This method makes it possible to analyze the human blood-brain barrier by measuring bacterial traversal upon different treatments. It may help to discover the pathogenesis of bacterial meningitis and postoperative delirium. It is a straightforward method and the effects of treatment are directly represented by the number of bacteria.
It can be modified to enable high throughput analysis of compounds on the endothelial cells. Begin by assembling the 12-well plate in a biological safety cabinet. Unpack the plate and each insert, then use sterilized forceps to grab the inserts at their base and put them into the wells.
Coat the porous membrane of each insert with 90 microliters of 10 micrograms per microliter collagen IV and fibronectin mixture and incubate the plate in a cell culture incubator at 37 degrees Celsius for 24 hours. After the incubation, wash the inserts twice by pipetting one milliliter of PBS into each insert and aspirating it with a vacuum pump. Then equilibrate the membranes by pipetting prewarmed DMEM F12 medium in the upper and lower chambers and incubate the plate at 37 degrees Celsius and 5%carbon dioxide.
To seed the human microvascular endothelial cells, aspirate the medium from the cell culture flask and wash the cells with 10 milliliters of PBS. Cover the cells completely with five milliliters of trypsin EDTA and incubate the flask for three to five minutes at 37 degrees Celsius. Transfer five milliliters of the cells into a 15 milliliter tube and add five milliliters of FCS containing medium to stop the enzymatic reaction.
Centrifuge the suspension for three minutes at 210 times g, remove the supernatant and resuspend the cell pellet in five milliliters of medium. Add 10 microliters of the cell suspension to 10 microliters of 0.4%trypan blue stain, then add 10 microliters of the mixture to a counting chamber. Put the counting chamber into the cell counter and start the appropriate program making sure that the counter is focused.
After counting, seed 200, 000 cells into each upper chamber on the 12-well plate and incubate the plate at 37 degrees Celsius for 14 days changing the media in the upper and lower chamber every two to three days. After 14 days, ensure that cell confluency is 100%by imaging them with a microscope. One day prior to permeability measurement, put one colony of E.coli strain GM2163 into three milliliters of LB medium.
Culture the cells at 37 degrees Celsius for 24 hours in an incubation shaker. To treat cells with a compound of interest, dilute it to its final concentration in DMEM F12 medium and add this mixture into the upper and lower chambers in the 12-well plate, then incubate the plate in a cell culture incubator. After the incubation, exchange the complete medium with antibiotic-free medium.
Determine the concentration of the overnight bacterial culture by measuring its optical density at 600 nanometers, then dilute it to an OD600 of 0.5 in a 50 milliliter Falcon tube. In a biological safety cabinet, add 450 microliters of the bacterial solution into each upper chamber of the 12-well plate, then incubate the plate for six hours at 37 degrees Celsius. After the incubation, remove the insert with forceps and sample 50 microliters of the medium from each lower chamber taking care not to spill the medium from the upper chambers into the lower ones.
It is important to strictly separate the media of the upper and lower chamber as a spillover from bacteria of the upper chamber would lead to false positive results. Drop each sample onto a separate agar plate and streak the solution with a cell spreader. Incubate the plates at 37 degrees Celsius for 24 hours, then count the colonies.
To investigate the effect of glycation on microbial traversal through the blood-brain barrier, cells were treated with a 0.5 and a 0.15 millimolar glyoxal solution for one hour with untreated cells serving as a control. The glycation was then detected via immunoblotting with anti-AGE antibodies. The obtained bacterial colonies were counted and represented as the absolute number of colonies or the relative number of colonies normalized to the control.
Samples treated with glyoxal displayed an increased number of colonies indicating that the treatment has an effect on cellular barrier density. To investigate glucose-induced protein glycation, THBMECs were cultivated in normal glucose and high glucose medium. The absolute number of colonies and the relative number of colonies normalized to the control indicated that glucose had no significant effect on the integrity of the blood-brain barrier.
Treated endothelial cells can be further analyzed by proteomics approaches, for example mass spectrometry, to analyze changes in the proteome. This technique helps in understanding the human blood-brain barrier and bacterial traversal. It may be used to discover aspects of aging and neurodegenerative diseases such as Alzheimer's disease.
