A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model

The evaluation of molecular profiles in local tissues is a critical step to understanding the mechanism of action of active pharmaceuticals as they are evaluated in disease-relevant preclinical models. In the field of arthritis research, the local tissue environment of interest is the small weight-bearing joints which are composed of a complex mixture of bone, cartilage, muscle, and immune cells. Here we describe a reliable robust method for the mechanical disruption and/or pulverization of this complex tissue environment in a cryogenically controlled environment.

This method can then be used to generate downstream proteomic and transcriptional profiling efforts for establishing molecular signatures of disease from a single uniform sample source. This method generates a homogenous powder unlike many other old techniques. Additionally, the old temperature isolation procedure allows for the isolation of high-quality RNA.

This method can then be used to generating downstream proteomic and transcriptional profiling enabling molecular characterization of relevant disease pathways of interest. This method can provide insights to any research field that is deriving molecular signatures from tissues. The method could be extended to any complex tissue system that has dense and difficult to dissociate constituents.

Although we have not evaluated to this point, we could see potential application to dense cartilage tissues like ears or bones. The time-critical nature of transferring the powder to tubes for weighing is something that takes practice. If too much time is taken, the powder will begin to thaw and become amorphous.

It is important to limit the number of samples to 10 to 15 until you're comfortable with the procedure. On the day of tissue processing, prechill all needed instruments on dry ice for the minimum of 10 minutes. Wear thermal gloves to avoid being harmed by the extreme cold.

Next, transfer each of the prepared mouse paw into a separate 1.5 milliliter microcentrifuge tube and immediately snap freeze in liquid nitrogen. Store the frozen paws at negative 80 degrees Celsius. When ready to continue, fill a freezer mill with liquid nitrogen and let it equilibrate for at least 10 minutes.

Place the unprocessed sample on dry ice and keep it there to avoid freeze/thaw cycles. Then transfer one hind paw into a pre-chilled large polycarbonate grinding vial with a bottom steel plug. Insert the pre-chilled steel impactor and close the polycarbonate grinding vial with the pre-chilled top steel plug.

Transfer the large polycarbonate grinding vials with the samples into the prefilled freezer mill and close the lid with the rubber clasp found at the front of the instrument. Let the samples cool in the liquid nitrogen for one minute. Next, set the freezer mill to the one minute program with 10 cycles per second.

Press the run button and wait for the freezer mill to complete its cycle. After this, open the lid of the freezer mill and take out the large polycarbonate grinding vial. Place the grinding vial into an extractor and remove the top steel plug by placing downward pressure on the black handle until the steel plug slides out of the polycarbonate tube.

Transfer the opened grinding vial onto dry ice and use pre-chilled forceps to remove the steel impactor. Transfer the frozen powder into a pre-chilled 50 milliliter conical tube. Weigh out between 30 and 50 milligrams of the frozen powder into a tiered 1.5 milliliter microcentrifuge tube for RNA extraction and between 100 and 200 milligrams for protein extraction.

Store the pulverized samples at negative 80 degrees Celsius and proceed to RNA and protein isolations within 24 hours. First, add 10 microliters of beta-mercaptoethanol for each milliliter of RLT buffer. Calculate the volume of RLT buffer corresponding to a ratio of 23.3 milliliters per milligram of tissue and add the appropriate volume of RLT buffer with beta-mercaptoethanol to the tube with frozen powder.

Vortex the sample at 3, 000 RPM for 10 seconds. Use a one milliliter pipetter to pipette up and down vigorously 10 times and vortex the sample again at 3, 000 RPM for 20 seconds. Centrifuge at 13, 000 times g for two minutes and transfer 700 microliters of the supernatant to a fresh tube.

Add 700 microliters of 70%ethanol to this tube and load 700 microliters of the sample onto an RNA purification column. Centrifuge the tube at a speed of at least 10, 000 times g for 30 seconds. Discard the flow-through and load the remaining part of the sample onto the RNeasy column and centrifuge the tube again at a speed of at least 10, 000 times g for 30 seconds.

