Name: three-dimensional imaging and analysis of mitochondria within human intraepidermal nerve fibers
Uploaded: 2017-09-29T12:30:00
Description: Watch this Scientific Journal Video about Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers at JoVE.com

This work was supported by National Institutes of Health Grants K08 NS061039-01A2, The Program for Neurology Research &#38; Discovery, and The A. Alfred Taubman Medical Research Institute at the University of Michigan. This work used the Morphology and Image Analysis Core of the Michigan Diabetes Research Center, funded by National Institutes of Health Grant 5P90 DK-20572 from the National Institute of Diabetes and Digestive and Kidney Diseases. The authors would like to thank J. Robinson Singleton and A. Gordon Smith (University of Utah) for their generous donation of human skin samples.

Skin punch biopsies were obtained from subjects that were recruited from a large community-based primary care network at the University of Utah Diabetes Center (Salt Lake City, UT). This study was approved by the University of Michigan Institutional Review Board and complied with the tenets of the Declaration of Helsinki. Written informed consent was obtained from each subject prior to testing.
1. Fluorescence Immunohistochemistry
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	<li>Prepare punch biopsies for intraepidermal n...

     

This protocol is designed to isolate, quantify and analyze the size and distribution of nerve-specific mitochondria within IENFs in 3D from human skin biopsies. There are several critical steps in the protocol. The free-floating fluorescence immunohistochemistry is designed to stain and analyze multiple signals in each sample, providing a more versatile methodology for explorative research44,45. This procedure allows for penetration of the antibodies into the tis...

Mitochondria serve vital cellular functions that include producing cell energy, buffering calcium, and regulating necrotic and apoptotic cell death1,2,3. The nervous system has a high metabolic rate compared to the body4 suggesting that neurons generate a high degree of cellular energy in the form of adenosine triphosphate (ATP) through mitochondrial respiration. A lot of evidence documents that neuronal functions are dependent on ATP5, especially at the synapses6. Therefore, the distribution of mitochondria within neurons is important.
Over the last 10 years a lot of information has shown that the trafficking and docking of neuronal mitochondria is highly regulated. Motor proteins are involved in distributing mitochondria to specific cellular compartments throughout the neuron. Trafficking of mitochondria is particularly important because neurons project axons and dendrites far away from the soma. Kinesin motor proteins primarily direct anterograde (away from the soma) trafficking of mitochondria along microtubules while dynein motor proteins direct retrograde (toward the soma) motility7,8,9,10. There are cellular signals such a mitochondrial membrane potential and impulse conduction that influence the presence and direction of mitochondrial trafficking11,12,13.
In addition to transporting mitochondria, there are specialized proteins to localize mitochondria to specific cellular compartments that have high energy demands, such as nodes of Ranvier and synapses8,14,17. In fact, the majority of mitochondria within axons are non-motile9,13,18. Specialized proteins like syntaphilin anchor mitochondria to microtubules along axons while other proteins anchor mitochondria to the actin cytoskeleton19-21. Growth factors and ions such as calcium have been reported to support the cessation of mitochondria movement to localize them to regions where they are needed21,22,23.
Taken together, the trafficking and docking of mitochondria are vital for proper function of neurons. In support of this, disruption in mitochondrial trafficking has been associated with several neurological conditions including Alzheimer&#39;s disease, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Huntington&#39;s disease, hereditary spastic paraparesis, and optic atrophy15,24,25,26,27. Recent studies have focused on mitochondrial dysfunction and pathology as a potential mechanism for diabetic neuropathy, the sensory loss associated with diabetes28,29,30,31,32,33. The hypothesis is that diabetes alters the distribution of mitochondria within the sensory projections of cutaneous nerve ending. Therefore, a technique was developed to visualize and quantify mitochondria within the intraepidermal nerve fibers (IENFs), the distal tips of dorsal root ganglion sensory afferents. The technique combines fluorescence immunohistochemistry of specific mitochondrial and nerve fiber labels with confocal microscopy z-series acquisition of signals with powerful 3D image analysis software to measure the distribution of nerve-specific mitochondria from human cutaneous punch biopsies to achieve this goal.

