Gli autori ringraziano i sig. e la Facoltà Core Facility per l'assistenza nell'imaging animale. Il lavoro è stato sostenuto dal Research Grant Council di Hong Kong (17300319).

Gli esperimenti condotti in questo studio sono stati approvati dal Committee on the Use of Live Animals for Teaching and Research (CULATR), The University of Hong Kong. Formulazioni in polvere secca preparate mediante liofilizzazione spray (SFD) contenenti lo 0,5% dell'RNA messaggero luciferasi (mRNA), il 5% di peptide sintetico PEG12KL4 e il 94,5% del mannitolo (w/w) sono utilizzate in questo studio per dimostrare l'espressione di mRNAnel polmone 16. Il diametro aerodinamico mediano di massa (MMAD) della polvere SFD è di 2,4 μm. La polvere di mannito essiccata a spruzzo (SD) viene utilizzata per studiare l'effetto del volume d'aria utilizzato nella dispersione dellapolvere 16. L'MMAD della polvere SD è di 1,5 μm.
1. Fabbricazione di insufflator a polvere secca e caricamento di polvere secca
<ol><li>(Facoltativo) Neutralizzare le cariche statiche della polvere secca (in una fiala) e la punta della pipetta di caricamento del gel rotonda non filtrante da 200 μL. Utilizzare una pistola antistatica o un equilibrio con funzione di deionizzazione secondo le istruzioni del produttore.</li>
	<li>Preparare una carta di pesatura con un formato di circa 4 cm x 4 cm. Piegare la carta a metà in diagonale e quindi aprirla.</li>
	<li>Pesare 1-2 mg di polvere secca sulla carta di pesatura.</li>
	<li>Riempire una punta di pipetta a caricamento in gel con polvere attraverso l'apertura più ampia della punta. Toccare delicatamente per imballare la polvere fino a quando la polvere non forma agglomerati sciolti vicino all'estremità stretta della punta(Figura 1A). Evitare di imballare la polvere troppo strettamente in quanto potrebbe ostacolare la dispersione della polvere.</li>
	<li>Collegare la punta caricata a polvere a una siringa da 1 ml attraverso un fermacock a tre velocità (Figura 1B). La dimensione della siringa può essere cambiata in base al volume d'aria utilizzato per disperdere la polvere. Tenere la punta e la siringa verticalmente durante il collegamento per evitare fuoriuscite di polvere. Se la somministrazione non viene eseguita immediatamente, utilizzare il parafilm per sigillare le aperture della punta e conservarla temporaneamente in condizioni adeguate fino alla somministrazione.</li>
</ol>2. Fabbricazione di dispositivi di intubazione
<ol><li>Sorgente luminosa(Figura 2)<ol>
<li>Preparare una sorgente luminosa su misura con una torcia a diodo emettitore di luce (LED) e una fibra ottica flessibile con un diametro di 0,8-1 mm.</li>
		<li>Crea un orifizio centrato sulla lente chiara della torcia a LED con un trapano a mano o una punta di perforazione in modo che la fibra ottica possa passare a malapena.</li>
		<li>Inserire la fibra ottica attraverso l'orifizio. Accendere la torcia a LED per regolare la posizione e la profondità di inserimento per la massima luminosità all'altra estremità della fibra ottica.</li>
		<li>Apporre la fibra ottica in posizione con colla epossidica chiara.</li>
	</ol>
</li>
	<li>Cannula guida(Figura 3)<ol>
<li>Prendere una pipetta Pasteur in plastica da 1 mL (Figura 3A) e tenere la pipetta ad entrambe le estremità.</li>
		<li>Utilizzare una lampada alcool (o altre fonti di calore in laboratorio come un bruciatore Bunsen) per riscaldare il centro della pipetta posizionandolo a 5-10 cm sopra la fiamma(Figura 3B). Ruotare la pipetta per assicurarsi che sia riscaldata uniformemente.</li>
		<li>Quando la plastica diventa morbida e deformabile, allontanare la pipetta dalla fiamma e allungare delicatamente la pipetta.</li>
		<li>Tagliare la pipetta allungata al centro con un paio di forbici nella parte A e nella parte B(Figura 3C-E). Utilizzate la parte A come pipetta a punta fine e la parte B come cannula guida. Per aumentare le possibilità di successo dell'intubazione con la cannula guida, fare una smussatura (non troppo affilata che può aumentare il rischio di ferire l'animale) alla fine della parte B(figura 3F). Quando una punta di pipetta a caricamento in gel da 200 μL (per il caricamento della polvere) viene inserita nella cannula guida, deve sporgere la cannula di 1-2 mm. NOTA: Una cannula guida (Parte B) con la dimensione appropriata (diametro interno ed esterno) per l'intubazione potrebbe avere un ago calibro 21 che si adatta al suo interno mentre può anche adattarsi all'interno di un ago calibro 17. Potrebbero essere necessari più tentativi per allungare le pipette per ottenere la quota appropriata.</li>
