The authors are grateful for the support from the Center for Predicative Medicine Animal Models (Carol Vanover, Ashley Biller and Jennifer Kraenzle) and Microbiology (Daniel Cramer and Julie Sotsky) Core Facilities. This work was supported by funding from the NIH (HHSN272201000033I to M.B.L and J.M.W.).

NOTE: All of the procedures described here were reviewed and approved by the University of Louisville Institutional Biosafety Committee (protocol # 13-056) and Institutional Animal Care and Use Committee (protocol # 13-064).
1. Preparation of Dye
<ol>
	<li>Dilute 0.1% (w/v) Coomassie Brilliant Blue in PBS and filter sterilize using a 0.45 &#181;M syringe filter.</li>
</ol>
2. Preparation of Pseudomonas aeruginosa Culture
<ol>
	<li>15 hrs before instillation, inoculate 3 ml of broth culture with a single bacterial colony.</li>
	<li>Grow the culture 15 hr at 37 &#176;C on a shaker (200 rpm).</li>
	<li>Centrifuge 1 ml of culture in a 1.5 ml microfuge tube 12,000 x g for 30 sec.</li>
	<li>Remove the medium and resuspend the pellet in 1ml of PBS.</li>
	<li>Dilute an aliquot of the bacterial stock suspension 1:10 in PBS and measure the OD600 of the diluted bacterial suspension to determine the bacterial concentration.</li>
	<li>Dilute the bacterial stock suspension in PBS to the desired concentration of bacterial inoculum, using a delivery volume of 50 &#181;l for IMIT inoculation.</li>
</ol>
3. IMIT Instillation
<ol>
	<li>Place a group of mice into isoflurane anesthesia induction chamber and anesthetize using a 2 - 3% isoflurane/oxygen mixture.</li>
	<li>At the initial onset of sedation, scruff the mouse, holding the mouse upright, and administer 10 &#181;l of a 2% lidocaine solution by gavage needle to the back of the throat and allow the solution to drain down to the epiglottis. Return the mouse to the anesthesia chamber.
	<ol>
		<li>Allow a minimum of 5 min to permit the lidocaine to take full effect as a local anesthetic.</li>
	</ol>
	</li>
	<li>When mice have achieved desired level of sedation (breathing rate of ~60 bpm), reduce the isoflurane to 2% to maintain sedation.</li>
	<li>Preload dye or bacterial inoculum into a 250 &#181;l gas-tight precision syringe fit with a 22 G long blunt needle.
	<ol>
		<li>First draw up 150 &#181;l of air, measured by the Teflon plunger of the syringe. Next, draw up 50 &#181;l of inoculum by advancing the Teflon plunger from the 150 &#181;l mark to the 200 &#181;l mark on the syringe body. 
		NOTE: When the sample is ejected into an intubated mouse, the 50 &#181;l suspension will be delivered first, followed by a 150 &#181;l air cushion which will distribute the inoculum throughout the lung.</li>
	</ol>
	</li>
	<li>Remove one mouse from the induction chamber and lay supine on an intubation platform. Secure the mouse to the platform by hooking its incisors with an O-ring attached to Velcro strip, and securing the Velcro to the platform. Raise the mouse to a 45&#176; incline.</li>
	<li>Using a micro cotton applicator, retract the tongue with a rolling motion, using the dominant hand.</li>
	<li>With the nondominant hand, use an operating otoscope fit with an intubation specula to both maintain tongue retraction and visualize the glottis.</li>
