modeling reactive oxygen species-induced neuronal death in mouse cerebellar granule neurons

Modeling Reactive Oxygen Species-Induced Neuronal Death in Mouse Cerebellar Granule Neurons

For ROS-induced cell death, treat the neurons with hydrogen peroxide at 75 to 100 micromolar for five minutes. After five minutes, switch it to the conditioned media from parallel cultures.
Due to the instability of hydrogen peroxide, the concentration must be optimized to a level that induces between 50% and 70% cell death after 24 hours. This concentration is usually between 75 and 100 micromolar.

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1. Experimental Preparation
NOTE: The following stock solutions can be prepared and maintained until use.
<ol>
	<li>Dissection Solution
	<ol>
		<li>Dissolve 3.62 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 0.069 g of sodium phosphate (NaH2PO4&#160;&#8729; H2O), and 1.306 g of D-(+)-Glucose in 500 mL of distilled H2O. Then, add 12.5 mL of HEPES Buffer, 450 &#181;L of Phenol red solution, and 20 mL of Bovine serum albumin (BSA) fraction V to the solution. Adjust to pH 7.4 using 1 N sodium hydroxide (NaOH).</li>
		<li>Sterilize with a 0.22 &#181;m filter and store at 4 &#176;C. This solution will remain stable for up to one week to 10 days.</li>
	</ol>
	</li>
	<li>Trypsin
	<ol>
		<li>Prepare a stock solution at a concentration of 25 g/L of trypsin in 0.9% sodium chloride. Store the solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Trypsin Inhibitor
	<ol>
		<li>Prepare a stock with a concentration of 10 mg/mL by dissolving 1 g of chicken egg white trypsin inhibitor in 100 mL of 1 mM HCl. Store this solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Deoxyribonuclease (DNase)
	<ol>
		<li>Dissolve 100 mg of DNase 1 in 10 mL of sterile water to generate a 10 mg/mL stock. Store the solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>MgSO4
	<ol>
		<li>Add 6.02 g of magnesium sulfate (MgSO4) to 50 mL of distilled water (H2O) to generate a 1 M stock. Store the solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>CaCl2
	<ol>
		<li>Dissolve 0.735 g of calcium chloride (CaCl2&#8729; 2H2O) in 50 mL of distilled H2O to a stock concentration of 100 mM. Store solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>KCl
	<ol>
		<li>Dissolve 3.7275 g of KCl in 50 mL of distilled H2O to a stock concentration of 1 M. Store solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>D-(+)-Glucose
	<ol>
		<li>Dissolve 1.802 g of D-(+)-glucose in 50 mL of distilled H2O to a stock concentration of 100 mM. Store solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Cell Culture Media
	<ol>
		<li>Prepare cell culture media as follows: 5 mL of heat inactivated dialyzed Fetal Bovine Serum, 500 &#181;L of 200 mM L-Glutamine, 100 &#181;L of 50 mg/mL Gentamycin and 1 mL of 1.0 M KCL should be added to 45 mL of filter sterilized Eagle&#39;s minimal essential media (E-MEM) that is supplemented with 1.125 g/L D-Glucose to generate 50 mL of cerebellar granule neuron (CGN) culture media. Store media at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Cytosine Beta-D- Arabino Furanoside
	<ol>
		<li>Dissolve 48 mg of cytosine beta-D-arabino furanoside in 10 mL of growth media to a stock concentration of 20 mM. Store solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Poly-D-Lysine
	<ol>
		<li>To generate a 2 mg/mL poly D-lysine stock, add 10 mL of sterile H2O to 20 mg of poly D-lysine. Stock poly D-lysine should be stored at -80 &#176;C in 100 &#181;L aliquots. 
		NOTE: The following solutions should be prepared prior to dissection and maintained at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>MgSO4&#160;Supplemented Dissection Solution
	<ol>
		<li>Add 300 &#181;L of stock MgSO4&#160;to 250 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Trypsin Dissection Solution
	<ol>
		<li>Add 100 &#181;L of stock trypsin and 12 &#181;L of stock MgSO4&#160;to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Trypsin Inhibitor Solution 1
	<ol>
		<li>Add 164 &#181;L of stock trypsin inhibitor, 250 &#181;L of stock DNase 1 and 12 &#181;L of stock MgSO4&#160;to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Trypsin Inhibitor Solution 2
	<ol>
		<li>Add 1040 &#181;L of stock trypsin inhibitor, 750 &#181;L of stock DNase 1 and 12 &#181;L of stock MgSO4 to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>CaCl2&#160;supplemented dissection solution
	<ol>
		<li>Add 10 &#181;L of stock CaCl2&#160;and 25 &#181;L of stock MgSO4&#160;to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Poly D-Lysine Coated Culture Dishes
	<ol>
