点击显示 收起
the Division of Strokology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Abstract
Background and Purpose— Neurons acquire tolerance to ischemic stress when preconditioning ischemia occurs a few days beforehand. We focused on collateral development after mild reduction of perfusion pressure to find an endogenous response of the vascular system that contributes to development of ischemic tolerance.
Methods— After attachment of a probe, the left common carotid artery (CCA) of C57BL/6 mice was occluded. The left middle cerebral artery (MCA) was subsequently occluded permanently on days 0, 1, 4, 14, and 28 (n=8 each). The change in cortical perfusion during MCA occlusion was recorded. A sham group of mice received only exposure of the CCA and MCA occlusion 14 days later. In apoE-knockout mice, the MCA was occluded 14 days after CCA occlusion or sham surgery. Infarct size and neurologic deficit were determined 4 days after MCA occlusion.
Results— Mice that had 45% to 65% of baseline perfusion after CCA occlusion were used. Cortical perfusion after MCA occlusion was significantly preserved in day 14 (47±16%) and day 28 (46±7%) groups compared with day 0 (28±8%), day 1 (33±19%), day 4 (29±16%), and sham groups (32±9%). Infarct size and neurologic deficits were also attenuated in day 14 and day 28 groups compared with other groups. In apoE-knockout mice, there was no significant difference in perfusion, neurologic deficits, or infarction size between groups with and without CCA occlusion.
Conclusion— Chronic mild reduction of perfusion pressure resulted in preservation of cortical perfusion and attenuation of infarct size after MCA occlusion. These responses of collaterals were impaired in apoE-knockout mice.
Key Words: chronic ischemia chronic perfusion collateral circulation focal ischemia ischemic tolerance
Introduction
Collateral circulation through leptomeningeal vessels may determine the severity of ischemic injury after occlusion of middle cerebral artery (MCA) in patients with stroke.1 Although chronic hypoperfusion is believed to be critical for collateral development,2 factors leading to it are uncertain. For better understanding of collateral circulation in cerebral ischemia, an animal model in which cerebral collaterals can be investigated is needed. Recently, Busch et al3 demonstrated that 3-vessel (one carotid plus both vertebral arteries) occlusion induced arteriogenesis at the circle of Willis in hypoperfused rat brain. They found that the diameter of the posterior cerebral artery enlarged significantly 1 week after 3-vessel occlusion,3 but the distal leptomeningeal collaterals that determine stroke outcome did not change. Although morphologic assessment of leptomeningeal collaterals requires multivessel angiography4 or latex perfusion methods,5 the level of collateral circulation after cerebral ischemia can be functionally examined by measuring the change of cerebral perfusion at the time of MCA occlusion. In this study, we examined the effect of a mild reduction of cerebral perfusion pressure by occlusion of the left common carotid artery (CCA) on collateral development and infarct size after MCA occlusion. Furthermore, we examined the effect of apoE deficiency on development of collateral circulation after CCA occlusion because arteriogenesis in knockout mice is impaired in a hindlimb ischemia model.6
Materials and Methods
C57BL/6 strain mice were obtained from Charles River (Yokohama, Kanagawa, Japan). apoE-knockout mice, originally produced by Zhang et al,7 were purchased from the Jackson Laboratory (Bar Harbor, Maine). All mice used in this study were mature males aged 12 to 16 weeks. Mice were given free access to food and water before surgery. The experimental procedures involving laboratory animals have been approved by the Institutional Animal Care and Use Committee of the Osaka University Graduate School of Medicine.
Surgery
General anesthesia was induced with 4.0% halothane and maintained with 0.5% halothane with an open facemask. A polyacrylamide column with an inner diameter of 0.8 mm for measurement of cortical microperfusion by laser-Doppler flowmetry (Advance laser Flowmetry, model ALF-21; Advance Co) was attached with dental cement to the intact skull 3.5mm lateral to the bregma. Laser Doppler flowmetry is not quantitative, but provides a reliable estimate of relative cerebral blood flow. The residual zero flow signal at the time of killing was <2% of baseline perfusion level in a preliminary experiment (n=10). Therefore, we did not determine it in each mouse in the following experiments. Body temperature was monitored with a rectal thermometer and maintained at 37.0°C with a heat lamp.