	<li>With the dominant hand, use a guide wire threaded through a 20 G catheter to intubate the mouse, seating the catheter to a 10 mm depth into the mouse trachea (catheter is fit with a silicon sleeve with 10 mm of exposed catheter). Remove the otoscope/specula.</li>
	<li>Confirm that the mouse has been correctly intubated by securing the catheter with the nondominant hand while briefly attaching a Luer connected length of 1/16&#8221; clear tubing containing a colored dye. 
	NOTE: The dye will rapidly migrate back and forth in response to breathing.</li>
	<li>Do not proceed with subsequent steps if confirmation of intubation has not been established at this point. If the intubation attempted failed, reset the catheter and guide wire for one additional attempt at intubation. 
	NOTE: It is unadvisable to attempt more than two intubations of a mouse in one session without causing trauma to the mouse.</li>
	<li>Continue to secure the catheter with the nondominant hand while inserting the precision syringe/blunt needle containing the liquid suspension/air cushion.</li>
	<li>Dispense the liquid/air directly into the lung in a single fluid motion and immediately remove the needle/catheter from the mouse.</li>
	<li>Return the mouse to a cage and allow recovery from anesthesia.</li>
</ol>
4. Characterization of IMT Delivery
<ol>
	<li>After IMIT instillation, euthanize the mouse by CO2 asphyxia at an appropriate time post-inoculation.</li>
	<li>Secure the euthanized mouse on a dissection board and soak the chest and abdomen with 70% EtOH using a squirt bottle.</li>
	<li>If evaluating distribution of an imaging agent throughout the lung, remove the lungs from the animal using sterile technique and display the lungs as appropriate for imaging. 
	NOTE: Lungs may be prepared for additional histological staining techniques through appropriate fixation or cryopreservation.</li>
	<li>If evaluating the bacterial burden of infected lung tissue, remove the lungs from the animal using sterile technique. Place lungs into a sterile, preweighed 1 oz sample bag. Weigh and record the weight of the sample bag + lungs.
	<ol>
		<li>Add 1 ml of sterile 1x PBS to each sample bag+ tissues. Reseal sample bag.</li>
		<li>Homogenize tissues by gentle rolling a 25 ml serological pipette over the sample bag + tissue.</li>
		<li>Generate serial dilutions of the lung homogenate in sterile PBS and plate on agar plate (LB, or as appropriate to the bacterial species being studied):
		<ol>
			<li>Conduct a six fold serial dilution in a U-bottom 96 well plate by multichannel pipettor then plate triplicate samples by multichannel on the agar plate.</li>
		</ol>
		</li>
		<li>Incubate agar plates overnight at 37 &#176;C and enumerate colony forming units the next day.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Reznik, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy,+physiology,+and+function+of+the+upper+respiratory+tract.">Comparative anatomy, physiology, and function of the upper respiratory tract.</a> Environ Health Perspect. 85, 171-176 (1990).</li><li>Hoyt, R. F. J., Hawkins, J. V., St. Clair, M. B., Kennet, M. B., Fox, J. G., 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=Chapter+2.">Chapter 2.</a> The Mouse in Biomedical Research, Volume 2, Second Edition: Diseases (American College of Laboratory Animal Medicine). 2, 23-90 (2007).