The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

The overall aim of this procedure is to simultaneously study changes of neurons and blood vessels in a tissue culture model of ischemic brain injury. This is accomplished by first setting up enter rhino hippocampal slice cultures. The second step is to mimic ischemic brain injury by exposing the cultures to hypoxic conditions.

The final step is to analyze and correlate neuronal survival and vascular changes. Ultimately, an in vitro approach based on organic hippocampal slice cultures is used to show the complex interactions between neurons and the vasculature under ischemic conditions. The main advantage of this technique is that after a hypoxic challenge, neuronal survival and changes of blood vessels can be studied simultaneously in a slice culture model system.

This method can help answer key questions in the neuro ischemia field, such as revealing the complex interactions between neurons and vasculature and the ischemic conditions. To begin, prepare, enter rhino hippocampal organotypic slice cultures from postnatal day form mouse pops. After removing the skull, identify the fissure between the cordal end of the cerebral hemispheres and the midbrain.

Carefully remove the parts of the brain rostral to the midbrain and place it in ice cold preparation medium consisting of supplemented MEM. Continue the dissection under a stereo microscope, keeping the brain immersed in the ice cold preparation medium. Remove remaining meninges from the cortical hemispheres.

Next, identify the hippocampus at the medial cordal surface of the hemisphere, and separate it together with the adjacent enter RH cortex from the remainder of the brain. Transfer the tissue to a tissue chopper and cut 400 micron thick slices transverse to the septal temporal axis of the hippocampus. Place the slices on cell culture inserts and incubate them in six well plates with one milliliter of incubation medium in a humidified atmosphere.

Change the medium the next day and then every other day up to one week Next, prepare the OGD medium. Add one milliliter of OGD medium to each well of two six. Well tissue culture plates perfuse the OGG medium with nitrogen for one hour in a hypoxia chamber, and before sealing and storing overnight In an incubator at 37 degrees Celsius, use anaerobic strips as hypoxic indicators.

Consider the medium to be oxygen free when the color of the strips changes from pink to white. When ready, select healthy cells by propidium iodide or PI staining. Add pi for 30 minutes to the culture medium at a concentration of two micrograms per milliliter.

View the live cultures with an inverted microscope equipped with fluorescence optics only. Slices with few labeled cells as shown here, should be selected for further study slices with many labeled cells as shown here. Should not be used for induction of OGD.

Transfer slices into OGD medium and keep for 15 minutes in the hypoxia chamber, ensure that all fluids that remain on the sides of the insert are aspirated away on the cultures of switch. From regular medium to OGD medium and back after OGD, replace the OGD medium with oxygenated serum free medium and allow cultures to recover for 3 24 or 48 hours. In serum free medium, add pi again prior to fixation for the determination of cell death.

If the study requires pharmacological treatments, begin them either during OGD or immediately after when slices are transferred to serum free. Medium for PI staining. In combination with immunohistochemistry, add the PI solution 30 minutes prior to fixation of the cultures.

After fixing the cells overnight, remove the PFA solution and wash the slices three to four times. With PBS, begin the blocking procedure by carefully detaching the slices from the culture inserts and transferring them into a well of a 96 well plate filled with blocking buffer. Keep the slices in the blocking buffer for two hours at room temperature under constant agitation by an orbital shaker.

After adding the primary antibodies of choice, incubate for 48 hours at four degrees Celsius with constant agitation after the incubation period. Wash them in PBS with 0.5%Triton X 103 to four times. Next, follow standard procedures throughout the secondary antibody before mounting the stain sections onto glass slides with cover slips.

View the stained slices either on a standard microscope with epi fluorescence equipment or with a confocal microscope. For some images, invert the fluorescence contrast in order to yield darkly stained vessels on a bright background using slices stained for laminin. Superimposed recorded images of 10 x magnification with a six by six grid overlay three such grids in the ca one, CA three DG and DC region.

And count the vessels crossing the lines of the grid to obtain results with good statistical relevance. Take measurements from at least three independent experiments each including data from three mouse puffs with three slices per mouse pup. Comparing control cultures with treated reveals that hypoxia induces neuronal death and blood vessel loss in the ca one region of the hippocampus.

In these examples, PI staining is shown in red and merged with laminin staining in green at three hours post hypoxia treatment, there is no PI staining visible and no vascular changes are evident. After 24 hours PI staining emerges and blood vessel loss is also evident. Both PI staining and blood vessel loss become more evident after 48 hours.

OGD alone induces both neuronal loss and loss of blood vessels. In ca one application of either TTX or CN QX prevented both neuronal loss as seen with PI staining and blood vessel loss revealed by laminin staining treatment with 100 micromolar A MPA for 30 minutes induced widespread neuronal loss in most areas at the hippocampus, but the loss of blood vessels remained restricted to area CA one. The quantification of blood vessels confirms the selective loss in the CA one area.

Following this procedure, drug treatments or genetic modifications can be performed to identify effective neuroprotective agents and to better understand the mechanisms of vascular remodeling. After watching this video, you should have a good understanding of how to set up interven hippocampus like cultures, and to induce OGD followed by analysis of both neuronal survival and vascular changes.