We thank Parkinson Schweiz and ABREOC (the Scientific Research Advisory Board of the Ente Ospedaliero Cantonale) for their financial support of this study.

The protocol has been approved by the Cantonal Ethics Committee and all enrolled subjects gave written informed consent to the study.
1. Skin Biopsy Collection
<ol>
	<li>Let a qualified physician perform the skin biopsy in an appropriate clinical setting.</li>
	<li>Choose the area to perform the skin biopsy and clean it with an alcohol swab.</li>
	<li>Prepare the anesthetic solution with 1 cc of lidocaine 2%.</li>
	<li>With the needle parallel to the area, inject local anesthesia subcutaneously.</li>
	<li>After checking the effect of anesthesia, take the 3 mm disposable punch and apply rotational and delicate downward pressure to rotate it down through the epidermis and dermis, until the subcutaneous fat is reached.</li>
	<li>Withdraw the punch.</li>
	<li>Use disposable forceps to gently pull the skin plug up.</li>
	<li>Use scissors to cut out the base of the specimen at the level of the fatty tissue.</li>
	<li>Place the specimen in a tube containing 10 mL of periodate-lysine-paraformaldehyde (PLP) fixative solution.</li>
	<li>Disinfect the area and cover it with an adhesive bandage.</li>
</ol>
2. Tissue Fixation and Storage
<ol>
	<li>Make fresh PLP fixative solution (see Table 1).</li>
	<li>Immediately after the collection of the skin biopsy, submerge it in a tube containing 10 mL of PLP fixative solution and incubate it overnight (O/N) at 4 &#176;C.</li>
	<li>The day after, under the fume hood, remove the PLP fixative gently and in the same tube, wash the biopsy 3 times for 5 min with 5 mL of 0.1 M Sorensen&#8217;s solution (Table 1).</li>
	<li>Discard the Sorensen&#8217;s solution and incubate the biopsy with 5 mL of cryo-protectant solution O/N at 4 &#176;C.</li>
	<li>Store the biopsy at 4 &#176;C if the cut with a cryotome is performed within 1 week; store the biopsy at -20 &#176;C if the cut is performed within 3 months; embed the biopsy in a cryomold (steps 3.2-3.4) and store it at -80 &#176;C to conserve it for a longer period of time.</li>
</ol>
3. Tissue Cut with a Cryotome
<ol>
	<li>Set the cryotome at -20 &#176;C.</li>
	<li>Take a cryomold and fill it up with the cryo-embedding medium. Avoid creating bubbles.</li>
	<li>Using tweezers immerse the biopsy into the cryo-embedding medium with the longitudinal axis (epidermis - dermis) parallel to the bottom of the cryomold.</li>
	<li>Snap freeze the sample with liquid nitrogen to obtain a solid cube of cryo-embedding medium containing the biopsy in the right orientation.</li>
	<li>Put the sample in the cryostat and wait for 30 min to allow the biopsy to acclimatize.</li>
	<li>Fix the sample on the cryostat and cut 50 &#181;m sections.</li>
	<li>With the help of a little brush, transfer the cryo-sections in a 96 well plate containing 200 &#181;L of antifreeze solution in each well. One section per well.</li>
	<li>Store at -20 &#176;C. 
	NOTE: If the analyze the entire biopsy is not analyzed, cut it only partially. In this case, the sections must be stored at -20 &#176;C and the remaining part of the biopsy at -80 &#176;C to conserve it for a longer period of time.</li>
</ol>
4. Immunofluorescence Staining
<ol>
	<li>Fill a 96 well plate, with 100 &#181;L of washing solution.</li>
	<li>Transfer the sections to be analyzed from the storage plate to the new one containing the washing solution. 1 section per each well (4 sections per anatomical site, per patient: usually a total of 12 sections per patient).</li>
	<li>Leave the section in the washing solution for 10 min at room temperature (RT). Transfer the section into another well containing the same solution and repeat the wash.</li>
	<li>Move the sections into new wells containing 100 &#181;L of Blocking solution and incubate for at least 90 min and for a maximum of 4 h at RT.</li>
	<li>Dilute the primary antibodies anti-PGP9.5 (Rabbit polyclonal, 1:1000) and anti-5G4 (Mouse monoclonal, 1:400) in the working solution.</li>
	<li>Transfer the sections into new wells containing 100 &#181;L of the working solution of the primary antibodies and incubate them O/N at RT.</li>
	<li>As step 4.3, wash the sections 2 times for at least 10 min at RT, with 100 &#181;L of washing solution.</li>
	<li>Dilute the secondary antibodies (conjugate with different fluorophores) Goat anti-Rabbit to detect PGP9.5 (1:700) and a Goat anti-Mouse to detect 5G4 (1:700) in the working solution.</li>