</li><li>Visweswaraiah, A., Novotny, L. A., Hjemdahl-Monsen, E. J., Bakaletz, L. O., Thanavala, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+tissue+distribution+of+marker+dye+following+intranasal+delivery+in+mice+and+chinchillas:+a+multifactorial+analysis+of+parameters+affecting+nasal+retention.">Tracking the tissue distribution of marker dye following intranasal delivery in mice and chinchillas: a multifactorial analysis of parameters affecting nasal retention.</a> Vaccine. 20, 3209-3220 (2002).</li><li>Eyles, J. E., Spiers, I. D., Williamson, E. D., Alpar, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+distribution+of+radioactivity+following+intranasal+administration+of+radioactive+microspheres.">Tissue distribution of radioactivity following intranasal administration of radioactive microspheres.</a> J Pharm Pharmacol. 53, 601-607 (2001).</li><li>Southam, D. S., Dolovich, M., O'Byrne, P. M., Inman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+intranasal+instillations+in+mice:+effects+of+volume,+time,+body+position,+and+anesthesia.">Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia.</a> Am J Physiol Lung Cell Mol Physiol. 282, 833-839 (2002).</li><li>Miller, M. 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=Visualization+of+murine+intranasal+dosing+efficiency+using+luminescent+Francisella+tularensis:+effect+of+instillation+volume+and+form+of+anesthesia.">Visualization of murine intranasal dosing efficiency using luminescent Francisella tularensis: effect of instillation volume and form of anesthesia.</a> PLoS ONE. 7, (2012).</li><li>Lathem, W. W., Crosby, S. D., Miller, V. L., Goldman, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+primary+pneumonic+plague:+a+mouse+model+of+infection,+pathology,+and+bacterial+transcriptional+activity.">Progression of primary pneumonic plague: a mouse model of infection, pathology, and bacterial transcriptional activity.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 17786-17791 (2005).</li><li>Warawa, J. M., Long, D., Rosenke, R., Gardner, D., Gherardini, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+diagnostic+imaging+to+characterize+altered+respiratory+tract+colonization+by+the+Burkholderia+pseudomallei+capsule+mutant.">Bioluminescent diagnostic imaging to characterize altered respiratory tract colonization by the Burkholderia pseudomallei capsule mutant.</a> Front Microbiol. 2, 133 (2011).</li><li>Gutierrez, M., Pfeffer, T. L., Warawa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+3+Secretion+System+cluster+3+is+a+critical+virulence+determinant+for+lung-specific+melioidosis.">Type 3 Secretion System cluster 3 is a critical virulence determinant for lung-specific melioidosis.</a> Submitted. , (2014).</li><li>Fodah, R. 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=Correlation+of+Klebsiella+pneumoniae+comparative+genetic+analyses+with+virulence+profiles+in+a+murine+respiratory+disease+model.">Correlation of Klebsiella pneumoniae comparative genetic analyses with virulence profiles in a murine respiratory disease model.</a> PLoS ONE. In revision, (2014).</li><li>Soto-Montenegro, M. L., 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=Assessment+of+airway+distribution+of+transnasal+solutions+in+mice+by+PET/CT+imaging.">Assessment of airway distribution of transnasal solutions in mice by PET/CT imaging.</a> Mol Imaging Biol. 11, 263-268 (2009).</li></ol>

