The overall goal of the following experiment is to observe the roles of secreted signals in the embryonic CSF on the development of the cerebral cortex. This is achieved by first isolating embryonic CSF to use as a culture medium. In the next steps, the developing cerebral cortical wall is dissected, placed on a polycarbonate membrane and floated on CSF.
The results show that the embryonic CSF provides instructive signals to the developing cerebral cortex based on the analysis of explants by immunos staining. This method can help answer key questions in the CSF field. For instance, what are the components of the CSF, what is its function at different points of development, and hopefully help determine how one day it may be used as a therapeutic vehicle for neurodegenerative diseases.
It is critical that CSF is collected without contamination and that damage to cortical explan is minimized. These procedures may require considerable practice because they involve manipulating small tissues with very fine dissection tools. Begin by preparing the micro pipette needles.
For example, on a Orica PC 10 vertical micro pipette puller pull 10 to 20 microliter micro capillary pipettes with the following settings, A one step pull heater Number two, set to 58 and a 100 gram pull weight. Snap the fine tip of the micro pipette using fine number 55 forceps. The average inner diameter of the resulting needle will be 85 micrometers.
Next, prepare the aspirator assembly. For the CSF aspiration, insert the micro plunger provided with the micro capillary pipettes into the capillary needle. Alternatively, attach a plastic disposable filter to the end of the aspirator tube assembly that is connected to the micro capillary pipette.
Next, on the opposite end of the aspirator assembly, push the needle through the gasket and into position. Now transfer an isolated mouse or rat embryo to a microdissection dish prepared with syl guard. Wash the embryo with sterile HBSS and remove the fluid surrounding the embryo by aspiration and padding with absorbent paper.
Visualize the embryo under a dissection microscope. If this is a mouse embryo, it should be laying on its side, providing a sagittal view. Now steadily insert the micro capillary pipette into the lateral or cephalic ventricle, mesocephalic ventricle or CI sternum.
Magna attempting not to contact the neuro epithelial cells. Once the pipette has been inserted, only insert the needle until the tissue is punctured. Now, carefully and gently begin aspirating the CSF into the pipette using gentle negative pressure created by the micro plunger or mouth suction.
The CSF should start to gently flow into the micro capillary pipette in a slow and controlled fashion. Keep applying negative pressure until the collection is complete. It is critical that the collection yields a clear, transparent fluid.
If there is any evidence of contamination from blood, such as red or yellow, tch to the solution, discard the collection in both mouse and rat embryos. At the E 16.5 to E 17 stage or earlier, it is possible to observe the ventricle collapse slightly, where the micro capillary pipette is positioned. The CSF flow will halt with this event when the flow ends, gently remove the micro capillary pipette.
Next, gently expel the collected CSF sample into a micro centrifuge tube chilled on ice from an E 14.5 mouse litter. Expect to collect between 10 to 15 microliters of CSF when all the collections are complete. Centrifuge the tube at 10, 000 Gs and four degrees Celsius for 10 minutes.
To remove any contaminating cells, transfer the clear CSF to a new tube. Microscopic examination for contaminants can now be performed if deemed pure. The sample can be used for analysis, pooled with other samples or snap frozen with liquid nitrogen and stored at minus 80 degrees Celsius To collect cortical explants for experimentation with CSF supplemented media.
In a cigar coated dish, expose the cortex of an E 14.5 embryo. Remove the duro with the developing skull, but leave the pia and arachnoid attached to the cortex using a scalpel. Bisect the brain along the midline in the midsagittal plane, separating the right and left cortical hemispheres.
Prepare each cortical hemisphere separately. Place the hemisphere medial side up so that you can see the ganglionic imminence and the developing cortex. Using the scalpel, make a coronal incision through the cortical neuro epithelium coddle to the developing olfactory bulb.
This incision should begin at the anterior boundary of the lateral and medial ganglionic eminences, and extend through the anterior cingulate region of the developing cortical rudiment. Make another coronal incision just coddled to the posterior boundary of the lateral ganglion eminence. This incision should extend from the lateral boundary of the ganglion eminence to the medial wall of the developing cortical rudiment.
Use the scalpel to retract the cortical flap created by the two incisions. This limits the risk of damaging the cortex. Now dissect away the developing hippocampus and cortical hem of the medial cephalon.
Do this from the apex of the neocortex where the lateral cortical surface meets the interhemispheric wall. Next, make a transverse incision along the boundary separating the lateral ganglionic eminence from the developing cortex. Remove any extra dissected tissue off the plate.
The dissected cortex is now prepared with the meningeal side facing down. To transfer the cortex, prepare a platinum wire loop connected to a glass pipette using a buns and burner melts the end of the pipette onto the platinum wire. Once assembled, sterilize the platinum wire loop and allow it to cool.
Remove the excess media from around the X explan with gentle aspiration. Leave a small amount of solution to use with the wire loop. Next place a one centimeter polycarbonate membrane in a four centimeter diameter imaging dish.
Now using the side of the platinum wire loop, gently flip the cortex so that the meningeal side is facing up. Then place the X explan in the middle of the loop and using intrinsic hydrostatic forces, lift the cortical X explan from the dish. Then gently place the explan on the polycarbonate membrane and lift the platinum wire loop away.
Next, load a pipette with 50 microliters of CSF or other media. Gently lift the membrane and detect the media below. Hello, cover the imaging dish.
Place it in a humidified secondary container and incubate at 37 degrees Celsius with 5%carbon dioxide for 24 hours. To support continued s proliferation and explan growth, CSF collection volumes from average litter sizes have been calculated for both mice and rats from various embryonic ages. When pure CSF samples have been collected, the CSF can be analyzed using a number of different techniques such as a silver stain of a few micrograms.
This CSF is from an E 14.5 mouse. 24 hour old explants proliferated with 100%CSF have tissue histology similar to rodent embryos at the same gestational age as shown by immuno staining. pH three.
A marker of cell division is expressed along the ventricular surface. B-R-D-U-A marker of DNA synthesis also shows incorporation along the ventricular zone. And T uj one, a neuronal marker is expressed in the developing cortical plate.
Before attempting these procedures, it's important to check your institutional policies regarding the techniques that we have demonstrated. This technique paved the way for researchers to study the secreted signals within the CSF in instructing the role of the cerebral cortex during development in vitro.
