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.

1. Animals Breeding and Trophic Peptide Treatment
<ol>
	<li>Breed C57BL/mice by placing the female mice (~6 week old, average weight 20 g) into male home cages for 2 hr.</li>
	<li>Check for a sperm plug and/or vaginal smear immediately afterwards.</li>
	<li>If positive, designate this time point as embryonic day 0 (E0).</li>
	<li>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 control group (PF); 3) Peptide treatment group, which should receive intraperitoneal (i.p.) injection of ADNF-9 peptide alongside alcohol exposure liquid diet (ALC/ADNF-9). The details of the preparation of ALC and PF diets and the procedure of i.p. injections can be found in our recent work 4.</li>
	<li>Measure daily alcohol liquid diet that can be provided as free access as the sole source of nutrients. PF liquid diet yoked individually to an ALC dam can be measured daily as well.</li>
	<li>The volume of liquid diet consumed during the previous 24 hr can be recorded from 30-ml graduated screw-cap tubes and freshly prepared diet should be provided daily from E7 to E13.</li>
</ol>
2. Dissection of Fetuses and Microdissection of Fetal Brains
<ol>
	<li>Sacrifice pregnant mice on E13 by deeply euthanizing the mice with a CO2 procedure followed by a cervical dislocation.</li>
	<li>Clean the abdominal site of incision with 70% ethanol.</li>
	<li>Make an abdominal incision using sterile scissors and forceps.</li>
	<li>Once the abdomen is open, dissect out the uterus by holding it with one forceps and using the other forceps to tear the mesometrium away from the uterus. Make sure to do this procedure slowly in order to avoid puncturing the embryos.</li>
	<li>Remove the embryos from the uterus and transfer them into a cell culture dish (60 mm x 15 mm) containing Hanks' Balanced Salt solution (HBSS) in ice cold water.</li>
	<li>Dissect fetal brains from the whole embryos under stereomicroscope for microdissection.</li>
	<li>Using ultra-fine forceps for microdissection, pull off the skin of the embryos at the top part of the head to expose the skull.</li>
	<li>Once the skull is exposed and clearly visualized under the microdissection stereomicroscope, then peel off the skull starting from the posterior part of the brain at the spinal cord and brainstem areas.</li>
	<li>Be sure to peel off the skull piece by piece from posterior to anterior parts of the head.</li>
	<li>Once fetal brains are visually exposed, proceed with extracting them.</li>
	<li>Dissect the fetal brains from the base of the primordium olfactory bulb to the base of the metencephalon.</li>
	<li>Postfix all dissected fetal brains in 4% paraformaldehyde for 2-3 days. You can also freeze other fetal brains from each dam if you are testing them for Western blot or enzymatic assays.</li>
</ol>
Note: The procedures for animal uses were approved by the Institutional Animal Care and Use Committee of University of Toledo which are in accordance with the guidelines of the Institutional Animal Care and Use Committee at the National Institutes of Health and the Guide for the Care and Use of Laboratory Animals.
3. Embedding Fetal Brains in Gelatin for Sectioning
<ol>
	<li>Prepare 10% of gelatin grade A.</li>
	<li>Fill a peel-away embedding mold halfway and wait until the gelatin solidifies.</li>
	<li>Place control and prenatally treated fetal brains side-by-side on top of the solidified gelatin. This is to keep consistent conditions of immunodetection of cell death between both the control and experimental groups.</li>
	<li>Add liquid gelatin on top of the fetal brains to make a complete mold (make sure the gelatin is not too hot).</li>
	<li>Refrigerate the prepared gelatin mold that holds fetal brains for 15 min.</li>
	<li>Peel off the embedding mold plastic and then transfer the pre-made gelatin containing fetal brains into 4% paraformaldehyde for two days.</li>
	<li>Place and section fixed fetal brains in gelatin using vibratome sectioning machine (Leica VT 1000S) at 50 &mu;m thickness for coronal sectioning. This thickness is tested in our studies because it provides better anatomical features. In addition, thicknesses of 25 &mu;m and higher can also be tested with this technique involving embedded of fetal brains in 10% gelatin. Note that coronal sections were cut using vibratome at the basal ganglia eminence level on the forebrain</li>