		<li>To coat culture dishes, dilute 100 &#181;L of stock poly D-lysine in 40 mL of sterile H2O. For 35 mm Nunc culture dishes, add 2 mL of diluted poly D-Lysine solution. For 4 well plates, add 300 &#181;L of diluted poly D-Lysine to each well.</li>
		<li>Let the poly D-lysine sit for 1 h before aspirating and washing each well with 4 mL or 600 &#181;L (for 35 mm culture dishes or 4 well plates, respectively) of sterile H2O. Then aspirate the H2O and let the dishes air dry for 1 h.</li>
	</ol>
	</li>
</ol>
2. Brain Extraction and Isolation of Cerebellum
<ol>
	<li>Decapitate a 6&#8211;7-day old mouse pup using decapitation scissors. Perform the dissection of the brain in a sterile tissue culture hood.</li>
	<li>To remove the brain, grasp the head using a pair of forceps and cut through the skin toward the anterior of the head using microdissection scissors as demonstrated in&#160;Figure 1. Then cut through the skull bone using a new pair of scissors to minimize risk of contamination. Remove the skull covering the brain using forceps and tease out the brain using forceps or a spatula.
	<ol>
		<li>As needed, cut through the optic nerve to facilitate getting the brain out as a whole.</li>
	</ol>
	</li>
	<li>Place the brain in the dissection solution, which is supplemented with MgSO4. Keep the solution and the brain on ice.</li>
	<li>Following brain removal, under a dissection microscope, dissect out the cerebellum from the brain while still in MgSO4&#160;supplemented dissection solution, and remove the meninges using fine forceps. Turn the cerebellum to its ventral side and insure removal of the choroid plexus.</li>
	<li>Pool the cerebella into a 35-mm dish containing ~1 mL of MgSO4&#160;supplemented dissection solution.</li>
	<li>Chop the tissue into small pieces and transfer into a 50 mL tube containing 30 mL of MgSO4&#160;buffered dissection solution.</li>
</ol>
3. Mouse Cerebellar Granule Neuron Isolation and Culturing
<ol>
	<li>Centrifuge the 50 mL tube containing the chopped cerebral tissue for 5 min at 644 x g and 4 &#176;C.</li>
	<li>Remove the supernatant and add 10 mL of trypsin dissection solution. Then, shake the tube at high speed for 15 min at 37 &#176;C.</li>
	<li>Add 10 mL of trypsin inhibitor solution 1 to the tube and rock gently for 2 min.</li>
	<li>Centrifuge the tube for 5 min at 644 x g and 4 &#176;C.</li>
	<li>Remove the supernatant and add 2 mL of trypsin inhibitor solution 2, and then transfer to a 15 mL tube.</li>
	<li>Triturate the tissue in the 15 mL tube until the solution becomes murky. Then let settle for 5 min.</li>
	<li>Remove the clear supernatant and transfer the supernatant to a new tube containing 1 mL of CaCl2&#160;supplemented dissection solution.</li>
	<li>Add another mL of trypsin inhibitor solution 2 to the bottom of the tube containing the pellet from step 3.6. Triturate again and let settle for 5 min. Remove the supernatant and add it to the tube containing the supernatant from step 3.7 (That is a repeat of steps 3.6-3.7). Repeat this process until most of the tissue is mechanically dissociated.</li>
	<li>Add 0.3 mL of CaCl2&#160;supplemented dissection solution to the supernatant collection for every mL of supernatant.</li>
	<li>Mix the contents of the tube and then centrifuge for 5 min at 644 x g.</li>
	<li>Remove the supernatant and add 10 mL of fresh media to the pellet and mix.</li>
	<li>Cells may then be counted and diluted to a concentration of 1.5 x 106&#160;cells/mL. Please note that in general 10 million cells are expected from each dissected brain, dependent on trypsinization time and efficiency of dissection. Plate cells on previously made poly D-lysine plates. For 4-well plates, plate 0.5 mL, giving 7.5 x 105&#160;cells per well. For 35-mm dishes, plate 4 mL, giving 6 x 106&#160;cells per plate.</li>
	<li>After 24 h, add cytosine-&#946;-arabino furanoside (AraC) to the plates to reduce glial contamination. For each mL of media 0.5 &#181;L of 20 mM AraC is required. Addition of AraC does not require a media change. If cells are to be maintained for 7-8 days, repeat this treatment on day 3.</li>
	<li>Maintain cultures in a 5% CO2&#160;incubator at 37 &#176;C and feed with 100 &#181;L of 100 mM glucose added to the culture for every 2 mL of media every 2 days past 5 days. A complete media change is not required.</li>
</ol>
4. Modeling Neuronal Injury
<ol>
	<li>To induce ROS-induced cell death (oxidative stress), treat neurons with 75-100 &#181;M hydrogen peroxide (H2O2) and switch to conditioned media from parallel cultures following 5 min exposure to H2O2. Note, due to the instability of H2O2, optimize the concentration to a level that induces 50-70% cell death in culture following 24 h of treatment (Figure 2).</li>
</ol>

