Apr 30, 2025
BBB Assessment with Evans Blue after CCI
This protocol is a draft, published without a DOI.
- 1University of Maryland School of Medicine;
- 2University of Maryland, Baltimore
Protocol Citation: Prajwal Ciryam, Timothy Zhang 2025. BBB Assessment with Evans Blue after CCI. protocols.io https://protocols.io/view/bbb-assessment-with-evans-blue-after-cci-drq955z6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it is working for Evans Blue extraction. We are still optimizing the acetone protein precipitation.
Created: November 15, 2024
Last Modified: April 30, 2025
Protocol Integer ID: 112129
Funders Acknowledgements:
GEn1E Lifesciences
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This is our approach to measure BBB barrier breakdown using Evans Blue in the controlled cortical impact model of TBI. A similar downstream approach could be applied to any brain injury model.
Materials
Evans Blue
Normal Saline
TCA
Glibenclamide
DMSO
1% lidocaine with epinephrine 1:100,000
Euthasol
Isoflurane
Acetone
RIPA
Protease inhibitor tablets (cOmplete)
5 mL brown tubes
5 mL clear tubes
1.9-mm ball diamond burr
Dremmel
Handheld electrical homogenizer
Stereotaxic frame
Leica Impact One Stereotaxic Impactor
Impactor tip (3-mm diameter)
Forceps
Scalpel
Camera
Microcentrifuge
BioMate 3 Spectrophotometer
Mettler-Toledo scale
Clear Eppendorf tubes (2 mL)
Brown Eppendorf tubes (5 mL)
Micropipettes
Pipette tips
-20ºC freezer
Controlled Cortical Impact
Controlled Cortical Impact
Sterilize the stainless steel impactor tip in a glass bleed sterilizer before first use.
The drill tip should be autoclaved before first use and the drill body/flexible shaft covered with sterile autoclaved aluminum foil for handling.
Anesthetize animal (typically male C57BL/6 mouse) with isoflurane and evaluate for reaching a surgical depth of anesthesia.
Place animal in a stereotaxic frame.
Infiltrate the site of planned incision with local anesthetic (1% lidocaine with epinephrine 1:100,000).
Using aseptic technique, create a 1-cm linear skin incision midline along the skull in a sagittal fashion, using a sterile surgical scalpel.
Remove a 4-mm diameter circular piece of skull on the left side in a location abutting the sagittal and lambdoid sutures ("the craniectomy") as described below.
Apply low-pressure circular arcing movements with the 1.9-mm ball diamond burr driven by high speed Dremmel tool to produce the craniectomy.
Every 10 seconds, dip the tip of the burr in cold sterile saline to prevent overheating. Failure to perform this step will cause thermal injury to the brain.
This procedure produces a circular groove with remaining paper-thin bone, which should be removed carefully with forceps to ensure that the dura is not penetrated.
Induce controlled cortical impact (CCI) using a Leica Impact One Stereotaxic impactor for CCI. (Omit this step for sham animals).
At the beginning of a day's use, the impactor should be calibrated with mock test impactions. The impactor is at risk for drift that can induce variability in the extent of injury.
Position the impactor (3mm in diameter) on the surface of the dura at a 20º angle.
Set the impactor deliver a 2.5 mm deep and 200 sec duration displacement of cortical tissue.
Activate the impactor.
Between animals, clean the impactor tip with soap and sterile water and then sterilize it in a glass bead sterilizer for a minimum of 20 seconds.
Drug Formulation
Drug Formulation
This is an example. Glibenclamide: Dilute 40 µL of glibenclamide stock (2.5 mg/mL, run DMSO) in 960 µL normal saline, to make 100 µg/mL stock solution. injection volume = 100 µL
Vehicle control for gibenclamide; 40 µL DMSO in 960 µL normal saline. injection volume = 100 µL
Drug Treatment
Drug Treatment
For this example, intraperitoneal injections of drug/vehicle occur immediately after surgery and at 6 hours after surgery.
Animals should also receive appropriate analgesia per IACUC protocol.
Evans Blue Treatment (at desired time point)
Evans Blue Treatment (at desired time point)
Prepare 2% Evans Blue
Weigh out 0.08g Evans Blue in a weigh boat on a Mettler scale. Take great care not to contaminate the space with Evans Blue dust and clean the space thoroughly if being used for downstream steps (as even a small amount of Evans Blue contaminant can interfere with later experimental results).
Add Evans Blue to 4 mL sterile normal saline in a brown (light-protected) 5 mL tube. Triturate and vortex to mix.
Using a 23g or larger bore needle, aspirate Evans Blue solution into a 5 mL syringe.
Remove needle and replace it with a 0.20-0.22 µm filter.
Inject the contents of the syringe through the filter and into a new brown (light-protected) 5 mL tube.
Vortex tube. Keep tube covered in aluminum foil.
Inject 200 µL of 2% Evans Blue I.P. into mice.
Three hours later, anesthetize the mice with euthasol in preparation for vital perfusion.
Photograph the mice (head, scalp, tail). If Evans Blue has perfused systemically, the mice should be blue-tinged. If they are not, then exclude from further analysis.
Perfuse mice with 15 mL ice-cold 0.9% saline solution.
