecche 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 Views.  Centers for Disease Control and Prevention. La raccolta di emolinfa zecca infetta, ghiandole salivari, e la saliva è importante studiare come zecche patogeni causare la malattia. In questo protocollo si dimostra come raccogliere emolinfa ghiandole salivari e di nutrirsi Ixodes scapularis Ninfe. Dimostriamo anche il prelievo della saliva da femmina I. scapularis Adulti.

Video: 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.

RNA interferenza nelle zecche

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

Ricerca

Immunologia e infezioni

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.

Ibridazione<em&gt; In situ</em&gt; Delle ghiandole salivari, ovaie, e gli embrioni di Vector zanzare

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.

Alimentazione di zecche su animali per trasmissione e Xenodiagnosis a 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 ...

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.

impennata<em&gt; Ixodes scapularis,</em&gt; Il Tick Black-legged: Alimentazione stadi immaturi sui topi

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.

Un metodo per la misurazione della funzione della ghiandola salivaria in topi

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.

Preparazione della ghiandola salivare sottomandibolare murino per microscopia Intravital 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.

Isolamento delle cellule epiteliali salivari dalle ghiandole salivari umane per la crescita in vitro come Salispheres o Monostrati

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.

Purificazione dei preparati vescicoli extracellulari ad alto rendimento lontano dal 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.

Rivelare la raccolta di hemolymph e l'inoculo di Metarhizium blastospores in Rhipicephalus MicroPlus zecche verso gli studi di patologia degli invertebrati

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

Imaging in tempo reale della rigenerazione della ghiandola sottomandibolare del topo

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.

Autore in primo piano: Studio delle interazioni tra macrofagi e cellule epiteliali nella rigenerazione delle ghiandole salivari dopo un infortunio 

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.

Interrogazione delle interazioni cellula-cellula nella ghiandola salivare tramite imaging di cellule vive ex vivo

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