This method can help answer key questions about the roles of streptococcus suis'components in many sites, such as the extracellular matrix from its biofilms. The main advantage of this technique is that it enables investigations of bacterial components that affect how the immune responses directly in a brain and mimic bacteria entrance into the central nervous system. Demonstrating the procedure will be Mister Shouming Zhang, a graduate student from my laboratory.
To collect strain P1/7 planktonic cells, first collect five milliliters of the cells from mid-lock phase culture. Centrifuge for three minutes at 8000 times G before washing the cells three times in PBS. Re-suspend the cells with five milliliters of 25%glycerol in THB and aliquot into five tubes.
Alternatively, to collect strain P1/7 biofilm state cells, take 20 milliliters of an overnight culture and add to 180 milliliters of fresh THB. Equally divide the diluted culture into 10 round culture plates, then incubate the plates at 37 degrees celsius in 5%carbon dioxide for 24 hours. Following incubation, shake the plate gently to re-suspend the bacteria that have not adhered to the plate and avoid re-suspending the sediment.
Then, discard the supernatant by aspiration. Add five milliliters of PBS to harvest biofilm state cells. Re-suspend the sediment completely and then transfer the sample to a new tube.
Sonicate the re-suspended biofilm state cells as listed in the text protocol. Next, centrifuge the sonicated cells for three minutes at 8000 times G and store the supernatant at minus 80 degrees celsius. The supernatant may contain biofilm components and it can be used to re-suspend the biofilm bacteria before infection.
Re-suspend the sediment with 10 milliliters of 25%glycerol in THB before observing the samples by scanning electron microscopy as described in the text protocol. Infect all mice using planktonic cells or biofilm state cells and perform euthanasia as described in the text protocol. Next, extract the total RNA from brain tissue using an RNA extraction kit.
For each mouse, use whole brain tissue for extracting RNA. Divide whole brain tissue into five tubes. Take up to 100 milligrams of brain tissue and add one milliliter of lysis solution to each tube containing the lysis matrix provided by the kit.
Extract the RNA following the steps listed in the text protocol. Next, perform CDMA synthesis, including elimination of genomic DNA, using a thermocycler with a reverse transcription reagent kit. Add one microgram of RNA to a tube containing two microliters of 5X genomic DNA elimination buffer and one microliter of geneomic DNA elimination enzyme.
Add RNAse free water until the volume reaches 10 microliters. Incubate for two minutes at 42 degrees celsius. Add 10 microliters of master mix to the reaction solution and then mix gently.
Proceed immediately with the reverse transcription reaction by placing the tube at 37 degrees celsius for 15 minutes, then transfer the reaction to 85 degrees celsius for five seconds. Perform the quantitative real-time PCR analysis using a real-time PCR machine with a cyber quantitative real-time PCR kit. Following the kit instructions, combine the enzyme, primers, 50X ROX reference dye two, and CDMA template, then add RNAse free water until a total volume of 20 microliters is reached.
Run each sample in triplicate using the thermal parameters listed in the text protocol and calculate the relative fold-change based on the two minus delta delta CT method. Also perform the observation and scoring of histological sections of the infected brain tissue as detailed in the text protocol. The effect of biofilms on inflammatory response in mirroring brain tissue is shown here.
At 12 hours post-infection, the expression of CCL2, IL6, TNF-alpha, and TLR2 from brains of infected mice was significantly higher for biofilm state cells as compared to planktonic cells. Far more severe pathological changes were observed in the meninges and cerebral cortex of mice infected with the biofilm bacteria as compared to mice infected with the planktonic bacteria. These changes included large areas of necrosis and intense inflammatory cell infiltration.
However, much less inflammatory cell infiltration was observed in the meninges from mice infected with the planktonic bacteria. Furthermore, there was far more intense inflammatory cell infiltration observed in ventricles from mice infected with biofilm bacteria as compared to mice infected with the planktonic bacteria. As shown here, much less inflammatory cell infiltration was observed in ventricles from mice infected with the planktonic bacteria.
While attempting this procedure, it is important to keep in mind that different strains may have different infective doses, so a preliminary experiment is strongly recommended to determine the appropriate infection dose. After watching this video, you should have a good understanding of how to collect biofilm cells and perform intercranial subretinal direct infection in mice.
