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

The aim of this procedure is to assess the neuroprotective effects of trophic peptides in a fetal alcohol exposure model. This is achieved by first mating the experimental animals and then treating the pregnant females with trophic peptides as a second step, the fetuses are dissected to isolate fetal brains for experimental uses. Next, the fetal brains are embedded in gelatin for sectioning, followed by tunnel staining to determine cell death results were obtained.

That show prenatal alcohol exposure increases the number of tunnel positive cells in the ganglionic eminence. Compared to the control group administration of the trophic peptide, a D and F nine significantly reduced the alcohol induced increase in tunnel positive cells compared to control alcohol treated animals. So visual demonstration of this metal is critical as the fetal brain dissection and embedded steps are difficult to learn because fetal brains are very small to dissect and process for sectioning First breed C 57 black six mice by placing six week old female mice of around 20 grams into male home cages for two hours immediately following removal.

Check each female for the presence of a sperm plug to confirm that mating has occurred. If a sperm plug is present, then designate this embryonic day zero on E seven weight match pregnant females into four groups. One alcohol liquid diet group provided as free access as the sole source of nutrients.

Two Perfed control group three peptide treatment group, which should receive intraperitoneal injection of a DNF nine peptide alongside alcohol exposure. Liquid diet. Record the volume of liquid diet consumed by the animals in 24 hours from 30 milliliter graduated screw cap tubes to determine the daily amount of alcohol, liquid diet, or paired liquid diet that can be provided via a free excess as the sole source of nutrients.

Ensure that freshly prepared liquid diet is provided daily from E seven to E 13 after sacrificing the pregnant Myer E 13 by carbon dioxide euthanization, followed by cervical dislocation. Lay the carcass on the dissection board in a supine position and clean the abdomen with 70%ethanol. Use sterile scissors and forceps to make an abdominal incision.

Once the abdomen is open, hold the uterus with forceps and use a second forceps to slowly and gently tear the me endometrium away from the uterus without puncturing the embryos. Carefully remove the embryos from the uterus and transfer them into Hank's balance sort solution In a 60 millimeter by 15 milliliter cell culture dish on wet ice, place the dish and ice container on the stage of a stereo microscope using ultra fine forceps for microdissection. Pull off the skin of the embryos at the top part of the head to expose the skull.

Once the skull is exposed and clearly visualized under the stereo microscope, grasp the base of the skull at the level of the spinal cord and brain stem with forceps and begin peeling off the skull. Continue peeling off the skull piece by piece from posterior to anterior parts of the head. Once the brain is fully exposed, use then forceps to extract it.

Then dissect the fetal brain from the base of the primordial or factory bulb to the base of the Met Postfix all dissected fetal brains in 4%paraldehyde for two to three days. Fetal brains can also be frozen if testing them for western blot or enzymatic assays. After preparing a 10%solution of grade A gelatin prepared in distilled water, fill a peel away embedding mold halfway and wait until the gelatin solidifies.

Place control and prenatally treated fetal brains side by side on top of the solidified gelatin. This is to keep consistent conditions between both the control and experimental groups. Ensure that the gelatin is not too hot and add liquid gelatin on top of the fetal brains to make a complete mold.

The final mold should resemble this image, refrigerate the prepared gelatin mold that holds the fetal brains for 15 minutes. Next, peel off the embedding mold plastic. The final mold should look like this and then transfer the gelatin containing fetal brains into 4%paraldehyde for two days.

On the third day, attach gelatin block containing fixed fetal brains to the stage of the viome. Then section the brains using a viome set at 25 to 50 micron thickness for coronal sectioning. Collect the fetal brain sections in PBS, then mount the fetal brain sections on super frosts plus slides in water and allow them to dry for 15 minutes.

After 15 minutes have elapsed. Place the slides in PBS for next day testing of tunnel reaction. First, immerse the slides in a solution of 20 micrograms per milliliter proteinase K and PBS for five minutes at 37 degrees Celsius.

Then rinse three times in PBS for five minutes each on an orbital shaker. Incubate the sections with 3%hydrogen peroxide in methanol for 10 minutes at room temperature without shaking. Then wash the slides as before.

Next, incubate sections in permeable solution for two minutes on ice. Following the incubation washed twice in PBS. As before, use a laboratory tissue to dry the area of the slide around the sections.

Then use a pat pen to draw around the sections on the slide. This prevents leakage of the staining solution, thus allowing a small volume to be used. Incubate one section in each pair with tunnel reaction mixture prepared according to the manufacturer's instructions.

Add a carrier solution without tunnel reagent to the negative control section. Then incubate the slides for one hour at 37 degrees Celsius. Once the incubation time has elapsed, wash the slides three times in PBS on an orbital shaker as before.

Then dry the area around the tissue with a laboratory wipe. Next, incubate the sections with converter POD solution for 30 minutes at 37 degrees Celsius without shaking. Then rinse the sections three times for five minutes each with tris HCL.

Transfer the slides to a holder and place them in a coplin jar containing 0.05%DAB and 0.003%Hydrogen peroxide. Cover the jar with aluminum foil and place it on an orbital shaker on ice for seven minutes after the incubation washing PBS as before if desired. The slides can be stored in PBS before proceeding to missile counter staining as detailed in the text portion of the protocol.

The following images, show effects of the neurotrophic peptide, A DNF nine against alcohol-induced apoptosis using tunnel assay in the ganglionic eminence as E 13 stage prenatal alcohol exposure induced increases in cell death as indicated by the arrowheads in the A LC group compared to the PF group. A D NF nine administration alongside prenatal alcohol exposure induced a decrease in tunnel positive cells. Statistical analysis demonstrated that AD and F nine administration prevented alcohol induced increases in tunnel positive cells.

Values are shown as means plus minus standard error of the mean. A single asterisk indicates a P value of less than 0.052. Asterisk indicates a P value of less than 0.01 by Newman Coles post hoc test.

Each group contained an N of four. So after its development, this technique way for researchers and the field of brain development to explore the effects of drugs or compound and brain development in mice.