	<li>Collect the fetal brain sections in phosphate-buffered saline (PBS) solution.</li>
	<li>Mount the fetal brain sections in Superfrost Plus slides in water.</li>
	<li>Dry the mounted sections for 15 min.</li>
	<li>Place the slides (mounted with the fetal brain sections) in PBS for next day testing of TUNEL reaction.</li>
</ol>
4. TUNEL Reaction for Detection of Cell Death or Apoptosis
(TUNEL: TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase). This assay is aimed to determine cell death.
<ol>
	<li>Treat fetal brain sections mounted in Superfrost Plus slides with Proteinase K (20 &mu;g/ml) for 5 min at 37 &deg;C (300 &mu;l of proteinase K mixed in 20 ml PBS). Slides are assembled in 5-Plend open maile).</li>
	<li>Rinse fetal brain sections (mounted in slides) with PBS three times for 5 min under Orbital shaker.</li>
	<li>Incubate the sections with 3% H2O2 in methanol for 10 min at room temperature (3 ml 30% H2O2 in 27 ml 100% methanol) (slides should not be in a shaker).</li>
	<li>Rinse sections with PBS three times for 5 min in orbital shaker.</li>
	<li>Incubate sections in permeabilization solution (0.1% TritonX-100 in 0.1% sodium citrate) for 2 min on ice (4 &deg;C) (100 &mu;l TritonX + 0.1 g sodium citrate + 99.9 ml distilled water).</li>
	<li>Rinse sections in PBS two times for 5 min in orbital shaker.</li>
	<li>Dry area around sections in the slides.</li>
	<li>Draw barrier on Superfrost Plus slides using PAP Pen. This is aimed to prevent any leaking of solution that is used to treat the sections for the detection of TUNEL-positive cells.</li>
	<li>Incubate the sections with TUNEL reaction mixture (50 &mu;l from bottle 1 and 450 &mu;l from bottle 2) for 1 hr at 37 &deg;C, the control will be used by incubation in solution from bottle 2.</li>
	<li>Rinse with PBS three times 5 min under Orbit shaker.</li>
	<li>Dry area around tissue.</li>
	<li>Incubate sections with converter-POD for 30 min at 37 &deg;C.</li>
	<li>Rinse the sections three times for 5 min with Tris HCl (pH 7.5, 0.05 M).</li>
	<li>Incubate the sections for 7 min in 0.05% 3'-3'-diaminobenzidine tetrahydrochloride (DAB) and 0.003% H2O2 (25 ml Tris HCl, pH7.5, 0.05 M + 25 &mu;l 3% H202 + 500 &mu;l DAB) under Oribtal shaker at 4 &deg;C (in ice) and in dark (make sure to do this procedure in new 5-Plend open mailer).</li>
	<li>Rinse the sections with PBS three times 5 min under Orbital shaker.</li>
	<li>Keep the slides sections in PBS for no more than 24 hr in PBS, then process them for Nissl staining as detailed in the next section protocol.</li>
</ol>
Note: DAB is a carcinogen. Use gloves and clean all dishes used with the bleach once finished with all the experiments.
5. Nissl staining for Preparation of Fetal Brain Sections for Microscopic Observation
<ol>
	<li>Nissl staining procedure should be done in a biological safety hood.</li>
	<li>Rinse the slide sections with deionized water for 2 min.</li>
	<li>Incubate the sections in 70% ethanol for 2 min.</li>
	<li>Incubate the sections in 95% ethanol for 6 min.</li>
	<li>Incubate the sections in 70% ethanol for 2 min.</li>
	<li>Rinse the sections in deionized water for 2 min.</li>
	<li>Incubate the sections in Cresyl Violet Stain for 3 min.</li>
	<li>Rinse the sections in deionized water for 2 min.</li>
	<li>Incubate the sections in 70% ethanol for 2 min.</li>
	<li>Incubate the sections in 95% ethanol + 3 ml glacial acetic acid for 3 min.</li>
	<li>Incubate the sections in 95% ethanol for 2 min.</li>
	<li>Incubate the sections in 100% ethanol for 2 min three times.</li>
	<li>Incubate the sections in Xylene for 10 min three times.</li>
	<li>Slides holding the fetal brain sections can be mounted using Permount and cover slip glass slides.</li>
	<li>Leave the mounted slides overnight prior to microscopic observation and analyses.</li>
	<li>Microscopic observation and photomicrographs can be performed under Leica upright microscope.</li>
</ol>