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	<tbody>
		<tr height="69">
			<td height="69" style="height:69px;width:267px;">2% Zamboni&#39;s Fixative</td>
			<td style="width:200px;">Newcomer Supply, Middleton, WI&#160;</td>
			<td style="width:199px;">1459A</td>
			<td style="width:363px;">2% paraformaldehyde, 0.2% saturated picric acid in phosphate buffered saline (PBS), pH 7.4</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:267px;">10X Phosphate Buffered Saline (PBS)&#160;</td>
			<td style="width:200px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:199px;">BP399-4</td>
			<td style="width:363px;">To make up 1X PBS</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:267px;">Image-iT FX Signal Enhancer</td>
			<td style="width:200px;">ThermoFisher Scientific, Waltham, Massachusetts</td>
			<td style="width:199px;">I36933</td>
			<td style="width:363px;">enhances Alexa Fluor dye signals by reducing nonspecific binding</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:267px;">Anti-Protein Gene Product 9.5 Antibody (Rabbit Polyclonal)</td>
			<td style="width:200px;">Proteintech Group Inc. Rosemont, IL</td>
			<td style="width:199px;">14730-1-AP</td>
			<td style="width:363px;">abbreviated as PGP9.5, replaces discontinued AbD Serotec (Cat. No. 7863-0504) antibody</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:267px;">Anti-Pyruvate Dehydrogenase E2/E3bp Antibody (Mouse Monoclonal)</td>
			<td style="width:200px;">abcam, Cambridge, MA</td>
			<td style="width:199px;">ab110333</td>
			<td style="width:363px;">abbreviated as PDH</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:267px;">Goat anti-mouse Secondary antibody Alexa Fluor 594 conjugate</td>
			<td style="width:200px;">ThermoFisher Scientific, Waltham, Massachusetts</td>
			<td style="width:199px;">A-11034</td>
			<td style="width:363px;">red-fluorescent conjugated secondaryantibody</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:267px;">Goat anti-rabbit Secondary antibody Alexa Fluor 488 conjugate</td>
			<td style="width:200px;">ThermoFisher Scientific, Waltham, Massachusetts</td>
			<td style="width:199px;">A-11032</td>
			<td style="width:363px;">green-fluorescent conjugated secondaryantibody</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:267px;">Albumin, from Bovine Serum</td>
			<td style="width:200px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:199px;">A7906-100</td>
			<td style="width:363px;">abbreviated as BSA</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:267px;">Triton X- 100</td>
			<td style="width:200px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:199px;">T9284</td>
			<td style="width:363px;">abbreviated as TX-100</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:267px;">0.22 &#181;m Filter</td>
			<td style="width:200px;">EMD Millipore, Billerica 
			MA</td>
			<td style="width:199px;">MILLEX GP SLGP 033NS</td>
			<td style="width:363px;">0.22 &#181;m Millipore filter</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:267px;">Parafilm M</td>
			<td style="width:200px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:199px;">13-374-10</td>
			<td style="width:363px;">Curwood Wisconsin LLC Parafilm M (PM-996)</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:267px;">Non-calibrated Loop</td>
			<td style="width:200px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:199px;">22-032092</td>
			<td style="width:363px;">inoculating Loop by Decon LeLoop (MP 199-25)</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:267px;">96-well Assay Plate</td>
			<td style="width:200px;">Corning Incorporated, Corning, NY</td>
			<td style="width:199px;">3603</td>
			<td style="width:363px;">96-well flat bottom plate</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:267px;">Prolong Gold antifade reagent with DAPI</td>
			<td style="width:200px;">ThermoFisher Scientific, Waltham, Massachusetts</td>
			<td style="width:199px;">P-36931</td>
			<td style="width:363px;">DAPI staining of nuclei</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:267px;">Microscope Cover Glass 50 x 24 mm</td>
			<td style="width:200px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:199px;">12-544E</td>
			<td style="width:363px;">Coverslips</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:267px;">Superfrost Plus Microscope Slides</td>
			<td style="width:200px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:199px;">12-550-15</td>
			<td style="width:363px;">Microscope Slides</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:267px;">Leica SP5 Laser Scanning Confocal Microscope</td>
			<td style="width:200px;">Leica Microsystems, Buffalo Grove, IL</td>
			<td style="width:199px;">SP5</td>
			<td style="width:363px;">Confocal Microscope</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:267px;">Volocity x64 Software&#160;</td>
			<td style="width:200px;">Perkin Elmer, Waltham , MA</td>
			<td style="width:199px;">version 4.4.0</td>
			<td style="width:363px;">Volocity software is used for Steps 3.1 and 3.2 in the protocol for image processing</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:267px;">Imaris x64 3 Dimensional Analysis Software</td>
			<td style="width:200px;">Bitplane, Concord, MA</td>
			<td style="width:199px;">version 7.7.1</td>
			<td style="width:363px;">Imaris software is used for Steps 3.3 through 3.5 in the protocol for image analysis</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:267px;">Excel</td>
			<td style="width:200px;">Microsoft, Redmond, WA</td>
			<td style="width:199px;">version Office 2013</td>
			<td style="width:363px;">Excel spreadsheet software is used for Step 3.6 in the protocol to summarize morphometric features</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:267px;">Optimum Cutting Temperature Compound</td>
			<td style="width:200px;">Sakura Finetek USA, Inc., Torrance, CA</td>
			<td style="width:199px;">4583</td>
			<td style="width:363px;">abbreviated as OCT</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Leica Cryostat</td>
			<td style="width:200px;">Leica Biosystems, Buffalo Grove, IL</td>
			<td style="width:199px;">CM1850</td>
			<td style="width:363px;">Cryostat for cutting 50 &#181;m sections</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:267px;">CellLight Mitochondria-GFP, BacMam 2.0</td>
			<td style="width:200px;">ThermoFisher Scientific, Waltham, Massachusetts</td>
			<td style="width:199px;">C10600</td>
			<td style="width:363px;">Used as a postive control to label mitochondria with a green fluorescent signal</td>
		</tr>
	</tbody>
</table>

