Minocycline attenuates white matter damage in a rat model of chronic cerebral hypoperfusion

White matter lesions are thought to result from chronic cerebral ischemia and constitute a core pathology of subcortical vascular dementia. This rarefaction has been known to be associated with microglial activation. We investigated whether minocycline, a microglial inhibitor, attenuates the white matter damage induced by chronic cerebral hypoperfusion that is used as a model of vascular dementia. Male Wistar rats were subjected to bilateral, permanent occlusion of the common carotid arteries (BCCAO) to induce chronic cerebral hypoperfusion. Minocycline or saline was injected daily for 2 weeks after BCCAO. In the corpus callosum and the optic tract, white matter damage observed with Klüver-Barrera staining was significantly attenuated in the minocycline-treated group compared to saline-treated controls. In control rats, immunoreactivities of major basic protein (MBP), Ox-42 as a microglial marker, and matrix metalloproteinase (MMP)-2 were increased in the corpus callosum. Minocycline significantly reduced these changes. Co-expression of Ox-42 and MMP-2 was confirmed by double immunofluorescence histochemistry. Our results suggest that chronic treatment with minocycline could be protective against at least some ischemic white matter damage, and its mechanism may be related to suppressing microglial activation.     

Phosphodiesterase III inhibition promotes differentiation and survival of oligodendrocyte progenitors and enhances regeneration of ischemic white matter lesions in the adult mammalian brain

Vascular dementia is caused by blockage of blood supply to the brain, which causes ischemia and subsequent lesions primarily in the white matter, a key characteristic of the disease. In this study, we used a chronic cerebral hypoperfusion rat model to show that the regeneration of white matter damaged by hypoperfusion is enhanced by inhibiting phosphodiesterase III. A rat model of chronic cerebral hypoperfusion was prepared by bilateral common carotid artery ligation. Performance at the Morris water-maze task, immunohistochemistry for bromodeoxyuridine, as well as serial neuronal and glial markers were analyzed until 28 days after hypoperfusion. There was a significant increase in the number of oligodendrocyte progenitor cells in the brains of patients with vascular dementia as well as in rats with cerebral hypoperfusion. The oligodendrocyte progenitor cells subsequently underwent cell death and the number of oligodendrocytes decreased. In the rat model, treatment with a phosphodiesterase III inhibitor prevented cell death, markedly increased the mature oligodendrocytes, and promoted restoration of white matter and recovery of cognitive decline. These effects were cancelled by using protein kinase A/C inhibitor in the phosphodiesterase III inhibitor group. The results of our study indicate that the mammalian brain white matter tissue has the capacity to regenerate after ischemic injury.     

Axonal damage and demyelination in the white matter after chronic cerebral hypoperfusion in the rat

Cerebral white matter (WM) lesions are observed frequently in human ischemic cerebrovascular disease and have been thought to contribute to cognitive impairment. This type of lesion can be experimentally induced in rat brains under chronic cerebral hypoperfusion by the permanent occlusion of both common carotid arteries. However, it remains uncertain whether chronic ischemia can damage both the gray and white matter, and whether it can induce demyelination with or without axonal damage. Therefore, we examined axonal damage using immunohistochemistry for the amyloid beta/A4 precursor protein (APP), chromogranin A (CgA) and demyelination using immunohistochemistry for the encephalitogenic peptide (EP) in this model. Severe WM lesions such as vacuolation and the loss of nerve fibers appeared in the optic nerve and optic tract after 3 days of ligation, and less intense changes were observed in the corpus callosum, internal capsule, and fiber bundles of the caudoputamen after 7 days with Klüver-Barrera and Bielschowsky staining. These WM lesions persisted even after 30 days. The APP, CgA, and EP-immunopositive fibers increased in number from 1 to 30 days after the ligation in the following WM regions: the optic nerve, optic tract, corpus callosum, internal capsule, and fiber bundles of the caudoputamen. In contrast, only a few APP, CgA, or EP-immunopositive fibers were detected in the gray matter regions, including the cerebral cortex and hippocampus. These results indicate that the WM is more susceptible to chronic cerebral hypoperfusion than the gray matter, with an involvement of both axonal and myelin components. Furthermore, immunohistochemistry for APP, CgA, and EP is far superior to routine histological staining in sensitivity and may become a useful tool to investigate WM lesions caused by various pathoetiologies.     

