Simplified Model of Krypton LaserInduced Thrombotic Distal Middle Cerebral Artery Occlusion in Spontaneously Hypertensive Rats
Background and Purpose The effects of thrombotic occlusion of the middle cerebral artery on compromised ischemic tissue may be different from and more severe than those of cerebral ischemia induced by mechanical occlusion of the artery. Photothrombosis, which is based on photochemical damage to the endothelium and subsequent platelet aggregation, is an efficient method to induce thrombosis in vivo. This study aimed to improve and simplify this unique method for an ischemia model of middle cerebral artery occlusion in rats.
Methods Male spontaneously hypertensive rats (5 to 6 months old, 300 to 450 g) were anesthetized with halothane, endotracheally intubated, and mechanically ventilated. A krypton laser operating at 568 nm was used to irradiate the exposed distal middle cerebral artery with an intact dura above the rhinal fissure. The photosensitizing dye rose bengal (20 mg/kg body wt) was administered intravenously over 90 seconds starting simultaneously with 4 minutes of laser irradiation at a power of 20 mW to cause thrombotic occlusion of this artery.
Results The irradiated middle cerebral artery was completely occluded by intraluminal thrombi within 3 minutes after simultaneous laser irradiation and rose bengal infusion. Thrombosed materials were not stained by phosphotungstic acid–hematoxylin stain (ie, aggregated platelets lacked apparent fibrin). The mean volume of 3-day-old infarction, indicated by the lack of staining with 2,3,5-triphenyltetrazolium chloride, was 84.8±17.4 mm3 (mean±SD, n=6).
Conclusions We demonstrated a reproducible and minimally traumatic model of brain infarction induced by the thrombotic distal middle cerebral artery occlusion in rats.
Ischemic stroke or brain infarction is defined as an insufficient blood supply to a focal region of the brain. Accordingly, focal ischemia models replicate the situation of human brain infarction.1 In the context of thrombosis, however, the effects of thrombotic stroke on compromised brain tissue may be different from those due to cerebral ischemia induced by mechanical occlusions of intracranial or extracranial brain arteries (see Watson et al2 for review). The neuronal and microvascular changes in rats with photothrombotic occlusion of the MCA were more rapid and severe in comparison to mechanical (clip) occlusion.3 Massive release of platelet-derived serotonin occurs during common carotid artery photothrombosis determined by intra-arterial microdialysis.4 Such blood-borne factors secreted during the photochemical process are considered to be responsible for a marked breakdown of the blood-brain barrier and abnormal CBF in locations distant from the thrombosed artery.5
Recently, a focal ischemia model of photochemically induced MCA occlusion was developed in normotensive rats6 ; the procedure was based on a previously described method by Chen et al,7 in which the occlusion of the distal MCA was combined with common carotid artery ligations. The major disadvantage of this model is thatconcurrent carotid occlusions may unnaturally suppress retrograde collateral flow to the “penumbra,” which could make the model more resistant to pharmacoprotection.8 9 To avoid such common carotid artery involvements, we attempted to use SHR as a thrombotic MCA occlusion model because SHR develop much larger cortical infarcts after MCA occlusion than normotensive rats because of hemodynamically vulnerable collateral function.1 Here we present a simplified model of distal MCA occlusion in SHR produced by an interaction of a krypton laser and the photosensitizing dye rose bengal (ie, photothrombosis2 ).
Materials and Methods
This study was conducted according to the guidelines of the local committee on animal research. Sixteen male SHR (5 to 6 months old, 300 to 450 g), which were allowed free access to food and tap water, were anesthetized with halothane (4% for induction, 1.5% during the surgical preparation with a face mask, 0.75% after intubation, and 0.5% for maintenance) in a mixture of 70% nitrous oxide and 30% oxygen. The right femoral artery and vein were cannulated with PE-50 tubing. The rats were endotracheally intubated with PE-240 tubing. Pancuronium bromide (an initial dose of 0.3 mg followed by 0.1 mg every 30 minutes) was injected, and the rats were mechanically ventilated. Blood pressure was continuously monitored, and physiological variables were determined before and after distal MCA occlusion. Rectal and head temperatures were maintained at 37.5°C and from 36.0°C to 36.5°C, respectively, by means of a warming lamp. Rats were placed on a stereotaxic head holder in the prone position, and a 2-cm incision was made vertically midway between the right orbit and the right external auditory canal. The temporalis muscle was separated and retracted, and a burr hole (3 mm in diameter) was made 1 mm rostral to the anterior junction of the zygoma and squamosal bones under an operating microscope, revealing the distal segment of the MCA above the rhinal fissure as described previously.6 8 9 10 The dura was thereby left intact.