		<li>(Facoltativo): Tagliare una piccola apertura all'estremità più ampia della cannula guida per renderla più flessibile in modo che sia più facile tenere la fibra ottica (Figura 3F). Questa apertura consente anche il montaggio di un microsprayer per la somministrazione di aerosol liquido.</li>
	</ol>
</li>
</ol>3. Intubazione
<ol><li>Anestetizzare il topo (BALB/c, 7-9 settimane) con chetamina (100 mg/kg) e xiazina (10 mg/kg) per iniezione intraperitoneale.</li>
	<li>Preparare una piattaforma in plexiglass e montarla su un supporto con un morsetto (Figura 4A). Posizionare il mouse anestetizzato sulla piattaforma (a circa 60° di inclinazione) in posizione supina. L'altezza e l'angolo di inclinazione della piattaforma potrebbero essere regolati dalla posizione del morsetto sul supporto.</li>
	<li>Sospendere il mouse agganciando i suoi incisivi su un filo interdentale di nylon (Figura 4B). Fissare la posizione del mouse con un pezzo di nastro o un elastico.</li>
	<li>Inserire la fibra ottica nella cannula guida prima dell'intubazione con la punta del livello della fibra con l'apertura della cannula guida. Accendere la torcia a LED per illuminare.</li>
	<li>Sporgere delicatamente la lingua del mouse con un paio di forcep per esporre la sua trachea.</li>
	<li>Utilizzare l'altra mano per tenere la cannula guida con fibra ottica all'interno. Inseriscili attraverso la cavità orale. Con l'illuminazione dalla fibra ottica, l'apertura della trachea può essere vista come un orifizio tra le corde vocali.</li>
	<li>Allineare la smussatura della cannula guida verso la linea mediana dell'apertura (Figura 5A). Intubare delicatamente la cannula guida con fibra ottica nella trachea puntando la punta più fine della cannula all'apertura tracheale.</li>
	<li>Al momento dell'intubazione, rimuovere rapidamente la fibra ottica e lasciare la cannula guida all'interno della trachea (Figura 5B). Si deve osservare una respirazione normale.</li>
	<li>Tenere la pipetta a punta fine (parte A) all'apertura della cannula guida e insufflate un piccolo sbuffo d'aria (circa 0,2 mL) nel polmone del topo. Una leggera inflazione nel petto del topo indica una corretta intubazione. Rimuovere la pipetta della punta fine prima della somministrazione della polvere.</li>
</ol>4. Somministrazione di polvere
<ol><li>Tenere la punta caricata in polvere collegata alla siringa come descritto al passaggio 1.5. Assicurarsi che il flusso d'aria tra la siringa e la punta sia scollegato.</li>
	<li>Tirare lo stantuffo della siringa all'indietro per prelevare 0,6 ml di aria. NOTA: Il volume d'aria utilizzato per disperdere la polvere dipende dalle proprietà della polvere e dalla quantità di polvere caricata. Questo è ulteriormente descritto nella sezione dei risultati.</li>
	<li>Ruotare la valvola del fermaflusso a tre vie per collegare il flusso d'aria tra la siringa e la punta carica di polvere.</li>
	<li>Inserire la punta caricata a polvere nella cannula guida che è già stata posizionata nella trachea del mouse (Figura 5C). Tenere la cannula guida e spingere il pistone della siringa con forza in un'azione continua per disperdere la polvere come aerosol nel polmone. NOTA: Qualsiasi movimento in avanti del dispositivo deve essere ridotto al minimo per evitare di ferire l'animale.</li>
	<li>Rimuovere la punta e verificare se la polvere all'interno della punta è stata svuotata. In caso di meno, ripetere il passaggio da 4.1 a 4.4. NOTA: Se la polvere è imballata troppo strettamente a causa di un'eccessiva maschiatura, potrebbe non essere dispersa correttamente.</li>
	<li>Una volta completata la somministrazione, rimuovere la cannula guida dalla trachea.</li>
	<li>Lasciare che il mouse si riprenda posizionandolo orizzontalmente in posizione supina con la lingua semiverotta per evitare il blocco delle vie aeree.</li>
</ol>

