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Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

Published: April 24th, 2013



1Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, University of Toledo

The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This is mostly due to the difficulty of microdissection of fetal brains and their sectioning for determination of apoptotic cells caused by prenatal exposure to alcohol. The experiments described here provide visualized techniques from mice breeding to the identification of cell death in fetal brain tissue. This study used C57BL/6 mice as the animal model for studying fetal alcohol exposure and the role of trophic peptide against alcohol-induced apoptosis. The breeding consists of a 2-hr matting window to determine the exact stage of embryonic age. An established fetal alcohol exposure model has been used in this study to determine the effects of prenatal alcohol exposure in fetal brains. This involves free access to alcohol or pair-fed liquid diets as the sole source of nutrients for the pregnant mice.

The techniques involving dissection of fetuses and microdissection of fetal brains are described carefully, since the latter can be challenging. Microdissection requires a stereomicroscope and ultra-fine forceps. Step-by-step procedures for dissecting the fetal brains are provided visually. The fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon.

For investigating apoptosis, fetal brains are first embedded in gelatin using a peel-away mold to facilitate their sectioning with a vibratome apparatus. Fetal brains embedded and fixed in paraformaldehyde are easily sectioned, and the free floating sections can be mounted in superfrost plus slides for determination of apoptosis or cell death.

TUNEL (TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase) assay has been used to identify cell death or apoptotic cells. It is noteworthy that apoptosis and cell-mediated cytotoxicity are characterized by DNA fragmentation. Thus, the visualized TUNEL-positive cells are indicative of cell death or apoptotic cells.

The experimental designs here provide information about the use of an established liquid diet for studying the effects of alcohol and the role of neurotrophic peptides during pregnancy in fetal brains. This involves breeding and feeding pregnant mice, microdissecting fetal brains, and determining apoptosis. Together, these visual and textual techniques might be a source for investigating prenatal exposure of harmful agents in fetal brains.

The goal of the experimental methods described here is to assess the neuroprotective effects of trophic peptides in a fetal alcohol exposure model using several techniques involving breeding, feeding, microdissecting, and detecting cell death. Work from our laboratory has involved a liquid diet mixed with alcohol as a model of moderate alcohol drinking, which might be similar to a human drinking paradigm in term of the amount of alcohol consumed 1-5. We and others have identified three peptide derivatives that are neuroprotective against the deleterious effects of fetal alcohol exposure. Studies investigating alcohol exposure during embryonic stages using a....

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1. Animals Breeding and Trophic Peptide Treatment

  1. Breed C57BL/mice by placing the female mice (~6 week old, average weight 20 g) into male home cages for 2 hr.
  2. Check for a sperm plug and/or vaginal smear immediately afterwards.
  3. If positive, designate this time point as embryonic day 0 (E0).
  4. On E7, weight-match pregnant females to control and treatment groups (3 groups) are assigned as described recently 4: 1) Alcohol (ethanol) liquid diet group (ALC); 2) pair-fed contr.......

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The experimental methods described here show that a 2-hr breeding window is essential to estimate accurate gestational stage. We have demonstrated the dissection of the embryos at the age of E13. We also demonstrated microdissection of fetal brains at E13. The procedure involves several stages (a-c), as described in Figure 1. Fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon (Figure 1). Fetal brains are then fixed in 4% paraformald.......

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The methodology and technology presented in this study demonstrate the effects of prenatal exposure to alcohol in fetal brains and the role of trophic peptides in the prevention of these effects. These may provide information on how to study other drugs of abuse or other toxic chemicals in fetal brains during different pregnancy stages.

In regards to the breeding paradigm, in some cases the detection of the sperm plug might not be observable. At this point, it is important to test the vaginal.......

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This research project was supported by Award Number R21AA017735 (Y.S.) from the National Institutes on Alcohol Abuse and Alcoholism. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institute on Alcohol Abuse and Alcoholism or the National Institutes of Health. The author would also like to thank Charisse Montgomery for editing this manuscript.


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Name Company Catalog Number Comments
Name of the reagent/equipment Company Catalogue number Comments (optional)
Superfrost Plus Slides VWR 48311-703
PAP PEN Research Products Int. Corp. 195506
5-Plend Open Mailer Heathrow Scientific, LLC HS 15983G
Peel away embedding molds Electron Microscopy Sciences 70182
In situ cell death detection kit, POD Roche Diagnostics, Inc 11684817910
Hanks' Balanced Salt Solution Invitrogen 14170-120
Gelatin Type A FisherScientific G8-500
Stereomicroscope W. NUHSBAUM, Inc Leica M60, KL 200 LED
Micro-Vibratome Leica, Inc Leica VT 1000S
Moria Ultra Fine Forceps Fine Science Tools 11370-40 2 pairs
Graduate 30 ml feeding tubes Dyets, Inc 900012
Vitamin Mix Bio-Serv. Inc. F8031
Mineral Mix Bio-Serv. Inc. F8598
Maltose Dextrin Bio-Serv. Inc. 3650
Ethyl alcohol 190 Proof, 5 gallons 111000190-SS05 Pharmco-AAPER
Upright microscope W. NUHSBAUM, Inc LEICA DM 4000

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