Our combined approach helps answer how ultrastructural neuroplasticity parameters change after early developmental interventions to the nascent nervous system in utero. The main advantage of our method is the acquisition of high-resolution images of macro-meso-micro-and nanostructures in the predefined coordinate system within a tissue atlas. The implication of our method is its translational therapeutic potential for congenital neurodegenerative diseases.
For example, treatment and rescue at embryonic stages using this in utero transduction technique. Our method can be applied to any other model. For example, the mouse species can be changed, as well as the area of interest.
Researchers trying our method for the first time need profound surgical training, as well as the knowledge of how to prepare the tissue for electron microscopic imaging. Begin by loading a customized capillary tip containing four times 10 to the 11 viral particles of adeno-associated virus type 1 coating for the desired target, and labeled with Fast Green dye onto an aspirator tube. Add 15 microliters of the labeled adeno-associated virus particles into the capillary.
Next, confirm a lack of response to toe pinch in an anesthetized embryonic day 14.5 pregnant mouse, and disinfect the skin. Use curved serrated iris forceps and straight tungsten carbide scissors to make a skin incision along the linea mediana. Using straight Dumont tweezers to grip the peritoneal wall, continue along the linea alba with straight Vannas scissors and place a piece of fenestrated paraffin film over the abdominal opening and use a spoon-like device to expose the uterine horns without damaging the embryos inside the horns.
After applying a few drops of 37-degree-Celsius PBS onto the uterine horns, inspect the embryos for damage or malformations inside the uterine sac. Turn the embryos carefully inside their sacs until the desired position for each injection is achieved. When the embryos are in place, inject one to two microliters of the Fast Green labeled virus particle solution into each embryo.
After all of the embryos have been injected, place the uterine horns back into the abdominal cavity, and add a few drops of 37-degree-Celsius PBS into the abdomen. Then use polyamide 6-0 sized sutures to close the peritoneal wall and polyamide 3-0 sized sutures and Halsted's Mosquito hemostatic forceps to close the skin. To embed isolated tissues of interest for tele-macro photography, place the tissue sample into a special frame with the reproducible sectioning angle, and document the coordinates.
Fill the frame with 30-degree-Celsius 3%agarose until the tissue is covered, and cover the frame with a plastic lid until the agarose has hardened. While the agarose is hardening, use the tele-macro graphic device to image the embedded tissue and its coordinates within the frame. When the agarose has solidified, transfer the agarose-embedded tissue into the frame with cutting gaps corresponding to the coordinates of the first frame.
Use a device with a thin and vibrating razor blade to cut the embedded tissue into sections of an appropriate experimental thickness. Then image each tissue section in PBS, and collect the images into a folder. To prepare the tissue samples for transmission electron microscopy, in a biosafety cabinet, wash the tissue sections with two 30-minute washes in PBS, followed by a two-hour incubation in 2%aqueous osmium tetroxide solution at room temperature.
At the end of the incubation, wash the osmicated sections two more times in PBS for 30 minutes per wash and dehydrate the sections in sequential 10-to 15-minute ethanol immersions as indicated. After the 70%ethanol immersion, place each sample into a black dish on a dull black background and image the specimens in 70%ethanol under LED RGB light applied to the samples from the left and right sides at 45-degree angles. Create an atlas of the section images with coordinates by collecting images of the samples in series in a folder.
Treat the specimens with two 30-minute 100%ethanol incubations, followed by two 30-minute 100%propylene oxide incubations, both at room temperature. While the samples are incubating, mix freshly prepared resin with propylene oxide at one-to-one and one-to-two ratios, and add 3%accelerator to both solutions. After the second propylene oxide incubation, transfer the samples into the one-to-two resin propylene oxide embedding medium solution for two hours at room temperature, followed by a two-hour incubation in the one-to-one resin propylene oxide embedding medium solution at room temperature.
At the end of the second incubation, transfer the tissues into flat polypropylene dishes, and cure the embedded tissues with fresh resin containing 3%accelerator for 12 to 24 hours at 65 to 85 degrees Celsius. Then cool the tissue to room temperature, and remove the resin-embedded specimens from the polypropylene dishes. To map an area of interest, select an image containing the area of interest from the previously prepared image atlas.
Sketch the borders of the area of interest onto the sectioned image, optically superimpose these borders onto the embedded specimen under the microscope, and use a one-inch 26 gauge needle to scratch these region borders onto the resin specimen. Then, heat the specimens to 95 degrees Celsius in order to soften the resin. Next, take out the warm resin specimens from the oven, and use a razor blade to excise the areas of interest.
Glue the specimens onto acrylic glass holding bars of the appropriate caliber, and trim the mounted specimens for semi-and ultra-thin sectioning. Use an ultramicrotome to acquire semi-thin 0.7-micrometer and ultra-thin 70-nanometer sections, collecting the semi-thin sections on glass carriers, and the ultra-thin sections on nickel grids. Stain the semi-thin sections with 1%Toluidine blue in PBS for four minutes, followed by several washes in deionized water, before examining the sections by light microscopy.
The ultra-thin sections can then be directly assessed by transmission electron microscopy at 180 kilovolts without further modification. After sectioning, the tissue slices are imaged by tele-macro photography as just demonstrated. After osmication and ethanol immersion, the sections are imaged again by interference light tele-macrography, revealing a specifically colored pattern for each tissue surface.
After additional dehydration and resin embedding, the areas of interest are selected and further processed for transmission electron microscopy. In the hippocampus and cerebellum, mossy fiber synapses are known to exhibit unique ultrastructural characteristics when compared to other synapses, including large presynaptic boutons. In their cross-section profiles, the boutons contain a vast number of vesicles and mitochondria, and very often enclose several dendritic spines.
In the spinal cord, the dorsal funiculus contains many axonal fibers that are myelinated by oligodendroglia. On transversal sections, the dorsal funiculus can be found between both posterior horns of the spinal cord. On transmission electron microscopy images, cross-sectioned myelinated axons appear round, and the myelin sheaths surrounding the axons are organized in the lamellae.
The prepared atlas and the micrographic transmission electron microscopy images of the ultrastructural parameters are not only helpful for comparison of the morphological neuroplasticity of different sections under various treatment conditions, but also for comparisons between parameters within the same area. Remember to thoroughly plan and prepare before starting the in utero transduction, and to be sure to have backup of materials and instruments in case of complications. Following our method, other techniques can be added such as immunogold labeling prior to electron microscopy for ultrastructural tracing of a defined molecule of interest.
Starting with a holistic view of the entire organism and zooming in towards the molecular topography, we are able to study and manipulate the relationship between tissue function and morphology. Osmium tetroxide is hazardous. Be sure to always work under the hood, and to wear protective glasses and hand-gloves when working with this reagent.
