Name: using an automated hirschberg test app to evaluate ocular alignment
Uploaded: 2020-03-24T15:00:00
Description: Watch this Scientific Journal Video about Using an Automated Hirschberg Test App to Evaluate Ocular Alignment at JoVE.com

Dette arbejde blev delvist støttet af NIH tilskud R44EY025902 og af Mass Eye & Ear Hærdning Kids Grant.

Denne undersøgelse blev udført i overensstemmelse med principperne i Helsinki-erklæringen, på Schepens Eye Research Institute (Boston, MA) og Spaulding Rehabilitation Hospital (Boston, MA). Alle deltagerne indhentede et informeret samtykke. Undersøgelsen blev godkendt af de lokale institutionelle review boards af Mass Eye and Ear (Boston, MA).
BEMÆRK: Kriterier for patientinklusion var tidligere diagnosticering af horisontalstrabismus (konstant eller intermitterende exotropi eller esotro...

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	<li>Kemper, A. R., Clark, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preschool+vision+screening+in+pediatric+practices.">Preschool vision screening in pediatric practices.</a> Clinical Pediatrics. 45 (3), 263-266 (2006).</li><li>Rowe, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+profile+of+strabismus+in+stroke+survivors.">The profile of strabismus in stroke survivors.</a> Eye. 24 (4), 682-685 (2010).</li><li>Black, K., McCarus, C., Collins, M. L. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+preference+and+visual+acuity+testing+in+a+population-based+cohort+of+preschool+children+with+amblyopia+risk+factors.">Fixation preference and visual acuity testing in a population-based cohort of preschool children with amblyopia risk factors.</a> Ophthalmology. 116 (1), 145-153 (2009).</li><li>Robaei, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+Associated+with+Childhood+Strabismus:+Findings+from+a+Population-Based+Study.">Factors Associated with Childhood Strabismus: Findings from a Population-Based Study.</a> Ophthalmology. 113 (7), 1146-1153 (2006).</li><li>Tarczy-Hornoch, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+decreased+visual+acuity+in+preschool+children:+the+multi-ethnic+pediatric+eye+disease+and+Baltimore+pediatric+eye+disease+studies.">Risk factors for decreased visual acuity in preschool children: the multi-ethnic pediatric eye disease and Baltimore pediatric eye disease studies.</a> Ophthalmology. 118 (11), 2262-2273 (2011).</li><li>Chan, K. W., Deng, L., Weissberg, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Strabismus+by+Non-Health+Care+Professionals+in+an+Ethnically+Diverse+Set+of+Images.">Detection of Strabismus by Non-Health Care Professionals in an Ethnically Diverse Set of Images.</a> JAMA Ophthalmology. 134 (1), 30-36 (2016).</li><li>Hunter, D. G., Guyton, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertical+location+of+the+corneal+light+reflex+in+strabismus+photography.">Vertical location of the corneal light reflex in strabismus photography.</a> Archives of Ophthalmology. 116 (6), 767-771 (1998).</li><li>Eskridge, J. B., Wick, B., Perrigin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hirschberg+test:+a+double-masked+clinical+evaluation.">The Hirschberg test: a double-masked clinical evaluation.</a> American Journal of Optometry and Physiological Optics. 65 (9), 745-750 (1988).</li><li>Hasebe, S., Ohtsuki, H., Tadokoro, Y., Okano, M., Furuse, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reliability+of+a+video-enhanced+Hirschberg+test+under+clinical+conditions.">The reliability of a video-enhanced Hirschberg test under clinical conditions.</a> Investigative Ophthalmology &amp; Visual Science. 36 (13), 2678-2685 (1995).</li><li>Pundlik, S., Tomasi, M., Liu, R., Houston, K., Luo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Preliminary+Evaluation+of+a+Smartphone+App+for+Measuring+Eye+Alignment.">Development and Preliminary Evaluation of a Smartphone App for Measuring Eye Alignment.</a> Translational Vision Science &amp; Technology. 8 (1), 19 (2019).</li><li>Cheng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EyeTurn+App+for+School+Vision+Screening.">The EyeTurn App for School Vision Screening.</a> American Academy of Optometry Annual Meeting. , (2018).</li><li>Committee on Practice and Ambulatory Medicine and Section on Ophthalmology. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Photoscreening+for+Children's+Vision+Screening.">Use of Photoscreening for Children's Vision Screening.