German Research Foundation (FOR2240 "(Lymph) Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye" to CC and LMH; HE 6743/2-1 and HE 7643/3-1 to LMH; CU47/6-1 to CC), German Cancer Aid (to LMH and CC), GEROK program University of Cologne (to SLS and LMH), and EU COST BM1302 "Joining Forces to Corneal Regeneration" (to CC).

L&#39;uso di tessuti umani deve essere esaminato e approvato da un comitato istituzionale di revisione o equivalente. Il lavoro qui descritto è stato approvato dal comitato etico locale e ha avuto l&#39;approvazione per l&#39;esame scientifico. Questo lavoro è stato eseguito in base alla Dichiarazione di Helsinki. I campioni sclerali umani sono stati ottenuti dagli occhi dei donatori globo (massimo post mortem 24 ore) presso la Banca degli Occhi del Dipartimento di Oftalmologia, Università di Colonia, in Germania. 1. Preparazione sperimentale <ol><li> Preparare 96% etanolo e tampone fosfato isotonico (PBS) in tubi diversi. Preparare gli anticorpi primari nella diluizione raccomandata di PBS contenente 2% di albumina sierica bovina (BSA) e mantenere tutte le soluzioni su ghiaccio. Preparare 100 microlitri per campione. </li><li> Pulire tutti gli strumenti e utilizzare bisturi taglienti. Dopo ripetute taglio con la stessa bisturi, cambiare il bisturi. </li><li> Ottenere 1-2 pinze Colibri, forbici dissezione micro scala, un # 10 bisturio oftalmica bisturi micro piuma, e quattro aghi 26 G. </li><li> Avvolgere un foglio di alluminio intorno a un piatto di polistirolo o utilizzare una piastra di sughero di apporre il tessuto. Lavorare sotto un stereomicroscopio binoculare. Lavorare sterile sotto un flusso laminare, se necessario. </li></ol> 2. Preparare la sclera <ol><li> Premere delicatamente la lampadina ed eseguire una trapanazione perforazione da parte corneosclerale, quindi ruotare il trapano. Ruotare il trapano con cura e in modo uniforme sulla superficie di eseguire un taglio altrettanto rotondo. Utilizzare un trapano di dimensioni 15,5 millimetri. Tagliare i restanti allegati con forbici curve. Rimuovere la trapanazione corneosclerale. Questo si tradurrà in una lampadina anteriormente aperto. <ol><li> Mettere la sclera su un tampone con la parte aperta verso l&#39;alto. Rimuovere la retina e uvea, con pinze Colibri di tirare fuori entrambi gli strati (retina e uvea) dalla sclera. Non lasciare uvea pigmentato sulla sclera interiore. Utilizzare forbici curve per rimuovere la retina e uvea dalla papilla. Rimuovere il remaining congiuntiva, muscoli estrinseci, e la capsula di tenone dalla sclera superficiale. </li></ol></li><li> Rimuovere il campione sclerale nella dimensione desiderata utilizzando le forbici straight-tipo e le pinze Colibri. Prendere circa 2 cm 2 campioni sclerali dimensioni da luoghi diversi. A causa delle piccole dimensioni, tenere la sclera delicatamente e tagliare fuori la dimensione richiesta come quadrati utilizzando le forbici straight-tipo. La trapanazione corneosclerale definisce il margine anteriore dei campioni sclerali. <ol><li> Evitare afferrando ripetuto con le pinze come l&#39;area in cui il tessuto è stato afferrato non è adatto per le analisi microscopia confocale. Preparare il numero di campioni in funzione del tipo di esperimento (es anteriormente vs. posteriormente, confrontare Figura 1) e la quantità di anticorpi utilizzati. </li></ol></li><li> Mettere i campioni in provette da 1,5 ml per un uso successivo. Fissare i campioni in 1,5 ml di etanolo 96% per 15 minuti, e poi lavarli tre volte ciascuno per 5 minin 1,5 ml di PBS sul shaker. Se si lavora con il tessuto congelato, eseguire questo passaggio prima di scatto di congelamento. </li><li> Utilizzare tessuto fresco. Tuttavia, se ciò non è possibile, a scatto congelare la sclera in azoto liquido e la tiene a -20 ° C per un uso successivo. Se si lavora con il tessuto congelato, scongelare