A krypton laser operating at 568 nm (Innova 301, Coherent Inc) was used to irradiate the distal MCA at a power of 20 mW. The laser beam was focused with a cylindrical lens and positioned with a mirror onto the distal MCA. The corresponding energy at the focal plane was about 64 mW/mm2. To establish a criterion for the irradiation of the distal MCA with the laser beam, the anatomy of the distal MCA had been recorded carefully by drawing under an operating microscope (×20) through the cranial window in 127 Sprague-Dawley rats, 42 Wistar rats, and 43 SHR; we have found that distal MCAs of SHR are very simple, and the lower half of the exposed distal MCA can be enveloped with an elliptical, almost “linear,” laser beam (approximately 2 mm long) as shown in the Figure⇓ (A). The photosensitizing dye rose bengal (15 mg/mL in 0.9% saline; Wako Pure Chemical Industries Ltd) was administered intravenously to a body dose of 20 mg/kg over 90 seconds starting simultaneously with 4 minutes of laser irradiation.
Two hours after distal MCA occlusion, the head wound was closed and the catheters were removed. The rats were carefully weaned from the respirator and returned to the home cage after regaining the ability to breathe independently. After 3 days, the rats were decapitated under amobarbital anesthesia (100 mg/kg IP), and the brains were rapidly removed. The entire brains were cooled in ice-cold saline for 10 minutes and cut into 2-mm-thick coronal sections in a cutting block, and the brain slices were then immersed in 2% TTC (Wako Pure Chemical Industries Ltd) at 37°C for 30 minutes in the dark.11 The posterior surface of each section was photographed, and the infarct area, indicated by the lack of staining, was calculated with National Institutes of Health Image software (version 1.56). The infarct volume of each rat was calculated as the product of the infarct area times the 2-mm thickness of each section.
In a separate group of rats, acute changes in cortical brain temperature during and immediately after the laser irradiation were monitored with a thermocouple probe that was inserted into the brain cortex very close (within 1 mm) to the irradiated distal MCA. These rats were used for PTAH staining12 of the thrombosed material. Regional CBF was determined with laser-Doppler flowmetry 2 mm posterior and 4 mm lateral to the bregma. Because visible light interferes with laser-Doppler flowmetry, the heating lamp was temporarily turned off during measurements of CBF. Changes in CBF were expressed as a percentage of the average of two to three baseline values.
Physiological variables recorded just before photochemically induced distal MCA occlusion were as follows (n=16, mean±SD): Paco2, 37.7±2.5 mm Hg; Pao2, 146±22 mm Hg; pH, 7.43±0.03; hematocrit, 0.42±0.03; and blood glucose, 5.8±1.0 mmol/L. These variables were stable during the follow-up period. The irradiated MCA was completely occluded by an intraluminal thrombus within 3 minutes after simultaneous laser irradiation and rose bengal infusion, which was confirmed by visual inspection under the operating microscope. Correspondingly, CBF showed little change at 1 minute but decreased to 36% of resting at 3 minutes and remained at the same level throughout the experiment (Table⇓). Continuous blood pressure monitoring revealed a transient drop during the 90 seconds of rose bengal infusion, but pressure soon returned to the resting level. In rats in which the brain temperature was measured before, during, and after laser irradiation, a modest rise in temperature from 36.5°C to 37.1°C was observed at 1 minute of irradiation. The distal MCA was successfully occluded with an intravascular thrombus as shown in panels B and C of the Figure⇑. Thrombosed materials were not stained by PTAH (ie, aggregated platelets lacked apparent fibrin) (panel C). A typical TTC-stained section is presented in panel D. The infarct was limited to the neocortex with a sharply marginated infarct rim. The mean volume of infarction in six rats was 84.8±17.4 mm3.
It is well known that the rat models of distal MCA occlusion are confounded by the remarkable variability in the distal part of this artery.1 2 To establish a criterion for laser irradiation of the distal MCA with a linear beam, we analyzed the anatomy of this artery in three strains of rats (Sprague-Dawley rats, Wistar rats, and SHR) and found that the lower half of the exposed distal MCA was acceptably linear in Sprague-Dawley rats and more simple in SHR but often was complicated in Wistar rats. Therefore, our “linear” hit model with a green laser beam (Figure⇑, A) can be applied to the former two strains of rats. We found, however, that the infarcts produced in four Sprague-Dawley rats by the same method were much smaller (11.6±2.2 mm3; H.Y., unpublished data, 1995) than those in SHR, despite the fact that the extent of thrombotic occlusion was essentially the same between the two strains. This finding is compatible with reported studies of ligated distal MCA occlusion in stroke-prone SHR and Wistar-Kyoto rats.13 14 An upward shift of the lower limit of CBF autoregulation due to chronic hypertension15 and impaired endothelium-mediated dilatation16 may be the basis for diminished luminal width14 and insufficient collateral vasodilator response in SHR, resulting in much larger infarcts in hypertensive rats than in normotensive rats. Taken together, the function of retrograde collaterals rather than anatomic variations (branching patterns) of the distal MCA17 appears to correlate with the severity of CBF reduction and the size of infarction produced by distal MCA occlusion.
