This work was supported by grants from the Fritz Thyssen Foundation (10.11.2.153 to R.K.), the German Research Council (DFG, KR2119/3-2 and KR2119/8-1 to R.K.), the Federal Ministry for Education and Research (BMBF, NGFNplus; 01GS08134 to R.K.) and by a doctoral scholarship from the charitable Hertie Foundation [to L.F.B]. We thank Carolin Obermaier and Julia Westermeier for their support during the video shoot.

1. Skin Biopsy and Culturing of Human Fibroblasts
<ol>
	<li>The skin biopsy has to be taken by an experienced physician. The procedure takes place under sterile conditions and requires local anesthesia. Typical sites used for biopsy are the inner side of the upper arm, shoulder or lower back.</li>
	<li>You can take 4x4mm or 6x6mm diameter specimen by punch biopsy to get enough tissue for culturing human fibroblasts.</li>
	<li>Cut the skin biopsy into equal small pieces under sterile conditions to separate them into 2-4 T25 flasks. Every flask should contain 2-3 pieces of epidermis. The culture media contains 15% FCS, 1.1% sodium pyruvate and 1% penicillin/streptomycin in Roswell Park Memorial Institute (RPMI) 1640 medium.</li>
	<li>Incubate the cells at 37 &deg;C and 5% CO2. The expected propagation time until first fibroblasts grow out of the epidermal sample is 1-2 weeks. If growth problems are encountered, you can make use of fibroblast growth factor (5-10 ng/ml FGF2) to enhance growth of cells.</li>
	<li>Once a cell confluency of 50-70% is reached, the primary human fibroblasts can be frozen. The time until 50-70% confluency is reached, differs among cell lines. Freeze the fibroblasts in 90% FCS plus 10% dimethyl sulfoxide (DMSO).</li>
</ol>
2. Preparation of Human Fibroblasts for Live Cell Imaging Microscopy
In general, experiments with human fibroblasts should be performed with passages below 10 as senescence-associated alterations can change properties of cells after multiple passages. In case of doubt, beta-galactosidase assays can be used to assess the induction of ageing processes in the respective cells. Generally, do not use aged fibroblasts(see above). Furthermore, to compare several cell lines, work with the same passage number of each cell line. Comparison of different genetically homogenous patients compared to healthy controls is recommended. However, as sometimes patients carrying specific mutations can be rare, this can mean that skin biopsies from only one patient with a certain passage number and different control lines have to be included - here it is essential to ensure the same passage number of all lines included. If a cell confluency of 50-70% is reached but no experiment planned, fibroblasts should be frozen.
<ol>
	<li>On the first day of the experiment, wash the attached fibroblasts once with PBS. Then release them from the flask bottom by using a cell detachment solution of proteolytic DNase enzymes (i.e. AccuMax).</li>
	<li>Count your cells and seed 50,000-70,000 cells in each well of a 4-chamber coverglass (1.8 cm2 culture area per well). Accordingly, for the 8-chamber coverglas, half of the cell number or a little less is appropriate (0.8 cm2 culture area per well). It is important to seed rather a small number of cells to perform real single-cell analysis. No specific coating for the chambers is needed.</li>
	<li>After 24-48 hr, the live cell imaging experiment can be performed. Ensure that the live cell imaging microscope is equipped with an incubator that controls temperature and CO2 levels. During image acquisition, an atmosphere of 37 &deg;C and 5% CO2 should be maintained.</li>
	<li>Each live cell imaging measurement should be carried out in at least three independent experiments. In total, a minimum of 50 cells per cell line should be imaged. The live cell imaging experiments as well as the off-line analyses have to be performed under blinded conditions to guarantee unbiased data collecting.</li>
</ol>
3. Measurement of Mitochondrial Function in Living Human Fibroblasts
For conventional measurement of mitochondrial function via mitochondrial membrane potential (MMP)-dependent dyes, fluorescent activated cell sorting (FACS) is the primary method. In the case of human fibroblasts, cellular autofluorescence is frequently observed and may cause false positive results by interference with the actual staining signal. Autofluorescence typically becomes more pronounced with higher passage numbers of fibroblasts. Therefore, we prefer to assess mitochondrial function of human fibroblasts by live cell imaging microscopy.
<ol>
	<li>24-48 hr after seeding, incubate the fibroblasts in 50 nM of the MMP-dependent dye tetramethylrodamine ethyl ester (TMRE) in RPMI medium. This RPMI medium should lack phenol red as this can negatively affect image quality. 1.6 &mu;M of Hoechst staining should be added to visualize nuclei.</li>
	<li>Incubate cells in prepared staining solution protected from light for 15 min at 37 &deg;C and 5% CO2.</li>
	<li>Replace the staining solution with RPMI medium (without phenol red). Proceed without a washing step and immediately image cells. Use 63x magnification and confocal laser scanning microscopy technique (alternatively fluorescence microscopy using Apotome technique) to define single layers of cells. Apply 37 &deg;C and 5% CO2 to cells during imaging process.</li>