prima dell&#39;uso. Evitare ripetuti cicli di congelamento scongelamento e. </li><li> Mantenere ogni campione in PBS contenente 1,5 ml 5% BSA a temperatura ambiente per 2 ore. Questo passo induce un rigonfiamento del campione che è utile per la laminazione e impedisce anche legame aspecifico. </li><li> Dopo che i campioni hanno gonfiato, preparare le piazze sclerali per la laminazione. Lavorare sotto un microscopio, ad esempio uno stereo microscopio binoculare. Fissare i campioni sclerali posteriori alla membrana in polistirolo avvolto in un foglio di alluminio o la piastra di sughero con gli aghi 26 g. </li><li> Piegare i bordi degli aghi in modo da non interferire con il campo visivo sotto il microscopio. </li><li> Laminare il tutto spessore SAMP scleraleles. <ol><li> In primo luogo, tenere il bordo sclerale anteriore con pinze Colibri. Poi accuratamente tagliato un sottile strato di sclera superficiale utilizzando il bisturi # 10, il oftalmica bisturi micro piuma, o una lama spatola. Tenere il bisturi orizzontale e tagliare lo strato superficiale più sottile possibile dallo strato sottostante. </li><li> Sezionare uno strato sottile sclerale dalla piazza sclerale. Questo metodo è simile alle tecniche chirurgiche standard per preparare un lembo durante trabeculectomia e dovrebbe tradursi in strati di spessore 30-80 micron (confronta figura 2). </li></ol></li><li> Impedire essiccamento della sclera aggiungendo piccole quantità di PBS al tessuto, per esempio 50 microlitri di PBS utilizzando una pipetta. </li><li> Mettere i livelli di taglio in 100 pl di PBS nella piastra da 96 pozzetti e l&#39;etichetta sia l&#39;orientamento (verso extern e intern) e lo strato (superficiale e profonda) ad esempio con un colore impermeabile. </li><li> Ripetere i passaggi 2,7-2,9 fino a quando il tessuto è completamente laminato. &lt;/ Li&gt; </ol> 3. Eseguire immunoistochimica <ol><li> Aggiungere 100 ml di anticorpi primari necessari alla diluizione raccomandata. Qui, per esempio CD utilizzo 31 (topo monoclonale anti umano) e anticorpi LYVE1 (coniglio anti umano), sia in una diluizione di 1: 100 in PBS (contenente 2% BSA) e incubare a 4 ° C durante la notte. Per cambiare il supporto nella piastra da 96 pozzetti, tenere la pipetta alla parete del pozzo e rimuovere il liquido. </li><li> Il giorno dopo, lavare i campioni per tre volte ciascuno per 5 min con 200-300 ml di PBS sul shaker. Aggiungere 100 pl di corrispondenti anticorpi secondari; qui utilizzare capra anti topo fluoresceina isotiocianato (FITC) e di capra anti coniglio cianina coloranti 3 (Cy3), e incubare i campioni a temperatura ambiente per 1-2 ore. Diluire gli anticorpi secondari 1: 300 in PBS contenente 2% di siero di capra. </li><li> Lavare i campioni di nuovo tre volte ciascuno per 5 min in 200-300 ml di PBS sul shaker. </li><li> Eseguire colorazione nucleo, ad esempio, </em&gt;. 4 &#39;, 6-diamidino-2-fenilindolo (DAPI) (diluizione 1: 2.000, 100 microlitri in ciascun pozzetto ed incubare 10 min a temperatura ambiente), poi lavare ancora 2 volte in PBS 200-300 microlitri l&#39;agitatore. </li><li> Trasferire i campioni su vetrini da microscopio, incorporare loro in 1-2 gocce di mezzo di montaggio fluorescente, aggiungere un vetrino, sigillarla coprendo i bordi con vernice trasparente, e conservare a 4 ° C per un uso successivo o esaminare direttamente i vetrini con il confocale microscopio. </li><li> Utilizzare un microscopio confocale (o equivalente) per analizzare i campioni. Utilizzare l&#39;ingrandimento desiderato, ad esempio 10-40X ingrandimento. </li><li> Per verificare lo spessore dei campioni, misurare con microscopia confocale utilizzando Z-Stack. La quantità di possibili sezioni sclerali dipende dalla posizione della sclera, come lo spessore diverso equatoriale e posteriormente (confrontare introduzione). </li></ol>