The authors have nothing to disclose.

IMIT instillation offers key improvements to existing respiratory disease models in the ability to reproducibly instill reagents directly into the lung. It is a rapid approach which is ideally situated for a team of two researchers, one of which manages the logistics of anesthesia and caging, and the other who performs the IMIT technique. Large studies may be conducted using IMIT with an average time commitment of 2 - 3 min per mouse. Because the approach makes use of isoflurane as an anesthetic, mice recover rapidly from the anesthesia, reducing the husbandry time of monitoring animals through recovery.
The most technically challenging aspect of the IMIT method is the initial step of intubating mice. Individuals learning to perform IMIT are able to focus on this first step of catheter placement and ensuring that intubation has been achieved through the visual confirmation of dye movement. The benefit of the approach is that lung-specific instillation is guaranteed through use of the confirmation of intubation, which increases the confidence of both the new researcher as well as the expert attempting to intubate a difficult animal. The key elements of optimizing the likelihood of a successful intubation are: i) achieving a deep sedation to allow sufficient working time, ii) correct placement of the specula in the mouth to allow good visualization of the epiglottis, iii) good depth placement of the specula so that the tongue remains retracted throughout the procedure, and iv) use of the tilting platform to support the researcher&#8217;s hands so that the procedure is conducted relaxed and with a steady approach.
One of the limitations of the IMIT procedure is related to frequency of IMIT instillation events. Due to the potential trauma associated with a missed intubation, it is not recommended that more than two intubation attempts be conducted in a single session (up to two misses). IMIT has an excellent potential in its ability to be used to deliver therapeutics into the murine lung, however therapeutic regimens which make use of very frequent delivery of reagent into the lung may not be suitable for IMIT. It may be possible that IMIT could be used daily to deliver reagents into a murine lung without causing significant trauma, but only when conducted by a highly skilled researcher, as the majority of trauma associated with intubation is thought to be associated with a missed intubation event. Such high-frequency IMIT should be discussed with local veterinarians and IACUC.
An additional potential limitation of IMIT is the size of the mouse which is being intubated. The IMIT procedure described above was developed using mice of approximately 17 - 22 g, where a 20 G catheter was found to be a suitable size for the trachea of mice in this size range. Larger catheters have been successfully used in older mice; the initial development of IMIT made use of an 18 G catheter in BALB/c mice which are &#62;20 g. Importantly, if alternate catheter sizes are used, blunt needles should be sourced which fit the lumen of the catheter and are trimmed to a length that extends just 1mm beyond the catheter tip. Intubation of mice smaller than 17 g may be possible but is not recommended due to the expertise required, and would require use of smaller catheters and specula than are described above.
We have used IMIT for the delivery of several respiratory pathogens in addition to P. aeruginosa, including B. pseudomallei9 and Klebsiella pneumoniae10. The IMIT model has made important advances to our studies of B. pseudomallei respiratory disease, having identified that intranasal inoculation causes an early, URT-related morbidity of mice rather than the systemic disease endpoint observed in human disease9. B. pseudomallei is a Tier 1 select agent of biodefense impact, and as such, respiratory disease models are being developed for aerosol exposure which models a potential biodefense related route of entry for weaponized pathogens. Because current aerosol models result in infection of both the URT and LRT, the same potential early morbidity phenotypes we have identified for the intranasal model of B. pseudomallei respiratory disease may apply to the aerosol model. A future adaptation of the IMIT model could be an intubation-mediated aerosol delivery (IMAD), in which mice are intubated for target aerosol delivery only into the lung. Mechanical ventilators are currently available to maintain isoflurane anesthesia, which could be adapted to deliver an aerosolized, rather than liquid based, pathogen challenge.
IMIT was developed initially as an approach to optimize the delivery of bacteria to the lung, but also has application for the delivery of other reagents into the mouse lung. As discussed above, intranasal delivery of compounds into mice results in a low efficiency, highly variable delivery of reagents into the target organ of the lung. Intranasal delivery of Positron Emission Tomography (PET) imaging reagents to the murine lung yielded a 40% delivery efficiency11, whereas we have demonstrated that IMIT offers an excellent alternative to other lung delivery approaches with its &#62;98% delivery efficacy and multilobar distribution. This improvement in targeted delivery to the lung has the potential to increase the reproducibility of therapeutic delivery for treatment of pulmonary disease. IMIT could similarly offer benefits to studies of: i) the impact of environmental pulmonary irritants, ii) lung cancer phenotypic studies, iii) lung-specific siRNA knock-down.