	<li>When imaging the fibroblasts, pay maximum attention to the applied exposure time of fluorescent light as TMRE staining can bleach out quickly. Define your appropriate exposure time between 200 and 300 ms and do not exceed this interval. The image acquisition under standardized settings is very important, as one would be unable to make comparisons based on intensity if the settings are different between cell lines.</li>
	<li>As a defined radius around the imaged cells is bleached out after light exposure, keep the next visual field adequately far from the photobleached section.</li>
	<li>Off-line analyses are performed using ImageJ Software (Wayne Rasband; National Institutes of Health, USA).</li>
	<li>Select your cell of interest by using a selection tool.</li>
	<li>Convert image to 8-bit (Image &lt; Type &lt; 8-bit).</li>
	<li>Reduce unspecific noise by using the "despeckle" function (Process &lt; Noise &lt; Despeckle).</li>
	<li>Choose the "Lookup Tables - Fire" condition (Image &lt; Lookup Tables &lt; Fire).</li>
	<li>Switch in the threshold function to the "Over/Under" function and adjust threshold (Image &lt; Adjust &lt; Threshold).</li>
	<li>Select the "analyze particles" option, which will give you the mean gray value for each mitochondrial particle detectable (Analyze &lt; Analyze Particles). Save the given result list as an Excel spreadsheet.</li>
	<li>Open result file in Excel program and calculate the average of the mean gray value of the mitochondrial structures (indicated as 'mean' in the given excel result list).</li>
	<li>To create a surface plot, choose the plugin "Interactive 3D surface plot" in the "3D" plugin section. Here, you can further adjust the plot using different display options. Changing the "Original Colors" to "Spectrum LUT" gives the intensity scale bar for the appropriate plot.</li>
	<li>Alternatively to using TMRE for analyzing the mitochondrial function of human fibroblasts via live cell imaging, a combination of the MMP-independent dye Mitotracker Green FM and the MMP-dependent dye Mitotracker CM-H2XRos can be used. Again, for human fibroblasts live cell imaging microscopy is preferred, but in general this alternative has also been used using FACS analysis in other kind of cells 12. When using this alternative approach to measure mitochondrial function in human fibroblasts, step 3-7 of the protocol section 5 "Measurement of mitochondrio-lysosomal colocalization in living human fibroblasts" should be applied. Therewith, the overlay of signals, both from mitochondria positive for Mitotracker Green FM and Mitotracker CM-H2XRos is calculated and indicates the amount of functional mitochondria as ratio to all mitochondria present.</li>
</ol>
4. Measurement of Mitochondrial Morphology in Living Human Fibroblasts
<ol>
	<li>24-48 hr after seeding, incubate cells in 200 nM Mitotracker Green FM in RPMI medium (no phenol red) protected from light for 15 min at 37 &deg;C and 5% CO2. 1.6 &mu;M of Hoechst staining should be used to highlight nuclei. Mitotracker Green FM is a MMP-independent dye, which stains all mitochondrial structures present.</li>
	<li>Gently wash the cells once with PBS.</li>
	<li>Add fresh RPMI medium (without phenol red) to cells and immediately image cells. Use 63x magnification and confocal laser scanning microscopy technique (alternatively fluorescence microscopy using Apotome technique) to define single layers of cells.</li>
	<li>Off-line analyses are taken out by the use of ImageJ Software.</li>
	<li>Select your cell of interest by using a selection tool.</li>
	<li>Convert image to 8-bit (Image &lt; Type &lt; 8-bit).</li>
	<li>Reduce unspecific noise by using the "despeckle" function (Process &lt; Noise &lt; Despeckle).</li>
	<li>Use the convolution filter to highlight mitochondrial structures (Process &lt; Filters &lt; Convolve).</li>
	<li>Adjust the threshold so that the signal-to-noise ratio is appropriate (Image &lt; Adjust &lt; Threshold).</li>
	<li>By using the "analyze particles" option, the automatic identification of mitochondrial structures is initiated based on a chosen pixel size (Analyze &lt; Analyze Particles). Several mitochondrial parameters are subsequently calculated such as area, perimeter, minor and major axes or circularity. These measurements can be set individually (Analyze &lt; Set Measurements). Save the given result list as an Excel spreadsheet.</li>
	<li>Open result file in Excel program. With use of such parameters, analyses of mitochondrial morphology can be carried out. This includes the definition of the form factor indicating the degree of branching of a mitochondrial network [formula: (perimeter2)/(4*PI()*area)] as well as the aspect ratio defining the length of mitochondria (formula: major axes / minor axes).</li>
</ol>
5. Measurement of Mitochondrio-lysosomal Colocalization in Living Human Fibroblasts
<ol>
	<li>24-48 hr after seeding, incubate cells in 200 nM Mitotracker Green FM and 100 nM Lyostracker Red DND-99 in RPMI medium (without phenol red) protected from light for 15 min at 37 &deg;C and 5% CO2. 1.6 &mu;M of Hoechst staining is used to highlight nuclei.</li>
	<li>Replace the medium with fresh RMPI medium (without phenol red) containing 100 nM Lyostracker Red DND-99. This dye has to be present during the entire imaging session. Do not wash the cells after incubation period. Immediately image the cells. Use 63x magnification and confocal technique (alternatively Apotome technique) to define single layers of cells.</li>
	<li>Off-line analyses are taken out by the use of ImageJ Software.</li>
	<li>Open the two respective images you would like to analyze in terms of colocalization status.</li>
	<li>Convert image to 8-bit (Image &lt; Type &lt; 8-bit).</li>
	<li>Reduce unspecific noise by using the "despeckle" function in both images (Process &lt; Noise &lt; Despeckle).</li>
	<li>Choose the plugin "JACoP" as colocalization analyzing tool, which gives you values for the respective channels and calculates the Pearson's coefficient, overlap coefficient and Manders' coefficient (Plugins &lt; JACoP).</li>
</ol>
6. Representative Results
Mitochondrial function in living human fibroblasts can be evaluated via live cell imaging technique. For functional assays, the TMRE fluorescence signal correlates with the mitochondrial membrane potential (MMP). Under normal MMP conditions, the TMRE fluorescence is significantly higher (Figure 1A, upper row) than under pathological conditions characterized by a reduced MMP (Figure 1A, lower row). This fluorescence intensity is measured by calculating the average of the mean gray value of every mitochondrial structure (Figure 1B). The fluorescence intensity can be measured via ImageJ software. Here, the use of the option "interactive surface plot" allows for the illustration of different peak intensities (Figure 1A, right side).
Next to the assessment of mitochondrial function in human fibroblasts, live cell imaging microscopy can be used to define different parameters of mitochondrial morphology. By using the Mitotracker Green FM dye mitochondrial structures are visualized independent of their respective MMP (Figure 2). This is important to ensure the inclusion of all mitochondrial structures present into the analyses. Using ImageJ Software, the morphology is analyzed on the basis of binary figures, as only here mitochondrial morphology parameters can be assessed (Figure 2, "binary"). Examples for mitochondrial morphology analyses in terms of form factor as well as aspect ratio were recently published 6, 13. Another important feature that can be measured in living human fibroblasts is the degradation of mitochondrial structures. The first step in this progress can be assessed by mitochondrio-lysosomal colocalization in live cell imaging microscopy. Certain pathological conditions result in an increased colocalization of mitochondria with lysosomes (Figure 3A). Again, Mitotracker Green FM is used to stain mitochondrial structures independent of their respective MMP. In addition, staining with Lysotracker Red DND-99 allows visualizing lysosomal structures. It is critical that the Lysotracker Red dye is present during the imaging process, as washing steps reduce the signal that becomes too low for visualization. Colocalization is represented by clear yellow fluorescence signals due to the overlap of the green (Mitotracker Green FM) and red (Lysotracker Red DND-99) staining (white arrows, Figure 3A) and are determined by calculation of the Pearson coefficient (Figure 3B).
In general, for off-line analyses with ImageJ software, it is helpful to visualize one cell per image, so that the definition of a specific region of interest (ROI) can be avoided.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/4228/4228fig1highres.jpg" src="/files/ftp_upload/4228/4228fig1.jpg" /> 
 Figure 1. Measurement of the TMRE fluorescence signal indicating the mitochondrial membrane potential (MMP) in living human fibroblasts by live cell imaging technique. (A) TMRE is a fluorescent MMP-dependent dye, whose fluorescence intensity correlates with the MMP. Hoechst dye was used to stain nuclei (blue). Under normal MMP conditions (upper row), the TMRE fluorescence is significantly higher than under pathological conditions characterized by a reduced MMP (lower row). Fluorescence intensity is additionally shown as intensity surface plot. Scale bar indicates 10 &mu;m. (B) Statistical analyses of the exemplary fibroblasts shown in (A). Under pathological conditions, a decreased TMRE fluorescence of mitochondria is measured compared to the normal MMP condition. <a href="https://www.jove.com/files/ftp_upload/4228/4228fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/4228/4228fig2.jpg" /> 
 Figure 2. Measurement of the mitochondrial morphology in living human fibroblasts by live cell imaging technique. Mitotracker Green FM (green) is used to visualize mitochondrial structures, for that it is independent of the MMP. Hoechst dye was used to stain nuclei (blue). Using ImageJ Software, the morphology can be analyzed on binary figures. Scale bar indicates 10 &mu;m. <a href="https://www.jove.com/files/ftp_upload/4228/4228fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/4228/4228fig3highres.jpg" src="/files/ftp_upload/4228/4228fig3.jpg" /> 
 Figure 3. Measurement of the colocalization of mitochondria with lysosomes in living human fibroblasts by live cell imaging technique. (A) Mitotracker Green FM (green) is used to stain mitochondrial structures, Lyostracker Red DND-99 (red) visualizes lysosomal structures. Hoechst dye was used to stain nuclei (blue). Successful colocalization under pathological conditions is indicated by a yellow signal due to overlap of Lysotracker Red and Mitotracker Green staining (white arrows). Scale bar indicates 10 &mu;m. (B) Statistical analyses of the exemplary fibroblasts shown in (A). A higher degree of mitochondrio-lysosomal colocalization results in an elevated Pearson coefficient value. <a href="https://www.jove.com/files/ftp_upload/4228/4228fig3large.jpg" target="_blank">Click here to view larger figure</a>.

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No conflicts of interest declared.

Patient skin fibroblasts as ex vivo models represent an important tool to characterize disease-associated genetic defects. In addition, skin-derived fibroblasts are easily accessible and can be expanded upon culturing. Therefore, primary cells obtained from patients carrying PD-associated genetic mutations are preferable over the use of tumor cell lines as they contain not only the endogenous disease-causing gene, but the whole genetic background of the affected individual. Moreover, the fibroblasts have ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue no.</td>
 </tr>
 <tr>
 <td>Roswell Park Memorial Institute (RPMI) 1640 medium</td>
 <td>Invitrogen</td>
 <td>52400-025</td>
 </tr>
 <tr>
 <td>RPMI 1640 medium, no Phenol Red</td>
 <td>Invitrogen</td>
 <td>11835-063</td>
 </tr>
 <tr>
 <td>Dulbecco's Phosphate-Buffered Saline (DPBS)</td>
 <td>Invitrogen</td>
 <td>14190-094</td>
 </tr>
 <tr>
 <td>Fibroblast growth factor 2 (FGF2)</td>
 <td>PeproTech</td>
 <td>100-18B</td>
 </tr>
 <tr>
 <td>AccuMax (detachment solution)</td>
 <td>PAA</td>
 <td>L11-008</td>
 </tr>
 <tr>
 <td>Lab-TekTMII chambered coverglasses</td>
 <td>Nalge Nunc International</td>
 <td>115382</td>
 </tr>
 <tr>
 <td>Tetramethylrodamine ethyl ester (TMRE)</td>
 <td>Invitrogen</td>
 <td>T-669</td>
 </tr>
 <tr>
 <td>Mitotracker Green FM</td>
 <td>Invitrogen</td>
 <td>M-7514</td>
 </tr>
 <tr>
 <td>Mitotracker CM-H2XRos</td>
 <td>Invitrogen</td>
 <td>M-7513</td>
 </tr>
 <tr>
 <td>Lyostracker Red DND-99</td>
 <td>Invitrogen</td>
 <td>L-7528</td>
 </tr>
 <tr>
 <td>Hoechst 33342</td>
 <td>Invitrogen</td>
 <td>H-3570</td>
 </tr>
 </tbody>
</table>
Table 1. Specific reagents and equipment.

the use of primary human fibroblasts for monitoring mitochondrial phenotypes in the field of parkinson's disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

Watch this Scientific Journal Video about The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease at JoVE.com

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

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JoVE Journal

Medicine

16.7K 조회수.  DZNE. 파킨슨 병을 일으키는 유전자에 변이를 가지고 환자의 섬유 아세포가 쉽게 접근 할 수를 나타냅니다 예 생체 모델입니다. 라이브 셀 이미징은 살아있는 세포에서 형태학 및 기능 매개 변수를 연구 할 수있는 기회를 제공합니다. 여기 인간 섬유 아세포와 mitochondrial phenotypes의 후속 모니터링의 준비에 대해 설명합니다.

비디오: The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson&#039;s Disease

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson&#039;s Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Controlling Parkinson&#039;s Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Developing Neuroimaging Phenotypes of the Default Mode Network in PTSD: Integrating the Resting State, Working Memory, and Structural Connectivity

Complementary structural and functional neuroimaging techniques used to examine the Default Mode Network (DMN) could potentially improve assessments of psychiatric illness severity and provide added validity to the clinical diagnostic process. Recent neuroimaging research suggests that DMN processes may be disrupted in a number of stress-related psychiatric illnesses, such as posttraumatic stress disorder (PTSD).
Although specific DMN functions remain under investigation, it is generally thought to be involved in introspection and self-processing. In healthy individuals it exhibits greatest activity during periods of rest, with less activity, observed as deactivation, during cognitive tasks, e.g., working memory. This network consists of the medial prefrontal cortex, posterior cingulate cortex/precuneus, lateral parietal cortices and medial temporal regions.
Multiple functional and structural imaging approaches have been developed to study the DMN. These have unprecedented potential to further the understanding of the function and dysfunction of this network. Functional approaches, such as the evaluation of resting state connectivity and task-induced deactivation, have excellent potential to identify targeted neurocognitive and neuroaffective (functional) diagnostic markers and may indicate illness severity and prognosis with increased accuracy or specificity. Structural approaches, such as evaluation of morphometry and connectivity, may provide unique markers of etiology and long-term outcomes. Combined, functional and structural methods provide strong multimodal, complementary and synergistic approaches to develop valid DMN-based imaging phenotypes in stress-related psychiatric conditions. This protocol aims to integrate these methods to investigate DMN structure and function in PTSD, relating findings to illness severity and relevant clinical factors.

This protocol describes the complementary neuroimaging techniques of resting state structural connectivity, task-induced deactivation, and structural connectivity analyses to examine the default network in post-traumatic stress disorder. The use of synergistic methods could potentially lead to improved diagnostics and assessments of severity, outcome, and other relevant clinical factors.

Complementary structural and functional neuroimaging techniques used to examine the Default Mode Network (DMN) could potentially improve assessments of ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...