M.C. and A.V.d.P. are doctoral fellows supported by grants from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). J.T. holds a part-time Clinical Research Fellowship (KOOR) from UZ Leuven. D.V. is a doctoral fellow supported by a grant from KU Leuven, DBOF/10/062. M.M.d.C is a doctoral fellow supported by a grant from Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) and Erasmus Mundus. Research was funded by IWT-Vlaanderen, by the EC grant DIMI (LSHB-CT-2005-512146) and by the In vivo Molecular Imaging Research group (IMIR) from the KU Leuven. We would like to acknowledge the UPenn Vector Core founded by James M. Wilson for their kind gift of the AAV6.2 packaging plasmid for rAAV vector production.

1. Mating of Mice to Obtain Desired Pregnancy Stage
Time mate pregnant NMRI mice so that they are 18 days (E18) pregnant (total gestation E19.5) at the time of surgery. Prior to and after surgery they are housed in filter top cages at normal room temperature and normal daylight with free access to water and chow.
2. Fetal Intratracheal (I.T.) Injection (Figure 1)
<ol>
 <li>First submit the pregnant NMRI mouse to general anesthesia with 1.5% isoflurane in a mixture o...

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Critical steps
 <ul>
 <li>The mouse strain we chose to work with is NMRI mice because they have an abundant number of pups (average litter size 14.4 &plusmn; 1.8, own data), tolerate interventions well and have good maternal characteristics.</li>
 <li>Placing the purse string through the uterine wall and the fetal membranes is a critical step as you only want to expose the fetal head and not the shoulders, otherwise repositioning is almost impossible without causing trauma.</li>
 <li>An optimal position of...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>NMRI mice</td>
 <td>Janvier, Le Genest St Isle, France</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane </td>
 <td>Isoba, Intervet / Schering-Plough Animal Health, Milton Keynes, UK</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Prolene 6-0</td>
 <td>Ethicon, Groot Bijgaarden, Belgium</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vicryl 5-0</td>
 <td>Ethicon, Groot Bijgaarden, Belgium</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>50 &mu;l Hamilton Glass Syringe, Model 1710.5 TLLX SYR</td>
 <td>Hamilton, Reno, NV, USA</td>
 <td>5495-20</td>
 <td></td>
 </tr>
 <tr>
 <td>30G sharp needle</td>
 <td>Hamilton, Reno, NV, USA</td>
 <td>7762-03</td>
 <td></td>
 </tr>
 <tr>
 <td>2% xylocaine </td>
 <td>AstraZeneca, Zoetermeer, The Netherlands</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model at JoVE.com

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

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.

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

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16.3K visualizações.  KU Leuven. Nós desenvolvemos uma nova abordagem cirúrgica para administração intratraqueal de agentes bioactivos para o feto de rato. A via de entrega é mais eficiente na segmentação dos pulmões do rato fetal do que o normalmente usado injecção intra-amniótica. Este procedimento tem a data ainda não foi descrita em um modelo de rato.