<ol>
	<li>Sari, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+neuroprotective+protein-derived+peptide,+NAP,+preventing+alcohol-induced+apoptosis+in+fetal+brain+of+C57BL/6+mouse.">Activity-dependent neuroprotective protein-derived peptide, NAP, preventing alcohol-induced apoptosis in fetal brain of C57BL/6 mouse.</a> Neuroscience. 158 (4), 1426-1435 (2009).</li><li>Sari, Y., Chiba, T., Yamada, M., Rebec, G. V., Aiso, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+peptide,+colivelin,+prevents+alcohol-induced+apoptosis+in+fetal+brain+of+C57BL/6+mice:+signaling+pathway+investigations.">A novel peptide, colivelin, prevents alcohol-induced apoptosis in fetal brain of C57BL/6 mice: signaling pathway investigations.</a> Neuroscience. 164 (4), 1653-1664 (2009).</li><li>Sari, Y., Hammad, L. A., Saleh, M. M., Rebec, G. V., Mechref, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+selective+neurotransmitters+in+fetal+brains+of+prenatally+alcohol-treated+C57BL/6+mice:+quantitative+analysis+using+liquid+chromatography/tandem+mass+spectrometry.">Alteration of selective neurotransmitters in fetal brains of prenatally alcohol-treated C57BL/6 mice: quantitative analysis using liquid chromatography/tandem mass spectrometry.</a> Int. J. Dev. Neurosci. 28 (3), 263-269 (2010).</li><li>Sari, Y., Weedman, J. M., Ge, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+neurotrophic+factor-derived+peptide+prevents+alcohol-induced+apoptosis,+in+part,+through+Bcl2+and+c-Jun+N-terminal+kinase+signaling+pathways+in+fetal+brain+of+C57BL/6+mouse.">Activity-dependent neurotrophic factor-derived peptide prevents alcohol-induced apoptosis, in part, through Bcl2 and c-Jun N-terminal kinase signaling pathways in fetal brain of C57BL/6 mouse.</a> Neuroscience. 202, 465-473 (2012).</li><li>Sari, Y., Zhang, M., Mechref, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+proteins+in+fetal+brains+of+alcohol-treated+prenatally+C57BL/6+mice:+a+proteomic+investigation.">Differential expression of proteins in fetal brains of alcohol-treated prenatally C57BL/6 mice: a proteomic investigation.</a> Electrophoresis. 31, 1-14 (2010).</li><li>Bassan, M., Zamostiano, R., Davidson, A., Pinhasov, A., Giladi, E., Perl, O., Bassan, H., Blat, C., Gibney, G., Glazner, G., Brenneman, D. E., Gozes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+sequence+of+a+novel+protein+containing+a+femtomolar-activity-dependent+neuroprotective+peptide.">Complete sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective peptide.</a> J. Neurochem. 72 (3), 1283-1293 (1999).</li><li>Brenneman, D. E., Hauser, J., Neale, E., Rubinraut, S., Fridkin, M., Davidson, A., Gozes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+neurotrophic+factor:+structure-activity+relationships+of+femtomolar-acting+peptides.">Activity-dependent neurotrophic factor: structure-activity relationships of femtomolar-acting peptides.</a> J. Pharmacol. Exp. Ther. 285 (2), 619-627 (1998).</li><li>Spong, C. Y., Abebe, D. T., Gozes, I., Brenneman, D. E., Hill, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+fetal+demise+and+growth+restriction+in+a+mouse+model+of+fetal+alcohol+syndrome.">Prevention of fetal demise and growth restriction in a mouse model of fetal alcohol syndrome.</a> J. Pharmacol. Exp. Ther. 297 (2), 774-779 (2001).</li><li>Sari, Y., Gozes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+deficits+associated+with+fetal+alcohol+exposure+may+be+protected,+in+part,+by+peptides+derived+from+activity-dependent+neurotrophic+factor+and+activity-dependent+neuroprotective+protein.">Brain deficits associated with fetal alcohol exposure may be protected, in part, by peptides derived from activity-dependent neurotrophic factor and activity-dependent neuroprotective protein.</a> Brain Res. Brain Res. Rev. 52 (1), 107-118 (2006).</li><li>Brenneman, D. E., Gozes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+femtomolar-acting+neuroprotective+peptide.">A femtomolar-acting neuroprotective peptide.</a> J. Clin. Invest. 97 (10), 2299-2307 (1996).</li><li>Zamostiano, R., Pinhasov, A., Gelber, E., Steingart, R. A., Seroussi, E., Giladi, E., Bassan, M., Wollman, Y., Eyre, H. J., Mulley, J. C., Brenneman, D. E., Gozes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+the+human+activity-dependent+neuroprotective+protein.">Cloning and characterization of the human activity-dependent neuroprotective protein.</a> J. Biol. Chem. 276 (1), 708-714 (2001).</li><li>Offen, D., Sherki, Y., Melamed, E., Fridkin, M., Brenneman, D. E., Gozes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasoactive+intestinal+peptide+(VIP)+prevents+neurotoxicity+in+neuronal+cultures:+relevance+to+neuroprotection+in+Parkinson's+disease.">Vasoactive intestinal peptide (VIP) prevents neurotoxicity in neuronal cultures: relevance to neuroprotection in Parkinson's disease.</a> Brain Res. 854 (1-2), 257-262 (2000).</li><li>Steingart, R. A., Solomon, B., Brenneman, D. E., Fridkin, M., Gozes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VIP+and+peptides+related+to+activity-dependent+neurotrophic+factor+protect+PC12+cells+against+oxidative+stress.">VIP and peptides related to activity-dependent neurotrophic factor protect PC12 cells against oxidative stress.</a> J. Mol. Neurosci. 15 (3), 137-145 (2000).</li></ol>