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In questo documento vengono presentati dispositivi su misura per l'insufflazione di polvere secca e l'intubazione intratracheale. Nella fase di caricamento della polvere, la polvere secca viene caricata in una punta di pipetta a caricamento in gel da 200 μL. È importante toccare delicatamente la punta per consentire l'impacchettamento sciolto della polvere all'estremità stretta della punta. Tuttavia, se la polvere è imballata troppo strettamente, rimarrà bloccata nella punta e non può essere adeguatamente dispersa....

La via polmonare di somministrazione offre vari vantaggi nel fornire terapie sia per le azioni locali che sistemiche. Per il trattamento delle malattie polmonari, l'alta concentrazione locale di farmaci può essere ottenuta con l'erogazione polmonare, riducendo così la dose richiesta e abbassando l'incidenza di effetti collaterali sistemici. Inoltre, le attività enzimatiche relativamente basse nel polmone possono ridurre il metabolismo precoce dei farmaci. I polmoni sono anche efficienti per l'assorbimento del farmaco per l'azione sistemica a causa della superficie ampia e ben perfusa, dello strato cellulare epiteliale estremamente sottile e dell'alto volume sanguigno nei capillaripolmonari 1.
Le formulazioni in polvere secca inalato sono state ampiamente studiate per la prevenzione e il trattamento di varie malattie come asma, broncopneumopatia cronica ostruttiva, diabete mellito e vaccinazionepolmonare 2,3,4. I farmaci allo stato solido sono generalmente più stabili che nella forma liquida e gli inalatori a polvere secca sono più portatili e facili da usare rispetto ai nebulizzatori5,6. Nello sviluppo di formulazioni in polvere secca inalato, la sicurezza, il profilo farmacocinetico e l'efficacia terapeutica devono essere valutati in modelli animali preclinici dopo somministrazione polmonare7. A differenza degli esseri umani che possono inalare attivamente polvere secca, la consegna polmonare di polvere secca ai piccoli animali è impegnativa. È necessario stabilire un protocollo efficiente per fornire polvere secca ai polmoni degli animali.
I topi sono ampiamente utilizzati come modelli animali da ricerca perché sono economici e si riproducono bene. Sono anche facili da maneggiare e molti modelli di malattia sono ben consolidati. Esistono due approcci principali per somministrare polvere secca al polmone del topo: inalazione e somministrazione intratracheale. Per l'inalazione, il topo viene posto in una camera per tutto il corpo o solo per il naso in cui la polvere secca viene aerosolizzata e gli animali respirano nell'aerosol senza sedazione8,9. Sono necessarie attrezzature costose e l'efficienza di somministrazione dei farmaci è bassa. Mentre la camera di tutto il corpo può essere tecnicamente meno impegnativa, la camera di esposizione solo al naso potrebbe ridurre al minimo l'esposizione dei farmaci alla superficie corporea. Indipendentemente da ciò, è ancora difficile controllare e determinare con precisione la dose somministrata ai polmoni. La polvere secca viene depositata principalmente nella regione del nasofaringe dove lo sgombero mucociliare è prominente10. Inoltre, i topi all'interno della camera sono sotto stress significativo durante il processo di somministrazione perché sono vincolati e privati dell'approvvigionamento di cibo eacqua 11. Per la somministrazione intratracheale, si riferisce generalmente all'introduzione della sostanza direttamente nella trachea. Ci sono due diverse tecniche per raggiungere questo obiettivo: la tracheotomia e l'intubazione orotracheale. Il primo richiede una procedura chirurgica che faccia un'incisione nella trachea, che è invasiva e raramente utilizzata per la somministrazione della polvere. Qui viene descritta solo la seconda tecnica. Rispetto al metodo di inalazione, la somministrazione intratracheale è il metodo più comunemente usato per la consegna polmonare nel topo a causa della sua elevata efficienza di consegna con una perdita minimadi farmaci 12,13. È un metodo semplice e veloce per fornire con precisione una piccola quantità di polvere entro pochi milligrammi al topo. Sebbene il topo sia anatomicamente e fisiologicamente distinto dall'uomo e l'anestesia sia richiesta durante il processo di intubazione, la somministrazione intratracheale aggira le vie respiratorie superiori e offre un modo più efficace per valutare le attività biologiche della formulazione della polvere secca come l'assorbimento polmonare, la biodisponibilità e gli effetti terapeutici14,15.
Per somministrare polvere secca intratrachealmente, il mouse deve essere intubato, il che potrebbe essere impegnativo. In questo documento viene descritta la fabbricazione di un insufflatore di polvere secca su misura e di un dispositivo di intubazione. Vengono dimostrate le procedure di intubazione e insufflazione di polvere secca nel polmone del topo.