Os ratinhos foram usados ​​para modelar numerosas manifestações de doenças humanas, incluindo uma miríade de doenças respiratórias. Modelos de doenças substitutos são frequentemente incapazes de recapitular todos os aspectos de uma doença modelado, normalmente devido a diferenças fisiológicas importantes ou imunes nos dois modelos de acolhimento. Assim, o objetivo de melhorar os sistemas de modelos é desenvolver abordagens que permitem substitutos para espelhar mais de perto um processo de doença ou resposta do hospedeiro, como observado no sistema host original. Existem várias diferenças fisiológicas principais entre os ratos e os seres humanos no mecanismo pelo qual eles inspirar ar. Incluídos nestes diferenças são diferenças raciométrica significativas de tamanho entre a URT e LRT. Estimou-se que os ratos possuem&gt; 100 vezes a área de superfície TRS relação aos seres humanos, normalizados contra 1,2 capacidade pulmonar total. Assim, os cornetos nasais do mouse permitem mais ampla filtragem do ar inspirado para facilitar uma maior taxa de breathing, o que pode ter um impacto significativo sobre os estudos de pneumonia se a infecção da cavidade nasal desempenha um papel significativo na progressão da doença. Várias abordagens diferentes têm sido empregues para incutir bactérias nos pulmões de ratos para estudar a doença respiratória semelhante à humana. A mais comum destas abordagens é a inoculação intranasal, no qual uma suspensão líquida é aplicada em uma ou ambas as narinas de um rato. Embora relativamente simples, advertências como volume instilação e tipo de anestesia utilizada pode afetar a eficiência da instilação no LRT via intranasal inoculação 3-5. Especificamente, Miller et al. demonstraram que a instilação intranasal de Francisella tularensis em volumes de menos de 50 ul não resultou em instilação das bactérias no LRT 6. Eles observaram ainda melhor LRT instilação ao usar isoforane ao contrário de injetado cetamina / xilazina para anestesia. No entanto, o nosso experience com Yersinia pestis inoculação intranasal indica a inoculação mais consistente pode ser conseguida utilizando cetamina / xilazina, em comparação com isoflurano (MBL, dados não publicados). Estas diferenças podem ser atribuídas ao patógeno utilizado ou à variação nos procedimentos de laboratório, mas importante destacar a variabilidade potencial desta técnica. Além disso, os pulmões colhidos logo após a instilação intranasal mostram que uma percentagem relativamente baixa do inoculo bacteriano inicial atinge o pulmão (no caso de Y. pestis, apenas 10% foram recuperados 1 h após a instilação 7), sugerindo que um grande número de bactérias pode ser retida no TRS (ou engolido no tracto GI). Em certos modelos de doenças, esta deposição significativa de bactérias na mucosa URT pode confundir o nosso entendimento da progressão da doença, se o organismo é capaz de colonizar a cavidade nasal murino de forma incompatível com a doença humana. Por exemplo, usando in vivo &lt;/ Em&gt; imagiologia, tem-se observado que Burkholderia pseudomallei, que não colonizar o TRS humano, provoca uma infecção oportunista esmagadora da cavidade nasal de murino quando entregue pelo método de instilação intranasal 8. Outros métodos de instilação de bactérias nos pulmões de ratos também foram empregues na pesquisa de doenças infecciosas. No entanto, em comparação com instilação intranasal estes métodos tendem a exigir mais conhecimentos técnicos e / ou equipamentos caros, sem eliminar o potencial de iniciação infecção em vários locais (por exemplo, aerosol [URT e LRT]; transoral [trato digestivo e LRT]; e intratraqueal cirúrgico [LRT e sangue]). Dadas as potenciais complicações que poderiam estar associados com sites secundários de infecção, buscou-se desenvolver uma abordagem intratraqueal que ignora a URT e entrega patógeno diretamente para os pulmões de ratos anestesiados, mas também limita inoculat inadvertidaíon na corrente sanguínea ou do trato gastrointestinal. Para este fim, intratraqueal (IMIT) instilação mediada por intubação foi desenvolvido como um procedimento não-cirúrgico que garante LRT instilação de inoculo por inclusão de um passo intermediário para verificar a colocação do cateter adequado antes da instilação. Este método é descrito usando corante de instilação para demonstrar visualmente ampla distribuição do inoculo em todo o pulmão, e P. aeruginosa instilação para demonstrar a entrega altamente eficaz (&gt; 98% do inoculo) do presente método para o pulmão. É importante ressaltar que, enquanto originalmente desenvolvido para entrega bacteriana, IMIT também oferece uma ferramenta eficaz para: i) a instilação de várias moléculas para o estudo de outros modelos de doenças respiratórias, ii) a entrega terapêutica específica do pulmão, e iii) os estudos de função pulmonar de base, incluindo alvo siRNA entrega ao pulmão.