	<li>Transfer the sections into new wells containing 100 &#181;L of the working solution of the secondary antibodies for 90 min at RT. From this point, cover the 96 well plate with aluminum foil to avoid the bleaching of fluorophore conjugated with secondary antibody/ies.</li>
	<li>As step 4.3, wash the sections 2 times for at least 10 min, with 100 &#181;L of the washing solution.</li>
	<li>Transfer the sections into new wells containing 100 &#181;L of DAPI (diluted 1:5,000 in PBS 1x) for 5 min at RT.</li>
	<li>As step 4.3, wash the sections 2 times for at least 10 min, with 100 &#181;L of the washing solution.</li>
	<li>Mount the sections on a slide in the correct position avoiding misfolding.</li>
	<li>Add a few drops of the mounting medium on the slide and cover with a coverslip.</li>
	<li>Let slides dry O/N before using the confocal/fluorescence microscope.</li>
	<li>Store the slides in an appropriate box at 4 &#176;C avoiding the light exposure. If accurately stored, the signal will be visible for about 6 months. 
	NOTE: The transfer of a section from the 96 well plate containing the newly cut slices to the 96 well plate containing the washing solution (step 4.1) is performed using a small brush that helps to pick up the biopsy. After the transfer of the section into the first well, every time the slice needs to be incubated with a different solution, move it from well to well with the help of the brush.</li>
</ol>
5. Immunofluorescence Imaging
<ol>
	<li>View sections under an inverted fluorescence microscope or a confocal microscope (20X, 40x or higher magnification, use successive frames of 2 &#181;m increments on a Z-stack plan for best results).</li>
	<li>Acquire images by a microscope connected camera</li>
	<li>Use an adequate imaging software (i.e. ImageJ) to analyze positive signals in sections in terms of spatial distribution and intensity of the signal. To do this perform the steps mentioned below.
	<ol>
		<li>Open the file with ImageJ software.</li>
		<li>Click on Image &#62; color &#62; split channels in order to analyze each color channel.</li>
		<li>Click Image &#62; stack &#62; Z project to obtain a merge of multiple section acquisitions.</li>
		<li>Click Image &#62; color &#62; merge channels to obtain a merge of different color channels of the same acquisition.</li>
	</ol>
	</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Antifreeze solution 
			(store at 4 &#176;C for up to 6 months)</td>
			<td>30% Glycerol 
			30% Ethylene glycol 
			30% dH2O 
			10% 2x Phosphate buffer</td>
		</tr>
		<tr>
			<td>Blocking solution 
			(prepare at the moment)</td>
			<td>4% Normal Goat Serum 
			1% Triton X-100 
			in Washing solution</td>
		</tr>
		<tr>
			<td>Cryo-protectant 
			(store at 4 &#176;C for up to 6 months)</td>
			<td>20% Glycerol 
			80% Sorensen&#39;s solution</td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate solution 
			(store at RT up to 6 months)</td>
			<td>0.45M Disodium hydrogen phosphate (Na2HPO4) in dH2O 
			Filter in a sterile bottle</td>
		</tr>
		<tr>
			<td>Lysine solution 
			(store at 4 &#176;C for up to 3 weeks)</td>
			<td>50% of 0.3M L-Lysine monohydrochloride solution 
			50% of 0.1M Sorensen&#39;s solution pH7.6 
			pH 7.4, filter in a sterile bottle</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA) 8% 
			(prepare under fume hood and store at 4 &#176;C for up to 1 month)</td>
			<td>2.6M PFA in dH2O (to 55&#176;C_do not exceed 60 &#176;C<nr> to avoid formic acid formation) 
			filter in a sterile bottle</nr></td>
		</tr>
		<tr>
			<td>Phosphate buffer 2x 
			(store at 4 &#176;C for up to 6 months)</td>
			<td>6% Monosodium phosphate (NaH2PO4) solution 
			45% Disodium phosphate (Na2HPO4) solution in dH2O</td>
		</tr>
		<tr>
			<td>PLP fixative solution 
			(prepare at the moment, under fume hood )</td>
			<td>25% Paraformaldehyde 8% 
			0.01M Sodium (meta)periodate 
			75% Lysine solution&#60;</td>
		</tr>
		<tr>
			<td>Sodium Dihydrogen Phosphate Monohydrate 
			(store at RT up to 6 months)</td>
			<td>0.52M Sodium Dihydrogen Phosphate Monohydrate (NaH2PO4*H2O) in dH2O 
			Filter in a sterile bottle.</td>
		</tr>
		<tr>
			<td>Sorensen&#39;s solution 
			(store at 4 &#176;C for up to 1 month)</td>
			<td>2.5% Monosodium phosphate solution 
			18.7% Disodium phosphate solution 
			pH7.6, in dH2O</td>
		</tr>
		<tr>
			<td>Washing solution 
			(prepare at the moment)</td>
			<td>0.25M Trizma base 
			0.26M NaCl 
			pH7.6, in dH2O</td>
		</tr>
		<tr>
			<td>Working solution 
			(prepare at the moment)</td>
			<td>50% Blocking solution 
			50% Washing solution</td>
		</tr>
	</tbody>
</table>
Table 1: Required solutions. List of required solutions for the protocol and brief description of how to prepare it.

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraepidermal+nerve+fiber+density+in+patients+with+painful+sensory+neuropathy.">Intraepidermal nerve fiber density in patients with painful sensory neuropathy.</a> Neurology. 48, 708-711 (1997).</li><li>McArthur, J. C., Stocks, E. A., Hauer, P., Cornblath, D. R., Griffin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+nerve+fiber+density:+normative+reference+range+and+diagnostic+efficiency.">Epidermal nerve fiber density: normative reference range and diagnostic efficiency.</a> Archives of Neurology. 55 (12), 1513-1520 (1998).</li><li>Lauria, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=European+Federation+of+Neurological+Societies/Peripheral+Nerve+Society+Guideline+on+the+use+of+skin+biopsy+in+the+diagnosis+of+small+fiber+neuropathy.+Report+of+a+joint+task+force+of+the+European+Federation+of+Neurological+Societies+and+the+Peripheral+Nerve+Society.">European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. 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The authors have nothing to disclose.

We describe a free-floating immunofluorescence assay for skin biopsies for the diagnosis of PD: it exploits double immunostaining with anti-PGP9.5 antibody, a panaxonal marker, and anti-5G4, a conformation specific antibody that recognizes the aggregated form of &#945;Syn.
The great advantages of skin biopsy for diagnostic purpose in PD and possibly in other protein conformational disorders are: 1) the direct access to nervous tissue prone to disease by a mildly invasive technique and thus wit...

Skin biopsy has acquired a great importance as the diagnostic and research tool in the field of neurological disorders1. Indeed, epidermis and dermis contain abundant somatic sensory nerve fibers (myelinated and unmyelinated), nociceptive free nerve endings, sensory receptors and autonomic innervation of sweat glands, vessels, sebaceous glands and muscle arrector pilorum2.
In the mid-20th century, the setup for immunohistochemistry of PGP9.5 antibody allowed the evidence of an extensive innervation of human epidermis mammalian skin3. PGP9.5 is a carboxyl-terminal hydrolase equally distributed along axons of both the central and peripheral nervous system (PNS). The availability of this antibody allowed not only to clarify the morphology and anatomy of PNS in the skin but also implemented the study of diseases associated with it3,4. Skin biopsy contributed to defining a new clinical entity: the small fiber neuropathy. Several international groups demonstrated the association between the loss of intraepidermal nerve fibers and symptoms/signs of small fiber neuropathy5 by skin biopsy analysis and provided standardized protocols for nerve morphometry as well as normative reference values to be used in the clinical practice6,7,8.
Recently a large amount of evidence has shown that neurodegenerative diseases, characterized by misfolded protein accumulations in the central nervous system, are multi-system pathologies9. Indeed, PD is characterized by &#945;Syn accumulation in the dopaminergic neuron of substantia nigra, but it has been demonstrated that &#945;Syn and its pathological form, phosphorylated &#945;Syn (P-&#945;Syn), could be detected also in the peripheral tissues. Gastro-intestinal mucosa10, salivary glands11, skin autonomic fibers surrounding sweat glands and pilomotor muscles12,13,14, show immunoreactivity to pathogenic forms of &#945;Syn, in accordance with Braak hypothesis that intriguingly postulate that &#945;Syn pathology may begin in PNS well in advance, before its accumulation in the brain15. Further, the presence of p-&#945;Syn has been demonstrated in skin nerves of patients with REM Behavior Disorders that are considered prodromal PD16,17 thus skin pathologic &#945;Syn can be considered a promising early peripheral histopathological marker of synucleinopathy.
The association of small fiber neuropathy in PD has been demonstrated previously and it has been found that intraepidermal nerve fibers density reflects disease progression18,19. Hence, the skin biopsy is a useful tool for studying neurodegeneration in PD and for establishing a pre-mortem histopathological diagnosis of the disease. Indeed, skin biopsy has a great advantage of being an easily accessible and minimally invasive procedure, allowing the analysis of nervous tissue prone to the pathology. Finally, the possibility of repeating the skin biopsy in the course of follow-up of the same patients allows studying the longitudinal correlation with disease progression.
In our laboratory, exploiting a double immuno-staining with PGP9.5 and the conformation-specific monoclonal 5G4 antibody, that recognizes disease specific forms of &#945;Syn including small aggregates20,21, we were able to show the presence of &#945;Syn aggregates in skin nerves with a promising high diagnostic efficiency19. Immunofluorescence analysis of the skin biopsy in conformational diseases stands out as a promising source of biomarkers by combining both the detection of protein aggregates and the measure of the neurodegeneration in vivo. Hereafter, we illustrate an easy and versatile protocol on handling the skin biopsy and performing the free-floating immunofluorescence staining for detecting &#945;Syn aggregates. Moreover, this protocol can be adapted for targeting any other protein of interest expressed in skin PNS.
The following study protocol has been used to evaluate the diagnostic utility of aggregated &#945;Syn analysis in the PNS of PD by skin biopsy19. Inclusion criteria for PD were: a definite clinical diagnosis according to the UK Brain Bank diagnostic criteria, disease duration at least 3 years, no family history, and no major cognitive impairment or major dysautonomic symptoms in the history. Exclusion criteria were known causes of neuropathy (glycated hemoglobin, creatinine, vitamin B12, TSH, serum immunofixation, HIV, HCV, syphilis, and borreliosis). Each subject underwent to 3 mm-diameter skin biopsies at three anatomical sites (neck at C8 dermatomal level, thigh 10 cm above the knee, leg 10 cm above lateral malleolus) on the side, which was clinically more affected. In general, the following protocol is about handling the skin biopsy and performing the free-floating immunofluorescence staining and analysis. Hence it can be adapted and used for the detection of other proteins of interest in skin tissue.

<table border="0" cellpadding="0" cellspacing="0" style="width:964px;" width="964">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:264px;">5G4 (anti human &#945;Syneclein 5G4)</td>
			<td style="width:161px;">Analytik Jena Roboscreen</td>
			<td style="width:344px;">847-0102004001</td>
			<td style="width:195px;">Mouse monoclonal&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AlexaFluor 488 Goat anti Rabbit IgG&#160;</td>
			<td>Invitrogen</td>
			<td>1971418</td>
			<td>2mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AlexaFluor 594 Goat anti Mouse IgG&#160;</td>
			<td>Invitrogen</td>
			<td>1922849</td>
			<td>2mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disodium hydrogen phosphate solution</td>
			<td>Merk Millipore</td>
			<td>106586</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethylene Glycol</td>
			<td>Sigma-Aldrich</td>
			<td>324558</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G7757</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Lysine monohydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>L5626</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde</td>
			<td>Aldrich Chemistry</td>
			<td>441244</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:264px;">PGP9.5</td>
			<td>Abcam</td>
			<td>ab15503</td>
			<td>Rabbit polyclonal</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Chloride</td>
			<td>Sigma&#160;</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Dihydrogen Phosphate Monohydrate</td>
			<td>Merck Millipore</td>
			<td>106346</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium (meta)periodate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S1878</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trizma Base</td>
			<td>Sigma&#160;</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tryton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vectashield&#160;</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Mounting medium</td>
		</tr>
	</tbody>
</table>

targeting alpha synuclein aggregates in cutaneous peripheral nerve fibers by free-floating immunofluorescence assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Following the described procedure (Figure 1), we detected &#945;Syn aggregates, labeled with 5G4 antibody, in dermal nerve fascicles innervating autonomic structures of PD patients. Morphology of alpha-synuclein deposits appears as a dotted signal along the axons of dermal nerves (Figure 2). Indeed, exploiting this protocol in 19 PD patients and 17 controls in skin biopsies at three anatomical site (cervical, thigh and distal leg) we found that 5G4 had 81% of se...

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Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

targeting-alpha-synuclein-aggregates-cutaneous-peripheral-nerve

Research

JoVE Journal

Neuroscience

8.1K Views.  Neurocenter of Southern Switzerland. Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

Video: Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.