</a> Pediatrics. 109 (3), 524-525 (2002).</li><li>Simons, B. D., Siatkowski, R. M., Schiffman, J. C., Berry, B. E., Flynn, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pediatric+photoscreening+for+strabismus+and+refractive+errors+in+a+high-risk+population.">Pediatric photoscreening for strabismus and refractive errors in a high-risk population.</a> Ophthalmology. 106 (6), 1073-1080 (1999).</li><li>Kerr, N. C., Somes, G., Enzenauer, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+developmentally-at-risk+status+on+the+reliability+of+the+iScreen(R)+photorefractive+device+in+young+children.">The effect of developmentally-at-risk status on the reliability of the iScreen(R) photorefractive device in young children.</a> The American Journal of Orthopedics. 61, 117-123 (2011).</li><li>Arnold, R. W., O'Neil, J. W., Cooper, K. L., Silbert, D. 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En person uden faglig uddannelse kan bruge EyeTurn-appen til at tage billeder af øjnene og få målinger af okulær justering, som kan fortolkes af en øjenspecialist på stedet eller eksternt. Appen giver kun omfanget af forskydningen i stedet for nogen fortolkning eller diagnose. Eye pleje fagfolk såsom optometrister eller øjenlæger bør afgøre, om forskydningen er signifikant eller ej, og stille en diagnose efter at have overvejet andre faktorer, herunder de betingelser, hvorunder målingen blev taget.
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Måling af øjenjustering udføres ofte i vision pleje klinikker. Cover test med prisme neutralisering er den almindeligt anvendte kliniske metode til kvantificering af graden af øjenforskydning (strabismus). Denne metode kræver en høj grad af uddannelse og erfaring. Nøjagtig måling bliver mere udfordrende, når patienter ikke fuldt ud kan deltage i eksamen såsom små børn1, personer med hjerneskader eller slagtilfælde2, eller udviklingsmæssige handicap3. Desuden er der behov for øjenjusteringstest i skolescreening, fordi strabismus udvikler sig i barndommen i ca. 5-8 % af den amerikanske befolkning4og er en væsentlig risikofaktor for amblyopi med ca. 30-40 % af tilfældene af amblyopi, der tilskrives strabismus5,6,7. Skolesygeplejersker er dog normalt ikke uddannet til at gennemføre standarddækningstesten med prisme neutralisering til en sådan screening. For ikke-øje pleje fagfolk, en yderligere udfordring i strabismus screening er, at intermitterende strabismus (forskydning er ikke altid manifesteret) og mindre størrelser af forskydning er ikke visuelt indlysende (<15 prisme dioptri [Δ])8.
I et forsøg på at løse udfordringerne i detektion og måling af strabismus, har vi udviklet en smartphone app (EyeTurn), der implementerer og automatiserer den fotografiske Hirschberg metode9 ved at sammenligne forskydning af hornhinde refleksioner mellem øjnene. Mens konventionelle fotografiske Hirschberg metode har vist sig at have god reproducerbarhed i klinikker10,11, omkostningerne for dedikerede, standalone enheder er en barriere for bred vedtagelse. Ved at give en nem at bruge værktøj til at måle øje tilpasning til standard smartphones, vi hypotesedet vil blive bredt vedtaget i skolen vision screening og anvendes af ikke-eye pleje fagfolk. Vores tidligere evalueringsundersøgelser har vist, at appmålingen er i overensstemmelse med den aktuelle kliniske standard for prisme og alternativ dækningstest12for strabismusstørrelser af esotropi og exotropi a op til 60Δ. I en pilot skole screening undersøgelse, vi viste også, at den app kan hjælpe skolesygeplejersken opdage børn med intermitterende exotropia, der blev savnet af standard skole vision screening protokoller13.
IOS-versionen af appen er i øjeblikket tilgængelig for forskere og klinikere efter anmodning til forskningsformål. De anmodere har hidtil inkluderet skole sygeplejersker, pædiatriske øjenlæger, optometrister, neuro-øjenlæger, og strabismus specialister. Formålet med denne artikel er at dele de detaljerede appprotokoller til brug af appen til at evaluere okulær justering under forskellige visningsforhold, nemlig nær og fjern fikseringsafstand. med og uden øjendækning for at bryde kikkertfusion.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EyeTurn</td>
			<td>EyeNexo</td>
			<td></td>
			<td>Smartphone app for measureing eye misalignment</td>
		</tr>
	</tbody>
</table>