Leukoencephalopathy in diffuse hemorrhagic cerebral amyloid angiopathy

We have studied 12 patients with diffuse hemorrhagic cerebral amyloid angiopathy clinically and at postmortem examination. The brains in 8 patients had diffuse bilateral loss of myelin in the hemispheric white matter sparing the U fibers, corpus callosum, and internal capsules. The periventricular areas were predominantly affected. Microscopic examination of the white matter showed an association with subacute or chronic edematous lesions: spongiosis, swollen oligodendroglia, widening of the perivascular spaces with edema fluid or siderophages, hyalinization of the blood vessel walls, incomplete myelin loss, and astrocytic gliosis. Three of 8 autopsied patients had undergone computed tomographic examination, which showed bilateral hypodensity of the hemispheric white matter. The brains of 4 patients with illnesses of shorter duration showed only discrete but similar lesions in the centrum semiovale. These white matter changes are similar to those observed in Binswanger's subcortical encephalopathy. We suggest that a common mechanism of hypoperfusion of the distal white matter causes the leukoencephalopathy.     

老龄大鼠慢性脑灌注不足脑内白细胞和T细胞的入侵

目的 探讨白细胞和Ｔ细胞在慢性脑灌注不足脑损害中的活动。方法 ７０只老龄Ｗｉｓｔａｒ大鼠持久性双侧颈总动脉结扎（２ＶＯ）,其中１２只接受环孢霉素Ａ（ＣｓＡ）治疗。免疫组化法检测白细胞和Ｔ细胞。实现研究为持久性２Ｖ０１～４月。结果 大鼠慢性脑灌注不足造成了明显的脑损害与白细胞和Ｔ细胞的入侵。１～４月,白细胞在皮层、白质和海马的活动均减少,而Ｔ细胞的活动在皮层下白质增多,在皮层和海马减少。同时脑损害加重。ＣｓＡ治疗后白细胞和Ｔ细胞的活动明显减弱,脑损害减轻。结论 慢性脑灌注不足的病理损害,尤其白质损害中,Ｔ细胞伴有重要作用,白细胞仅起次要作用。ＣｓＡ能抑制白细胞和Ｔ细胞的活动,从而防治了脑损害。     

Quercetin,a natural flavonoid,attenuates vacuolar formation in the optic tract in rat chronic cerebral hypoperfusion model

Epidemiological studies suggest that the intake of flavonoids is inversely associated with risk of cardiovascular diseases and stroke, but there is no evidence showing the effect of flavonoids on vascular dementia. Because quercetin, a natural flavonoid, is known to scavenge free radicals, we investigated whether quercetin attenuates white matter damage in rats with chronic cerebral hypoperfusion, as a model of vascular dementia. Chronic hypoperfusion was induced by ligation of the bilateral carotid arteries in male Wistar rats, which received vehicle alone, 100 mg/kg quercetin, or 200 mg/kg quercetin intraperitoneally at 4-day intervals for 8 weeks after operation. Sham-operated rats were also studied. The area of vacuoles in the optic tract observed after hematoxylin and eosin staining was significantly reduced in the 200 mg/kg quercetin-treated hypoperfusion group versus the vehicle-treated hypoperfusion group (1.7+/-0.2% versus 3.9+/-0.3%; P<0.05). The present results are consistent with the idea that chronic treatment with quercetin could be protective against at least a part of ischemic white matter damage.     

Expression of S100 protein and protective effect of arundic acid on the rat brain in chronic cerebral hypoperfusion

