This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.
One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.
One of the most common causes of morbidity and mortality worldwide is stroke. Globally, ischemic stroke represents 68% of all stroke cases, while in the United States ischemic stroke accounts for 87% of stroke cases1,2. It is estimated that the economic burden of stroke reaches $34 billion in the United States2 and €45 billion in the European Union3. Animal models of stroke are necessary to study its pathophysiology, develop new methods for evaluation, and propose new therapeutic options4.
Ischemic stroke occurs with occlusion of a major cerebral artery, usually the middle cerebral artery or one of its branches5. Thus, models of ischemic stroke have historically involved middle cerebral artery occlusion (MCAO)6,7,8,9,10,11,12. Following MCAO, neurological injury is most commonly assessed by measuring infarct zone (IZ) using a 2,3,5-triphenyltetrazolium chloride (TTC) staining method13, brain edema (BE) using drying or calculating hemispheric volumes14,15,16, and blood brain barrier (BBB) permeability by a spectrometry technique using Evans blue staining17,18,19.
The traditional MCAO method uses separate sets of brains for each of the three brain measurements. For a large sample size, this results in a significant number of euthanized animals, with added ethical and financial considerations. An alternative method to alleviate these costs would involve measurements of all three parameters in a single set of post-MCAO rodent brains.
Previous attempts have been made to measure combinations of parameters in the same brain sample. Simultaneous immunofluorescent staining methods20 as well as other molecular and biochemical analyses21 have been described after TTC staining in the same brain sample. We have previously calculated brain hemisphere volumes to assess brain edema and performed TTC staining to calculate infarct zone in the same brain set15.
In the present protocol, we present a modified MCAO technique that measures ischemic brain injury through determining IZ, BE, and BBB permeability in the same set of rodent brains. IZ is measured by TTC staining, BE is determined by calculating hemispheric volume, and BBB permeability is obtained by spectrometry methods19. In this protocol, we used a modified MCAO model, based on direct insertion and fixation of the monofilament catheter into the internal carotid artery (ICA) and further blocking of blood flow to the middle cerebral artery (MCA)22. This modified method shows a decreased rate of mortality and morbidity compared to the traditional MCAO method16,22.
This new approach provides a financially-sound and ethical model for measuring neurological injury after MCAO. This assessment of the main parameters of ischemic brain injury will help to comprehensively investigate its pathophysiology.
The following procedures were conducted according to the recommendations of the Declaration of Helsinki and Tokyo and the Guidelines for the Use of Experimental Animals of the European Community. The experiments were also approved by the Animal Care Committee at the Ben-Gurion University of the Negev.
1. Preparing rats for the experimental procedure
2. Preparing rats for surgery
3. Right side middle cerebral artery occlusion
NOTE: MCAO is performed by a modified technique, as previously described16,22,23, with the use of instruments described by McGarry et al.24 and Uluç et al.25.
4. Determination of infarct zone
5. Determination of brain edema31
NOTE: Use ImageJ 1.37v for measurement of BE32,33.
6. Determination of BBB disruption
Infarct zone measurement
An independent-sample t-test indicated that 19 rats that underwent permanent MCAO demonstrated a significant increase in brain infarct volume compared to the 16 sham-operated rats (MCAO=7.49% ± 3.57 vs. Sham = 0.31% ± 1.9, t(28.49) = 7.56, p < 0.01 (see Figure 2A)). The data is expressed as a mean percentage of the contralateral hemisphere ± SD.
Brain edema measurement
An independent-sample t-test indicated that 19 rats that underwent permanent MCAO demonstrated significant increase in the extent of brain edema after 24 h compared to the 16 sham-operated rats (MCAO=12.31% ± 8.6 vs. Sham = 0.64% ± 10.2, t(29.37) = 3.61, p = 0.01, d = 1.23 (see Figure 2B)). The data is expressed as a mean percentage of the contralateral hemisphere ± SD.
Blood brain barrier permeability
An independent-sample t-test indicated that 19 rats that underwent permanent MCAO demonstrated significant increase in the extent of BBB breakdown after 24 h compared to the 16 sham-operated rats (MCAO=2235 ng/g ± 1101 vs. Sham = 94 ng/g ± 36, t(18.05) = 8.47 p < 0.01, d = 2.7 (see Figure 2C)). The data are measured in ng/g of brain tissue and presented as mean ± SD.
Group | Time | Procedures |
Sham operated (16 rats) | 0 | Induction of MCAO and insertion of filament for sham operated group |
MCAO (19 rats) | ||
Sham operated (16 rats) | 23h | Injection of Evans blue |
MCAO (19 rats) | ||
Sham operated (16 rats) | 24h | Brain collection for measurements of IZ, BE, and BBB disruption |
MCAO (19 rats) |
Table 1: Protocol timeline. At 23 h after MCAO, the Evans blue solution was injected. One hour later (24 h after MCAO), brain collection was performed, and IZ, BE and BBB permeability were measured in all groups.