Common Carotid Artery Occlusion
In the first experiment, in each of 50 C57BL/6 mice, the CCA was ligated and the change in cortical perfusion after CCA occlusion was recorded. Seven days later, mice were killed with an overdose of pentobarbital. The brains were removed, fixed by immersion in an alcohol-5% acetic acid solution for 5 hours at 4°C, dehydrated, and embedded in paraffin. Tissue sections (5 μm) were obtained every 1mm, beginning at the frontal pole, and were examined after conventional staining with hematoxylin and eosin (H&E). In the sections including the hippocampus, the terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) procedure was performed using the Apoptag in situ Detection Kit (Chemicon). In another 8 mice that had 45% to 65% of baseline perfusion after CCA occlusion, the cortical perfusion recordings were measured at 2 hours and 1, 4, 14, and 28 days after CCA occlusion. The change of cortical perfusion after sham surgery was also recorded in the other 8 mice. The level of cortical perfusion was expressed as a percentage of the baseline value.
Middle Cerebral Artery Occlusion Subsequent to Common Carotid Artery Occlusion
In the second experiment, the CCA was ligated under halothane anesthesia. In mice that had 45% to 65% of baseline cortical perfusion after CCA occlusion, the MCA was occluded using electrocoagulation as described previously8 on days 0, 1, 4, 14, and 28 (n=8 each). Sham-group mice (n=8) received only exposure of CCA and MCA occlusion 14 days later. Mice were placed in the recumbent position, and a vertical skin incision was made at the midpoint between the left orbit and the external auditory canal. The mandible was pulled downward to expose the skull base. A small burr hole was made in the skull over the left MCA. The MCA was permanently occluded with a microbipolar electrocoagulator just proximal to the point where the olfactory branch came off. Cortical perfusion was monitored under halothane anesthesia for 15 minutes after MCA occlusion. Four days after MCA occlusion, mice were evaluated for neurologic deficits by a blind observer. The neurologic deficit score assignment of 0 to 4 was based on methods described previously by Yang et al9: 0, no neurologic deficits; 1, failed to extend right forepaw while held by the tail; 2, circled to the right; 3, fell to the right; or 4, unable to walk spontaneously. Then, mice were killed under pentobarbital anesthesia, and their brains were removed, fixed, and embedded in paraffin. The volume of infarction was measured using MCID Image Analysis Software (Imaging Research). The volume (mm3) was determined by integrating the appropriate area and the section thickness. To visualize the capillary perfusion, we labeled plasma with dichrolotriazinyl amino fluorescein (DTAF; excitation 489 nm, emission 515 nm; Sigma-Aldrich) as described previously.8 After MCA occlusion for 6 hours in mice with CCA occlusion or sham operation 14 days earlier, 50 μL of DTAF conjugated to mouse serum was injected for 10 seconds into the saphenous vein. Thirty seconds after completion of the injection, each mouse was decapitated and the brains were fixed in 80% ethanol for 24 hours. Brain slices, 50 μm in thickness, were prepared with a vibratome and examined under a fluorescence microscope. In mice with CCA occlusion or sham operation 14 days earlier (n=5 each), a femoral artery was cannulated with a PE-10 polyethylene tube at the time of MCA occlusion. Blood pH, PaO2, and PaCO2 were measured using the Acid-Base Laboratory system (ABL550; Radiometer).
Effect of Common Carotid Artery Occlusion in apoE-Knockout Mice
In the last experiment, we used apoE-knockout mice. The left CCA of apoE-knockout mice (n=8) was ligated under halothane anesthesia. Fourteen days later, the left MCA was permanently occluded. Sham-group mice (n=8) received only exposure of the left CCA and occlusion of the left MCA 14 days later. Four days after MCA occlusion, mice were evaluated for neurologic deficits, and they were killed and their brains were examined for infarction size. The experiment schedule is shown in Figure 1.
Statistics
All values are presented as mean±standard deviation. One-way analysis of variance (ANOVA) was performed with Scheffe’s multiple comparisons test to assess differences in perfusion change, neurologic deficit, score and infarct size between day 0, day 1, day 4, day 14, day 28, and sham groups. The perfusion values after CCA occlusion were analyzed by repeated-measures ANOVA followed by a post hoc Dunnett test. A Mann–Whitney U test was performed to assess differences between 2 groups of apoE-knockout mice. Pearson’s test was used to evaluate the relationship between perfusion change after CCA occlusion and that after MCA occlusion. P<0.05 was considered significant. All statistical analyses were conducted using SPSS/Windows software, version 11.5J (SPSS Japan Inc).
