Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia. The goal of the work presented in this video is to examine the effect of cold preconditioning on rat neural immune signaling as a means to identify novel targets for therapeutics to protect the brain before injury onset. This is achieved by producing healthy and long-lived adult rat hippocampal slice cultures.

The cultures are exposed to cold preconditioning to induce immune-based neuroprotection. Then anci toxin is added and the slice cultures are imaged. Image analysis suggests that cold preconditioning leads to a dramatic reduction in the level of selective neuronal loss.

My name is Richard Craig, and this is Heidi Mitchell. We're from the University of Chicago in the Department of Neurology, and today we're going to describe for you how the slice culture preparation can be used to answer questions about neuroprotection involving low level pro-inflammatory immune signaling as a means to make the brain stronger against brain disease. Generally speaking, individuals new to this procedure struggle because of the need to ensure long-term viability and healthy cultures that are capable of showing low level pro-inflammatory signaling.

Heidi will now show you a number of procedures that we use in the laboratory to overcome these hurdles so that low level pro-inflammatory signaling can be measured as a means to make neuroprotectants for brain. The following experiments should be performed wearing a clean lab coat and sterile gloves stretched over the lab.Coat. Sleeves Begin by placing all items needed for the procedure in a BSL one lamina flow hood, including a mcil wane tissue chopper related Teflon inserts, a razor blade, surgical tools, and dissection petri dishes.

The hood should be located in a HIPAA filtered positive pressure culture room with the air purified by self-contained ultraviolet light air cleansing fans at least 30 minutes prior to starting the experiments. Place a dissection cold plate in the hood and connect it to a nearby water bath in the cell culture hood. Place a milli cell insert into each well of a six Well plate add 1.1 milliliters of culture medium to each.

Well place the tray in an incubator at 36 degrees Celsius, 5%carbon dioxide, and 95%humidity for at least 20 minutes. Keep rat pups warm until they are needed by placing them under a heat lamp anesthetize pup by placing it in 100%carbon dioxide. Once anesthetized, dip the pup in 100%ethanol, then decapitate it and place the body in one 100 millimeter Petri dish and the head in another.

Next, use Curved iris scissors to open the skull with a midline incision followed by two lateral cuts to the left and right behind the eyes. Use angled forceps to peel back the overlying skull And a spatula to begin scooping out the brain. Remove the brain by cutting the trigeminal Nerves with the spatula and place the brain into the bottom half of a 60 millimeter Petri dish containing 10 milliliters of sterile cold gaze balanced salt solution supplemented with the Glucose.

Next, use the number five IOx forceps under a number two iris spatula to dissect out the hippo campi from each hemisphere and place them onto a Teflon disc for the McIlwain chopper. Using the spatula, spread the medium away from the brain tissue using the chopper with a section thickness set at 350 to 400 micrometers. Section the hippo campi perpendicular to their long axis with a one milliliter perpet briskly.

Wash the fresh cut slices off of the Teflon inserts and into the top half of a 60 millimeter Petri dish containing cold gaze BSS. Place the slices under a stereo microscope and examine them. Identify any slices that have an intact dentate gyrus and a parametal cell layer.

Next gently place the largest three slices onto an insert, preparing a total of 12 slices per pop. Place the inserts into a six world tray and incubate at 36 degrees Celsius every three to four days. Refresh the growth medium by replacing the old medium with fresh, warm, medium.

Return the cells to the 36 degree incubator after seven days. Replace the medium with 1.1 milliliters of serum free supplemented neuro Basal medium. On day 18, examine the Vitality of the slice cultures.

Begin by placing 1.1 milliliters of ox poel in six well culture dishes. Place the dish in the incubator to warm it up to 36 degrees Celsius. Meanwhile, turn on a fluorescent light source with a fitzy filter for the microscope and allow it to warm up for at least 20 minutes.

This will ensure uniform light intensity. Next, use forceps to place the slice culture inserts inox media for 10 minutes. Then transfer them back to the SIM free medium.

Place the culture insert on the microscope stage and mark the orientation to screen for irreversible. CA one parametal layer injury. Place the cultures on an aseptic surface of an inverted microscope under five x magnification with slices maintained at an orientation similar to that used for pre-screen.

Switch to the FIT CX citation emission cube. Select cultures that have fewer than 30 cytx positive cells in the ca one area For Cold preconditioning. Place 1.1 milliliters of serum free medium in a six well culture dish and place the dish in a 30 degree incubator with 5%carbon dioxide and 95%humidity for at least 20 minutes to equilibriate the medium after 20 minutes have passed.

Remove the equilibrating medium from the incubator. Transfer the culture inserts with the slices from the normal serum free medium to the 30 degree medium. Then place them in the 30 degree incubator for 90 minutes following the incubation.

Return the slice cultures back to the normal serum free medium and incubate 36 degrees For 24 hours. Begin the pre-screen for slice culture experimental use by transferring the culture inserts to cyt media for 20 minutes. While the slicers are incubating, turn on the ultraviolet lamp and a CCD camera and allow them to warm up for 20 minutes.

If needed, use the software to clear the CCD by exposing it to light for 50 cycles. Then pepeta solution of 10, microliters of fluorescein in 90 microliters of PBS onto a 100 micrometer deep hemo cytometer and place it on the microscope stage. Using the software, adjust the exposure time so that the dynamic range is 1000 outta 4, 096.