Discard the flow-through and wash the column by adding 700 microliters of RW1 buffer and centrifuging at at least the speed of 10, 000 times g for 30 seconds. If performing option DNAse digestion, 350 microliters of RW1 is added prior to DNAse treatment. And after DNAse treatment, an additional 350 microliters of RW1 is added.

Then wash the column twice by adding 500 microliters of RPE buffer and centrifuge at a speed of at least 10, 000 times g for 30 seconds making sure to discard the flow-through each time. After this, dry the column by centrifuging it at a speed of at least 10, 000 times g for two minutes. Elute the RNA by adding 50 microliters of water and centrifuging at a speed of at least 10, 000 times g for two minutes.

Collect the flow-through and transfer it to a new 1.5 milliliter tube. Determine the quantity of RNA using the researcher's method of choice and store at negative 80 degrees Celsius before further analysis. Dilute the 10X cell lysis stock solution to 1X cell lysis stock solution with cell culture grade water.

Reconstitute the protease inhibitor cocktail set one with one milliliter of water to make a 100X protease inhibitor stock. Add 100 milliliters of protease inhibitor to 9.9 milliliters of 1X cell lysis buffer to obtain a 1X final stock solution. Add four microliters of ice cold cell lysis buffer for each milligram of tissue powder and add one five millimeter stainless steel bead to the tube.

Vortex the tube at 3, 000 RPM for 60 seconds. Transfer the tube to wet ice and continue to the next sample. Additionally, researchers may find that placing the box vertical can facilitate better mixing with the bead.

After one hour, centrifuge the tubes at 10, 000 times g and at four degrees Celsius for 15 minutes. Transfer the supernatants into a fresh tube making sure to avoid the fat layer on the top. Determine the total protein concentration.

Aliquot each sample into 1.5 milliliter microcentrifuge tubes and store them at negative 80 degrees Celsius until ready to perform further analysis. In this study, a cryogenic pulverization method is used to process murine paws in order to improve the yield and quality of RNA or protein extracted from the tissues and enable the analysis of molecular profiles associated with inflammatory responses. A representative gel image visualization shows the RNA extracted from front paws of CIA mice.

The 28S rRNA and the 18S rRNA bands indicate that all of the samples have sufficient amount of intact RNA. A representative scatter plot of the total protein concentrations based on protein BCA analysis is shown here. Total protein concentrations from naive mice, CIA mice, or CIA mice under various treatments are comparable across groups.

Luminex analyses are then conducted to determine the concentrations of inflammatory cytokines and chemokines in the protein extract. A representative scatter plot of the concentrations of normalized cytokines and chemokines reveals that when compared to naive mice, several cytokines are elevated in CIA mice and treatment with anti-IL17A antibody significantly inhibits production of several cytokines. Microarray analysis is performed to evaluate transcriptome changes in CIA and treatment-related effects.

A representative heat map plot of genes significantly increased in CIA mice compared to naive mice is shown here. When performing this procedure, it is critical for researchers to minimize the time that the tube and powder are kept out of dry ice. This helps prevent thawing of the powder and subsequent problems to the degraded RNA and protein.

The high quality of the molecular signatures generated by this technique allows researchers to interrogate the unique profile a therapeutic will impart and further allows for the differentiation of mechanisms that can be used to treat arthritic conditions. This technique could be used to extract a number of dense tissues like ears and bones to further evaluate molecular signatures in these tissues of interest. Understanding how therapeutics modulate the molecular signatures of disease enable us to better evaluate what specific signaling pathways are modulated.

And perhaps even more importantly, what pathways are not regulated with the therapeutic mechanism being evaluated. When working with liquid nitrogen and dry ice, it is imperative to use personal protective equipment including properly rated gloves and face shields. Exposure of skin for more than a few seconds can cause severe burns.

When working with large quantities of dry ice, it is important to work in a well-ventilated area as dry ice releases carbon dioxide.