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

Summary

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.

Abstract

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> 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.

Introduction

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> 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 </ Em> 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 (> 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.

Protocol

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 Dilute 0.1% (w/v) Coomassie Brilliant Blue in PBS and filter sterilize using a 0.45 µM syringe filter. 2. Preparation of Pseudomonas aeruginosa Culture 15 hrs before instillation, inoculate 3 ml of broth culture with a single bacterial colony. Grow the culture 15 hr at 37 °C on a shaker (200 rpm). Centrifuge 1 ml of culture in a 1.5 ml microfuge tube 12,000 x g for 30 sec. Remove the medium and resuspend the pellet in 1ml of PBS. 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. Dilute the bacterial stock suspension in PBS to the desired concentration of bacterial inoculum, using a delivery volume of 50 µl for IMIT inoculation. 3. IMIT Instillation Place a group of mice into isoflurane anesthesia induction chamber and anesthetize using a 2 – 3% isoflurane/oxygen mixture. At the initial onset of sedation, scruff the mouse, holding the mouse upright, and administer 10 µl 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. Allow a minimum of 5 min to permit the lidocaine to take full effect as a local anesthetic. When mice have achieved desired level of sedation (breathing rate of ~60 bpm), reduce the isoflurane to 2% to maintain sedation. Preload dye or bacterial inoculum into a 250 µl gas-tight precision syringe fit with a 22 G long blunt needle. First draw up 150 µl of air, measured by the Teflon plunger of the syringe. Next, draw up 50 µl of inoculum by advancing the Teflon plunger from the 150 µl mark to the 200 µl mark on the syringe body. NOTE: When the sample is ejected into an intubated mouse, the 50 µl suspension will be delivered first, followed by a 150 µl air cushion which will distribute the inoculum throughout the lung. 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° incline. Using a micro cotton applicator, retract the tongue with a rolling motion, using the dominant hand. With the nondominant hand, use an operating otoscope fit with an intubation specula to both maintain tongue retraction and visualize the glottis. 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. 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” clear tubing containing a colored dye. NOTE: The dye will rapidly migrate back and forth in response to breathing. 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. Continue to secure the catheter with the nondominant hand while inserting the precision syringe/blunt needle containing the liquid suspension/air cushion. Dispense the liquid/air directly into the lung in a single fluid motion and immediately remove the needle/catheter from the mouse. Return the mouse to a cage and allow recovery from anesthesia. 4. Characterization of IMT Delivery After IMIT instillation, euthanize the mouse by CO2 asphyxia at an appropriate time post-inoculation. Secure the euthanized mouse on a dissection board and soak the chest and abdomen with 70% EtOH using a squirt bottle. 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. 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. Add 1 ml of sterile 1x PBS to each sample bag+ tissues. Reseal sample bag. Homogenize tissues by gentle rolling a 25 ml serological pipette over the sample bag + tissue. 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): 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. Incubate agar plates overnight at 37 °C and enumerate colony forming units the next day.

Representative Results

To visualize the distribution of instilled material via the IMIT method, 50 µl 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, 1.21×107, and 1.21 x 106 colony forming units [CFU] per 50 µl). 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 >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). Figure 1: IMIT instillation distributes inoculum throughout the lungs. Lungs from mice instilled with 50 µl of 0.1% Coomassie Brilliant Blue show blue dye distributed in all lobes. 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 – recovered) were compared to the estimated inoculum (Log10 CFU – delivered). Each circle represents the CFU/lung of an individual mouse (n = 3 for each bacterial dose).

Discussion

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’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 >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 >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.

Materials

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