three-dimensional imaging and analysis of mitochondria within human intraepidermal nerve fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

Visualization and quantification of mitochondria within human IENFs
Fluorescence immunohistochemistry allows for the simultaneous labeling of multiple signals within human skin biopsies to visualize nerves, mitochondria, and nuclei. A 96-well plate is a convenient way to organize the steps in the immunohistochemistry procedure. Figure 1 shows that this configuration accounts for up ...

Watch this Scientific Journal Video about Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers at JoVE.com

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

The overall goal of this imaging and analysis technique is to isolate specific information from a complex signal. Specifically, this protocol describes how to visualize and quantify nerve-specific mitochondria with other mitochondria within the epidermis. This method can help answer key questions in the field of neurology such as how does mitochondria within intraepidermal nerve fibers of healthy individuals compare to nerve-specific mitochondria of patients with neurological complications.The main advantage of this technique is that we take a complex signal and extract specific information from it. This protocol helps us to isolate a specific signal such as the signal in these straws from the signals around them. Prepare for fluorescence immunohistochemistry staining of skin biopsies by first labeling a 96-well plate according to this schematic.Then pipette 150 microliters of stock signal enhancer solution to reduce nonspecific binding of secondary antibodies into each well of the top row. Prepare rinse wells by adding 150 microliters of 1x phosphate buffered saline into each well in rows two and three of the 96-well plate. Then add 150 microliters of 5%BSA blocking solution into the wells of row four.Dilute the primary antibodies PGP9.5 and PDH in 1, 500 microliters of 1%BSA rinsing solution and add 150 microliters to each well in row five. Use an inoculating loop to transfer the sections into the signal enhancer solution in row one. Once the sections are in the primary antibody solution in row five, wrap the plate tightly with laboratory film to prevent evaporation and then agitate the samples on a flat rocker at room temperature for one hour.Incubate with rocking overnight at four degrees Celsius. Begin day two of the procedure by pipetting 150 microliters of 1%BSA rinsing solution to the wells in rows six, seven, and eight of the 96-well plate. Add the fluorophore-conjugated secondary antibodies to 1, 500 microliters of 1%BSA.Then pipette 150 microliters of the secondary antibody solution into each well of row nine. Rinse the sections by passing through wells six, seven, and eight. Once the sections are in the secondary antibody solution in row nine, wrap the plate in parafilm and cover with aluminum foil to protect the fluorescence signals.Incubate with rocking as before. On day three, pipette 150 microliters of sterile filtered 1x PBS into rows 10, 11, and 12. Transfer the samples to row 10 then cover the plate with aluminum foil.Rinse for one hour at room temperature with rocking. Next, prepare a microscope slide for mounting the sections by pipetting 50 microliters of filtered 1x PBS onto the slide. Once the rinses are complete, transfer a section from row 12 onto the slide then add one to two drops of mounting reagent containing DAPI directly on top of the section.Gently place a 50 millimeter by 24 millimeter 1.5 microscope glass coverslip over the section. Place the slides in the dark overnight at room temperature to cure the mounting medium before storing or imaging the slides. Select the 40X oil immersion objective on an inverted laser scanning confocal microscope.Select the appropriate lasers and detectors to image the nuclei, nerve fibers, and mitochondria. Enter the following scan parameters into the microscope software, 12-bit intensity resolution, scan rate of 500 Hertz with two frame averaging, and zoom of 2.2. Set the