Chlamydia Trais is a gram-negative obligate intracellular bacterium. Different chlamydial serovars are known that cause infections of the eye or the genital tract. Genital tracted infections with chlamydia trais are the most frequent sexually transmitted disease worldwide.
They are found with a prevalence of nearly 3 million reported infections in the European Union from 1990 to 2009. Almost 400, 000 cases were reported in 2009. Although public health and sexual education are constantly improving in increasing rate of new genital chlamydial infections from 70, 000 cases in 1990 to 400, 000 cases in 2009 is detected.
75%of all new infections are reported in young people at the age of 15 to 24 years. Clinical sequela are supposed to occur due to inefficient clearance of the pathogens, which may lead to ascending infections from the lower to the upper genital tract, and subsequently cause chronic inflammatory damage to the infected tissue. 10 to 30%of women who are infected with chlamydia tracheitis develop a pelvic inflammatory disease PID, and up to 50%of PID cases are attributed to chlamydia tracheitis infections.
As a consequence, in 50%of patients with tubal infertility and in 40%of patients with ectopic pregnancy chlamydia, trais is found as the causative pathogen. In a healthy non-infected woman, the fertilized egg migrates through the fallopian tube into the uterus where it develops until delivery. In infected patients, chlamydia may ascend from the lower genital tract to the fallopian tube triggering chronic inflammatory processes.
Pathogen induced epithelial cell damage and loss of central epithelial cell functions are supposed to finally cause tubal occlusion resulting in reduced or completely abrogated egg transport from the ovaries to the uterus. For This purpose, human fallopian tubes from women undergoing hysterectomy are collected and stored. The tubes are dissected and carefully opened.
According to the experimental setup, they are cut into small specimen and infected with infectious chlamydia tracheitis elementary bodies for 24 to 48 hours after respective time points. Specimen are fixed with Monty's fixative for at least three days until further preparation. Store human fallopian tubes HFT from women undergoing hysterectomy immediately after surgery.
In RPMI containing 5%FCS without antibiotics in four degrees Celsius until preparation. Dissect the human fallopian tube in a Petri dish containing RPMI with 5%FCS without antibiotics during the whole processing. The tube should be submerged with media to prevent drying, cut off and discard connective tissue and tissue destroyed during surgery.
After dissection, open the HFT carefully with a small scalpel. Avoid tissue damage through incautious handling with forceps and scalpel. Prepare small pieces of tissue in a size of five by five millimeters for infection store HFT specimen in one milliliter RPMI containing 5%FCS without antibiotics and add C track.
OMAS elementary bodies HFT specimen are incubated for up to 48 hours in 37 degrees Celsius, 5%CO2 and 20%O2 within a normal incubator or in a low oxygen environment, less than 5%O2 in a hypoxia chamber. After respective incubation time points, specimen are collected and stored in Monty's Fixative for at least three days at four degrees Celsius until SEM or TEM preparation for transmission electron microscopy. The fixated tissue is cut into small pieces washed and incubated with 1%osse and tetroxide overnight.
After several washing and drying steps, the dehydrated specimens are transferred into freshly prepared aite and polymerized for 48 hours at 60 degrees Celsius. After trimming semit thin and ultra thin sections are cut and transferred on slides or grids respectively. After staining, semi thin sections can be examined using a light microscope for transmission electron microscopy.
Remove specimen from Monty's fixative and cut in up to two by two by five millimeter pieces. After washing and sodium CCO dilate buffer incubation and osmium tetroxide washing and dehydration and ascending concentrations of ethanol transfer specimen into a mixture of 50%propylene oxide and aite Overnight. The next day transfer into freshly prepared aite for at least one hour.
Then transfer into flat molds and fill with aite and let aerd dite polymerize for 48 hours at 60 degrees Celsius. After trimming of the embedded sample, cut semi thin sections of approximately 700 nanometers or ultra thin sections of 70 nanometers using an ultra microtome equipped with a glass knife or preferably a diamond knife stain. Semi thin and ultra thin sections analyze the specimen by light and transmission electron microscopy For scanning electron microscopy.
The specimen are pinned with epithelium facing upwards on cork plates, washed and dehydrated using increasing concentrations of acetone. After drying in a critical point dryer, the specimen are transferred to an SEM specimen mount and coated with platinum, gold or palladium using a sputter coder. Then they're ready to be analyzed in a scanning electron microscope for scanning electron microscopy.
Remove specimen from Monty's fixative and orient tissue piece with epithelium facing upwards on a cork sheet and fixed position with insect needles. After washing sodium caco dilate buffer an acetone treatment dry specimen in a critical point dryer. After transfer of specimen from cork to an SEM specimen mount, prepare a conductive platinum gold or palladium coating of the specimen by using a sputter coter transfer sputtered specimen into a scanning electron microscope for further analysis.
The unchanged High incidence of lumus infections in women and tremendous clinical consequences of ascending al infections are the rational for intensifying basic and translational research in this area. We made use of a previously by the group of St.Perus and who established exvivo tissue model of the human fallopian tube to morphologically characterize chlamydia induced cell damage in the fallopian tube. By the use of this method, formation of intracellular inclusions is observed in different developmental stages, seeing both replicating and persistent infections.
However, experimental evidence for the presence of persistent chlamydia in the human fallopian tube tissue is still missing and requires further analysis for chlamydia, progeny and transcription upregulation of persistence genes. One of the major limitations of this model is the absence of inflammatory cells that hampers the analysis of secondary effects to due to the initial KLA glaucomatous infection. However, this model seems appropriate to analyze environmental conditions such as oxygen content in the context of fallopian tube epithelial cell damage, and to analyze the initial host immune response against acute glaucomatous infection.
One of the future aims is to establish a more elaborate model for MIC testing of CTUs in the pH human fallopian tube tissue model, and to use this model to really define the different developmental stages of CLIs metabolically making use of the two photon laser scanning Microscopy technique.