using an automated hirschberg test app to evaluate ocular alignment

En smartphone app er blevet udviklet til at udføre den automatiserede fotografiske Hirschberg test for objektiv måling af okulær forskydning. Ved at beregne forskellen i hornhinde refleksion genereret af telefonens kamera flash i forhold til iris center baseret på billeder i høj opløsning, kan app måle forskydning med en langt højere præcision end det blotte øje udfører Hirschberg test. Det er blevet valideret i en tidligere klinisk evalueringsundersøgelse ved at sammenligne med den kliniske guld standard-prisme og suppleant dækning test. Målet med denne artikel er at beskrive testteknikkerne for, hvordan du bruger appen til at måle okulær justering for forskellige fikseringsafstande, uden eller med dækning for at bryde fusion, samt vinkel kappa, så brugerne kan bruge appen til at udføre tilsvarende test, der typisk udføres på klinikken ved hjælp af prismer.

I dette arbejde beskriver vi protokollerne til evaluering af okulær justering ved hjælp af en smartphone-app, der udfører den fotografiske Hirschberg-test. Appens grænseflade vises i figur 1. Brugerne kan vælge at udføre dækning test eller måle en patient med begge øjne fiksering på et mål samtidigt, enten på nær eller fjern fiksering afstande. Når visningsbetingelserne er bestemt afhængigt af testformål, kan brugerne følge protokollerne og t...

Watch this Scientific Journal Video about Using an Automated Hirschberg Test App to Evaluate Ocular Alignment at JoVE.com

Using an Automated Hirschberg Test App to Evaluate Ocular Alignment

Vi præsenterer en protokol for at bruge en smartphone app til at udføre Hirschberg test til måling af manifest og intermitterende okulær forskydning (strabismus) under nær og fjern fiksering betingelser.

Brug af en automatiseret Hirschberg-testapp til at evaluere okulær justering

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing ...

Den mobilapp, som vi har udviklet, kan bruges til at måle strabismus til forskning eller til klinisk brug. Denne metode, letter en nem strabismus måling, som i øjeblikket kræver omfattende uddannelse og erfaring, der skal udføres ved hjælp af kun en standard telefon kamera og app software. I app-grænsefladen kan brugerne vælge at udføre en covertest eller at måle en patient med begge øjne fikseret på et mål samtidigt, enten ved en nær eller fjern fikseringsafstand.Hvis du vil måle tropia med et enkelt øjebliksbillede med en næsten fiksering, skal du starte appen og indstille tilstanden til No Cover. Vælg Nær og placer telefonen i en liggende retning omkring 40 centimeter fra patienten i øjenhøjde, med det bageste kamera mod patienten. Instruer patienten i at fiksere på lommelygten, som skal være slukket på dette tidspunkt.Til test, der kræver en præcis indkvartering, skal du indsætte et fikseringsmål på bagsiden af telefonen direkte under eller over lommelygten. Når patienten bekræfter, at de er fiksering, skal du trykke på den runde knap for at tage et øjebliksbillede, der skal analyseres af app. Når analysen er færdig, vil appen vise den registrerede limbus grænse, midten af øjet, og placeringen af hornhinde refleksion.Når du har kontrolleret, at disse funktioner er blevet opdaget uden åbenlyse fejl, skal du observere målingerne i forbindelse med øjenjusteringen. Tryk derefter på knappen Gem for at gemme den aktuelle test i telefonen, eller tryk på pil tilbage for at teste igen. Hvis du vil måle periodisk okulære forskydning for langt fiksering i appen, skal du indstille tilstanden til Cover Test og vælge Far.Hold telefonen i en liggende retning, omkring 40 centimeter fra patienten i øjenhøjde med flashkameraet placeret mellem patientens øjne. Instruere patienten til at se lige over telefonen, til et mål omkring seks meter væk. Få patienten til at dække det ene øje og trykke på den runde knap, vil app begynde at opdage.Samtidig med at sikre, at patienten er fiksering korrekt, har dem hurtigt fjerne okklusion. Appen tager automatisk et billede, så snart okklusionen er fjernet. Når analysen er færdig, vil appen vise den registrerede limbus grænse, midten af øjet, og placeringen af hornhinde refleksion.Når du har kontrolleret, at disse funktioner registreres uden åbenlyse fejl, skal du gennemgå måleresultaterne for øjenjusteringen i prisme dioptrere, før du gemmer dataene eller gentager testen. Vælg Måler Kappa-vinkel for vinkel Kappa-måling i appen. Placer telefonen i en liggende retning omkring 40 centimeter fra patienten i øjenhøjde, og instruere patienten til at bruge øjet, der skal testes for at fiksere på lommelygten, mens der dækker det andet øje.Mens du sikrer dig, at patienten fikserer korrekt, skal du trykke på den runde knap for at tage et øjebliksbillede, som vil blive analyseret af appen. Når analysen er færdig, vil appen vise den registrerede limbus grænse, midten af øjet, og placeringen af hornhinde refleksion. Når du har kontrolleret, at disse funktioner registreres uden åbenlyse fejl, skal du se måleresultaterne for Kappa-vinklen i grader.I denne repræsentative analyse, limbus grænser for de to øjne og hornhinde afspejling af lommelygten, blev opdaget korrekt, hvilket tyder på, at den okulære tilpasning foranstaltning ikke er genstand for Image Analysis fejl. I dette særlige tilfælde, patienten havde forladt exotropia som er indlysende fra de større hornhinde refleksion offset i venstre øje. Den app ikke rapportere, hvilke øjne blev afveget dog, fordi der i tilfælde af en lille strabismus vinkel og ukendt Kappa vinkel, ville det være upålidelige for app til at bestemme den afvigende øje.Til sammenligning vises et eksempel uden strabismus. I denne analyse kan der observeres en fejlagtig limbus-detektion. Mens detektion af hornhinden refleksion er korrekt, den grønne cirkel klart ikke svarer til limbus grænse, hvilket indikerer, at testen skal gentages.I denne analyse af 14 forsøgspersoner, 10 emner udstillet exotropia og fire emner præsenteret med exotropia. Lineær regressionsanalyse, viste, at øjenmålingerne af strabismusvinklerne var i overensstemmelse med kliniske dæktestmålinger. Når du bruger appen, er der to vigtige ting, du skal huske på.Den ene skal dækprøven og fikseringstilstanden indstilles korrekt for hver enkelt prøvning. To, kontroller, om registreringen af øjenfunktionen er korrekt. Den app kun tillader strabismus måling, og det giver ikke en diagnose.Klinikere bliver nødt til at kombinere den okulære måling med andre vision målinger for at stille en diagnose.