Figure 1: Representative brain slices of sham-operated and MCAO rats.
(A-B) Original scanned image. (C-D) Transformation to greyscale. (E-G) Threshold function. (G-H) Application of a blue filter. (I-J) Threshold function after blue filter application. (K-L) Using threshold function to assess brain edema. Please click here to view a larger version of this figure.
Figure 2: Histological outcomes of MCAO rats compared to sham-operated rats.
(A) Infarct zone. The infarct zone volume in 19 rats after MCAO was significantly increased compared to the 16 sham-operated rats 24 h after surgery (*p < 0.01). (B) Brain edema. The brain edema volume in 19 rats after MCAO was significantly increased compared to the 16 sham-operated rats 24 h after surgery (*p < 0.01). (C) Blood brain barrier permeability. The blood brain barrier permeability in 19 rats after MCAO was significantly increased compared to the 16 sham-operated rats 24 h after surgery (*p < 0.01). Values were expressed as a mean percentage of the contralateral hemisphere ± SD and mean Evans blue extravasation index in ng/g of brain tissue ± SD according to independent-samples t-test. Results were considered statistically significant when p< 0.05, and highly significant when p < 0.01. This figure has been modified from Kuts et al.23 Please click here to view a larger version of this figure.
Supplement 1: Example scan of brain slices. Please click here to download this figure.
Supplement 2: Macros that may be used in ImageJ software for the auto threshold function and measuring pixels. Please click here to download this file.
Supplement 3: Example auto threshold. Please click here to download this figure.
Supplement 4: Example of measured pixels on each hemisphere. Please click here to download this figure.
Supplement 5: Sample analysis. Please click here to download this figure.
The principal goal of the present protocol was to demonstrate consistent measurements of three main parameters of ischemic injury: IZ, BE and BBB permeability. Previous studies in this field have demonstrated the possibility of performing one or two of these parameters together in the same sample. Besides the cost reduction that this three-part method offers, it also provides a more desirable bioethical model that limits the number of animals that must be operated on and subsequently euthanized. As in all histological techniques, the method is limited by the inability to observe ischemic injuries dynamically.
Four computer programs were used in the image analysis: ImageJ 1.37v, Adobe Photoshop CS2, Microsoft Excel 365, and and IBM SPSS Statistics 22. ImageJ was used to measure the extension of infarct zone and brain edema. Adobe Photoshop was used to limit the effect of Evans blue on brain tissue, since a blue color indicates BBB breakdown. Before calculating the infarct zone, it is necessary to remove the blue color from the image, since the color does not allow for accurate measurements of the infarct zone (see Figure 1B, 1D, 1F). Excel and SPSS was used for data processing.
The technique for assessing an infarct zone with ImageJ computer software is based on a comparison of black and white pixels in a healthy hemisphere to pixels in an infarcted hemisphere. In the infarct hemisphere, the infarct zone is not stained with TTC; therefore, it is indicated by a white region in Figure 1B that is measured. In order for the program to correctly calculate the infarct zone, it is necessary to convert the pixels to shades of varying intensities of gray colors (see Figure 1F, 1J). The blue color, which is caused by Evans blue (see Figure 1B), may affect the assessment of the infarct zone (see Figure 1F). Therefore, the first step is to remove the blue color using a blue filter and then convert the image to a black and white image (see Figure 1J). We used Adobe Photoshop, but other computer programs can also be used for this purpose, such as RawTherapeePortable.
We then established uniform parameters for calculating the infarct zone in all 6 slices of one brain set using ImageJ in order to standardize the measurement procedure. This is necessary because all 6 slices from each set were stained and scanned under the same conditions and require a unified cut-off parameter. The cut off is a critical parameter for determining which pixels to convert to white and which to convert to black depending on the shade of gray (see Figure 1D, 1H). We used the Threshold function from the main menu for this purpose. The final step in the image analysis was the calculation of the infarct zone and brain edema.
The infarct zone measurement can be performed by various techniques, including histological staining or radiologic techniques such as a computed tomograph36, positron emission tomography, and magnetic resonance imaging23,36. Previous studies in the lab have demonstrated the assessment of infarct volume using staining with TTC15,26. This method is based on a chemical reaction between TTC and mitochondrial dehydrogenases of neurons. The healthy tissues, rich with dehydrogenases, are colored with red with this staining. However, in necrotic cells, this color change does not occur due to damage in the system that participates in the oxidation of organic compounds37. In our previous studies, we demonstrated a high correlation between this histological technique and results from brain image scanning of this area23.
Measurements of cerebral edema can be assessed both in vivo and in vitro. Cerebral edema results from pathological changes in the activities of sodium and calcium ion channels and transporters that lead to an increase in intracellular water38,39,40,41. Alternatively, swelling can occur by BBB damage that increases extracellular water.42 In previous studies, cerebral edema was determined based on wet and dry techniques followed by the calculation of tissue water content43. An advantage of the method we present in this protocol is its simplicity and accuracy comparing to other existing techniques23,44,45.
