This protocol is significant because it allowed us to identify whether a patient sample contained pathogenic antibodies. The main advantage of this technique lies in its ability to differentiate reactivity between surface and intracellular antigens, that cannot easily be discriminated by other methods. The cultural hippocampal neurons are used to identify new antibodies.
The identification of these new antibodies eventually allows the initiation of a specific in monotherapies to improve patient outcomes. This technique is important because it can be extended to the whole field of the diseases related to pathogenic antibodies directed to neuronal surface proteins. Holding the embryo head with fine curved forceps, pierce the orbits with a pair of fine straight forceps at a 45 degree angle.
Then release the fine curved forceps. With the help of surgery scissors, dissect the skin and skull starting from the occipital bone to the frontal bone. Then remove the brain with a pair of fine curved forceps and collect it in the six centimeter dish with hibernate plus B27.
Once the brains from all the embryos are recovered, use the fine straight forceps to sagittally separate the telencephalons. Next, prepare a 10 centimeter dish by placing drops of hibernate plus B27 at one centimeter distances in a circle, and place one telencephalon per drop to visualize through the dissecting scope at a 1.25 X magnification. While working under the microscope, carefully remove the thalamus, and peel the meninges to better visualize the hippocampus.
Then dissect the hippocampus with the precision spring scissors. And place it in the 3.5 centimeter dish with hibernate plus B27 and istrate two. Repeat the procedure to collect all the hippocampi.
For the enzymatic dissociation of the hippocampus, add one milliliter of 2.5%trypsin in the 50 milliliter tube containing the hippocampi. After bringing the volume in the tube to five milliliter with HBSS, incubate the tube for 15 minutes in the water bath at 37 degrees Celsius. To further dilute the trypsin, add 10 milliliters of preheated HBSS, and place the tube back in the water bath, set at 37 degrees Celsius for five minutes.
Next, use a 1000 microliter micro pipette to transfer the hippocampi appearing as a mucus mass to a 50 milliliter tube. And incubate the tissue with six milliliters of preheated HBSS for five minutes, as demonstrated. To dissociate the tissue mechanically, transfer the hippocampal mass to a two milliliter tube with conical bottom.
After adding one milliliter of preheated DMEM media, homogenize the pellet with a 1000 microliter micro pipette by gently aspirating up and down. Ensuring not to create bubbles, repeat the up and down aspiration 10 to 20 times with a pre-pulled glass pipette with its tip in contact with the conical bottom of the tube until the mixture is translucent. Count the cells before distributing them evenly on a 3.5 centimeter dish with crossed shaking movements.
Once done, place the dish in the incubator with 5%carbon dioxide. For fluorescent immunostaining of 14 days in vitro cells, add the sample containing Anti-NMDAR antibodies to the cover slips and incubate at 37 degrees Celsius and 5%carbon dioxide. After one hour, treat the sample with 4%formaldehyde and PBS as a fixation solution at room temperature for five minutes.
Then wash the sample thrust with PBS for five minutes each, followed by addition of secondary antibody goat anti-human Alexa Fluor 488 at one to 1000 dilution for one hour at room temperature. Next, mount the cover slips containing the sample with a liquid mounting medium and aspirate any remaining liquid. Visualize the neurons with fluorescence imaging.
For calcium imaging, use 14 to 18 days in vitro cells that have been grown on cover slips. Prior to imaging, treat the cells with the viral vector coding for pAAV2 CAG GCaMP 5G at 2.5 times 10 to the 10th genome copies per milliliter for five to seven days. One day before imaging, incubate the cells with the patient sample for 24 hours.
On the imaging day, prepare the setup and set the microscope cell chamber to 37 degrees Celsius. Fill the lane with distilled water, infuse it with 5%carbon dioxide to maintain cell physiological conditions. After covering the cells with extracellular physiological solution, transfer them to the microscope cell chamber.
Add 10 micromolar NBQX to block the AMPA and KA receptors, and visualize the NMDA receptor response. Acquire the movie of four minutes with the frames recorded every 100 milliseconds. Shortly after starting the acquisition, add stimulation solution to the dish.
Upon stimulation, extract the fluorescent signal over time from the culture's incubated with the control and patient's cerebral spinal fluid or CSF samples using processing software such as Image J.It was observed that the cells on one day in vitro were evenly distributed and have adhered to the plate with a lamella developing around the cell body and minor neurites beginning to extend. After some days in culture, the neurites extended a short distance and the cells showed a significant polarization. The neuronal network kept growing and became complex.
At 18 days, neurons were mature, interconnected, and built the neuronal network. The representative fluorescent images show mature neurons at 18 days using selective markers. Incubation of the cultures with the patient samples produced an intense fluorescence signal as it contains the auto antibodies that recognized the NMDA receptor present on the surface of the neurons.
In contrast, the control samples produced no fluorescent signal when administered to the neuronal cultures. The application of NMDA increased the fluorescence intensity as indicated by a change in the intracellular green fluorescence. When the stimulator was applied, the neurons treated with the control CSF sample showed a higher difference in fluorescence intensity than the cells treated with the patients CSF sample.
The property identification and dissection of the hippocampus determine the purity of the cultures. Cell is aggregation is a key factor, especially when using the cultures for imaging purposes. Additionally, cultured hippocampal neurons can be used to identify novel antibodies and their target antigens when combined with immunoprecipitation and mass spectrometry techniques.
This technique has allowed the characterization of a new category of antibody mediated diseases. Therefore, it is essential in the study of the novel field of nano immunology.