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The authors declare no financial disclosures. The contents of this article are solely the responsibility of the authors.

Laminazione la sclera umana è un metodo per eseguire la microscopia confocale in questo tessuto. Un punto critico in questo processo è l&#39;uso di etanolo invece di formalina per l&#39;apposizione del tessuto. Nella nostra esperienza, i risultati migliori si ottengono quando si utilizza etanolo invece di formalina per fissare. bisturi Blunt aggravano la procedura e deve essere evitato. Analogamente, il prosciugamento della sclera dovrebbe essere evitata, in quanto complica la procedura e riduce la qualità delle imma...

La sclera è lo strato esterno rigido che copre l&#39;occhio, che è fatto di tessuto connettivo denso. Aiuta a proteggere le strutture intraoculari e di mantenere la pressione intraoculare. Così, la sclera è essenziale per una visione chiara. E &#39;privo di vasi linfatici 1,2 e, quindi, forma un bordo esterno libero-linfatica tra essa e l&#39;occhio interiore senza linfatica 3-7. Esso fornisce inoltre i siti di attacco per i muscoli estrinseci, condividendo in tal modo somiglianze anatomiche con tendini. Poiché la sclera consiste principalmente di densi fasci di collagene di tipo I e ha un numero inferiore di collagene di tipo III, IV, V, VI, VIII 8,9 ed elastina 10,11, questo tessuto non è facile da usare per immunoistochimica. Anatomicamente, la sclera può essere suddiviso in tre strati principali: (1) il superficiale episclera vascolarizzato, trovati sotto la congiuntiva e la capsula di tenone e verso i lati e the posteriore dell&#39;occhio rivolto verso l&#39;orbita; (2) stroma sclerale, la parte principale della sclera; e (3) il fusca lamina, che è un sottile strato pigmentato situato direttamente sopra la uvea. La nostra conoscenza anatomica sulla sclera deriva principalmente dalla prima metà del 20 ° secolo. A quel tempo, i ricercatori hanno studiato l&#39;anatomia del sistema vascolare prevalentemente utilizzando iniezioni di inchiostro India 12 e vascolare fusione 13-15. Successivamente, è stato studiato in studi angiografici 16-19. Da quel momento, le tecniche più vecchie sono state migliorate e nuove sono state sviluppate che ci hanno permesso di completare una precedente conoscenza anatomica. Ad esempio, è stato solo di circa un decennio da quando abbiamo avuto tali marcatori linfatici affidabili come linfatico vascolare acido ialuronico specifico recettore-1 (LYVE1) 20 o 21 podoplanin. La microscopia confocale offre nuove possibilità per lo studio delle caratteristiche anatomiche dei diversi tiUESTIONI dell&#39;occhio. Esso consente più macchie da utilizzare per differenziare marcatori di cellule o per la localizzazione delle cellule in relazione ai vasi sanguigni e altre strutture anatomiche. Esso fornisce una panoramica quando il campione è di dimensioni maggiori e ci permette di scandiamo un campione quando alla ricerca di un tipo specifico di cellula. Con la tecnologia Z-Stack, microscopia confocale può essere utilizzato per i campioni fino a 100-200 micron. La sclera differisce di spessore tra 0,3 mm dietro le inserzioni muscolari e 1 mm al polo posteriore 11. A causa sia suo spessore e opacità, la sclera non è adatto per la microscopia confocale con metodi tradizionali. Per rimediare a questo, i tessuti sclerali sono stati stratificati per consentire la loro analisi con microscopia confocale. Questa tecnologia è utile per una migliore comprensione di entrambe le situazioni fisiologiche e patologiche nella sclera umana.

<table>
	<tbody>
		<tr>
			<td>96% ethanol</td>
			<td>Merck Chemicals, Darmstadt, Germany</td>
			<td>P075.4</td>
			<td></td>
		</tr>
		<tr>
			<td>binocular stereo microscope&#160;</td>
			<td>Motic, Hongkong, China</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>26G needles&#160;</td>
			<td>Terumo, Leuven, Belgium</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr>
			<td>15.5mm trepan</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>no.10 scalpel&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td>2E+08</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic scalpel micro feather&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td colspan="2">no. 7657BR</td>
		</tr>
		<tr>
			<td>CD 31 antibody (monoclonal mouse anti human)</td>
			<td>Dako, USA</td>
			<td>IR610</td>
			<td></td>
		</tr>
		<tr>
			<td>LYVE1 antibody&#160; (polyclonal rabbit anti human)</td>
			<td>Zytomed, Germany</td>
			<td colspan="2">RBK014-05</td>
		</tr>
		<tr>
			<td>goat anti mouse FITC antibody</td>
			<td>Sigma Aldrich, Steinheim, Germany</td>
			<td>F0257</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rabbit Cy3 antibody</td>
			<td>Dianova, Germany</td>
			<td colspan="2">111-165-003</td>
		</tr>
		<tr>
			<td>Goat Serum normal</td>
			<td>Dako, Glostrup, Denmark</td>
			<td colspan="2">X090710-8</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Carl Roth GmbH, Karlsruhe, Germany</td>
			<td>6335.1</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides&#160;</td>
			<td>Engelbrecht, Ederm&#252;nde, Germany</td>
			<td colspan="2">WC7695002</td>
		</tr>
		<tr>
			<td>Coverslips 24x24mm</td>
			<td>Th. Gayer, Lohmar, Germany</td>
			<td>7695026</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO fluorescent mounting medium&#160;</td>
			<td>DAKO, USA</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM Meta 510 confocal microscopy&#160;</td>
			<td>Carl Zeiss AG, Jena, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a laminating technique to perform confocal microscopy of the human sclera

