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
<ol>
	<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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		<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 ...

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