using-an-automated-hirschberg-test-app-to-evaluate-ocular-alignment

Research

JoVE Journal

Medicine

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

En<em&gt; In vivo</em&gt; Assay til Test Blood Vessel Permeabilitet

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Brug af kvantitativ real-time PCR til Bestem donorcelle indpodning i en konkurrencepræget murin knoglemarvstransplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Ved hjælp af en knæ Arthrometer at vurdere væv-specifikke bidrag til knæ fleksion kontraktur i rotter

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral studies in animal models. In this protocol, to study depression-like and anhedonic behavior in rats, the established sucrose preference and novelty-induced hypophagia tests are combined with an automated food and liquid intake monitoring system. Prior to testing, in the sucrose preference paradigm, male rats are trained for at least 2 days to consume a sucrose solution in addition to tap water. During the test, rats are again exposed to water and sucrose solution. Consumption is registered every second by the automated system. The ratio of sucrose to total water intake (sucrose preference ratio) is a surrogate parameter for anhedonia. In the novelty-induced hypophagia test, male rats undergo a training period in which they are exposed to a palatable snack. During training, rodents show a stable baseline snack intake. On test day, the animals are transferred from home cages into a fresh, empty cage representing a novel unknown environment with access to the known palatable snack. The automated system records the total intake and its underlying microstructure (e.g., latency to approaching the snack), providing insight into anhedonic and anxious behaviors. The combination of these paradigms with an automated measuring system provides more detailed information, along with higher accuracy by reducing measuring errors. However, the tests use surrogate parameters and only depict depression and anhedonia in an indirect manner.

Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters.

Saccharose præference og nyhed-induceret hypofagi test hos rotter ved hjælp af en automatiseret fødeindtagelse overvågningssystem

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

Endotelcelletranscytoseanalyse som en in vitro-model til evaluering af indre blod-retinal barrierepermeabilitet

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

Semi-Automated Planimetric Quantification of Dental Plaque Using an Intraoral Fluorescence Camera

