Neurodenegration in many neurological disorders is a chronic process of which neuronal fiber loss often occurs prior to the cell body loss. The stereological quantification of neuronal fibers in three dimensions provides a sensitive and accurate method for detecting early neurodegenerative change. This technology utilizes the reliable and efficient computer-based unbiased fractionation of space balls to measure the lengths of line-like structures such as neuronal fibers in 3D.
Demonstrating the procedure will be done by Prabhakar Singh, a post-doc and our expert in stereology, and David Peng, the research assistant in my lab. After confirming a lack of response to pedal reflex in the anesthetized mouse, transcardially perfuse with approximately 50 milliliters of ice cold 0.1 molar DPBS, followed by approximately 25 milliliters of 4%PFA in 0.1 molar PBS. At the end of the perfusion, post-fix the brain in a container of fresh 4%PFA for 24 hours at four degrees Celsius.
The next day, wash the brain with three one to two-minute washes in fresh DPBS per wash and transfer the brain into a 15%sucrose in 0.1 DPBS solution overnight. The next morning, place the brain into a 30%sucrose solution and 0.1 molar DPBS for 48 hours at four degrees Celsius. At the end of the incubation, transfer the brain to a cryotome chamber pre-set to minus 20 degrees Celsius.
Brains can be kept in sealed tubes and stored at minus 80 degrees Celsius. For sectioning, embed the tissue sample in optimum cutting temperature embedding medium and mount the sample on a specimen disc. Then acquire serial 30 micrometer thick sections in a coronal plane in the cryotome, transferring each section into individual wells of a 24-well culture plate containing cryoprotectant solution in preparation for minus 20 degrees Celsius storage.
To identify the first and last sections of each brain, compare the morphological features with a standard mouse brain atlas. NBM begins at bregma minus 0.0 millimeters and ends at minus 1.6 millimeters. The selection of every eighth section yields a total of six to seven sections and allows an appropriate coefficient of error for both a volume and fiber length estimation.
For immunohistochemical labeling of the tissue sections, after blocking any nonspecific binding with 10%normal bovine serum in 0.1%Triton X-100 in Tris-buffered saline, label the sections with the primary antibody of interest for 48 hours at four degrees Celsius. At the end of the incubation, wash the sections three times for three minutes in fresh Tris-buffered saline per wash, followed by incubation with an appropriate biotinylated secondary antibody for one hour at room temperature. At the end of the incubation, wash the sections three times in fresh Tris-buffered saline as demonstrated, followed by staining with Avidin biotin peroxidase complex and DAB peroxidase substrate solution according to the manufacturer's protocols.
After washing, mount all of the sections from one brain tissue sample onto one gelatinized slide and allow the samples to air dry. To dehydrate the sections, immerse the samples two times for five minutes per dilution in sequential ascending ethanol solutions as indicated, followed by clearing with two 10-minute xylene washes. Then use an appropriate mounting medium to place coverslips onto the slides.
For stereology, open a new study in the software. A study initialization dialogue box will open. Fill out the study information and confirm that multi-level fraction-based is selected.
Double-click on volume to open the volume dialogue box. Name the feature of interest and select the region point counting probe and click next. Double-click on length and provide a name of the feature.
Select sphere probe and click next. In the case initialization box, to code the groups before starting the study, set the total number of sections starting from the first section containing the area of interest to the last section containing the area of interest and the section sampling interval to eight. Click next to open the probe initialization dialogue box which will automatically set the lowest magnification for the region selection and volume estimation and confirm the settings.
Click preview to check if the region of interest can be identified at the selected lower magnification. Enter 50, 000 micrometers cube per point for the region volume fraction and click preview and done. Under object high magnification, set the length to 63X and double-click length to open the length sphere dialogue box.
Set the diameter of this sphere to 10 micrometers and click done. Set appropriate values for the frame area, frame height, guard zone, and frame spacing. Then click next and follow the instructions provided by the software.
To insert the first section, place the slide onto the stage of the microscope under a 5X objective and click the green arrow to draw an arbitrary boundary around the NBM to define the region of interest. Click the green arrow and done to change the 63X objective to obtain the fiber measurements of the current fraction. The section thickness dialogue box will appear.
Set the top and bottom surface of the section to measure the actual thickness of the section using the manual z-axis focus knob and click done. Move the z-axis slowly from top to bottom in the frame height of the section to mark all of the intersecting fibers on the surface of the virtual sphere probe. When all of the fibers have been marked, click next to advance to the next location.
When all of the fractions have been completed, click the green arrow. The software will ask to have the next section inserted. Bring the next sample on the slide into focus using the 5X objective and repeat the fiber measurement as just demonstrated.
When all of the sections have been measured, the software will generate a result for the case showing the coefficient of error values. If the coefficient of error is acceptable, proceed to the next case. If the coefficient of error is not acceptable, the software will provide recommendations to change some of the parameters.
When all of the cases have been completed, generate results for each case and group. In this representative experiment, the Swedish mutant beta amyloid precursor protein group demonstrated a significantly lower fiber length and fiber length density compared to their wild-type litter mates. The results also showed that there was no significant difference in the volume of the NBM between the two groups analyzed.
This technique requires proper the acquisition in the correct orientation and a good staining method with the incubation period. The stereology protocol can be used for the estimation of any linear profiles such as other neuronal fibers, astrocyte processes, or even vascular profiles.
