Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Podsumowanie

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury. This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Streszczenie

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or 'neuronal-like' cell culture systems are commonly utilized. While these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Wprowadzenie

The most common form of stroke is ischemic stroke, which occurs when a cerebral blood vessel becomes occluded. The tissue ischemia which results from cessation of blood flow causes widespread depolarization of membranes, release of excitatory neurotransmitters, and sustained elevation of intracellular calcium, which leads to the activation of cell death pathways ¹. This process has been termed 'excitotoxicity', and is a common pathway involved in neuronal death produced by a variety of pathologies, including stroke ². Inhibition of the signaling pathways involved in excitotoxicity and other neuronal cell death cascades is an appealing approach to limit neuronal damage following stroke.

Identifying the precise molecular mechanisms involved in excitotoxicity and ischemic stroke can be difficult when using whole animal systems. As such, primary embryonic and 'neuronal-like' (e.g. neuroblastoma and adenocarcinoma immortalized lines) cell culture systems are often used. The main advantages of these models are that they are easy to manipulate, relatively cost-effective, and cell death can be readily measured and quantitated. However, signaling pathways can be altered by the culturing conditions used ^3,4, and immature neurons and immortalized lines can express different receptors and signaling molecules when compared to mature brain ^5-8. Furthermore, cultured neurons only allow the examination of one cell type (or two, if a coculture system is used), whereas intact brain tissue is heterogeneous, containing a variety of cell types that interact with each other. Organotypic slice culture systems (thin explants of brain tissue) are also used, and these models allow the study of heterogeneous populations of cells as they are found in vivo. However, only a limited amount of tissue can be obtained from each animal when using this technique, slices cannot be cultured for as long as immortalized cell lines, and medium to long-term culture can result in alterations in signaling pathways and receptors in the slices. Whilst mature brain can be used to generate organotypic slices, slices from immature brain are more amenable to culture, and are more commonly utilized. There is therefore the need to develop models which mimic or represent intact mature brain, that are easy to use, in which neuronal signaling pathways can be examined.

Herein, an in vitro technique involving intact nervous tissue that can be used to elucidate molecular mechanisms involved in cell death following an excitotoxic or ischemic insult is described. This technique reduces the number of animals required to perform an experiment, is reproducible, and generates viable tissue that behaves in a metabolically similar fashion to larger organotypic slices. Additionally, the neuronal circuitry, cellular interactions, and postsynaptic compartment remains partly intact. The physiological buffer used allows the cell membranes to 'reseal', and enables cells to recover their original membrane resistance ⁹. This brain slice model is able to faithfully mimic responses observed following excitotoxicity mediated brain injuries ¹⁰, and can be used to examine the molecular mechanisms involved in stroke.

Protokół

All procedures are performed with approval from the University of Newcastle Animal Care and Ethics Committee, as well as in accordance with the relevant guidelines and regulations, including the NSW Animal Research Act, the NSW Animal Research Regulation, and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

1. Dissection of Brain Tissue

1. Sacrifice twelve week old male rats by decapitation. Rats can either be sacrificed by stunning and decapitation, or can be first anesthetized with isoflurane (5% induction, 1.5-2% maintenance) in 70% N₂ and 30% O₂, and then decapitated.
2. Remove the brain from the skull as rapidly as possible (ideally, this should be performed in less than 2 min).
3. Remove the cortex by free-hand dissection. The dissection should be performed in the minimum time possible, with as little damage to the brain as possible so as to maintain the integrity of the slices.
4. In order to remove any residual skin, fur, blood, etc., immerse the brain in a small amount (3-5 ml) of 37 °C Krebs buffer (118 mM sodium chloride, 4.7 mM potassium chloride, 1.3 mM calcium chloride, 1.2 mM potassium dihydrogen phosphate, 1.2 mM magnesium sulfate, 25 mM sodium hydrogen carbonate, 11.7 mM glucose, 0.03 mM disodium ethylenediaminetetraacetic acid (EDTA), equilibrated with 95% oxygen/5% carbon dioxide (carbogen), pH 7.4).

