The aim of this procedure is to assess the impact of a progressive colonization on the host hepatic metabolism. This is accomplished by first progressively colonizing germ-free mice. The colonization process is monitored by evaluating the urinary excretion of microbial co metabolites through NMR spectroscopy.
Subsequent to monitoring of the colonization process, a liver biopsy is collected from the mouse and prepared for metabolic profiling. The hepatic metabolic profile can then be acquired from the intact biopsy using non-destructive high resolution magic angle spinning NMR spectroscopy. Ultimately, this use of proton NMR spectroscopy reveals various metabolites involved in metabolic pathways such as energy and oxidative stress pathways.
So this experiment can provide insight into the liver functions during colonization. It can also be applied through the system such as kidney. To colonize the animals, remove germ-free mice from isolators and house them in a conventional husbandry room in cages, equipped with a filter in front of the conventional animals which serve as controls.
Then mix half of a three day old litter taken from the control conventional cage with the litter of the germ-Free animals collect urine in a 1.5 milliliter micro tube by handling the mouse over the tube and help tion by gently massaging the bowel. A minimum of 20 microliters is required for acquisition with a five millimeter NMR probe, but it is recommended to use 30 microliters to improve the quality of metabolic profiling. Snap freeze the urine immediately in liquid nitrogen store at at least minus 40 degrees Celsius until NMR analysis.
Prior to NMR acquisition, prepare 0.2 molar sodium phosphate buffer solution in heavy water. pH 7.4 containing one millimolar TSP mix 30 microliters of the th urine with 30 microliters of sodium phosphate buffer. Transfer 50 microliters of the mixed solution into a 1.7 millimeter NMR capillary tube using a 50 microliter glass syringe equipped with a metal needle.
Take care to avoid bubbles. Fit the capillary adapter on top of the capillary containing the urine sample and place it into a 2.5 millimeter NMR micro tube for a five millimeter NMR probe. This combination of tubes can be used as a standard five millimeter NMR tube for spectral acquisition.
In this demonstration, a bruer advanced three 700 megahertz NMR spectrometer is used to collect the data. Acquire one dimensional proton NMR Spectra using the parameters discussed in the written protocol. Following NMR acquisition, use the extraction rod to remove the capillary from the 2.5 millimeter NMR tube by screwing the capillary adapter gently to pull it out following euthanasia of animals as discussed in the written protocol.
Prepare for the liver biopsy. Do not use any product containing alcohol to avoid contamination and wash tools using water or saline solution. Collect liver biopsies from the left lobe for reproducible biopsies.
Collect consistently in the center of the left lobe, avoiding the peripheral areas where tissue is thinner. Take care not to perforate the gallbladder in case of bile leak. Wash tissue immediately with water or saline solution.
Snap freeze biopsies in liquid nitrogen immediately and store them at minus 80 degrees Celsius until NMR analysis To set up for H-R-M-A-S-N-M-R, insert the third biopsy into a zirconium rotor and fill the rest of the volume with pure heavy water for NMR lock. Be careful not to make any bubbles as they could alter the quality of subsequent shimming and data acquisition. Insert a 50 microliter Teflon spacer using the cylindrical screw.
Unscrew it and calibrate it using the depth gauge on the short side. Place the thread pin on the spacer and screw it gently with the screwdriver. Dry house, any residual water with a piece of tissue.
Next, place the cap at the top of the rotor and insert it in the rotor packer. Press firmly until the cap is in place. There should not be any space left between the rotor and its cap mark half of the bottom of the rotor using black marker pen to allow optical spin rate detection.
Once the rotor has been sufficiently packed, place it inside the NMR spectrometer and start spinning up five kilohertz.Here. A bru advanced three 500 megahertz spectrometer is used. Acquire proton NMR spectrum using a CP MG pulse sequence.
According to the manufacturer's guidelines, the abundant alpha and numeric glucose doublet peak at 5.22 parts per million could be used to calibrate the resulting NMR spectra. To unpack the rotor, proceed by removing the cap, using the cap remover, unscrew the thread pin, and remove the Teflon spacer. Using the cylindrical screw, the apparatus can be washed thoroughly using water and detergent.
The appearance of gut microbial co metabolites over the colonization process is clearly illustrated in this proton spectrum of a urinary metabolic profile for an animal colonized 20 days. This animal did not excrete any in docile sulfate and very small amounts of phenyl acetyl glycine, abbreviated PAG and que cell sulfate at the germ-free state. As colonization progresses, these three markers of protein metabolism by the gut microbiota increase considerably and reach an equilibrium at day 20.
By integrating the specific PAG triplet peak, it was possible to quantify the increase in this particular marker over time. This diagram represents the average PAG concentration during colonization for a group of seven animals using proton H-R-M-A-S-N-M-R spectroscopy, the hepatic metabolic profile of two mice before and after colonization was obtained. This figure illustrates well the information that can be derived from A-M-A-S-N-M-R based metabolic profile.
Numerous amino acids as well as metabolites derived from energetic metabolism can be visualized. These profiles also contain information relevant to oxidative stress, nucleotide metabolism, and methyl amine metabolism. In this example, it is very clear that the germ-free mouse displays almost no glycogen and very low amounts of glucose and triglycerides.
So following this procedure, so multiple statistical tools such as chemo can be applied in order to cluster logical samples according to metabolic profile, and highlight biomarkers associated with the metabolic status.