La sclera è un tessuto connettivo denso che copre e protegge l&#39;occhio. Essa consiste essenzialmente di fasci di collagene denso (tipo I, III, IV, V, VI, e VII). Grazie alla sua autofluorescenza, opacità e spessore, non è stato trovato adatto per la microscopia confocale. Un approccio alternativo a quello qui presentato, che utilizza sclera fissati in formalina inclusi in paraffina per immunoistochimica, ha problemi tecnici, in particolare per preriscaldare il tessuto per il recupero antigene. Dal momento che la sclera è relativamente povero di entrambe le cellule e dei vasi, l&#39;uso di campioni di tessuto più grandi è stato esplorato per aiutare a prevenire le cellule che si affacciano e per capire la loro localizzazione in relazione alle navi e altri siti anatomici. Per consentire l&#39;analisi di campioni di tessuto grandi al microscopio confocale, una tecnica di laminazione è stata eseguita per creare strati sottili dalla sclera. Dopo l&#39;analisi dei risultati dei vasi sanguigni CD31 e vasi linfatici endoteliale hyaluRonan recettore 1 (LYVE1) cellule positive, per le quali è stato ottenuto l&#39;approvazione per l&#39;esame scientifico, i vantaggi ei limiti di questo metodo sono discussi.

Negli esperimenti rappresentativi condotti qui, ci sono benefici dimostrabili derivanti dall&#39;uso di questa particolare tecnica di laminazione. Il primo esperimento illustra la rete diversificata del plesso vaso sanguigno episclerale in tre immagini rappresentative (Figura 3). I vasi sono positivi per CD31. Il secondo esperimento mostra cellule immunitarie, in particolare le cellule LYVE1 + del episclera e ...

Watch this Scientific Journal Video about Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera at JoVE.com

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

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.

Utilizzando una tecnica di laminazione per l&#39;esecuzione di Microscopia confocale della sclera umana


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 ...

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Research

JoVE Journal

Medicine

7.5K Views.  University of Cologne. 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. 

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

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

Microscopia corneale confocale: A Novel tecnica non invasiva per quantificare Piccole Fibre Patologia neuropatie periferiche

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

Ricerca

Medicina

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Utilizzando la Stimolazione Magnetica Transcranica per studiare il sistema neuromuscolare umano

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

Probe-based Endomicroscopy laser confocale del tratto urinario: La Tecnica

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Eccitotossica stimolazione del cervello Microslices come<em&gt; In vitro</em&gt; Modello di Corsa

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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.

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

Scheletrici Procedure biopsia muscolare dell&#39;uomo con la tecnica Bergström Modificato

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

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Costruzione di vapore Chambers utilizzato per esporre topi da alcol Durante l&#39;equivalente di tutti i tre trimestri di Sviluppo Umano

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rosa Bengala Photothrombosis da confocale Optical Imaging<em&gt; In Vivo</em&gt;: Un modello di singolo vaso Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

Sviluppo di un algoritmo di effettuare uno studio globale di autonomica disreflessia in animali con alta ferita del midollo spinale Uso di un dispositivo di telemetria

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

Topico Airway Anestesia per intubazione da sveglio-endoscopica Utilizzando la Spray-as-you-go Tecnica con alto flusso di ossigeno

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Seconda generazione di armoniche segnali coniglio sclera come strumento per la valutazione del tessuto terapeutico reticolazione (TXL) per la miopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Utilizzando una tecnica di laminazione per l'esecuzione di Microscopia confocale della sclera umana

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Capitoli in questo video

Microscopia corneale confocale: A Novel tecnica non invasiva per quantificare Piccole Fibre Patologia neuropatie periferiche

Utilizzando la Stimolazione Magnetica Transcranica per studiare il sistema neuromuscolare umano

Probe-based Endomicroscopy laser confocale del tratto urinario: La Tecnica

Eccitotossica stimolazione del cervello Microslices come

Scheletrici Procedure biopsia muscolare dell'uomo con la tecnica Bergström Modificato

Costruzione di vapore Chambers utilizzato per esporre topi da alcol Durante l'equivalente di tutti i tre trimestri di Sviluppo Umano

Rosa Bengala Photothrombosis da confocale Optical Imaging

Sviluppo di un algoritmo di effettuare uno studio globale di autonomica disreflessia in animali con alta ferita del midollo spinale Uso di un dispositivo di telemetria

Topico Airway Anestesia per intubazione da sveglio-endoscopica Utilizzando la Spray-as-you-go Tecnica con alto flusso di ossigeno

Seconda generazione di armoniche segnali coniglio sclera come strumento per la valutazione del tessuto terapeutico reticolazione (TXL) per la miopia