2. Preparation of Microslices

1. Place the brain on the stage of a McIlwain chopper on which two slices of filter paper have been placed and moistened with warm Krebs buffer. Keep the brain and filter paper moistened with warm Krebs buffer, but not so wet that there is free liquid on the surface of the paper that can cause the brain to slide around.
2. Slice the brain into 250 µm coronal sections. As nutrients, oxygen, and waste materials that are normally exchanged with the blood supply are exchanged with a fluid which surrounds the tissue in isolated neural tissue, the size of the slices should not be more than 400 µm in thickness to allow adequate oxygenation and nutrient exchange to occur.
3. Turn the McIlwain stage 90°.
4. Slice brains into 250 µm sagittal slices. Herein, these brain sections (250 µm x 250 µm slices of variable length depending on the thickness of the tissue) are referred to as microslices.
5. Place the microslices into a round bottom tube with 10-15 ml 37 °C Krebs buffer (the amount of Krebs buffer used will depend on the amount of tissue).

3. Equilibration of Microslices

1. Gently agitate until individual microslices are suspended, and then allow them to settle under gravity.
2. Carefully remove the supernatant, which will be cloudy due to suspended cell debris, and add 10 ml fresh 37 °C Krebs buffer to the tube.
3. Repeat this washing procedure four times, which will result in the removal of the majority of debris.
4. Following this, equilibrate the microslices at 37 °C with gentle shaking/inversion, and change the Krebs buffer every 15 min for 1 hr in total.
5. At this time, add 2 ml fresh 37 °C Krebs buffer, and transfer 15-20 microslices (150 µl of microslice suspension) to flat bottom polystyrene 5 ml tubes using an extra wide bore pipette tip with smoothed edges (which can be created by cutting ~2 mm from the end of a P1000 tip). Spread the microslices evenly over the base of the tube in a single layer, so that each microslice is not further than a few millimeters from the oxygenated atmosphere.

4. Excitotoxic Stimulation

1. Incubate the microslices at 37 °C, and continually pass carbogen over the surface of the 150 µl microslice suspension, using a gas line that is positioned just above the surface of the microslice suspension (to avoid mechanical disruption).
2. Treat microslices with either 150 µl 5 mM glutamate + 100 µM glycine in Krebs buffer (excitotoxic stimulus: final concentration 2.5 mM glutamate + 50 µM glycine) or 150 µl Krebs buffer alone (control nonstimulated). A variety of stimuli can be used at this step (including, but not limited to, AMPA, NMDA + glycine, KCl depolarization, and oxygen/glucose deprivation).
3. Remove the incubation solution at various times post-stimulation (0, 30, 60, 90, 120, and 300 sec), and replace it with 300 µl of ice-cold homogenization buffer (30 mM Tris, pH 7.4, 1 mM ethylene glycol tetraacetic acid, 4 mM EDTA, 100 µM ammonium molybdate, 5 mM sodium pyrophosphate, 25 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonylfluoride, complete protease inhibitor cocktail).
4. Homogenize microslices on ice, using a glass-Teflon Dounce Homogenizer (20 strokes; 700 rpm).
5. Store homogenates at -80 °C, and various signaling molecules, death molecules, etc. can be measured by western blot.

Wyniki

Microslices generated using this procedure are viable, and a variety of species (for example, rat, mouse, and chicken) can be used to produce microslices. Three independent measures of viability have been utilized: respiration rate (Figure 1), adenine nucleotide ratios (Figure 2), and tissue potassium content (Figure 3). Using these measures, it has been demonstrated that microslices remain viable for at least 2 hr post-generation.

Brain micro...

Dyskusje

Herein, an in vitro technique for the generation of microslices that can be used to examine the molecular mechanisms involved in excitotoxicity and ischemia-mediated cell death in intact mature brain tissue is described. This technique produces viable tissue (Figures 1-3), that is metabolically similar to larger organotypic slices¹⁵. Furthermore, this microslice model closely corresponds to the response observed following excitotoxicity mediated brain injuries in vivo^10<...

Materiały

Name	Company	Catalog Number	Comments
Guillotine			Used to decapitate animal
Surgical equipment			Forceps, scissors, tweezers, etc., for brain removal and dissection
McIlwain chopper	McIlwain choppers are manufactured/distributed by a range of companies including Mickle Engineering, Harvard Apparatus, Campden Instruments and Ted Pella.		Used to generate 100-400 µm brain sections.
Round bottom plastic tubes	Greiner
Water bath			For keeping tissue at 37 °C
Humidifier/aerating apparatus			Used to keep microslices in a humidified, oxygenated environment
Flat bottomed polystyrene tubes	Nunc
Dounce Homogenizer