Results
Unilateral occlusion of the left CCA resulted in 40% to 70% of baseline microperfusion over the MCA area in most mice. However, in 6 mice, cortical microperfusion was reduced to less than 35% of baseline (Figure 2). On the basis of histologic examination, one mouse showed infarction in the cortex, caudoputamen, and hippocampus, and 2 mice showed ischemic neuronal damage in the hippocampus with fragmented DNA detected by the TUNEL method (Figure 2B). In the other 47 mice, no ischemic damage was found (Figure 2B). Because more than 70% of mice had 45% to 65% of baseline cortical perfusion, we used those mice in the subsequent experiment. In the control group, the mean cortical perfusion after the sham operation varied from 90% to 111% without any significant changes between any time intervals. The cortical perfusion values decreased to 59.0±4.6%, 59.3±6.9%, and 55.4±9.0% at 2 hour and 1 and 4 days, respectively, and remained significantly lower at 14 (61.4±11.6%) and 28 days (63.2±14.0%) after CCA occlusion as compared with the baseline value (Figure 2C).
The change in the cortical perfusion after MCA occlusion compared with that before CCA operation, the perfusion change at the time of MCA occlusion, neurologic deficit score, and infarct size 4 days after MCA occlusion are shown for each group in Figure 3. CCA occlusion and subsequent MCA occlusion reduced perfusion to 15% to 20% in day 0, day 1, and day 4 groups (Figure 3A). However, in day 14 and day 28 groups, the change in perfusion after CCA and MCA occlusion were approximately 30% and were significantly higher than those of day 0, day 1, and day 4 groups (Figure 3A). In the sham-operation group, the change in perfusion after sham CCA operation and MCA occlusion was 31.8±9.0%, higher than those in day 0, day 1, and day 4 groups but not significant different compared with those in day 14 and day 28 groups (Figure 3A). At the time of MCA occlusion, cortical perfusion reduced to approximately 30% of baseline in day 0, day 1, day 4, and sham CCA exposure groups (Figure 3B). However, in day 14 and day 28 groups, the changes in perfusion at the time of MCA occlusion were 47.6±15.8% and 46.1±6.7%, respectively, and were significantly preserved compared with other groups (Figure 3B). In day 14 and day 28 groups (n=16), there was significantly negative correlation between the perfusion change after CCA occlusion and that at the time of MCA occlusion (r=–0.686, P<0.01). Both neurologic deficit score (Figure 3C) and infarction size (Figures 3D and 4) were significantly attenuated in day 14 and day 28 groups compared with other groups. Reduction of infarction size was markedly observed in the cerebral cortex (Figures 3D and 4). Capillary fluorescence was hardly observed in the center of the MCA territory in sham operation group (Figure 4). In contrast, capillary perfusion in the same territory was better preserved in day 14 group (Figure 4). The physiological variables were determined for mean arterial blood pressure (day 14 group, 76.6±4.3 mm Hg; sham operation group, 77.0±3.4 mm Hg), PaCO2 (day 14 group, 35.2±3.3 mm Hg; sham operation group, 34.4±2.8 mm Hg), PaO2 (day 14 group, 119.6±9.2 mm Hg; sham operation group, 120.2±8.8 mm Hg), and pH (day 14 group, 7.29±0.07; sham operation group, 7.32±0.04 mm Hg). There were no significant differences in any of the variables between day 14 and sham-operation groups.
In apoE-knockout mice, the change in cortical perfusion, the neurologic score, and infarct size are shown in Figure 5. In apoE-knockout mice, the CCA occlusion treatment group (n=8) and the sham CCA exposure group (n=8) showed no significant difference in cortical microperfusion after CCA occlusion and subsequent MCA occlusion (29.5±9.0% compared with 30.4±8.5%), perfusion change at the time of MCA occlusion (36.4±11.8% compared with 30.2±7.8%), in neurologic scores (1.5±0.8 compared with 1.9±1.0), or in infarct size (31.5±8.2 mm3 compared with 37.0±5.3 mm3, P=0.14).
