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

Este trabalho foi apoiado em parte pela bolsa NIH R44EY025902 e pelo Mass Eye & Ear Curing Kids Grant.

Este estudo foi conduzido de acordo com os princípios da Declaração de Helsinque, no Schepens Eye Research Institute (Boston, MA) e no Spaulding Reabilitation Hospital (Boston, MA). O consentimento informado foi obtido de todos os participantes. O estudo foi aprovado pelos conselhos locais de revisão institucional de Mass Eye and Ear (Boston, MA).
NOTA: Os critérios de inclusão do paciente foram o diagnóstico prévio do estrabismo horizontal (exotropia ou esotropia constante ou intermit...

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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. I., Donahue, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+smartphone+photoscreening+app+to+detect+refractive+amblyopia+risk+factors+in+children+aged+1-6+years.">Evaluation of a smartphone photoscreening app to detect refractive amblyopia risk factors in children aged 1-6 years.</a> Clinical Ophthalmology. 12, 1533-1537 (2018).</li></ol>

Uma pessoa sem treinamento profissional pode usar o aplicativo EyeTurn para capturar imagens dos olhos e obter medidas de alinhamento ocular, que podem ser interpretadas por um especialista em cuidados oculares no local ou remotamente. O aplicativo apenas fornece magnitude do desalinhamento, em vez de qualquer interpretação ou diagnóstico. Profissionais de cuidados oculares, como optometristas ou oftalmologistas, devem determinar se o desalinhamento é significativo ou não, e fazer um diagnóstico após considerar ou...

A medição do alinhamento ocular é realizada frequentemente em clínicas de cuidados com a visão. O teste de cobertura com neutralização de prisma é o método clínico comumente utilizado para quantificar o grau de desalinhamento dos olhos (estrabismo). Este método requer um alto grau de treinamento e experiência. A medição precisa torna-se mais desafiadora quando os pacientes não podem participar totalmente do exame, como crianças pequenas1, indivíduos com lesões cerebrais ou acidente vascular cerebral2, ou deficiências de desenvolvimento3. Além disso, há necessidade de testes de alinhamento ocular na triagem escolar, pois o estrabismo se desenvolve durante a infância em uma população estimada de 5 a 8% da população dos EUA4, e é um fator de risco substancial para ambliopia com cerca de 30-40% dos casos de ambliopia atribuídos ao estrabismo5,6,7. No entanto, os enfermeiros escolares normalmente não são treinados para realizar o teste de cobertura padrão com neutralização de prisma para tal triagem. Para os profissionais não-oculares, um desafio adicional na triagem do estrabismo é que o estrabismo intermitente (o desalinhamento nem sempre se manifesta) e magnitudes menores de desalinhamento não são visualmente óbvios (<15 dioptrios prisma [Δ])8.
Na tentativa de enfrentar os desafios na detecção e medição do estrabismo, desenvolvemos um aplicativo para smartphone (EyeTurn) que implementa e automatiza o método fotográfico Hirschberg9, comparando o deslocamento das reflexões córneas entre os olhos. Embora o método convencional fotográfico Hirschberg tenha se mostrado ter boa reprodutibilidade nas clínicas10,11, o custo para dispositivos dedicados e autônomos é uma barreira para a ampla adoção. Ao fornecer uma ferramenta fácil de usar para medir o alinhamento dos olhos com smartphones padrão, temos a hipótese de que ela será amplamente adotada na triagem de visão escolar e usada por profissionais não-oculares. Nossos estudos de avaliação anteriores mostraram que a medição do aplicativo é consistente com o padrão clínico atual de prisma e teste de cobertura alternativo12, para magnitudes estrabismo de esotropia e exotropia até 60Δ. Em um estudo de triagem escolar piloto, também mostramos que o aplicativo pode ajudar a enfermeira da escola a detectar crianças com exotropia intermitente que foram perdidas pelos protocolos padrão de triagem da visão escolar13.
A versão iOS do aplicativo está atualmente disponível para pesquisadores e médicos mediante solicitação para fins de pesquisa. Os solicitantes incluíram até agora enfermeiros escolares, oftalmologistas pediátricos, optometristas, neurooftalmologistas e especialistas em estrabismo. O objetivo deste artigo é compartilhar os protocolos detalhados do aplicativo para usar o aplicativo para avaliar o alinhamento ocular em diferentes condições de visualização, ou seja, distância de fixação próxima e distante; com e sem cobertura ocular para quebrar a fusão binocular.

