Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

Summary

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.

Abstract

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.

Introduction

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 cases^1,2. 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 volumes^14,15,16, and blood brain barrier (BBB) permeability by a spectrometry technique using Evans blue staining^17,18,19.

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 method^16,22.

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.

Protocol

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

1. Select adult male Sprague-Dawley rats without overt pathology, each weighing between 300 and 350 g.
2. Maintain all rats at room temperature at 22 °C, with 12 hours of light and dark cycles before experiment.
3. Ensure that food and water are available ad libitum.
4. Perform all procedures between 6:00 a.m. and 2:00 p.m.

2. Preparing rats for surgery

1. Anesthetize the rats for 30 min with isoflurane (4% for induction and 2% for maintenance) and 24% oxygen (1.5 L/min).
   1. Test the level of anesthesia in the rats by ensuring they do not have a pedal withdrawal reflex.
2. Insert the 24-gauge catheter into the tail vein.
   NOTE: Tail warming for vasodilation is not performed.
   1. Place the rats on the table in a supine position. Use medical tape to affix all four of the rats’ limbs.
3. Place the probe for temperature measurement into the rat rectum before surgery.
4. During the procedure, maintain a heating plate to support a 37 °C core body temperature.
5. Add ointment in both of the rat’s eyes for protection.
6. Shave the surgical area and disinfect with three applications of 10% povidone-iodine followed by 70% isopropyl alcohol.

3. Right side middle cerebral artery occlusion

NOTE: MCAO is performed by a modified technique, as previously described^16,22,23, with the use of instruments described by McGarry et al.²⁴ and Uluç et al.²⁵.

1. Dissect the skin and superficial fascia at the ventral midline of neck with surgical tweezers and scissors with curved blades.
2. Identify the muscle triangle, consisting of the ICA, external carotid artery (ECA) and common carotid artery (CCA).
3. Carefully separate the right CCA and ICA from the vagus nerve with microforceps for vascular surgery.
4. Expose the right CCA and the ICA. Block the blood flow coming from the CCA to the ICA using either micro-clips or special tourniquets for vascular surgery. Make an incision (approximately 1 mm) on the ICA using microscissors for vascular surgery.
5. Insert a monofilament catheter (4-0 nylon) directly through the ICA, about 18.5-19 mm from the bifurcation point of the right CCA into the circle of Willis until reaching a mild resistance, to occlude the MCA²⁶.
6. Ligate around the ICA above the bifurcation of CCA.
7. For the sham-operated control group, perform an insertion of nylon thread instead of steps 3.5 and 3.6^16,22.
8. Administer 5 mL of 0.9% sodium chloride by intraperitoneal injection.
9. Close the wound by suture and take the rat to a recovery area.
   NOTE: A few minutes after the end of anesthesia, the rat will wake up and move independently around the cage.
10. At 23 h after MCAO, inject 2% Evans blue in saline (4 mL/kg)^23,26 into the tail vein for both operated groups via a cannula²⁷.
    NOTE: This is used as a blood-brain permeability tracer. Allow to circulate for 60 minutes.