<table border="0" cellpadding="0" cellspacing="0" width="1035">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Name of Material/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:447px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Rodent, Tilting WorkStand</td>
			<td style="width:160px;">Hallowell EMC</td>
			<td style="width:119px;">000A3467</td>
			<td>Base should be detached when working in a BSC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Operating Otoscope Head</td>
			<td style="width:160px;">Welch Allyn</td>
			<td style="width:119px;">21700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Otoscope 3.5 V Li Battery</td>
			<td style="width:160px;">Welch Allyn</td>
			<td style="width:119px;">71900</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:311px;">Mouse Intubation Specula short, Autoclaveable</td>
			<td style="width:160px;">Hallowell EMC</td>
			<td style="width:119px;">200A3589S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Incisor Loops</td>
			<td style="width:160px;">Hallowell EMC</td>
			<td style="width:119px;">210A3490A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Cotton fine tip applicator</td>
			<td style="width:160px;">Puritan</td>
			<td style="width:119px;">871-PC DBL&#160;</td>
			<td>Used for tongue retraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">I.V. Catheter, 20G</td>
			<td style="width:160px;">Exel Int</td>
			<td style="width:119px;">26741</td>
			<td>Optional: fit a silicon sleeve with 10mm exposed catheter surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Gas tight syringe, 250ul</td>
			<td style="width:160px;">Hamilton</td>
			<td style="width:119px;">81120</td>
			<td>Used for delivery of liquid inoculum by IMIT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Blunt Needle, 22G</td>
			<td style="width:160px;">Hamilton</td>
			<td style="width:119px;">91022</td>
			<td>Trim to length to protrude 1mm from 20G catheter</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Guide wire (Fiber optic wire, 0.5mm)</td>
			<td style="width:160px;">TheFiberOpticStore.com</td>
			<td style="width:119px;">FOF .50</td>
			<td>Cut to 6&#34; length: used as guide wire for intubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Tuberculin syringe, 1ml</td>
			<td style="width:160px;">Becton Dickinson</td>
			<td style="width:119px;">309659</td>
			<td>Assemble with fiber optic wire as guide wire</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Brilliant Blue R (Coomassie)</td>
			<td style="width:160px;">Sigma</td>
			<td style="width:119px;">B0149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Tygon tubing, 1/16&#34;</td>
			<td style="width:160px;">Saint Gobain</td>
			<td style="width:119px;">ALC00002&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Male luer 1/16&#34; barb</td>
			<td style="width:160px;">Cole Parmer</td>
			<td style="width:119px;">45503-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Female luer 1/16&#34; barb</td>
			<td style="width:160px;">Cole Parmer</td>
			<td style="width:119px;">45500-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Lidocaine, USP</td>
			<td style="width:160px;">Spectrum</td>
			<td style="width:119px;">LI102</td>
			<td>pH lidocaine into solution at 2%(w/v) pH7.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Sample bag, 1oz</td>
			<td style="width:160px;">Whirl-Pak</td>
			<td style="width:119px;">B01067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">U-bottom 96 well plate, sterile</td>
			<td style="width:160px;">Greiner</td>
			<td style="width:119px;">650161</td>
			<td></td>
		</tr>
	</tbody>
</table>

intubation-mediated intratracheal (imit) instillation: a noninvasive, lung-specific delivery system

Estudos de doenças respiratórias envolvem tipicamente a utilização de sistemas de modelos murinos como substitutos. No entanto, existem diferenças fisiológicas significativas entre os sistemas respiratório humano e murino, especialmente em suas vias respiratórias superiores (URT). Nalguns modelos, estas diferenças na cavidade nasal de murino podem ter um impacto significativo sobre a progressão da doença e na apresentação do trato respiratório inferior (LRT) quando se utiliza técnicas de instilação intranasal, limitando potencialmente a utilidade do modelo de rato para estudar estas doenças. Por estas razões, seria vantajoso desenvolver uma técnica para instilar bactérias directamente para os pulmões do rato, a fim de estudar a doença de LRT na ausência de envolvimento do TRS. Temos chamado este específico intratraqueal técnica entrega intubação mediada (IMIT) instilação de pulmão. Esta técnica não invasiva minimiza o potencial para a instilação para a corrente sanguínea, o que pode ocorrer durante traditi mais invasivaonal abordagens cirúrgicas intratraqueais infecção, e limita a possibilidade de entrega do aparelho digestivo incidental. IMIT é um processo de duas etapas em que ratinhos são entubados em primeiro lugar, com um passo intermédio para assegurar a colocação correcta do cateter para dentro da traqueia, seguido da inserção de uma agulha romba para dentro do cateter para mediar a entrega directa das bactérias para o pulmão. Esta abordagem facilita uma eficácia de&gt; 98% de entrega para os pulmões com uma excelente distribuição de reagente ao longo pulmão. Assim, IMIT representa uma nova abordagem para estudar a doença de LRT e entrega terapêutica directamente no pulmão, após a melhorar a capacidade de usar ratos como substitutos para estudar a doença respiratória humana. Além disso, a precisão e reprodutibilidade deste sistema de entrega também faz com que seja passível de Boas Práticas de Laboratório (BPL) Normas, bem como a entrega de uma ampla gama de reagentes que requerem a entrega de alta eficiência para o pulmão.

