The overall goal of this experiment is to observe and quantify the therapeutic effects of water soluble mitochondrial activators on cerebellar behavior and tissue in spinal cerebellar ataxia type one mice. This method can help answer key questions in the spinal cerebellar ataxia fields such as do mitochondrial deficits contribute to disease pathology and could disease be impeded by a water soluble mitochondrial activator. The main advantage of this technique is that succinic acid is an FDA approved inexpensive water soluble compound that can be delivered directly into the brain via the drinking water.
The implications of this technique may extend to a therapy of other neurodegenerative diseases because deficits in mitochondrial oxidative phosphorylation complexes are found in multiple neurodegenerative diseases including Alzheimer's and Huntington's disease. Generally individuals new to this method will struggle with the initial set up of the behavioral paradigms which may have to be adjusted in order to achieve consistent results. Visual demonstration of this method is beneficial because the behavioral steps may be difficult to reproduce from a written method section, but are easy to replicate with the aid of a visual recording.
To begin this procedure, set the runway at a slight upward angle towards the home box, lined with bedding and reward food. Then line a sheet of white paper on the runway. Allow the mouse to acclimate to the home box for five minutes.
Next pick up a single subject and paint left and right hind feet with blue and red non-toxic water based paint respectively. Afterward place the subject at the beginning of the runway and allow it to walk to the home box. Once the subject has reached the home box, close the trap door and allow it to stay within the home box for one minute before returning to the respective home cage.
Then clean the runway and home box with 30%ethanol and replace the bedding, reward food and runway paper for the next trial. At the same time, collect the runway paper after the experiment and inspect the trial. A successful footprint trial is defined as a minimum of five sequential footprints in which the subject walks towards the gold box without turning around or stopping.
Now set up the beam apparatus according to the bar cross specifications with six different beams. At one end of the beam set up a darkened home box with reward food. To train subjects on the beam, assemble the apparatus with beam three.
Gently place the subject on the end of the beam opposite to the home box. And allow it to walk across the beam. Allow each subject up to three two minute trials to achieve a successful run.
Which is defined as crossing the beam and entering the home box within two minutes. On day four of beam training, assemble the apparatus with beam one. And set up the home box.
Place a video camera in front of the apparatus and verify that the full beam apparatus can be clearly captured on video. Allow the mouse to traverse the beam, videotaping each trial and recording the number of successful trials per subject per beam. In this step, acclimate the subjects to their lane reservoir for 10 minutes prior to the start of the trial.
During this acclimation period, prepare the rotarod apparatus cleaning each lane of the apparatus with 30%ethanol. Next, open the rotarod software. Set the start settings to four RPM, acceleration settings to five minutes and final speed settings to 40 RPM to provide a constant acceleration from four to 40 RPM over a period of five minutes.
After acclimation period, place the subjects onto the rotarod in their respective lanes while the rod is rotating at four RPM. Once all the subjects are in their respective lanes, begin the first trial. Record the latency to fall using the instrument software.
A secondary timer can be employed as a backup. Following the trial completion, allow each subject to rest in their respective lane reservoirs for 10 minutes prior to the next trial to prevent fatigue. In this procedure, attach the temperature control box tubing of a sledge microtome from the stage to a sink faucet.
Use the temperature control box to cool the microtome stage to 28 degrees Celsius. Then set the sectioning thickness dial to 50 micrometers. Next dab a dime sized portion of OTC solution on to the stage.
Before the OTC freezes, use forceps to position the hemisphere so that the mid sagittal cut surface is facedown on the OTC layer. Quickly and gently orient the tissue so that the lateral tip of the hemisphere points toward the blade. Superior to the stage.
Working in layers, add additional OTC around the tissue and allow the OTC to freeze fully before adding the next layer. Then add a thin layer of OTC on top of the tissue and freeze it. Subsequently, section the tissue with equal body weight distributed on to the cutting blade.
Collect the sections using a fine artist paint brush. And place them into an appropriately labeled well plate containing one XPBS. Between sections, reset the cutting thickness to 50 micrometers if necessary.
After sectioning, replace the PBS in each well of the plate with urea solution. And place the plate on ice. When ready, transfer the plate to a heating block set to boiling temperature to boil the sections in urea solution in order to unmask the epitopes for immunolabeling.
Between each boiling step, cool the plate containing the tissue sections on ice. After boiling, wash the sections three times, replacing the present solution with one XPBS and agitating the sections for 10 minutes. Then block the sections with donkey serum solution by incubating them in serum for one hour with gentle agitation at room temperature.
Sections can now be stained. Once labeled, mount the sections on to 2%gelatin coated slides with mounting medium to reduce photo-bleaching. Cover slip and dry overnight at four degrees Celsius.
Subsequently seal the cover slips with nail polish and store at four degrees Celsius. Using a scanning confocal microscope, locate the primary fissure of the cerebellum under the bright field light. The single most critical step of the amino assay is to accurately locate the cerebellar tissue in modules five and six A that flank the primary fissure.
Analyzing staining patterns and inconsistent lobules will ensure variability into your result. With a 10X objective, scan and sequentially excite DAPI, Alexa Fluor 488 and Alexa Fluor 594 channels. Using a 405 nanometer diode laser, a 488 nanometer argon gas laser and a 559 nanometer diode laser respectively, in conjunction with a DM 488559 nanometer dichroic beam splitter.
Then separate and collect the emitted light using SDM 490 and SDM 560 beam splitters and a BA 575675 bandpass filter respectively. Succinic acid was dissolved in home cage drinking water of SCA1 mice for four weeks beginning at 17 weeks of age. SCA1 mice begin to display the ataxia phenotype at five weeks of age.
Behavioral assessments were conducted during the treatment period as follows. Footprint assay during week 17. Beam assay during week 18 and accelerating rotarod during week 19.
At 20 weeks of age mice were sacrificed and cerebellar tissue was harvested for amino pathology assays, HPLC assays and respiration assays. Here is the representative data from the footprint assay showing a vehicle treated wild type gate, a vehicle treated SCA1 gate and a succinic acid treated SCA1 gate. Here beam analysis data shows the improvement in beam performance of SCA1 mice with succinic acid treatment.
Succinic acid treatment significantly improves the accelerating rotarod performance of SCA1 mice as shown by increased latency to fall. While attempting this procedure it's important to remember to apply the technique consistently to each subject over multiple trials. Following this procedure, other methods like mitochondrial respiration can be performed in order to answer additional questions like is respiration through individual oxidative phosphorylation complexes improved following succinic acid delivery.
After watching this video, you should have a good understanding of how to perform behavioral and neuro-pathological assays. That assess the improvement of cerebellar behavior and cytoarchitecture.