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area of a tooth that is covered by plaque deposits. Compared to clinical indices, the PPI has a higher discriminatory power, but traditional planimetry is a time-consuming analysis, as the plaque-covered and clean tooth areas have to be determined manually for each image using image-processing software. Here, we present a method for the semi-automated planimetric quantification of dental plaque, which allows for the rapid processing of up to 1,000 images simultaneously. The method exploits the enhanced contrast between disclosed plaque, sound tooth surfaces, and soft tissues in fluorescence images acquired with an intraoral camera. Careful execution of the clinical procedures and accurate image acquisition are crucial steps for the successful semi-automated identification of the plaque-covered areas. The method is suitable for planimetry on sound facial and oral tooth surfaces, on most composite resin restorations, and on teeth with orthodontic brackets, but not on metallic restorations. Compared to traditional PPI recordings, semi-automated planimetry considerably reduces the amount of time spent on the analysis, as well as the subjective human input, thus increasing the reproducibility of planimetric measurements.

This study presents a semi-automated digital image analysis procedure for the planimetric quantification of disclosed dental plaque based on images acquired with an intraoral fluorescence camera. The method allows for the rapid and reliable quantification of dental plaque in the research environment.

Halvautomatisk planimetrisk kvantificering af plak ved hjælp af et intraoralt fluorescenskamera

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area ...

Forebyggelse af posterior kapselopacificering gennem IOL-rotation ved kataraktoperationer

Rotating the Intraocular Lens to Prevent Posterior Capsular Opacification in Cataract Surgeries

Posterior capsule opacification (PCO) is a common postoperative complication of extracapsular cataract surgery, which is caused by the proliferation and migration of lens epithelial cells and can affect long-term visual outcomes significantly. The most effective treatment for PCO is neodymium-doped yttrium aluminum garnet (Nd:YAG) laser capsulotomy; however, this treatment is associated with posterior segment complication and can break the stability of capsular bag, affecting the position and function of trifocal or toric intraocular lenses (IOLs). Advances in surgical procedures, IOL design, and pharmacy have reduced the rate of PCO in recent years, concentrating on the inhibition of proliferative lens epithelial cells (LECs). This protocol aimed to clear LECs more thoroughly during phacoemulsification and IOL implantation. The first several steps, including clear corneal incision, continuous circular capsulorhexis, hydrodissection, hydrodelineation, and phacoemulsification, were completed as conventional procedures. After placing the IOL into the capsular bag, rotation of the IOL by at least 360&#176; was performed using an irrigation/aspiration tip or a hook, with slight stress on the posterior capsule. Some residuals occurred in the originally transparent capsular bag after rotation of the IOLs. Then, these materials and the viscoelastic were cleared completely using an irrigation/aspiration system. A clear posterior capsule was observed after the surgery in patients undergoing this method. This method of rotating IOLs is a simple, effective, and safe way to prevent PCO by clearing residual LECs and can be carried out without extra tools or skills.

Forfatter Spotlight: Forbedring af visuelle resultater ved grå stærkirurgi: En ny teknik til at forhindre posterior kapselopacificering gennem IOL-rotation 

The present protocol describes removing residual epithelial cells by rotating the intraocular lens in extracapsular cataract surgery without extra tools for preventing posterior capsular opacification.

Rotation af den intraokulære linse for at forhindre posterior kapselopacificering ved grå stæroperationer

Posterior capsule opacification (PCO) is a common postoperative complication of extracapsular cataract surgery, which is caused by the proliferation and ...

Automatiseret processor til fremstilling af CAR-T-celler i klinisk skala

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination. The CliniMACS Prodigy, an automated cell processor, allows for manufacturing cell therapy products at a clinical scale in a closed system, minimizing the risk of contamination. Processing occurs semi-automatically under the control of a computer and thus&#160;minimizes human involvement in the process, which saves time and reduces variability and errors.
This manuscript and video describes the T cell transduction (TCT) process for manufacturing CAR-T cells using this processor. The TCT process involves CD4+/CD8+ T cell enrichment, activation, transduction with a viral vector, expansion, and harvest. Using the Activity Matrix, a functionality that allows ordering and timing of these steps, the TCT process can be customized extensively. We provide a walk-through of CAR-T cell manufacturing in compliance with current Good Manufacturing Practice&#160;(cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstrate&#160;the feasibility and discuss the advantages and disadvantages of using a&#160;semi-automatic process for clinical CAR-T cell manufacturing. Finally, we describe an ongoing investigator-initiated clinical trial that targets pediatric B-cell malignancies [NCT05480449] as an example of how this manufacturing process can be applied in a clinical setting.

Forfatter Spotlight: Fremskridt inden for CAR-T-cellefremstilling og genterapiproduktion 

This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial.