Name: a human fallopian tube model for investigation of c. trachomatis infections
Uploaded: 2012-08-11T14:00:00
Duration: 9 min 11 s
Description: Watch this Scientific Journal Video about A Human Fallopian Tube Model for Investigation of C. trachomatis Infections at JoVE.com

This work was supported by the DFG-Cluster of Excellence "Inflammation at Interfaces" (RA-If, RA-D). We are grateful for the excellent technical assistance of Kristin Wischnat.

1. Preparation of Chlamydia trachomatis Serovar D Stock
<ol>
 <li> Infect confluent 175 cm2 cell culture flask containing HeLa-229 cells with 4 ml 
 C. trachomatis D in SPG buffer. </li>
 <li> Incubate for one hour at 37 &deg;C during constant shaking. </li>
 <li> Add 20 ml DMEM with 10% FCS and 240 &mu;l Cycloheximide. </li>
 <li> Incubate in a humidified incubator for 48 h at 37 &deg;C and 5% CO2. </li>
 <li> Add 5 ml sterile glass beads, strong shake to detach and destroy C. trachomatis harboring HeLa-229 cells. </li>
 <li> Collect supernatant and add another 5 ml of sterile glass beads. </li>
 <li> Rock three times for 1 min to destroy all HeLa-229 cells and release 
 C. trachomatis. </li>
 <li> Centrifuge for 5 min at 1000 rpmi and 4 &deg;C to get rid of cellular debris. </li>
 <li> Collect the supernatant for infection of 8 to 10 175 cm2 cell culture flasks confluent grown with HeLa 229 cells. </li>
 <li> Incubate every flask with 4 ml SPG buffer and 4 ml supernatant from previous infection for one hour at 37 &deg;C with constant shaking. </li>
 <li> After one hour add 15 ml DMEM with 10% FCS and 230 &mu;l cycloheximide followed by 48 h incubation at 37 &deg;C and 5% CO2. </li>
 <li> Add 5 ml sterile glass beads, shake to detach and destroy C. trachomatis harboring HeLa-229 cells. </li>
 <li> Collect the supernatant and add another 5 ml of sterile glass beads. </li>
 <li> Rock three times for 1 min to destroy all HeLa-229 cells and release 
 C. trachomatis. </li>
 <li> Centrifuge for 5 min at 1000 rpmi at 4 &deg;C to get rid of cellular debris. </li>
 <li> Collect the supernatant. </li>
 <li> Fill 40 ml of supernatant in 50 ml Falcon tubes, centrifuge for 99 min with 
 11000 rpmi. </li>
 <li> After centrifugation discard the supernatant and wash the pellet with 1 ml SPG buffer. </li>
 <li> Homogenize the pellet in 1 ml SPG and aliquot 20 &mu;l in a 1, 5 ml Eppendorf tube, store at -70 &deg;C until use. Biological activity of the prepared stock was confirmed in an established HeLa-229 cell infection model.</li>
</ol>
2. Preparation of Human Fallopian Tube 
<ol>
 <li> Store human fallopian tubes (HFT) from women undergoing hysterectomy immediately after surgery in RPMI containing 5% FCS without antibiotics in 4 &deg;C until preparation. The protocol was approved by the ethical committee of the University of L&uuml;beck (09-153). Women included in the study (age 34 until 53) had no history of prior C. trachomatis infection. Tissue of the Fallopian tubes was collected at peripartum sterilisation on maternal request during caesarean sections or during hysterectomies due to symptomatic uterine fibroids in the luteal phase. All HFT were tested negative for C. trachomatis by PCR. </li>
 <li> Dissect the HFT in a Petri dish containing RPMI with 5% FCS without antibiotics. During the whole processing the tissue should be submerged in media to prevent drying. </li>
 <li> Cut off and discard connective tissue and tissue destroyed during surgery. </li>
 <li> After dissection open the HFT carefully with a small scalpel. </li>
 <li> For further experiments prepare pieces of tissue in the size of 0.5 cm x 0.5 cm up to 1 cm x 1 cm. </li>
 <li> For infection store HFT specimen in 1 ml RPMI containing 5% FCS without antibiotics. HFT specimens were infected with 5.5x105 IFU (inclusion forming units) of C. trachomatis. </li>
 <li> Incubate HFT specimen in 37 &deg;C, 5% CO2 and 21% O2 as well as in 37 &deg;C, 5% CO2 and 2% O2 for 24 h. </li>
 <li> After incubation time collect specimen and store in Monti's fixative for at least three days at 4 &deg;C until SEM/TEM preparation. </li>
</ol>
3. Preparation of HFT Specimen for TEM
<ol>
 <li> For TEM remove specimen from Monti's fixative and cut 2 x 2 mm to 4 x 5 mm pieces. The remaining tissue can be used for SEM. </li>
 <li> Wash pieces with sodium cacodylate buffer, pH 7.35 for 1 h. </li>
 <li> Incubate in 1% (w/v) osmium tetroxide in aqua dest. overnight. </li>
 <li> Remove excess osmium tetroxide by washing 6x5 min in sodium cacodylate buffer, pH 7.35.</li>
 <li> Remove water by incubating in increasing concentrations of ethanol in water (v/v) (30% for 2 h, 40% for 2 h, 50% for 2 h, 60% for 2 h, 70% over night, 80% for 2 h , 90% for 1 h, 95% for 1 h, 100% for 1 h. </li>
 <li> Wash out ethanol for 2 x 15 min in propylene oxide and subsequently transfer to a mixture of 50% (v/v) freshly prepared araldite (including all components) in propylene and incubate overnight. </li>
 <li> Transfer to freshly prepared araldite (including all components) for at least 1 h. </li>
 <li> Transfer to flat embedding molds and fill with araldite. </li>
 <li> Let araldite harden for at least 48 h at 60 &deg;C </li>
 <li> After trimming of embedded sample cut semi thin sections (700 nm) on an ultramicrotome using glass knifes or a histo diamond knife and stain with Richardson's stain. </li>
 <li> Cut ultra-thin sections (approx. 70 nm) using a diamond knife and transfer to copper grids (e.g. 150 square mesh). </li>
 <li> Stain ultra-thin sections with 0.5% (w/v) uranyl acetate in aqua dest. followed by 3% (w/v) lead citrate in aqua dest. in an automatic section stainer. Alternatively, sections can be stained manually by leaving grids 15 min on a saturated centrifuged solution of uranyl acetate in aqua dest. followed by 0.3% lead citrate (w/v) in aqua dest. </li>
</ol>
4. Preparation of HFT specimen for SEM
<ol>
 <li> For SEM remove specimen from Monti's fixative, orient with epithelium facing upwards on a 1.5 x 1 cm cork sheet and fix position using insect needles. </li>
 <li> Transfer specimen on cork to suitable metal baskets and wash for 30 min in sodium cacodylate buffer, pH 7.35. </li>
 <li> Remove water by incubating specimen in increasing concentration of acetone/water mixtures (v/v) 30% for 6 h, 40% for 6 h, 60% for 8 h, 70% over night, 80% for 2 h, 90% for 2 h and 100% over night (be extremely cautious to keep specimen continuously submerged). </li>
 <li> Transfer to fresh 100% acetone and dry specimen by critical point drying (temperature 40 &deg;C, pressure 80 bar). </li>
 <li> Remove needles, cork and glue specimen with the epithelium facing upwards on SEM specimen mount equipped with a conductive carbon tab using conductive carbon cement. Use liquid conductive silver to enhance conductivity between the mount and the conductive carbon tab.</li>
 <li> Prepare a conductive platinum, gold or palladium coating of the specimen using a sputter coater. </li>
</ol>
5. Staining of Semi Thin Sections with Richardson's Stain
<ol>
 <li> Let semi thin sections dry on slide. </li>
 <li> Stain sections with staining solution at 60 &deg;C for 1-2 min. </li>
 <li> Wash in aqua dest.</li>
 <li> Dry sections and coverslip. </li>
</ol>
6. Representative Results
Within the human fallopian tube infection model we were able to visualize differences in the epithelial morphology between the non-infected controls (Figure 1) and the C. trachomatis- infected tubes (Figure 2) under normoxic condition. Characteristic morphological differences implied pathogen- induced swelling and lysis of fallopian tube epithelial cells that could not be detected in non-infected controls. Using semi thin sections and transmission electron microscopy we were able to prove intracellular C. trachomatis inclusion formation in the ex vivo infected human fallopian tube tissue (Figure 3, 5) but not in non-infected controls (Figure 4). As oxygen concentrations in the female genital tract are already low under physiological conditions 11, and further decrease during an inflammatory process we also investigated our model under hypoxic conditions. Preliminary results indicate the model is useful to analyze chlamydial growth and progeny under varying oxygen concentrations. Chlamydia- induced epithelial cell damage has to be separated from artifacts that are mediated through incautious preparation and handling of the fallopian tubes before infection (Figure 6).
<img src="/files/ftp_upload/4036/4036fig1.jpg" alt="Figure 1" /> 
Figure 1. Scanning electron microscopy (SEM) of a non-infected human fallopian after one day incubation in control medium (green arrow show ciliated cell, blue arrow show non- ciliated cell).
<img src="/files/ftp_upload/4036/4036fig2.jpg" alt="Figure 2" /> 
Figure 2. Scanning electron microscopy (SEM) of an ex vivo C. trachomatis infected human fallopian tube one day post infection (white arrow mark ruptured inclusion). <img src="/files/ftp_upload/4036/4036fig3.jpg" alt="Figure 3" /> 
Figure 3. Semi thin sections show typical intracellular inclusions (white arrows) 1d after infection of the human fallopian tube with C. trachomatis ex vivo.
<img src="/files/ftp_upload/4036/4036fig4.jpg" alt="Figure 4" /> 
Figure 4. Transmission electron microscopy (TEM) of a non-infected human fallopian tube after one day incubation in control medium.
<img src="/files/ftp_upload/4036/4036fig5.jpg" alt="Figure 5" /> 
Figure 5. Transmission electron microscopy (TEM) of an ex vivo C. trachomatis infected human fallopian tube one day post infection (white arrows mark chlamydial inclusions).
<img src="/files/ftp_upload/4036/4036fig6.jpg" alt="Figure 6" /> 
Figure 6. Scanning electron microscopy (SEM) of fallopian tube epithelium damaged through incautious preparation and handling of specimen (white arrows mark destructed epithelial tissue).

<ol>
	<li>Dean, D., Suchland, R. J., Stamm, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+long-term+cervical+persistence+of+Chlamydia+trachomatis+by+omp1+genotyping.">Evidence for long-term cervical persistence of Chlamydia trachomatis by omp1 genotyping.</a> J. Infect. Dis. 182, 909-916 (2000).</li><li>Campbell, L. A., Patton, D. L., Moore, D. E., Cappuccio, A. L., Mueller, B. A., Wang, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Chlamydia+trachomatis+deoxyribonucleic+acid+in+women+with+tubal+infertility.">Detection of Chlamydia trachomatis deoxyribonucleic acid in women with tubal infertility.</a> Fertil. Steril. 59, 45-50 (1993).</li><li>Peipert, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Genital+chlamydial+infections.">Clinical practice. Genital chlamydial infections.</a> N. Engl. J. Med. 349, 2424-2430 (2003).</li><li>Mardh, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubal+factor+infertility,+with+special+regard+to+chlamydial+salpingitis.">Tubal factor infertility, with special regard to chlamydial salpingitis.</a> Curr. Opin. Infect. Dis. 17, 49-52 (2004).</li><li>Ertel, A., Verghese, A., Byers, S. W., Ochs, M., Tozeren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway-specific+differences+between+tumor+cell+lines+and+normal+and+tumor+tissue+cells.">Pathway-specific differences between tumor cell lines and normal and tumor tissue cells.</a> Mol. Cancer. 5, 55 (2006).</li><li>Roshick, C., Wood, H., Caldwell, H. D., McClarty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+gamma+interferon-mediated+antichlamydial+defense+mechanisms+in+human+and+mouse+cells.">Comparison of gamma interferon-mediated antichlamydial defense mechanisms in human and mouse cells.</a> Infect. Immun. 74, 225-238 (2006).</li><li>Mestas, J., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+not+men:+differences+between+mouse+and+human+immunology.">Of mice and not men: differences between mouse and human immunology.</a> J. Immunol. 172, 2731-2738 (2004).</li><li>Hvid, M., Baczynska, A., Deleuran, B., Fedder, J., Knudsen, H. J., Christiansen, G., Birkelund, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1+is+the+initiator+of+Fallopian+tube+destruction+during+Chlamydia+trachomatis+infection.">Interleukin-1 is the initiator of Fallopian tube destruction during Chlamydia trachomatis infection.</a> Cell Microbiol. 9, 2795-2803 (2007).</li><li>Roth, A., Konig, P., van, Z. G., Klinger, M., Hellwig-Burgel, T., Daubener, W., Bohlmann, M. K., Rupp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+abrogates+antichlamydial+properties+of+IFN-gamma+in+human+fallopian+tube+cells+in+vitro+and+ex+vivo.">Hypoxia abrogates antichlamydial properties of IFN-gamma in human fallopian tube cells in vitro and ex vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 19502-19507 (2010).</li><li>Wyrick, P. B., Knight, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-exposure+of+infected+human+endometrial+epithelial+cells+to+penicillin+in+vitro+renders+Chlamydia+trachomatis+refractory+to+azithromycin.">Pre-exposure of infected human endometrial epithelial cells to penicillin in vitro renders Chlamydia trachomatis refractory to azithromycin.</a> J. Antimicrob. Chemother. 54, 79-85 (2004).</li><li>Van Voorhis, W. C., Barrett, L. K., Sweeney, Y. T., Kuo, C. C., Patton, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+Chlamydia+trachomatis+infection+of+Macaca+nemestrina+fallopian+tubes+produces+a+Th1-like+cytokine+response+associated+with+fibrosis+and+scarring.">Repeated Chlamydia trachomatis infection of Macaca nemestrina fallopian tubes produces a Th1-like cytokine response associated with fibrosis and scarring.</a> Infect. Immun. 65, 2175-2182 (1997).</li></ol>

No conflicts of interest declared.

Visualization of pathogen- induced harm to the infected tissue is arduous and often limited to experimental procedures in mice. We established an ex vivo infection model in human fallopian tubes to analyze C. trachomatis infections of the upper female genital tract. By the use of this method, we were able to visualize C. trachomatis- induced damage to human fallopian tube epithelium within one day post infection. Cellular swelling and lysis were typical morphological changes that were observed in C. trachomatis- infected fallopian tubes but not in non- infected controls. TEM analyses revealed that chlamydiae implement a typical intracellular developmental cycle within infected tissues, showing large inclusions with re-differentiated elementary bodies but also smaller inclusions with enlarged reticulate bodies that might indicate persistence. However, the morphological picture alone does not suffice to discriminate between replicating and persistent infections, which are characterized by reduced metabolism and increased resistance to antimicrobials 10.
Destruction of the epithelium in infected fallopian tubes is either due to disruption of C. trachomatis- infected epithelial cells after completion of the intracellular developmental cycle and release of infectious elementary bodies or the release of pro- inflammatory cytokines 8. Pathogen- induced tissue damage and host- induced immune reactions against C. trachomatis are supposed to be the major factors leading to loss of epithelial cell function, fibroblast remodeling and finally tubal infertility in vivo 8,11. 
One of the major limitations of this model is the absence of inflammatory cells which hampers the analysis of secondary effects to the initial C. trachomatis infection of the fallopian tube epithelium. However, the model is appropriate to investigate different environmental conditions (e.g. oxygen content) and the initial host-immune response in acute C. trachomatis infections 8,9. Further plans are to establish a model for testing of antimicrobials against C. trachomatis in whole human fallopian tubes and to characterize the metabolic states of the different developmental stages observed in the tubal epithelium by 2-photon laser scanning microscopy.