To visualize the distribution of instilled material via the IMIT method, 50 &#181;l of 0.1% Coomassie Brilliant Blue dye was instilled into the lungs of an anesthetized mouse. The mouse was immediately euthanized and the lungs were removed by sterile necropsy. Figure 1 shows that the dye was delivered to all lobes of the lung.
To determine the amount of bacteria delivered to the lungs via the IMIT method, three groups of mice (n = 3) were instilled with three different concentrations of P. aeruginosa (1.21 x 108, 1.21x107, and 1.21 x 106 colony forming units [CFU] per 50 &#181;l). Immediately after IMIT instillation, mice were euthanized, lungs removed, and bacterial numbers were enumerated and compared to the inoculums (Figure 2). Delivery of the inoculum is highly efficient via this method, with &#62;98% of the inoculum recovered from the lungs of instilled animals. Furthermore, IMIT instillation was highly reproducible regardless of the concentration of the inoculum (R2 = 0.9951).
<img alt="Figure 1" src="/files/ftp_upload/52261/52261fig1highres.jpg" /> 
Figure 1: IMIT instillation distributes inoculum throughout the lungs. Lungs from mice instilled with 50 &#181;l of 0.1% Coomassie Brilliant Blue show blue dye distributed in all lobes.
<img alt="Figure 2" src="/files/ftp_upload/52261/52261fig2highres.jpg" /> 
Figure 2: IMIT instillation of bacteria into lungs. Mice were instilled with P. aeruginosa and the number of bacteria instilled into the lungs (Log10 CFU &#8211; recovered) were compared to the estimated inoculum (Log10 CFU &#8211; delivered). Each circle represents the CFU/lung of an individual mouse (n = 3 for each bacterial dose).

Watch this Scientific Journal Video about Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System at JoVE.com

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Intratraqueal (IMIT) instilação mediada por intubação: A não-invasiva, sistema de entrega Lung-specific

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

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Medicine

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

24.1K visualizações.  University of Louisville Medical School. Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Vídeo: Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Uma nova abordagem cirúrgica para administração intratraqueal de agentes bioativos em um modelo de camundongo Fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Medicina

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Intraoperatória Detecção de Endometriose Sutil: um novo paradigma para a detecção e tratamento de dor pélvica associada com a perda de integridade Peritoneal

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

Não invasiva Intratraqueal intubação para estudar a Patologia e Fisiologia do Rato Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Implantação de fibrina Gel no tapete do pulmão para o Estudo de angiogênese Lung-specific

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

Instilação e fixação métodos úteis no mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

A Multimodal Imaging- e baseada Estimulação método de avaliação de conectividade relacionadas com o cérebro Excitabilidade em doentes com epilepsia

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Retroductal submandibular Glândula instilação e localizada fracionado irradiação em um modelo de rato de salivar Hipofunção

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

Um modelo de derrame Pleural em ratos por instilação intratraqueal de poliacrilato/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Induzindo lesão pulmonar aguda em camundongos por instilação de lipopolissacárido intratraqueal direto

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Desenvolvimento de um modelo experimental não invasivo, assistido a laser de perda de células endoteliais córneas

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...