Transuterine Fetal Tracheal Occlusion Model in Mice

Podsumowanie

Various animal models of congenital diaphragmatic hernia and fetal tracheal occlusion present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and availability of genetic tools. This model provides a new tool to study the impact of both tracheal occlusion and increased luminal pressure on lung development.

Streszczenie

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

Wprowadzenie

Congenital diaphragmatic hernia (CDH) occurs in 1:2500 pregnancies and results in pulmonary hypoplasia and neonatal pulmonary hypertension^1,2,3,4,5,6. Fetal tracheal occlusion (TO) is an established prenatal therapy in severe CDH patients involving fetoscopy in the 26-30^th gestational week in which a balloon is placed just above the carina and then removed in the 32^nd gestational week. This temporary TO induces fetal lung growth and improves survival.....

Protokół

All experiments have complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80023, revised 1978). The procedure was approved with IACUC protocol #2016-0068 by the Cincinnati Children's Research Foundation Institutional Animal Care and Use Committee.

1. Preparation

1. To mate age-matched wild-type (WT) C57BL/6 mice, place them in the same cage at 6:00 p.m. and separate them at 9:00 a.m. the next day.
2. To determine embryonic day 0 (E0), look at the vaginal plug, which has a homogeneous outer zone attached to the vaginal wall and an inner zone that is fibrous....

Wyniki

This study examined 37 fetuses: 20 (54.1%) as TO vs. 17 (45.9%) as control. As the trachea could not be occluded in 4 fetuses in the TO group, they were excluded from the study. There was no significant difference in mortality in both groups: 4 fetuses (25%) in the TO group and 2 fetuses (12%) in the control group (p=0.334, odds ratio (OR) 2.5, 95% confidence interval (CI) 0.39-16.05). The mean body weight, lung weight, and lung to body weight ratio (LBWR) were higher in the TO group than.......

Dyskusje

This method describes a surgical procedure of fetal tracheal occlusion in mice and its impact on lung development. There are some critical steps in the protocol that should be carefully performed for successful TO. The warmth of the platform on which the surgery takes place and the saline introduced into the peritoneal cavity is crucial for the progression of the pregnancy. In addition, a slight pressure has to be applied to the head of the pups to ensure exposure of the neck.

A 6.0 polypropyl.......

Materiały

Name	Company	Catalog Number	Comments
Buprenorphine	Par Pharmaceutical	NDC 42023-179-05	For regional anesthesia
Isoflurane	Halocarbon Life Sciences	NDC 66794-017-25	For general anesthesia
Magnification glasses	USA Medical-Surgical	SLR-250LBLK	At least 2.5x
Nikon 90i microscope	Nikon	3417	Motorized Fluorescence
Nucleospin Tissue Kit	Macherey-Nagel, Düren, Germany	740952.5	DNA isolation
Pierce BCA Protein Assay Kit	Thermo Fisher, IL, USA	23225	Protein quantification
Polyglactin suture	Ethicon	VCP451H	4-0, 24 mm, cutting
Polylysine slides	VWR	48382-117	Microscope adhesion slides
Polypropylene suture	Ethicon	Y432H	6-0, 13 mm 1/2c Taperpoint
RIPA buffer	Sigma-Aldrich, Missouri, USA	R0278-50ml	Protein isolation
Silk suture	Ethicon	VCP682G	4-0, 24 mm, cutting
Trizol	Invitrogen	15596026	RNA isolation