The overall goal of this procedure is to generate organotypic hippocampal slice cultures that can be used to model neuronal injury in response to a variety of stimuli, including NMDA, excitotoxicity oxygen, glucose deprivation, or inflammatory stimuli such as lipopolysaccharide or amyloid beta peptide. This is accomplished by first isolating the postnatal brain and slicing each hemisphere to generate coronal sucs. The second step of the procedure is to isolate the hippocampal regions and culture them on membrane inserts in six well plates After culturing the sections, the sections are stained with propidium iodide and the level of spontaneous neuronal death is calculated.
Finally, the slices are treated with a neurotoxic agent and restain to measure the toxicity response to the treatment. Ultimately, results can be obtained that show the percent neuronal death in the CA one, CA three, and dentate regions of the hippocampus across experimental conditions through quantification of propidium iodide fluorescence using immunofluorescence microscopy. The main advantage of least technique over existing miser, like a primary neuronal culture, is the mechanism of neuronal injury is examined in, in which preparation in which the basic architecture of the hippocampus is relatively intact.
And the third type include exercise. Microglia and ROY are preserved Before beginning the dissection. Insert a millipore permeable membrane into each.
Well add one milliliter of culture medium to each well of several. Six well tissue culture plates. Warm the plates in an incubator at 37 degrees Celsius.
Briefly dip the decapitated head of a day, seven rat pup in 70%ethanol. To disinfect it used micro dissecting scissors to make a sagittal incision through the skull to expose the brain. Use forceps to quickly remove the brain and transfer it to a 60 millimeter dish of dissecting medium on ice.
Use forceps to separate the brain into two hemispheres. Place each hemisphere medial side down on a dry cover sponge for two to three seconds to collect any excess moisture. Transfer the brain to a square of a clar film and place it on the platform of a tissue chopper slice coronal in 350 micron sections beginning at the rosland.
And continuing coddly. Gently lift the A clar square and submerge the slices in a fresh dish of ice cold dissecting medium. Under a dissecting microscope, identify the hippocampus, a CS shaped structure lying below the cerebral cortex, coddled to the striatum.
Use forceps to separate out the hippocampus from each coronal section. Use a Pasteur pipette modified as described in the text to transfer five hippocampal slices to each membrane. Insert in the prepared warm six well plates.
Remove any excess medium on the surface of the membrane. Incubate the plates at 37 degrees Celsius and 5%carbon dioxide for 12 to 14 days. Changing the medium as detailed in the accompanying text after 12 to 14 days in culture, exchange the medium and add five micrograms per milliliter per pum iodide, abbreviated pi to allow visualization and quantification of basal cell death.
Incubate the slices overnight at 37 degrees Celsius and 5%carbon dioxide under an inverted fluorescence microscope connected to A CCD camera Capture images of the slice at 40 x magnification this time. Point captures PI fluorescence corresponding to basal levels of cell death at time equals zero hours or T zero immediately after imaging. The slices induce neuronal injury with the preferred method for the current experiment.
Here slices are incubated with fresh medium containing 10 micromolar NMDA for one hour at 37 degrees Celsius and 5%carbon dioxide. After neurotoxic treatment. Replace the medium with fresh culture medium containing five micrograms per milliliter pi.
Incubate the slices overnight at 37 degrees Celsius and 5%carbon dioxide image of the PI fluorescence after neurotoxic treatment. The mean fluorescence measurements for this time point represent levels of cell death. After 24 hours of experimental injury, or T 24 hours, immediately replace the medium with fresh culture medium containing 10 micromolar NMDA and five micrograms per milliliter.
PI incubate slices overnight at 37 degrees Celsius and 5%carbon dioxide to achieve maximum neuronal death. Acquire the final set of images of PI fluorescence, the mean fluorescence measurements for this time point. Represent maximal levels of cell death or Tmax.
Quantify PI fluorescence in the region of interest using image analysis software as described in the accompanying text. This is a representative image of PI staining of basal neuronal death in the ca one region of the hippocampus. Here is a representative image of PI stained neuronal death.
24 hours after a one hour treatment of 10 micromolar NMDA, notice the more intense staining indicating a greater degree of cell death. This image shows maximal PI staining after an overnight incubation with 10 micromolar NMDA. After watching this video, you should have a good understanding how to generate organic hippocampus slice and the measuring level of a neuronal injury.