The most useful method for BBB breakdown detection is luminescence spectroscopy after Evans blue injection. The measurement of BBB permeability is based on the injection of Evans blue, which binds to albumin. In turn, the molecular mass of albumin is 66 kDa and much more significant than the molecular weight of Evans blue 961 Da. Thus, the measurement of the BBB permeability is determined precisely by the molecular mass of albumin which penetrates through the damaged BBB, thereby transferring the Evans blue. In addition to the techniques described above, there are other techniques, in particular those based on a combination of various dextrans and radioactive molecules, which together give more accurate results. Measurement of BBB breakdown by luminescence spectrometry is cheaper and easier to use, compared to more accurate but more expensive techniques. We used this method for the evaluations of BBB disruption together with measurements of infarct zone and brain edema. Injection of Evans blue for assessment of BBB permeability prior to induction of MCAO does not affect the accuracy of measuring these two parameters23.
The present protocol presents a novel technique for measuring the three most important determinants of ischemic brain injury on the same brain sample. This method can be also applied to models of other brain injuries. This protocol will contribute to the study of the pathophysiology of ischemic injury.
The authors have nothing to disclose.
We thank Maryna Kuscheriava, Maksym Kryvonosov, Daryna Yakumenko and Evgenia Goncharyk of the Department of Physiology, Faculty of Biology, Ecology, and Medicine, Oles Honchar, Dnipro University, Dnipro, Ukraine for their support and helpful contributions to our discussions. The data obtained are part of Ruslan Kuts’s PhD thesis.
2 mL Syringe | Braun | 4606027V | |
2% chlorhexidine in 70% alcohol solution | Sigma-Aldrich | 500 cc | Provides general antisepsis of the skin in the operatory field |
27 G Needle with Syringe | Braun | 305620 | |
3-0 Silk sutures | Henry Schein | 1007842 | |
4-0 Nylon suture | 4-00 | ||
Brain & Tissue Matrices | Sigma-Aldrich | 15013 | |
Cannula Venflon 22 G | KD-FIX | 183603985447 | |
Centrifuge Sigma 2-16P | Sigma-Aldrich | Sigma 2-16P | |
Compact Analytical Balances | Sigma-Aldrich | HR-AZ/HR-A | |
Digital weighing scale | Sigma-Aldrich | Rs 4,000 | |
Dissecting scissors | Sigma-Aldrich | Z265969 | |
Eppendorf pipette | Sigma-Aldrich | Z683884 | |
Eppendorf tube | Sigma-Aldrich | EP0030119460 | |
Fluorescence detector | Tecan, Männedorf Switzerland | Model: Infinite 200 PRO multimode reader | Optional. |
Fluorescence detector | Molecular Devices LLC | VWR cat. # 10822 512 SpectraMax Paradigm Multi Mode Microplate Reader Base Instrument | Optional. |
Gauze sponges | Fisher | 22-362-178 | |
Heater with thermometer | Heatingpad-1 | Model: HEATINGPAD-1/2 | |
Hemostatic microclips | Sigma-Aldrich | ||
Horizon-XL | Mennen Medical Ltd | ||
Infusion cuff | ABN | IC-500 | |
Micro forceps | Sigma-Aldrich | ||
Micro scissors | Sigma-Aldrich | ||
Multiset | Teva Medical | 998702 | |
Olympus BX 40 microscope | Olympus | ||
Operating forceps | Sigma-Aldrich | ||
Operating scissors | Sigma-Aldrich | ||
Optical scanner | Canon | Cano Scan 4200F | Resolution 3200 x 6400 dpi |
Petri dishes | Sigma-Aldrich | P5606 | |
Purina Chow | Purina | 5001 | Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical research for over 5 decades. Provided to rats ad libitum in this experiment. |
Rat cages | Techniplast | 2000P | Conventional housing for rodents. Cages were used for housing rats throughout the experiment |
Scalpel blades #11 | Sigma-Aldrich | S2771 | |
Software | |||
Adobe Photoshop CS2 for Windows | Adobe | ||
ImageJ 1.37v | NIH | The source code is freely available. The author, Wayne Rasband (wayne@codon.nih.gov), is at the Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA | |
Office 365 ProPlus | Microsoft | – | Microsoft Office Excel |
Windows 10 | Microsoft | ||
Reagents | |||
2,3,5-Triphenyltetrazolium chloride | Sigma-Aldrich | 298-96-4 | |
50% trichloroacetic acid | Sigma-Aldrich | 76-03-9 | |
Ethanol 96 % | Romical | Flammable liquid | |
Evans blue 2% | Sigma-Aldrich | 314-13-6 | |
Isoflurane, USP 100% | Piramamal Critical Care, Inc | NDC 66794-017 |