Prepare Tissue
Prepare Tissue
Surgically extract the brain and, optionally, liver for downstream analysis. Inspection of the peritoneum can be helpful in cases where there was inadequate perfusion, as occasionally perforation of the intestine can result in deposition of all the dye there.
Prepare brain tissue.
Dissect brain as illustrated below. First cut caudal to area of contusion, dissecting away cerebellum and brain stem (this generates sample #3). Second cut rostral to contusion (discard tissue rostral to contusion). Third cut bisecting remainder of cerebral hemispheres (ipsilateral is sample #1, contralateral is sample #2).
Place tissue in tared 2 mL Eppendorf tube and weigh. Record weight.
Clean the razor blade between brains with ethanol and distilled water to avoid cross-contamination of Evans Blue. N.B.: Evans Blue very easily cross-contaminates samples. Ensure working space, balance, gloves, ice, instruments are clear of Evans Blue.
Place tissue on ice until all dissections are complete. Protect from light.
TCA Extraction
TCA Extraction
To each sample, add 1000 µL of ice-cold 50% TCA (diluted in milliQ H2O).
Vortex samples for 5-10 seconds.
Use a handheld homogenizer to homogenize each sample. This typically requires 30 seconds at about half maximal speed. Rotatory movements and up-and-down movements through the Eppendorf tube help, but avoid spillage. N.B.: As a cross-check, when homogenization appears complete, turn off homogenizer and attempt to fish out large brain chunks from bottom tube. If none are apparent, homogenization is likely adequate.
Between samples, clean homogenizer probe by running briefly at maximal speed first in 70% ethanol then in milliQ H2O, then drying off with clean paper towel.
Centrifuge samples at 10,000 rpm for 20 minutes at 4ºC.
Photograph #1 and #2 sample tubes. The way we have done this most easily is to load them into a magnetic tube holder and to slide a piece of white printer paper behind them, and then photograph. See example below.
Collect supernatant in new tubes. For #1 and #2 samples, these should be individual tubes. For #3 samples, these can be pooled into one 5 mL tube.
Protein Precipitation with Acetone (optional and not optimized)
Protein Precipitation with Acetone (optional and not optimized)
Add 4 volumes of -20ºC acetone to the pellet.
Vortex the samples for an additional 5-10 seconds.
Homogenize the samples with hand-held homogenizer for 30s at half-maximal speed.
Incubate samples at -20ºC for 60 minutes.
Centrifuge samples at 15,000g for 10 minutes at 4ºC.
Discard supernatant.
Air dry samples, but not too much! (Room temperature, ~30 minutes) N.B.: Over-drying will make them difficult to re-solubilize.
Resuspend samples in RIPA with protease/phosphatase inhibitor. N.B.: Depending on your target, you may consider an alternate detergent like 1% SDS or 2% SDS.
Vortex the samples for 5-10 seconds.
Re-homogenize with hand-held homogenizer for 30s at half-maximal speed.
Incubate at 4ºC in RIPA for ~20 minutes.
Centrifuge at 10,000 rpm for 20 minutes at 4ºC
Collect supernatant.
Quantify protein concentration using a BCA assay.
Flash freeze samples in liquid N2 and store them at -20ºC.
Prepare Standards
Prepare Standards
Prepare TCA standards
| Label | Final Conc. (mg/mL) | Stock Volume | Diluent Volume | Initial Volume | Final Volume | Abs Target | |
| TCA6 | 0.2 | 20 µL of 20 mg/mL stock | 1980 µL of 50% TCA | 2000 µL | 1800 µL | N/A | |
| TCA5 | 1.5•10^-2 | 200 µL of TCA6 | 2467 µL of 50% TCA | 2667 µL | 1167 µL | 1.125 | |
| TCA4 | 1•10^-2 | 1500 µL of TCA5 | 750 µL of 50% TCA | 2250 µL | 1050 µL | 0.75 | |
| TCA3 | 5•10^-3 | 1200 µL of TCA4 | 1200 µL of 50% TCA | 2400 µL | 1200 µL | 0.375 | |
| TCA2 | 2.5•10^-3 | 1200 µL of TCA3 | 1200 µL of 50% TCA | 2400 µL | 1400 µL | 0.1875 | |
| TCA1 | 1*10^-3 | 1000 µL of TCA2 | 1500 µL of 50% TCA | 2500 µL | 2500 µL | 0.075 | |
| TCA0 | 0 | N/A | 1000 µL of 50% TCA | 1000 µL | 1000 µL | 0 |
TCA Standards.
N.B.: If large amount of tissue, can consider also making a standard by spiking in Evans Blue into the cerebellar tissue + TCA tube and then homogenizing. However, in our experience, for the number of mice used per run, there is often insufficient sample to do this reliably, so we have stopped doing this.
Run Absorbance
Run Absorbance
Measure absorbance at 620 nm of supernatants and standards on a BioMate 3 Spectrophotometer (or alternate instrument), with ~1000 mL per cuvette (except for extract standards, in which you can use 500 µL to save sample). Blank using milliQ water.
Quantification
Quantification
Prepare a standard curve.
Convert sample absorbances to mg. Divide this by the brain mass of the sample.
Multiply by a convenient scaling factor.
Protocol references
Acknowledgements
Orest Tsymbalyuk
Kaspar Keledjian
Jesse Stokum
Volodymyr Gerzanich
J. Marc Simard