4. Determination of infarct zone

1. Measure IZ at 24 h after MCAO as described previously^{9,15,18,19,26}.
   NOTE: Rats that lost more than 20% of their weight or developed seizures or hemiplegia are excluded from the experiment.
2. Euthanize the rat by replacing the inspired gas mixture with 20% oxygen and 80% carbon dioxide until the rat ceases to breathe spontaneously.
3. Open the chest with a 5-6 cm lateral incision through the abdominal wall under the rib cage using scissors and surgical forceps.
4. Perform a diaphragmatic incision along the entire length of the rib cage with scissors and surgical forceps.
5. Carefully displacing the lungs, cut through the rib cage up to the collarbone on the right and left sides²⁸.
6. Perfuse with 200 mL of normal saline through the left ventricle of the heart.
7. Puncture or incise the right atrium of the heart with scissors.
8. Perform decapitation using a guillotine and collect brain tissue.
9. Using iris scissors, cut from the foramen magnum to the distal edge of the posterior skull surface on both sides.
10. Separate the olfactory bulbs, nervous connections along the ventral surface and dorsal surface of the skull from the brain.
11. Remove the brain from the head.
12. Produce 6 brain slices by creating 2 mm thick horizontal sections with a .009" stainless steel, uncoated, single edge razor blade.
13. Incubate for 30 min at 37 °C in 0.05% TTC.
14. Place the brain tissue on the microscope slides and perform optical scanning of these 6 brain-slices with a resolution of 1600x1600 dpi (see Supplement 1 for example).
15. Add a blue filter with a photo editor (e.g., Adobe Photoshop CS2) using the Channel Mixer function (Image > Adjustments > Channel Mixer) and save the image as a JPEG file format.
    NOTE: After applying the blue filter, the image will appear greyscale.
16. Open the saved image in ImageJ 1.37v^29,30.
    NOTE: This computer program uses a threshold function to isolate and calculate the pixels that are either black or white (see Figure 1).
17. For each of the 6 brain slices of the image, select and save each hemisphere (right injured ipsilateral and left uninjured contralateral) as a separate image file using the “polygon selection” tool from the main menu.
18. Set the cut-off for determining IZ by using an auto threshold function from the main menu of the ImageJ software by selecting Image > Adjust > Threshold, and measure the number of pixels in each hemisphere of a single brain set.
    NOTE: Macros may be used for this step in ImageJ software (see Supplement 2 for the code). 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 Supplement 3 and Supplement 4 as examples). ImageJ then compares white and black pixels to determine IZ. Based on the staining protocol and scanner settings, we used a constant cut-off value of 0.220.
19. Perform measurement of IZ correcting for tissue swelling using the Ratios of Ipsilateral and Contralateral Cerebral Hemispheres (RICH) method^13,23 (see example in Supplement 5).
    
    NOTE: Infarct size is assessed as a percentage of the contralateral hemisphere.

5. Determination of brain edema³¹

NOTE: Use ImageJ 1.37v for measurement of BE^32,33.

1. Measure BE 24 h after MCAO. For calculation of BE, use the data from left and right hemisphere volume (in units).
2. Perform optical scanning with a resolution of 1600x1600 dpi (see Supplement 1 for example).
3. Select brain hemispheres and set the cut-off for determining BE with ImageJ 1.37v, as described above in sections 4.17-4.19.
4. Express the BE area as a percentage of the standard areas of the unaffected contralateral hemisphere, calculated by the RICH method using following equation (see example in Supplement 5)^23,34.
   
   NOTE: Extent of BE is assessed as a percentage of the contralateral hemisphere.

6. Determination of BBB disruption

1. Measure BBB disruption 24 h after MCAO.
2. Divide right and left hemispheres into six slices and put each one into a microcentrifuge tube.
3. Homogenize each slice of the brain tissue in trichloroacetic acid, based on the calculation of 1 g of brain tissue in 4 mL of 50% trichloroacetic acid.
4. Centrifuge at 10,000 x g for 20 min.
5. Dilute supernatant liquid 1:3 with 96% ethanol.
6. Perform luminescence spectrophotometry by utilizing spectrophotometry software, installing the plate, and performing a sample reading using the following parameters: Fluorescence intensity excitation wavelength of 620 nm (bandwidth 10 nm) and an emission wavelength of 680 nm (bandwidth 10 nm)^23,35 ; Mod top; Number Flesh 25; Manual 100; Shaking 1 sec, 1 mm.
   NOTE: Use an excitation wavelength of 620 nm (bandwidth 10 nm) and an emission wavelength of 680 nm (bandwidth 10 nm).^23,35

Results

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...

Discussion

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 te...

Materials

Name	Company	Catalog Number	Comments
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