<table>
	<tbody>
		<tr>
			<td>BALB/c mouse</td>
			<td></td>
			<td></td>
			<td>Female; 7-9 weeks old; Body weight 20-25 g</td>
		</tr>
		<tr>
			<td>CleanCap Firefly Luciferase mRNA</td>
			<td>TriLink Biotechnology</td>
			<td>L-7602</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Powder Insufflator</td>
			<td>PennCentury</td>
			<td>Model DP-4M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine 10%</td>
			<td>Alfasan International B.V.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Light emitting diode (LED) torch</td>
			<td>Unilite Internation</td>
			<td>PS-K1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol (Pearlitol 160C)</td>
			<td>Roquette</td>
			<td>450001</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-filter round gel loading pipette tip (200 &#181;L)</td>
			<td>Labcon</td>
			<td>1034-800-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon floss</td>
			<td>Reach</td>
			<td>30017050</td>
			<td></td>
		</tr>
		<tr>
			<td>One milliliter syringe without needle</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fibre</td>
			<td>Fibre Data</td>
			<td>OMPF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG12KL4 peptide</td>
			<td>EZ Biolab</td>
			<td></td>
			<td>(PEG12)-KLLLLKLLLLKLLLLKLLLLK-NH2</td>
		</tr>
		<tr>
			<td>Plastic Pasteur fine tip pipette</td>
			<td>Alpha Labotatories</td>
			<td>LW4061</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcock</td>
			<td>Braun</td>
			<td>D201</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 2%</td>
			<td>Alfasan International B.V.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Zerostat 3 anti-static gun</td>
			<td>MILTY</td>
			<td>5036694022153</td>
			<td></td>
		</tr>
	</tbody>
</table>

intratracheal administration of dry powder formulation in mice

Nello sviluppo di formulazioni in polvere secca inalabile, è essenziale valutare le loro attività biologiche in modelli animali preclinici. Questo documento introduce un metodo non invasivo di somministrazione intratracheale della formulazione della polvere secca nei topi. Viene presentato un dispositivo di caricamento a polvere secca costituito da una punta di pipetta di caricamento in gel da 200 μL collegata a una siringa da 1 ml tramite un fermacock a tre vie. Una piccola quantità di polvere secca (1-2 mg) viene caricata nella punta della pipetta e dispersa di 0,6 ml di aria nella siringa. Poiché le punte delle pipette sono usa e getta ed economiche, diverse formulazioni di polvere secca possono essere caricate in diverse punte in anticipo. Varie formulazioni possono essere valutate nello stesso esperimento animale senza pulizia del dispositivo e riempimento della dose, risparmiando così tempo ed eliminando il rischio di contaminazione incrociata dalla polvere residua. L'entità della dispersione della polvere può essere ispezionata dalla quantità di polvere rimanente nella punta della pipetta. È incluso un protocollo di intubazione nel mouse con una sorgente luminosa su misura e una cannula guida. La corretta intubazione è uno dei fattori chiave che influenza l'erogazione intratracheale della formulazione della polvere secca nella regione polmonare profonda del topo.

Quando un insufflatore di polvere secca viene utilizzato per fornire aerosol di polvere al polmone di un animale, il volume d'aria utilizzato è fondamentale in quanto influisce sulla sicurezza e sull'efficienza di dispersione della polvere. Per ottimizzare il metodo, sono stati utilizzati diversi volumi d'aria (0,3 mL, 0,6 mL e 1,0 mL) per disperdere la polvere secca (1 mg di mannito essiccato a spruzzo) e il peso dei topi è stato monitorato per 48 ore dopo lasomministrazione (figura 6). L...