Discussion
Leptomeningeal collaterals that may maintain perfusion beyond the site of an arterial occlusion have been appreciated as an important factor both in modifying the size of infarction and in clinical outcome after embolic occlusion of the MCA.1,10 Angiogenesis after MCA occlusion has been intensively investigated.11,12 However, little is known about the factors that determine collateral development at the time of embolic occlusion. MCA occlusion has been shown to induce growth and enlargement of surface collaterals in the ischemic border.13 Chronic hypoperfusion resulting from extracranial and intracranial occlusive disease promotes leptomeningeal collateral formation, although collaterals require time to develop.5,10,13 The precise role of chronic hypoperfusion in collateral development remains unclear, although it is known that the efficacy of collateral vessels also depends on age, hypertension, and associated comorbidities.14
Recently, arteriogenesis at the circle of Willis has been demonstrated clearly in a 3-vessel (1 carotid plus both vertebral arteries) occlusion model.3 Although reduction of perfusion pressure promotes arteriogenesis at the circle of Willis and in the distal anastomoses that have been described as "pre-existing collateral arterioles,"15 the diameters of leptomeningeal anastomoses in the 3-vessel occlusion model did not change.3 The estimation of vessel diameters may be insufficient for assessing collateral development, particularly in the ischemic condition. Resting cortical perfusion level remained 60% of baseline up to day 28 after CCA occlusion (Figure 2C), suggesting us to use a functional evaluation of collateral circulation by measuring the cortical perfusion at the time of MCA occlusion. We first examined the effect of unilateral CCA occlusion on cortical perfusion and histologic damage. Very few mice (6 of 50) showed moderate to severe ischemia (<35% of baseline) after unilateral CCA occlusion, and only 3 of them had histologic damage in the affected hemisphere. Therefore, in the present study, we used mice that showed 45% to 65% of baseline cortical perfusion.
Our results demonstrated that unilateral CCA occlusion treatment given 14 days before MCA occlusion preserved cortical perfusion and reduced infarct size markedly in the cerebral cortex after MCA occlusion. Negative correlation between changes in perfusion after the first CCA occlusion and at the time of subsequent MCA occlusion in day 14 and day 28 groups suggests that sufficient reduction of perfusion pressure is needed for collateral development. The time required to develop the effects in the present study is in agreement with the findings about collateral vessel development in hindlimb ischemia models.16 In general, the development of preexisting collateral arterioles into large conductance vessels may take days to weeks after a critical stenosis of the proximal artery.15 A direct neuroprotective preconditioning effect after chronic ischemia as shown in the previous study17 may in part contribute to the effect in day 14 and day 28 groups. Furthermore, coagulation status such as platelet accumulation and fibrin deposition may change after chronic ischemia as suggested in hypoxia-tolerant states.18
Because several studies demonstrated that disordered lipid metabolism may impair collateral vessel growth and angiogenesis in other organs,6 we examined the effect of unilateral CCA occlusion in apoE-knockout mice. We found that collateral development was impaired in apoE-knockout mice, suggesting that common mechanisms underlie arteriogenesis in the brain and systemic circulation. Based on the previous findings, possible mechanisms of collateral development after chronic hypoperfusion would be (1) fluid shear stress leading to endothelial activation, (2) monocyte invasion, and (3) proliferation of smooth muscle cells and vessel enlargement.19 Involvement of growth factors or cytokines such as VEGF6 and granulocyte-macrophage colony-stimulating factor20 in collateral development in the brain will need to be investigated further.
Endogenous adaptive responses to ischemic insults such as ischemic tolerance21 have received much attention, and it is widely believed that this phenomenon is largely the result of an endogenous response such as gene expression in the preconditioned neuron.22,23 However, vessel components should also be targets for investigation of endogenous response. Thus, we have clarified that chronic mild reduction of perfusion pressure leads to collateral development and brain protection after focal cerebral ischemia.
Acknowledgments
The authors thank Ms. A. Kanzawa and S. Higa for secretarial assistance. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, and Culture in Japan.
References
Ringelstein EB, Biniek R, Weiller C, Ammeling B, Nolte PN, Thron A. Type and extent of hemispheric brain infarctions and clinical outcome in early and delayed middle cerebral artery recanalization. Neurology. 1992; 42: 289–298.
Meyer JS, Denny-Brown D. The cerebral collateral circulation, I: factors influencing collateral blood flow. Neurology. 1957; 7: 447–458.
Busch HJ, Bushmann IR, Mies G, Bode C, Hossmann KA. Arteriogenesis in hypoperfused rat brain. J Cereb Blood Flow Metab. 2003; 23: 621–628.