No disclosures to be made.

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...

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 animal models may lead to the identification of potential mechanisms of neuroprotection and allow for the development of intervention procedures. This may provide ample information about the neuroprotective effects and attenuation of the deleterious effects of alcohol exposure during pregnancy.
Possible prevention of the effects of prenatal alcohol exposure may involve the treatment of pregnant mice with peptides that have been shown to be involved in neuroprotection in vitro 6,7 and in vivo 1,2,4,8,9. Among these peptides, SALLRSIPA, known as ADNF-9 or SAL, is derived from activity-dependent neurotrophic factor (ADNF) 7,10. Another peptide with the sequence NAPVSIPQ peptide, termed NAP, derived from activity-dependent neuroprotective protein (ADNP) 6,11, demonstrated a potent protective effect against oxidative stress associated with alcohol exposure 12,13. In addition, we have recently identified a new synthesized peptide, colivelin that appears to play a key neuroprotective role in the fetal alcohol exposure model 2. Colivelin is composed of ADNF-9 and AGA-(C8R)HNG17 (PAGASRLLLLTGEIDLP). The importance of the use of these peptides is that they have the capability to cross the brain-blood barrier to prevent the effects of alcohol-induced apoptosis and brain growth deficits.
The experimental methods used C57BL/6 mice for testing the neuroprotective effects against alcohol-induced apoptosis. We have adopted a 2-hr window instead of overnight breeding in order to accurately estimate the embryonic day 0 (E0), as it can be revealed by detection of a sperm plug and vaginal smear 1-5. We have used a liquid diet with feeding tubes, as this is considered free access instead of delivery of alcohol through gavage route, which may induce stress to pregnant mice. On the other hand, microdissection can be challenging due to the softness of the fetal brain tissue, which might be encountered when dissecting fetal brains at early embryonic stages. Here, we show visualized techniques to deal with all the challenges involving microdissection. Furthermore, since the fetal brain sectioning can also be challenging, we have adopted a technique that involves embedding the fetal brains in gelatin using peel-away embedding molds. We have been successful in cutting the fetal brains in free floating sections at 50 &mu;m thickness. This allows us to investigate any alterations of content protein in fetal brain sections, including the identification of cell death.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Superfrost Plus Slides</td>
 <td>VWR</td>
 <td>48311-703</td>
 <td></td>
 </tr>
 <tr>
 <td>PAP PEN</td>
 <td>Research Products Int. Corp.</td>
 <td>195506</td>
 <td></td>
 </tr>
 <tr>
 <td>5-Plend Open Mailer </td>
 <td>Heathrow Scientific, LLC</td>
 <td>HS 15983G</td>
 <td></td>
 </tr>
 <tr>
 <td>Peel away embedding molds</td>
 <td>Electron Microscopy Sciences</td>
 <td>70182</td>
 <td></td>
 </tr>
 <tr>
 <td>In situ cell death detection kit, POD</td>
 <td>Roche Diagnostics, Inc</td>
 <td>11684817910</td>
 <td></td>
 </tr>
 <tr>
 <td>Hanks' Balanced Salt Solution</td>
 <td>Invitrogen</td>
 <td>14170-120 </td>
 <td></td>
 </tr>
 <tr>
 <td>Gelatin Type A</td>
 <td>FisherScientific</td>
 <td>G8-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>W. NUHSBAUM, Inc </td>
 <td>Leica M60, KL 200 LED</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro-Vibratome</td>
 <td>Leica, Inc </td>
 <td>Leica VT 1000S</td>
 <td></td>
 </tr>
 <tr>
 <td>Moria Ultra Fine Forceps </td>
 <td>Fine Science Tools</td>
 <td>11370-40</td>
 <td>2 pairs</td>
 </tr>
 <tr>
 <td>Graduate 30 ml feeding tubes</td>
 <td>Dyets, Inc </td>
 <td>900012 </td>
 <td></td>
 </tr>
 <tr>
 <td>Vitamin Mix </td>
 <td>Bio-Serv. Inc. </td>
 <td>F8031 </td>
 <td></td>
 </tr>
 <tr>
 <td>Mineral Mix</td>
 <td>Bio-Serv. Inc. </td>
 <td>F8598 </td>
 <td></td>
 </tr>
 <tr>
 <td>Maltose Dextrin</td>
 <td>Bio-Serv. Inc. </td>
 <td>3650 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ethyl alcohol 190 Proof, 5 gallons</td>
 <td>111000190-SS05 </td>
 <td>Pharmco-AAPER</td>
 <td></td>
 </tr>
 <tr>
 <td>Upright microscope</td>
 <td>W. NUHSBAUM, Inc</td>
 <td>LEICA DM 4000 </td>
 <td></td>
 </tr>
 </tbody>
</table>