<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

Um aplicativo para smartphone foi desenvolvido para realizar o teste automatizado de Hirschberg fotográfico para medição objetiva do desalinhamento ocular. Ao calcular a diferença na reflexão córnea gerada pelo flash da câmera do telefone em relação ao centro de íris com base em imagens de alta resolução, o aplicativo pode medir o desalinhamento com uma precisão muito maior do que o olho nu realizando o teste de Hirschberg. Foi validado em estudo de avaliação clínica prévia, comparando-se com o padrão-prisma padrão-ouro clínico e o teste de cobertura alternativo. O objetivo deste artigo é descrever as técnicas de teste sobre como usar o aplicativo para medir o alinhamento ocular para diferentes distâncias de fixação, sem ou com cobertura para quebrar fusão, bem como o ângulo kappa, para que os usuários possam usar o aplicativo para realizar o equivalente testes tipicamente feitos em clínica usando prismas.

Neste trabalho, descrevemos os protocolos para avaliar o alinhamento ocular usando um aplicativo de smartphone que realiza o teste fotográfico de Hirschberg. A interface do aplicativo é mostrada na Figura 1. Os usuários podem optar por realizar o teste de cobertura ou medir um paciente com ambos os olhos fixando-se simultaneamente em um alvo, seja a distâncias de fixação próximas ou longas. Uma vez que as condições de visualização são determinadas...

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

Apresentamos um protocolo de uso de um aplicativo de smartphone para realizar o teste de Hirschberg para medir o desalinhamento ocular manifesto e intermitente (estrabismo) em condições próximas e distantes de fixação.

Usando um aplicativo de teste hirschberg automatizado para avaliar o alinhamento ocular