Next place the dish with cold precondition slice cultures on the microscope and acquire images. These will be used to determine the background fluorescence level, which will be subtracted from the fluorescence emitted from injured slices. If the cold preconditioning itself caused injury cells with increased fluorescence will be seen next.

To examine the effect of ExoToxic injury, transfer the inserts into serum free medium containing 20 to 50 micromolar and MDA that had been equated to 36 degrees Celsius for at least 20 minutes. Incubate for 60 minutes at 36 degrees Celsius, NMDA mimics the effects of the neurotransmitter glutamate following the incubation. Rinse the NMDA off the inserts by dipping each insert three times in three separate 60 millimeter dishes.

Each containing 10 milliliters of neuro basal medium warm to 36 degrees Celsius. Do not use more than three inserts for each set of 360 millimeter wash dishes. After washing, place the inserts into a tray containing fresh media and return them to the 36 degree incubator.

After 24 hours, calibrate the camera with a fluorescein standard as before. Then place the cultures in OX media for 20 minutes images before. If injury levels are low one day post-injury, extend the experiment to two to three days as needed to quantify the injury using metamorph software.

Define an area of interest around the CA one region to measure the intensity of a selected region for injury. Copy and paste the area of interest from injury to background image. Next, enter values from injury and background into excel Using statistical software.

Quantify the numerator minus background for control and cold preconditioned cultures run the appropriate statistical test to compare injury in the experimental group versus a control group, which should always be included with each experimental Run. This image shows a typical mature hippocampal slice culture stain after 21 days of in vitro culture. New end labeling shown in green is used to illustrate the cyto architecture of the principle neurons.

Parametal neurons are shown in areas ca one and ca three of the hippocampus and dentate gyrus or DG neurons are seen on the left here. The CA one area shown at higher power illustrates the branched quality of quiescent microglia. Within mature slice cultures, cells were labeled with the microglia surface marker CD 11 B.This figure shows the effects of cold preconditioning, neuroprotection, and hippocampal slice cultures incubated with cytx green marker for dead cells.

The prescreen images on the left show no ca one injury here. Sham control cultures are shown at the top and cold preconditioned cultures are shown at the bottom. The middle row images show relative slice culture injury 24 hours after exposure to 20 micromolar NMDA.

Notice that sham control injury is greater than that of cultures exposed to cold preconditioning labeled cp. The relative injury color calibration scale is shown in the upper left image. Traditionally, cultures are then exposed to a maximal level of injury, such as 20 micromolar and MDA overnight to maximize CA one neuronal injury levels and relative injury of cold preconditioning versus sham noted as a ratio of injury to maximal injury.

However, exposure to maximal injury stimuli may not be sufficient to overcome neuroprotection from preconditioning. Accordingly, use of ratios of injury to maximal injury may not accurately reflect neuroprotection from preconditioning. This is evident in the right hand images that show that cold preconditioning maximal levels are less than those of sham controls.

In this study, it was found that the use of injury to maximal injury ratios could obscure neuroprotection from cold preconditioning. This can be seen from the schematic where sham injury is shown in red and that after cold preconditioning in blue one day after NMDA exposure, cold preconditioning, the level of injury was determined to be at three compared to a level five, which was seen in the sham control. This is consistent with 40%neuroprotection.

However, if a traditional format using ratios such as injury to maximal injury is used, no protection is evident. In other words, 50%for sham versus 50%for cold preconditioning. Quantitative PCR was performed using mRNA samples as shown here.

Levels of TNF alpha after cold preconditioning represented by the blue line were higher than controls, which are represented by the green, yellow, and purple lines. This suggests that cold preconditioning triggers increased production of TNF alpha in the brain to confirm quantitative PCR and quantitative PCR array results immuno staining was performed. This slice culture was processed for interleukin 11 shown in red, A new end to show neurons shown in green.

Notice that some parametal neurons, which are indicated by the arrows show interleukin 11 and new end staining and are shown in yellow. The arrows point to a few smaller cells that are presumed to be astrocytes and show only increase interleukin 11 staining. In delineating The cytokine signaling system involved in cold preconditioning neuroprotection, it is important to note a few fundamentally important concepts.

Although cytokines are at extremely low concentrations in the normal brain, small changes in cytokine concentrations alter gene expression. Thus, even at low concentrations, cytokines have an immense potential to alter brain structure and function and therefore the phenotype of an organism. This concept is represented here by the inverse of distance, beginning with a cat whisker as a reference point and sodium potassium pH and calcium compared to TNF alpha.

It is also important to keep in mind that cytokines are highly redundant and plyo tropic multiple cytokines can have similar effects and a single cytokine can have variable effects. Thus, to accurately establish the innate cytokine basis for neuroprotection from cold preconditioning, composite analysis of related signaling variables must be determined. This is accomplished via multiplex assay strategies, which will establish the cytokine signature of cold preconditioning neuroprotection Once mastered, these cultures can be prepared in approximately three hours and manipulations to study.

The response of cold preconditioning can be performed over approximately three days while performing these procedures, it is essential to remember that sterile techniques and minimal handling is necessary to preserve the ability to detect low level pro-inflammatory signaling following these procedures. Other methods such as proteomic and molecular biological analyses can be applied to the cultures to decipher the means by which cold preconditioning has its nutritive and protective effect on brain.