experimental methods for testing the effects of neurotrophic peptide, adnf-9, against alcohol-induced apoptosis during pregnancy in c57bl/6 mice

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 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...

Watch this Scientific Journal Video about Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice at JoVE.com

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

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 ...

experimental-methods-for-testing-effects-neurotrophic-peptide-adnf-9

Research

JoVE Journal

Neuroscience

11.7K visualizações.  University of Toledo. O delineamento experimental proposto aqui se concentrar em estudar os efeitos da exposição ao álcool na apoptose e da aplicação de peptídeo neurotrófico durante a gravidez no cérebro fetal. Uma descrição detalhada da criação para a coleção de cérebros fetais é descrito. As técnicas para a determinação da apoptose são também descritos em detalhe.

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

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Murine Corneal Nerve Abrasion for the Induction of Neurotrophic Keratitis

To begin corneal abrasion, place an anesthetized mouse on a table. Apply a drop of artificial tear on the eye undergoing the surgery. Apply a drop of ocular gel on the contralateral eye.Next, position the mouse on its side on a heated plate. Gently use a cotton swab to absorb the artificial tear, and brush aside the mouse's eyelashes without making contact with the eye. Turn on the ocular burr, and carefully open the mouse's eyelid with two fingers.Simultaneously shield the vibrancy to prevent them from entangling in the burr. Locate the pupil or the eye center and gently apply the ocular burr to the eye's surface in circular motions until the epithelium is removed. Alternatively, perform about 20 circular movements on the cornea.Now apply a drop of fluorescent staining solution on the abraided eye. After 20 seconds, dab the eye with a tissue to absorb the solution. Then rinse it with a drop of artificial tear.Finally, absorb the artificial tear with a tissue, and illuminate the eye using a blue cobalt lamp. Prior to the surgery, the basal epithelium was intact and the nerve fibers in the sub basal plexus formed a vortex. The burr tip removed the outer corneal layer, leaving the stroma and endothelium intact with a visible gap at the basal epithelium and sub basal plexus.Regeneration of the epithelium occurred over a week post-surgery. However, the vortex remained absent from the corneal center.

Adolescent Social Stress Paradigm in C57BL&amp;#47;6 Mice for Behavioral Research

Social Defeat Stress Model for Adolescent C57BL&#47;6 Male and Female Mice

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female rodents is needed to understand its effects on ongoing brain development and behavioral outcomes. The chronic social defeat stress paradigm (CSDS) has been widely used to model social stress in adult C57BL/6 male mice by leveraging on the aggressive behavior displayed by an adult male rodent to an intruder invading its territory. An advantage of this paradigm is that it allows to categorize defeated mice into resilient and susceptible groups based on their individual differences in social behavior 24 h after the last defeat session. Implementing this model in adolescent C57BL/6 mice has been challenging because adult or adolescent mice do not typically attack early adolescent male or female mice and because adolescence is a short period of life, encompassing discreet temporal windows of vulnerability. This limitation was overcome by adapting an accelerated version of the CSDS to be used for adolescent male and female mice. This 4-day stress paradigm with 2 physical attack sessions per day uses a C57BL/6 male adult to prime the CD-1 mouse for aggressiveness such that it readily attacks the male or female adolescent mouse. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Adolescent exposure to AcSD induces social avoidance 24 h later in both males and females, but only in a subset of defeated mice. This vulnerability occurs despite the number of attacks being consistent across sessions between resilient and susceptible groups. The AcSD model is short enough to allow exposure during discrete periods within adolescence, allows the segregation of mice according to the presence or absence of social avoidance behavior, and is the first model available to study social defeat stress in adolescent C57BL/6 female mice.

Author Spotlight: Understanding Adolescent Social Adversity Effects on Neurodevelopment in Mice

We developed an accelerated social defeat stress model for adolescent C57BL/6 mice, which works in both males and females and allows exposure during discrete adolescent periods. Exposure to this model induces social avoidance, but only in a subset of defeated male and female mice.

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female ...