<table border="1">
<tbody>
 <tr>
 <td>Name 			of the reagent</td>
 <td>Company</td>
 <td>Catalogue 			number</td>
 <td>Comments 			</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>PAA</td>
 <td>E15-843</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RPMI </td>
 <td>PAA</td>
 <td>R15-802</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FCS</td>
 <td>PAA</td>
 <td>A15-101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cycloheximide </td>
 <td>Sigma 			- Aldrich</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> SPG 			Buffer</td>
 <td>various</td>
 <td>&nbsp;</td>
 <td>see 			recipe below</td>
 </tr>
 <tr>
 <td>Monti's 			fixative </td>
 <td>various</td>
 <td>&nbsp;</td>
 <td>see 			recipe below</td>
 </tr>
 <tr>
 <td>sodium 			cacodylate buffer</td>
 <td>various</td>
 <td>&nbsp;</td>
 <td>see 			recipe below</td>
 </tr>
 <tr>
 <td>Richardson's 			stain</td>
 <td>various</td>
 <td>&nbsp;</td>
 <td>see 			recipe below</td>
 </tr>
 </tbody>
</table>

Recipes: 
1.	SPG Buffer
<ol>
 <li>	Add 75 g sucrose. </li>
 <li>	Add 2.47 g disodium phosphate. </li>
 <li>	Add 0.36 g monosodium phosphate. </li>
 <li>	Add 0.72 g glutamic acid. </li>
 <li>	Fill to 1 l with aqua dest. </li>
 <li>	Adjust pH to 7.3. </li>
</ol>
2.	0.1 M sodium cacodylate buffer, pH 7.35.
<ol>
 <li>	Add 42.80 g dimethylarsenic acid sodium salt. </li>
 <li>	Add 68.46 g sucrose.</li>
 <li>	Fill to 1 l with aqua dest. to get 0.2 M sodium cacodylate buffer. </li>
 <li>	Before use dilute solution with aqua dest. to desired concentration and adjust pH to 7.35 if necessary. </li>
</ol>
3.	Monti's fixative
<ol>
 <li>	Add 156 ml 0.12 M sodium cacodylate buffer.
 3.2	Add 25 ml 25% glutardialdehyde in aqua dest. </li>
 <li>	Add 19 ml 10% paraformaldehyde in aqua dest. </li>
 <li>	Add 3 ml 3% CaCl2 in aqua dest. </li>
 <li>	Adjust pH to 7.35 and fill with aqua dest. to 312 ml final solution. </li>
</ol>
4.	Richardson's stain
<ol>
 <li>	Mix equal parts of 1% methylene blue in 1% aqueous borax solution and 1% Azur II in aqua dest.</li>
</ol>

a human fallopian tube model for investigation of c. trachomatis infections

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues 1,2. As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur 3,4. 
Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell- culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo 5. Further limitations are given by the fact that central signaling cascades (e.g. IFN-&gamma; mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans 6,7. We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo 8,9.
For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue.

Watch this Scientific Journal Video about A Human Fallopian Tube Model for Investigation of C. trachomatis Infections at JoVE.com

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

We describe an ex vivo infection model for visualisation of direct interactions from bacterial pathogens with human fallopian tube cells. The whole organ tissue model was established to investigate C. trachomatis induced pathology to the female fallopian tube under "life-like" conditions.

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient ...

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Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...