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

O aplicativo móvel que desenvolvemos pode ser usado para medir o estrabismo para pesquisa ou para uso clínico. Este método facilita uma medição fácil de estrabismo, que atualmente requer treinamento e experiência extensiva para ser realizado usando apenas uma câmera de telefone padrão e o software do aplicativo. Na interface do aplicativo, os usuários podem optar por realizar um teste de cobertura ou medir um paciente com ambos os olhos fixados em um alvo simultaneamente, a uma distância de fixação próxima ou distante.Para medir a tropia, com um único instantâneo com uma fixação próxima, inicie o aplicativo e defina o modo como No Cover. Selecione Perto e posicione o telefone em uma orientação paisagística a cerca de 40 centímetros do paciente ao nível dos olhos, com a câmera traseira voltada para o paciente. Instrua o paciente a se fixar na lanterna, que deve estar desligada neste momento.Para testes que requerem uma acomodação precisa, cole um alvo de fixação na parte de trás do telefone diretamente abaixo ou acima da lanterna. Quando o paciente confirmar que está se fixando, pressione o botão redondo para tirar uma foto para ser analisado pelo aplicativo. Quando a análise estiver concluída, o aplicativo mostrará o limite de limbus detectado, o centro do olho e a localização da reflexão corneal.Após verificar se essas características foram detectadas sem erros óbvios, observe as medidas relacionadas ao alinhamento ocular. Em seguida, pressione o botão Salvar para salvar o teste atual no telefone ou pressione o botão de seta traseira para testar novamente. Para medir o desalinhamento ocular intermitente para fixação de longe no aplicativo, defina o modo de cobrir o teste e selecione Far.Segure o telefone em uma orientação paisagística, cerca de 40 centímetros do paciente ao nível dos olhos com a câmera flash posicionada entre os olhos do paciente. Instrua o paciente a olhar logo acima do telefone, para um alvo a cerca de seis metros de distância. Que o paciente cubra um olho e pressione o botão redondo, o aplicativo começará a detecção.Ao mesmo tempo em que certifique-se de que o paciente está se fixando corretamente, certifique-o rapidamente de remover a oclusão. O aplicativo tirará automaticamente uma foto assim que a oclusão for removida. Quando a análise estiver concluída, o aplicativo mostrará o limite de limbus detectado, o centro do olho e a localização da reflexão corneal.Depois de verificar se esses recursos são detectados sem erros óbvios, revise os resultados de medição para o alinhamento ocular em dicómetros prisma, antes de salvar os dados ou repetir o teste. Para a medição angle Kappa no aplicativo, selecione medir o ângulo Kappa. Posicione o telefone em uma orientação paisagística a cerca de 40 centímetros do paciente ao nível dos olhos, e instrua o paciente a usar o olho para ser testado para fixar-se na lanterna enquanto cobre o outro olho.Ao certificar-se de que o paciente está se fixando corretamente, toque no botão redondo para tirar um instantâneo, que será analisado pelo aplicativo. Quando a análise estiver concluída, o aplicativo mostrará o limite de limbus detectado, o centro do olho e a localização da reflexão corneal. Depois de verificar se esses recursos são detectados sem erros óbvios, visualize os resultados de medição para o ângulo Kappa em graus.Nesta análise representativa, os limites de limbus dos dois olhos e o reflexo corneal da lanterna, foram detectados corretamente, sugerindo que a medida de alinhamento ocular não está sujeita ao erro de Análise de Imagem. Neste caso em particular, o paciente deixou exotropia, como é óbvio a partir do maior reflexo da córnea compensado no olho esquerdo. O aplicativo não informou qual olho foi desviado, no entanto, porque em casos de um pequeno ângulo de estrabismo e ângulo Kappa desconhecido, não seria confiável para o aplicativo determinar o olho desviado.Para comparação, aqui é mostrado um exemplo sem estrabismo. Nesta análise, pode-se observar a detecção errônea de limbus. Embora a detecção do reflexo da córnea esteja correta, o círculo verde claramente não corresponde ao limite do limbus, indicando que o teste deve ser repetido.Nesta análise de 14 sujeitos, 10 sujeitos apresentaram exotropia e quatro sujeitos apresentados com exotropia. A análise de regressão linear revelou que as medidas oculares dos ângulos de estrabismo foram consistentes com as medidas de teste de cobertura clínica. Ao usar o aplicativo, há duas coisas importantes para ter em mente.Primeiro, defina o teste de cobertura e o modo de fixação corretamente para cada teste específico. Segundo, verifique se a detecção do recurso ocular está correta. O aplicativo só permite a medição do estrabismo e não dá um diagnóstico.Os médicos precisarão combinar a medição ocular com outras medidas de visão para fazer um diagnóstico.

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.

Um<em&gt; In vivo</em&gt; Ensaio para ensaio de permeabilidade dos vasos sangüíneos

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 ...

Medicina

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.

Usando Quantitative PCR em tempo real para determinar enxerto célula dadora num modelo murino de osso Competitiva Marrow Transplantation

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.

Usando um artrômetro de joelho para avaliar as contribuições de tecido-específica para contratura de flexão do joelho em ratos

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.

Preferência por sacarose e testes de hipofagia induzidos por novidades em ratos usando um sistema automatizado de monitoramento da ingestão de alimentos

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.

Ensaio de Transcitose de Células Endoteliais como Modelo In Vitro para Avaliar a Permeabilidade da Barreira Interna Sangue-Retina

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.

Quantificação Planimétrica Semi-Automatizada de Placa Dentária Utilizando Câmera de Fluorescência Intraoral

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

Prevenindo a opacificação capsular posterior por meio da rotação da LIO em cirurgias de catarata

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.

Autor em destaque: Melhorando os resultados visuais na cirurgia de catarata: uma nova técnica para prevenir a opacificação capsular posterior por meio da rotação da LIO 

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.

Rotação da lente intraocular para prevenir opacificação da cápsula posterior em cirurgias de catarata

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

Processador automatizado para fabricação de células CAR-T em escala clínica

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.

Autor em destaque: Avanços na fabricação de células CAR-T e produção de terapia gênica 

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.