Name: impression cytology of the lid wiper area
Uploaded: 2016-08-09T15:00:00
Description: Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

We would like to acknowledge our colleague Dr. Jalaiah Varikooty who provided insight and expertise on the topic of the lid wiper, that greatly assisted this research.

declaração de ética: Antes da coleta de células de seres humanos, consentimento informado e aprovação ética deve ser obtida. 1. Prepare Stain NOTA: Prepare mancha no dia do experimento. <ol><li> Adicionar 5 uL de etídio (ou 4 mM) e 5 ul de calceína AM (ou 4 mM) a 2,5 ml de tampão fosfato salino (PBS) em 15 ml de tubo de centrifugação; misturar. Embrulhe em papel alumínio para proteger da luz e armazenar a RT até o uso. </li></ol><p class="jove_tit...

<ol>
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citologia de impressão é tipicamente realizada na conjuntiva bulbar, utilizando membranas de ésteres de celulose misturados. As amostras são cortadas ao tamanho a partir de folhas de material a granel, esterilizado, aplicado e removido da superfície conjuntival utilizando fórceps. Usando o mesmo material de membrana, um dispositivo de IC controlado-pistão foi recentemente concebido para coincidir com a geometria da superfície da conjuntiva bulbar, e manter a pressão constante entre 13 aplicações. E...

A pálpebra superior humano executa cerca de 10.000 pisca a cada dia 1, com apenas um 1 - região conjuntival 2 milímetros estreita opostos do globo ocular. Devido ao seu movimento de limpeza durante o pestanejar, esta porção da margem da pálpebra foi denominada a &quot;tampa do limpador&quot; região 2. Supõe-se que o atrito é aumentada nessa região durante a piscar habitual, devido à tampa limpar todo o globo. Isto pode desempenhar um papel significativo no conforto ocular. Lente de contato usuários em particular experiência de desconforto ocular e esta é uma das razões principais para cessar o uso das lentes 3. Quando uma lente de contacto é colocada sobre o olho, o coeficiente de atrito entre o material da lente e a superfície ocular foi mostrado para alterar 4. Há evidências que sugerem que os sintomas de secura pode estar relacionado a este atrito alterado 2,5,6. Esta associação também pode ser refletida em fenômenos observáveis ​​clinicamente, nomeadamente wi tampapor epiteliopatia (LWE), também chamado superior da tampa margem de coloração (ULMS) 6. LWE é um sinal precoce de irritação ocular e um preditor potencial para a doença do olho seco. Ele é observado e medido pela coloração vital das regiões superiores e inferiores margem palpebral. Embora este sistema de classificação 2 e sua validade clínica (ou seja, correlação com sintomas subjetivos) ainda estão em debate, desconforto ocular permanece um enigma para os médicos e cientistas e, mais importante, um inconveniente para os pacientes. Até à data, pouco se sabe sobre as variações clinicamente relevantes no (sub) anatomia celular e fisiologia da área do limpador a tampa 7 e apenas um relatório faz uso de métodos citológicos 8. Citologia de impressão (CI) foi utilizada há mais de 40 anos para recolher as células a partir da superfície do epitélio da conjuntiva tarsal bulbar ou por aplicação de uma membrana de 9,10. Após remoção, as células aderentes submeter Cytological coloração e de imagem microscópica. Devido às diferenças anatómicos celulares e na região do limpador a tampa, esta técnica requer a optimização.

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			<td height="20" style="height:20px;width:349px;">Alcian Blue, 1% in 3% Acetic Acid</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">B8438-250ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin, Gill 1</td>
			<td style="width:119px;">Sigma</td>
			<td>GHS116-500ML</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, OG-6</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40116-500 ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, Modified EA</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40232-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Life Technologies</td>
			<td>L-3224</td>
			<td>contains ethidium homodimer-1 and Calcein AM dyes</td>
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			<td height="20" style="height:20px;">Alcaine (0.5% proparacaine hydrochloride)</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Millicell Cell Culture Insert, 12&#160;mm, hydrophilic PTFE, 0.4&#160;&#181;m</td>
			<td>EMD Millipore</td>
			<td>PICM01250&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Glass Bottom Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-20-C</td>
			<td></td>
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impression cytology of the lid wiper area

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

A membrana de cultura de células durou mais o suporte de plástico é uma ferramenta conveniente para a recolha de mão de células epiteliais da conjuntiva tampa do limpador. Este método elimina a necessidade de instrumentos esterilizados adicionais tipicamente utilizados para preparar e manipular membranas IC. A Figura 1 representa a área de IC limpador a tampa usando uma inserção de cultura de células. Nós otimizamos dois protocolos de coloração diferentes qu...

Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

Impression Cytology of the Lid Wiper Area

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

A citologia de impressão da Área Wiper a tampa

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The ...

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