Watch this Scientific Journal Video about Intratracheal Administration of Dry Powder Formulation in Mice at JoVE.com

Intratracheal Administration of Dry Powder Formulation in Mice

Le formulazioni in polvere secca per inalazione hanno un grande potenziale nel trattamento delle malattie respiratorie. Prima di entrare in studi sull'uomo, è necessario valutare l'efficacia della formulazione della polvere secca in studi preclinici. Viene presentato un metodo semplice e non invasivo per la somministrazione di polvere secca nei topi attraverso la via intratracheale.

Somministrazione intratracheale della formulazione di polvere secca nei topi

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

intratracheal-administration-of-dry-powder-formulation-in-mice

Research

JoVE Journal

Medicine

9.7K Views.  The University of Hong Kong. Le formulazioni in polvere secca per inalazione hanno un grande potenziale nel trattamento delle malattie respiratorie. Prima di entrare in studi sull'uomo, è necessario valutare l'efficacia della formulazione della polvere secca in studi preclinici. Viene presentato un metodo semplice e non invasivo per la somministrazione di polvere secca nei topi attraverso la via intratracheale.

Video: Intratracheal Administration of Dry Powder Formulation in Mice

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.

Un nuovo approccio chirurgico per somministrazione endotracheale di agenti bioattivi in ​​un modello murino fetale

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

Ricerca

Medicina

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Somministrazione intranasale di terapeutica del sistema nervoso centrale di topi Awake

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Polvere secca e nebulizzata Aerosol L&#39;inalazione di prodotti farmaceutici distribuito nelle topo, utilizzando un naso-only Sistema di esposizione

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

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.

Un modello di effusione pleurica in ratti mediante instillazione intratracheale di poliacrilato/nanosilice

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.

Indurre lesioni polmonari acute nei topi mediante instillazione diretta del lipopolioaccaride intracheale

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

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Istituzione di un modello di occhio secco severo utilizzando la dacrigofectomia completa nei conigli

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Instillazione intratracheale delle cellule staminali nei " ratti neooatali

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.

Un metodo minimamente invasivo per l'instillazione intratracheale di farmaci nei roditori neonatali per il trattamento delle malattie polmonari

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered ...

Customizable Disposable Dosators to Deliver Dry Powder Aerosol Drugs to Mice

Disposable Dosators Intended for Dry Powder Delivery to Mice

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus metering and dosing, and a propellant-free dispersal mechanism. To develop pharmaceutical dry powder aerosol products, robust in vivo testing is essential. Typically, initial studies involve using a murine model for preliminary evaluation before conducting formal studies in larger animal species. However, a significant limitation in this approach is the lack of suitable device technology to accurately and reproducibly deliver dry powders to small animals, hindering such models&#39; utility. To address these challenges, disposable syringe dosators were developed specifically for intrapulmonary delivery of dry powders in doses appropriate for mice. These dosators load and deliver a predetermined amount of powder obtained from a uniform bulk density powder bed. This discrete control is achieved by inserting a blunt needle to a fixed depth (tamping) into the powder bed, removing a fixed quantity each time. Notably, this dosing pattern has proven effective for a range of spray-dried powders. In experiments involving four different model spray-dried powders, the dosators demonstrated the ability to achieve doses within the range of 30 to 1100 &#181;g. The achieved dose was influenced by factors such as the number of tamps, the size of the dosator needle, and the specific formulation used. One of the key benefits of these dosators is their ease of manufacturing, making them accessible and cost-effective for delivering dry powders to mice during initial proof-of-concept studies. The disposable nature of the dosators facilitates use in animal procedure rooms, where cleaning and refilling reusable systems and weighing materials is inconvenient. Thus, developing disposable syringe dosators has addressed a significant hurdle in murine dry powder delivery for proof-of-concept studies, enabling researchers to conduct more accurate and reproducible preliminary studies in small animal models for pulmonary drug delivery.

Author Spotlight: Developing a Disposable Dosator for Preclinical Testing of Dry Powder Inhalers in Small Animal Models 

Pharmaceutical dry powder development necessitates reliable in vivo testing, often using a murine model. Device technology for accurately and reproducibly delivering dry powder aerosols to mice is restricted. This study presents disposable dosators for pulmonary drug delivery at mouse-relevant doses, aiding initial proof-of-concept research.

Dosatori monouso destinati alla somministrazione di polvere secca ai topi

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus ...