We thank Wangchen Tsering and Pamela Walter for their critical reading and editing of this manuscript. This work was supported by the National Institute of Neurological Disorders and Stroke (NINDS) (NS054773 to C.J. L. and NS098393 to H.Z.) and the Department of Neuroscience at Thomas Jefferson University (Startup Funds to H.Z.).

All the procedures including animal work were conducted according to national and international guidelines and were approved by the Animal Care and Use Committee of Thomas Jefferson University. Male FVB/NTac mice at the age of 3 to 14 months were used. The following steps were performed in a non-sterile setting, but caution should be taken to keep everything as clean as possible.
NOTE: The method presented here was established and used in the research reported by Zhi et al.24...

<ol>
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The method we developed allowed an XF analyzer to be used for measuring OCR in striatal slices from adult mice over a time span of 4 h. This method provides a new way to measure cellular bioenergetics in punches excised from anatomically defined brain structures. Since the tissue samples being analyzed are rather small, the metabolic parameters of specific brain areas involved in a disease can be investigated. In addition, using acute slices more closely mimics the physiological cellular environment, which cannot be achi...

Mitochondrial dysfunction has been implicated in several neurological diseases, including Parkinson&#39;s disease (PD), Huntington&#39;s disease, and Alzheimer&#39;s disease1,2,3. PD models such as PINK1 knockout (KO) mice and rats display impaired mitochondrial function4,5,6,7,8,9,10,11. Mitochondria isolated from the striatum (STR) or whole-brain of aged PINK1 KO mouse exhibit defects in complex I7,10,12,13. Directly measuring the oxygen consumption rate (OCR) is one of the most common methods to evaluate mitochondrial function since OCR is coupled with ATP production, the principal function of mitochondria14. Therefore, measuring OCR in disease models or patient-derived samples/tissue can help investigate how mitochondrial dysfunction leads to disease.
Currently, there are several ways to measure mitochondrial OCR, including the Clark electrode and other O2 electrodes, O2 fluorescent dye, and the extracellular flux analyzer15,16,17,18,19. As an advantage, O2 electrode-based methods allow various substrates to be easily added. However, they are insufficient for simultaneously measuring several samples. Compared to traditional O2 electrode-based methods, the extracellular flux analyzer, a commonly used tool for OCR in cell cultures or purified mitochondria, offers improved throughput15,18,20. Nevertheless, all these methods are usually applied to measure OCR in isolated mitochondria or cell cultures6,16,17,19,20,21. The isolation of mitochondria causes inadvertent damage, and extracted mitochondria or cell cultures are less physiologically relevant than intact brain slices22. Even when microelectrodes are used in slices, they are less sensitive and more difficult to operate than in cultured cells23.
To meet these challenges, we developed a method using the XF24 extracellular flux analyzer, which allows for the analysis of multiple metabolic parameters from acute striatal brain slices of mice24. This technique provides continuous direct quantification of mitochondrial respiration via the OCR. In brief, small sections of striatal brain slices are placed into wells of the islet plate, and the analyzer uses oxygen and proton fluorescent-based biosensors to measure the OCR and extracellular acidification rate, respectively17,21,25.
One of the unique features of the analyzer is the four injection wells, which allow the continued measurement of OCR while sequentially injecting up to four compounds or reagents; this enables the measurement of several cellular respiration parameters, such as basal mitochondrial OCR, ATP-linked OCR, and maximal mitochondrial OCR. The compounds injected during the measurements for the protocol shown here were working concentrations of 10 mM pyruvate in the first solution well (port A), 20 &#181;M oligomycin in the second solution well (port B), 10 &#181;M carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) in the third well (port C), and 20 &#181;M antimycin A in the fourth well (port D), based on Fried et al.25. It must be noted that these concentrations were working concentrations, and stock solutions of 10x, 11x, 12x, and 13x were injected into the solution ports A through D, respectively. The purpose of using each solution was as follows: 1) Pyruvate was necessary since, without it, the addition of FCCP would have a decreased OCR response caused by a limitation of available substrates; 2) Oligomycin inhibits ATP synthase and allows the measurement of ATP linked respiration; 3) FCCP uncouples oxidation from phosphorylation and allows the measurement of maximal mitochondrial capacity; 4) Antimycin A inhibits complex III in the electron transport chain and, therefore, allows the measurement of OCR not linked to the mitochondria.
The concentration of oligomycin used was determined based on the following reasons: 1) The recommended dose of oligomycin for most cell types (isolated mitochondria or cell cultures) is 1.5 &#181;M. From experience, usually 3x-10x of the dissociated cells dose is used for the slice experiments since there might be a gradient, and the penetration of solution in the slices takes time. Therefore, the concentration should be in the range of 5 &#181;M to 25 &#181;M. 2) A 20 &#181;M concentration was selected based on Fried et al.25. Higher concentrations were not tried due to the non-specific toxicity of oligomycin. 3) In the report by Underwood et al.26, the authors did a titration experiment for oligomycin and found that doses at 6.25, 12.5, 25, and 50 &#181;g/mL resulted in similar inhibition. The higher concentration of oligomycin (50 &#181;g/mL) did not inhibit more but had a bigger variance. 4) In our observation, the determining factor seems to be the penetrating ability of oligomycin. It is difficult for oligomycin to penetrate the tissue, and that is why it takes at least 7 to 8 cycles to reach the plateau, the maximum response. As long as it reaches the plateau, the inhibition is assumed to be maximal.
A key technical challenge of adapting the extracellular flux analyzer for measuring OCR in striatal slices is to prevent tissue hypoxia. Since the buffer was not oxygenated during the entire duration of measurements (about 4 h), hypoxia was a central issue. This is especially true for thicker tissue samples, where oxygen cannot diffuse throughout the samples. To overcome this problem, slices were sectioned at 150 &#181;m thickness, so that ambient oxygen could penetrate the middle of the brain slices. In addition, 4 mg/mL bovine serum albumin (BSA) was added to the pre-oxygenated artificial cerebrospinal fluid (ACSF) buffer, which facilitated the determination of maximal OCR, as previously suggested23. We examined whether cells were alive. First, Hoechst 33258 (10 &#181;M) and propidium iodide (10 &#181;M) were used to examine whether cells were healthy under these conditions. We then examined whether medium spiny neurons were functionally healthy using patch-clamp recording. We further evaluated whether dopamine (DA) terminals in the striatal slices were functionally healthy by measuring DA release using fast-scan voltammetry. The results showed that striatal slices that were not oxygenated (ACSF/BSA group) were as healthy as the oxygenated control group24.
We then tested different combinations of slice thickness and punch size to determine optimal striatal slice conditions for the flux respiration assay. Dorsal striatal slices with different thicknesses (150 &#181;m and 200 &#181;m) and punch sizes (1.0 mm, 1.5 mm, and 2.0 mm in diameter) were used for OCR analysis using the analyzer. Striatal slices that were 150 &#181;m thick with a punch size of 1.5 mm in diameter had the highest coupling efficiency and OCRs within an optimal range for the analyzer24.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accumet AB150 pH benchtop meter</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-636-AB150</td>
			<td>To measure pH</td>
		</tr>
		<tr>
			<td>Antimycin A from streptomyces sp.</td>
			<td>SIGMA</td>
			<td>A8674</td>
			<td>To inhibit complex III of the mitochondria</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>SIGMA</td>
			<td>A6003</td>
			<td>To make modified artificial cerebrospinal fluid (BSA-ACSF)</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)</td>
			<td>SIGMA</td>
			<td>C2920</td>
			<td>To uncouple mitochondrial respiration</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>SIGMA</td>
			<td>G8270</td>
			<td>To make artificial cerebrospinal fluid (ACSF)</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>SIGMA</td>
			<td>D8418</td>
			<td>To dissovle compounds</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SIGMA</td>
			<td>H3375</td>
			<td>To make artificial cerebrospinal fluid (ACSF)</td>
		</tr>
		<tr>
			<td>Humidified non-CO2 incubator</td>
			<td>Fisher Scientific</td>
			<td>11-683-230D</td>
			<td>To hydrate plates at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Oligomycin from Streptomyces diastatochromogenes</td>
			<td>SIGMA</td>
			<td>O4876</td>
			<td>To inhibit mitochondrial ATP synthase</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>SIGMA-ALDRICH</td>
			<td></td>
			<td>sealing film</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Tocris</td>
			<td>3616</td>
			<td>To inhibit complex I of the mitochondria</td>
		</tr>
		<tr>
			<td>Seahorse XF Calibrant Solution 500 mL</td>
			<td>Seahorse Bioscience</td>
			<td>103681-100</td>
			<td>Solution for seahorse calibration</td>
		</tr>
		<tr>
			<td>Seahorse XF Extracellular Flux Analyzer</td>
			<td>Seahorse Bioscience</td>
			<td></td>
			<td>Equipment used to analyze oxygen consumption rate, old generation</td>
		</tr>
		<tr>
			<td>Seahorse XFe24 Extracellular Flux Analyzer</td>
			<td>Seahorse Bioscience</td>
			<td></td>
			<td>Equipment used to analyze oxygen consumption rate, new generation</td>
		</tr>
		<tr>
			<td>Seahorse XF24 FluxPaks</td>
			<td>Seahorse Bioscience</td>
			<td>101174-100</td>
			<td>Package of flux analyzer sensor cartridges, tissue culture plates, capture screens,&#160; calibrant solution and calibration plates; assay kit.</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>SIGMA</td>
			<td>P2256</td>
			<td>To prevent any substrate-limiting constraints of substrate supply</td>
		</tr>
		<tr>
			<td>Stainless steel biopsy punches</td>
			<td>Miltex</td>
			<td></td>
			<td>Device used to punch slices</td>
		</tr>
		<tr>
			<td>Sterile cell culture dish, 35 x 10 mm</td>
			<td>Eppendrof</td>
			<td>0030700102</td>
			<td>Used for slice punch</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td>To slice brain tissue</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Thermo Scientific Precision</td>
			<td>282-115</td>
			<td>To heat buffer and solutions</td>
		</tr>
	</tbody>
</table>

measurement of oxygen consumption rate in acute striatal slices from adult mice

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca2+ concentration control. Mitochondrial dysfunction has been implicated in the pathogenesis of multiple neurodegenerative diseases, including Parkinson&#39;s disease (PD), Huntington&#39;s disease, and Alzheimer&#39;s disease. To study the role of mitochondria in models of these diseases, we can measure mitochondrial respiration via oxygen consumption rate (OCR) as a proxy for mitochondrial function. OCR has already been successfully measured in cell cultures, as well as isolated mitochondria. However, these techniques are less physiologically relevant than measuring OCR in acute brain slices. To overcome this limitation, the authors developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice. The technique is optimized with a focus on the striatum, a brain area involved in PD and Huntington&#39;s disease. The analyzer performs a live cell assay using a 24-well plate, which allows the simultaneous kinetic measurement of 24 samples. The method uses circular-punched pieces of striatal brain slices as samples. We demonstrate the effectiveness of this technique by identifying a lower basal OCR in striatal slices of a mouse model of PD. This method will be of broad interest to researchers working in the field of PD and Huntington&#39;s disease.

The first step of this study was to optimize the slice thickness and punch size used to excise a section of the striatum from the slice (Figure 3A). A slice at 150 &#181;m thickness and a 1.5 mm punch size gave the best results determined by the coupling efficiency (Figure 3B-C). As shown in Figure 3B, OCR is relatively stable for 5 h with less than 10% run down. In addition, functional measurements...

Watch this Scientific Journal Video about Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice at JoVE.com

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice

Oxygen consumption rate (OCR) is a common proxy for mitochondrial function and can be used to study different disease models. We developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice that is more physiologically relevant than other methods.

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca2+ concentration control. Mitochondrial ...

We developed a new method using a seahorse XF analyzer to directly measure the oxygen consumption rate. But common proxy for mitochondrial function in acute slide slice from adult mice. This method measures a cellular by energetics in punctures from anatomically defined brain structures.Using acute slices more closely mimics the physiological cellular environment which cannot be achieved the ways isolated mitochondrial or culture cells. This method will be of broad interest to researchers working in the field of Parkinson's disease and Huntington's disease. To begin, open the extracellular flux assay kit and remove both the sensor cartridge and the utility plate.Then place the sensor cartridge aside and do not touch the sensors. Next, add 600 microliters of calibrate solution to each wall of the utility plate. Place the sensor cartridge on top of the utility plate and submerge the sensors in the calibrate solution, ensuring that the triangular notch of the utility and sensor cartridge plate are correctly aligned.Afterward, seal the extracellular flux assay kit with sealing film to prevent evaporation of the calibrate solution and then place it in a 37 degree Celsius incubator not supplemented with carbon dioxide or oxygen, overnight. Open the eyelet capture micro plate and take out the eyelet plate for tissue sitting. Then warm an appropriate volume of pre-oxygenated modified artificial cerebral spinal fluid buffer to 37 degree Celsius in a 50 milliliter tube.Next add BSA to a final concentration of four milligrams per milliliter to prepare the respiration buffer. Add 625 microliters of respiration buffer to each well of the eyelet plate carefully while avoiding shaking the plate, ensuring no air bubbles are present in the buffer of each well. Immediately dissect the brain in 10 milliliters of ice-cold pre-oxygenated cutting solution.Then, using a vibratome section coronal striatal slices, following the manufacturer's instruction at a thickness of 150 micrometer in ice-cold pre-oxygenated cutting solution. Next, recover the slices in 50 milliliters of oxygenated artificial cerebral spinal fluid. And keep them in solution for up to 30 minutes at room temperature.After recovery, transfer the slices to a 35 by 10 millimeters Petri dish with five millimeters of respiration buffer. Using a stainless steel biopsy punch create a circular piece of tissue in the desired area of the sliced brain by gently pressing down the punch while keeping the slice in the buffer. Then remove the rest of the tissue, lift the punch away and remove the circular piece of tissue into the buffer.Next, cut the very end of a one milliliter pipette tip to make a hole with a one-point-five to two-point-zero millimeter diameter and use it to hold and transfer the punched slice to the top of the capture screen. Suction one piece of punched brain tissue and carefully place the tissue onto the mesh side of the capture screen, a circular piece of plastic with the mesh attached to one side. Using a paper tissue gently dry the capture screen and remove the moisture, which allows the tissue to become sticky and attached to the center of the mesh.Next, hold the capture screen slice-side down with tweezers and place it into one of the wells of the incubating eyelet plate. Then incubate the eyelet plate at 37 degrees Celsius in an incubator for at least 30 minutes to allow temperature and pH equilibration before running the assay. Dilute the desired compounds and modified artificial cerebral spinal fluid without BSA to the final stock concentration of the working concentration for ports A to D, respectively.Gently preload 75 microliters of the diluted compounds into the appropriate injection ports of the sensor cartridge by placing the tips halfway into the injection ports at a 45 degree angle with the tip against the wall of the injection port, as complete insertion may cause compound leakage through the port. Afterward, withdraw the tips from the ports carefully without creating air bubbles and do not tap any portion of the cartridge to avoid alleviating air bubbles. Then visually inspect the injection ports for even loading ensuring all liquid is in the port and no residual drops are present on top of the cartridge.After placing the sensor cartridge onto the utility plate put it into an incubator for 30 minutes to allow it to heat up to 37 degrees Celsius while handling carefully by only holding onto the utility plate and moving as little as possible. First load the assay template in the software and then press the green start button. Then load the sensor cartridge on the utility plate into the instrument tray, ensuring that the plate sits correctly and is flat, loading the drug-filled sensor cartridge into the analyzer for calibration.Afterward, follow the instructions on the screen in order to calibrate. And equilibrate the sensors. Once the equilibration step is done, remove the calibration plate and replace it with the eyelet plate containing the mesh and tissue slices.Then measure the oxygen consumption rate in each well of the plate, using the assay protocols as described in the manuscript. Following, analyze the oxygen consumption rate measurement data and the coupling efficiency data. Oxygen consumption rates for different thicknesses and punch sizes of slices in the control group showed stable basal respiration over the whole measurement and were proportional to the volume of the slice.Furthermore, oxygen consumption rates were relatively stable for five hours with less than 10%rundown. The mitochondrial coupling efficiency was compared and the slice at the 150 micrometer thickness and a 1.5 millimeter punch size showed the highest coupling efficiency. The oxygen consumption rates were measured for young and old groups of Pink1 knockout and their age-matched wild-type mice.The basal oxygen consumption rate was similar in both the knockout and wild-type groups for young mice. However it decreased for the knockout mice in the old group. However, the mitochondrial dysfunction in the knockout mice was observed for the young age group, indicated by the decreased coupling efficiency caused by the knockout of Pink1.The critical steps in this protocol includes preparing brain slices, transferring them to the top of capture screen, placing slides into wells and the keeping them attached to the capture screen during the measurements.

measurement-oxygen-consumption-rate-acute-striatal-slices-from-adult

Research

JoVE Journal

Neuroscience

3.0K visualizações.  Thomas Jefferson University. Desenvolvemos um novo método usando um analisador XF de cavalo marinho para medir diretamente a taxa de consumo de oxigênio. Mas proxy comum para função mitocondrial em fatia de slide aguda de camundongos adultos. Este método mede um celular por energia em perfurações de estruturas cerebrais anatomicamente definidas.O uso de fatias agudas imita mais de perto o ambiente celular fisiológico que não pode ser alcançado das formas isoladas de células mitocondriais ou culturais. E...