Henderson RD, Eliasziw M, Fox AJ, Rothwell PM, Barnett HJ; for the North American Symptomatic Carotid Endarterectomy Trial (NASCET) Group. Angiographically defined collateral circulation and risk of stroke in patients with severe carotid artery stenosis. Stroke. 2000; 31: 128–132.
Coyle P. Diameter and length changes in cerebral collaterals after middle cerebral artery occlusion in the young rat. Anat Rec. 1984; 210: 357–364.
Couffinhal T, Silver M, Kearney M, Sullivan A, Witzenbichler B, Magner M, Annex B, Peters K, Isner JM. Impaired collateral vessel development associated with reduced expression of vascular endothelial growth factor in ApoE-/- mice. Circulation. 1999; 99: 3188–3198.
Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992; 258: 468–471.
Kitagawa K, Matsumoto M, Mabuchi T, Yagita Y, Ohtsuki T, Hori M, Yanagihara T. Deficiency of intercellular adhesion molecule 1 attenuates microcirculatory disturbance and infarct size in focal cerebral ischemia. J Cereb Blood Flow Metab. 1998; 18: 1336–1345.
Yang G, Chan PH, Chen J, Carlson E, Chen SF, Weinstein P, Epstein CJ, Kamii H. Human copper–zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia. Stroke. 1994; 25: 165–170.
Liebeskind DS. Collateral circulation. Stroke. 2003; 34: 2279–2284.
del Zoppo GJ, Mabuchi T. Cerebral microvessel responses to focal ischemia. J Cereb Blood Flow Metab. 2003; 23: 879–894.
Taguchi A, Soma T, Tanaka H, Kanda T, Nishimura H, Yoshikawa H, Tsukamoto Y, Iso H, Fujimori Y, Stern DM, Naritomi H, Matsuyama T. Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model. J Clin Invest. 2004; 114: 330–338.
Wei L, Erinjeri JP, Rovainen CM, Woolsey TA. Collateral growth and angiogenesis around cortical stroke. Stroke. 2001; 32: 2179–2184.
Coyle P, Heistad DD. Development of collaterals in the cerebral circulation. Blood Vessels. 1991; 28: 183–189.
Buschmann I, Schaper W. The pathophysiology of the collateral circulation (arteriogenesis). J Pathol. 2000; 190: 338–342.
Couffinhal T, Silver M, Zheng LP, Kearney M, Witzenbichler B, Isner JM. A mouse model of angiogenesis. Am J Pathol. 1998; 152: 1667–1679.
Ohtsuki T, Matsumoto M, Kitagawa K, Taguchi A, Maeda Y, Hata R, Ogawa S, Ueda H, Handa N, Kamada T. Induced resistance and susceptibility to cerebral ischemia in gerbil hippocampal neurons by prolonged but mild hypoperfusion. Brain Res. 1993; 614: 279–284.
Stenzel-Poore MP, Stevens SL, Xiong Z, Lessov NS, Harrington CA, Mori M, Meller R, Rosenzweig HL, Tobar E, Shaw TE, Chu X, Simon RP. Effect of ischemic preconditioning on genome response to cerebral ischemia: similarity to neuroprotective strategies in hibernation and hypoxia-tolerant states. Lancet. 2003; 362: 1028–1037.
Heil M, Schaper W. Influence of mechanical, cellular, and molecular factors on collateral artery growth (arteriogenesis). Circ Res. 2004; 95: 449–458.
Schneeloch E, Mies G, Busch HJ, Buschmann IR, Hossmann KA. Granulocyte-macrophage colony-stimulating factor-induced arteriogenesis reduces energy failure in hemodynamic stroke. Proc Natl Acad Sci U S A. 2004; 101: 12730–12735.
Kitagawa K, Matsumoto M, Tagaya M, Hata R, Ueda H, Niinobe M, Handa N, Fukunaga R, Kimura K, Mikoshiba K, et al. ‘Ischemic tolerance’ phenomenon found in the brain. Brain Res. 1990; 528: 21–24.
Kirino T. Ischemic tolerance. J Cereb Blood Flow Metab. 2002; 22: 1283–1296.
Dirnagl U, Simon RP, Hallenbeck JM. Ischemic tolerance and endogenous neuroprotection. Trends Neurosci. 2003; 26: 248–254.