Source: Laaper, M. et al., <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video outlines the generation of a neuronal injury model using reactive oxygen species (ROS) in mouse primary cerebellar granule neurons. It details the process of inducing oxidative stress by adding high concentrations of hydrogen peroxide. ROS exposure causes DNA strand breaks and genomic instability. Additionally, the oxidation of cellular proteins impairs their functions, while lipid peroxidation compromises membrane integrity. This cascade leads to cytochrome c release and caspase activation, resulting in neuronal apoptosis.

<img alt="Figure 1" src="/files/ftp_upload/22982/22982_Figure1.jpg" /> 
Figure 1: Removal of mouse brain and dissection of cerebellum.&#160;(A) To extract the brain of a 6-7 day old mouse, using a pair of forceps, grasp the head and cut the skin anteriorly along the dotted lines using a pair of microdissection scissors. Be careful to cut only the skin and connective tissue, too deep an incision may puncture the skull and damage the brain. These three...

1. Experimental Preparation
NOTE: The following stock solutions can be prepared and maintained until use.

	Dissection Solution
	
		Dissolve 3.62 g of ...

Take a culture of mouse primary cerebellar granule neurons.
Add media containing hydrogen peroxide, a reactive oxygen species or ROS, and incubate briefly.
Hydrogen peroxide diffuses into the cells and is converted into highly reactive free radicals.
These radicals induce lipid peroxidation, compromising the integrity of the cell membrane.
Additionally, the radicals cause oxidative modifications in cellular proteins, impairing their function.
Furthermore, they induce DNA breaks, leading to genomic instability.
The radicals also cause oxidative damage to intracellular organelles, including mitochondria.
In response, the damaged mitochondria release cytochrome c, which binds to the apoptotic protease activating factor-1, triggering apoptosome formation and the conversion of pro-caspase-9 to active caspase-9.
Caspase 9 activates executioner caspases, further cleaving cellular proteins and leading to apoptotic neuronal death.
Replace the media with fresh, hydrogen peroxide-free media to halt the signaling cascade.

11 Views. Modeling Reactive Oxygen Species-Induced Neuronal Death in Mouse Cerebellar Granule Neurons

Modeling Reactive Oxygen Species-Induced Neuronal Death in Mouse Cerebellar Granule Neurons

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