The wavelength of maximum absorption for protein- or membrane-bound rose bengal is 562 nm.2 18 The 568-nm krypton laser does not require the high-maintenance dye laser, but theoretically it still excites the intravascular rose bengal almost as efficiently as the 562-nm argon/dye laser and generates singlet oxygen, which peroxidizes lipid molecules within the vascular endothelium.2 This process of endothelial damage gives rise to prominent platelet aggregation, resulting in vascular occlusion. Actually, the irradiated MCA was consistently occluded within 3 minutes, and the thrombus was stable during the 2- to 3-hour-long observation period. The combination of a 568-nm krypton laser line and rose bengal may be second best in terms of efficacy in photochemical reaction compared with the 562-nm argon/dye laser, but in practice our method seems to be very efficient and may be the most simplified system for producing thrombotic MCA occlusion.
Laser-based methods of thrombus formation have the possible disadvantage of generating local heat. Even a 2°C to 3°C rise in brain temperature aggravates neuronal injury in ischemic brain.19 Furthermore, the issue of temperature is important because thrombus composition is likely to be affected by increased temperature. Under elevated temperatures, the thrombus shows mixed character, containing fibrin in addition to pure platelets. In contrast, mild hypothermia (core temperature of 32.0°C and arm-skin temperature of 27.3°C) attenuated platelet function, resulting in significantly longer bleeding time.20 Although we could not determine the actual rise in endothelial temperature, a very modest temperature increase from 37.2°C to 37.7°C in blood measured downstream from the irradiated site was observed during the 10-minute irradiation at a power of 325 mW (220 mW/mm2)4 ; the present study showed that an increase in brain temperature during laser irradiation at a power of 20 mW was small and the MCA thrombi were lacking fibrin,12 indicating no temperature-related artifacts in the formed thrombi. However, we cannot exclude the possibility that heat in addition to singlet oxygen may play a role in damaging or changing the functional property of the endothelium, encouraging platelet adhesion and aggregation.
The present results indicate that the infarcts produced by single segmental occlusion of the distal MCA with a linear laser beam in SHR were localized reproducibly with an acceptable coefficient of variation (CV=21%) while being surrounded by potential retrograde collateral flow. The method of MCA photothrombosis without additional carotid manipulation has been reported by Prado et al.21 The fact that the infarction observed in the present study was smaller than those usually reported in SHR after distal MCA occlusion22 may be accounted for by our exclusion of common carotid artery (tandem) occlusion and an absence of anesthesia-related hypotension. This moderate-sized infarction would be fitting for trials of pharmacological substances, since ischemic insults of submaximal severity are more likely to reveal beneficial effects of therapeutic interventions. This model does not entail extensive surgery. Animals are able to eat and drink soon after surgery and survive well. This laser-driven photochemical occlusion of the distal MCA is much easier and less traumatic than standard electrocautery or even ligature methods.
In summary, we have demonstrated a reproducible and minimally traumatic model of MCA territory infarction produced by a simplified method of photothrombosis in SHR. This model will be useful for biochemical studies of evolving thrombotic infarction.
Selected Abbreviations and Acronyms
|CBF||=||cerebral blood flow|
|MCA||=||middle cerebral artery|
|PTAH||=||phosphotungstic acid–hematoxylin stain|
|SHR||=||spontaneously hypertensive rats|
This study was partly supported by the Social Insurance Agency Contract Fund commissioned by the Japanese Health Sciences Foundation. We thank Brant D. Watson, PhD, and Ricardo Prado, MD, for their valuable advice during the course of this study and Hideko Noguchi for her technical assistance. Brant D. Watson suggested the use of krypton laser to one of the authors (H.Y.), and the method of krypton laser use for arterial occlusion has previously been discussed in the review by Watson and colleagues (Reference 2).
- Received July 17, 1995.
- Revision received October 24, 1995.
- Accepted October 27, 1995.
- Copyright © 1996 by American Heart Association
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