microscope software for optimized lateral resolution by selecting a scan resolution of 1024 by 1024.Optimize axial resolution and optical sectioning by selecting a confocal aperture of one air unit with a Z step size of 210 nanometers. Scan each signal separately and adjust the detector voltage and offset to remove any over and under saturated pixels. Activate a live scan for the nerve signal and adjust the Z focus control to find and set the upper and lower focal planes in the microscope software that encompass the nerve signal within the tissue section.Scan the final Z series with sequential scanning to eliminate fluorescence signal crosstalk. Isolate the epidermis from the stratum corneum and the dermis by drawing a region around the epidermis using a selection tool on a duplicate image of the original image. Then crop the image to the selection.Calculate point spread functions for the green and red fluorescence confocal signals using the calculate spread function feature. Then set the parameters for medium refractive index for oil at 1.515 and the numerical aperture for 40X oil objective at 1.25. Set the detector pinhole at one air unit and choose a laser excitation wavelength.Use iterative restoration set to 100%confidence, iteration limit of 10 cycles, and the green and red PSFs for the deconvulation of the green and red fluorescence signals. Use the create surface tool to make a surface around the deconvolved nerve signals selecting uncheck smooth feature, absolute intensity for thresholding, a lower threshold of 3, 000 and an upper threshold of 65, 535. Keep the surfaces above 10 voxels.Remove the nonnerve surfaces by using the edit tab in nerve surface to select single nonnerve surfaces or hold down the Control key to select multiple nonnerve surfaces. Delete the selected nonnerve surfaces by pressing the Delete button. Use the edit tab in nerve surface and press the mask all button in the mask properties.In the new window, select channel five mitochondria deconvolved under the channel selection and then check duplicate channel before applying the mask. Press the radio button for constant inside/outside and check the set voxels outside surface to 0.0 and press the OK button. Finally, create mitochondria-specific surfaces using the create surface tool to make a surface around the masked deconvolved mitochondrial signals by selecting uncheck smooth feature and using background subtraction for thresholding.Set the diameter of the largest sphere to 1.50 micrometers, the lower threshold to 2, 000 and the upper threshold to maximum 65, 535. Be sure to keep the surfaces above 1.0 voxels. This representative 3D confocal microscopy image illustrates the nerve-specific green fluorescence signal.A 3D surface shown in cyan is created for the nerve signal. Then the nerve-specific mitochondrial signal is isolated from the rest of the epidermal mitochondrial signals by using the nerve surface as a masking tool. The resulting nerve-specific mitochondrial red fluorescence signal is used to create surfaces shown as magenta around the mitochondria within the nerve surface which is shown in cyan.This allows the mitochondria in the nerve fibers to be seen in detail. Here, mitochondrial surface data are presented as a size frequency histogram to visualize the percentage of mitochondria that are present in each of the various bends according to their volume. While attempting this procedure, it's important to care for the tissue during the staining process to ensure that the tissue is fully submerged in the solutions to provide even and consistent staining throughout the sections.After watching this video, you should have a good understanding of how to visualize and quantify mitochondria within human intraepidermal nerve fibers. Our detailed protocol was designed to teach other investigators how to stain, image, process, and analyze human skin biopsies with the goal of understanding the mechanisms underlying the pathogenesis of mitochondrial-based neurologic diseases such as neuropathy.

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