S100 protein is expressed primarily by astroglia in the brain, and accumulates in and around the ischemic lesions. Arundic acid, a novel astroglia-modulating agent, is neuroprotective in acute cerebral infarction, whereas the protective effects remain unknown during chronic cerebral hypoperfusion. Rats undergoing chronic cerebral hypoperfusion were subjected to a bilateral ligation of the common carotid arteries, and were allowed to survive for 3, 7 and 14 days. The animals received a daily intraperitoneal injection of 5.0, 10.0 or 20.0 mg/kg of arundic acid, or vehicle, for 14 days. Alternatively, other groups of rats received a delayed intraperitoneal injection of 20.0 mg/kg of arundic acid or vehicle, which started from 1, 3 or 7 days after ligation and continued to 14 days. The degree of white matter (WM) lesions and the numerical density of S100 protein-immunoreactive astroglia were estimated. In the WM of rats with vehicle injections, the number of S100 protein-immunoreactive astroglia increased significantly after chronic cerebral hypoperfusion as compared to the sham-operation. A dosage of 10.0 and 20.0 mg/kg of arundic acid suppressed the numerical increase in S100 protein-immunoreactive astroglia and the WM lesions. These pathological changes were suppressed with delayed treatment up to 7 days in terms of astroglial activation, and up to 3 days in terms of the WM lesions. The protective effects of arundic acid against WM lesions were demonstrated in a dose-dependent manner, and even after postischemic treatments. These results suggest the potential usefulness of arundic acid in the treatment of cerebrovascular WM lesions.     

Chronic cerebral hypoperfusion induced by right unilateral common carotid artery occlusion causes delayed white matter lesions and cognitive impairment in adult mice

Some lines of evidence have suggested that subcortical ischemic vascular dementia (SIVD) is a common form of vascular dementia (VaD), and that its pathological changes are the development of ischemic white matter (WM) lesions under chronic hypoperfusion and lacunes. Here, we have developed a novel mouse model of VaD with WM lesions, which was induced by right unilateral common carotid artery occlusion (rUCCAO). The mice subjected to rUCCAO exhibited chronic cerebral hypoperfusion in the cerebral hemisphere ipsilateral to rUCCAO monitored using a laser-Doppler flow meter (p<0.01), and significant WM damage in the corpus callosum (p<0.05) and deficits in object recognition test correlated with the damage of frontal-subcortical circuits (p<0.01). However, no differences in spontaneous alternation or spontaneous motor activity were observed. Furthermore, the levels of pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta) and interleukin-6 (IL-6), significantly increased (p<0.01), and those of anti-inflammatory cytokines, such as interleukin-4 (IL-4) and interleukin-10 (IL-10), significantly decreased in the ischemic brain (p<0.05). These results suggest that this model is a useful tool for investigating the associations among inflammatory reactions, cognitive impairment, and WM damage, which may help elucidating the pathomechanism of VaD, particularly SIVD.     

氧化应激在慢性缺血性脑白质损伤中的作用

目的 阐明氧化应激是否参与大鼠慢性脑缺血所致的脑白质损伤.方法 健康雄性Wistar大鼠按照完全随机数字表法分为假手术组,持久性双侧颈总动脉结扎3 d组、7 d组、3周组及6周组,每组6只.应用大鼠双侧颈总动脉结扎制备慢性脑缺血模型,检测大鼠脑白质内超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性、谷胱甘肽(GSH)含量以及脂质过氧化产物丙二醛(MDA)和4-羟基壬烯醛(4-HNE)加合物的变化.结果 与假手术组比较,慢性脑缺血大鼠脑白质内MDA含量在手术后3周明显增加,手术后6周进一步增高,差异有统计学意义(P<0.05).手术后3d至6周,慢性脑缺血大鼠脑白质内4-HNE蛋白加合物逐渐增高,与假手术组比较有差异有统计学意K(P<0.05).SOD活性在手术后3周和6周才明显降低,与假手术组比较差异有统计学意义(P<0.05).此外,慢性脑缺血大鼠脑白质内GSH含量在手术后7d即开始降低,而在手术后3周及6周则进一步下降,与假手术组比较差异有统计学意义(P<0.05).结论 慢性脑缺血导致大鼠脑白质氧化性损伤增加,抗氧化防御能力降低:氧化性损伤的增加和抗氧化防御能力的降低与慢性脑缺血所致的脑白质损伤密切相关.     

MCP-1-mediated activation of microglia promotes white matter lesions and cognitive deficits by chronic cerebral hypoperfusion in mice

Microglia activation played a vital role in the pathogenesis of white matter lesions (WMLs) by chronic cerebral hypoperfusion. In addition, hypoxia induced up-regulated expression of MCP-1, promotes the activation of microglia. However, the role of MCP-1-mediated microglia activation in chronic cerebral ischemia is still unknown. To explore that, chronic cerebral hypoperfusion model was established by permanent stenosis of bilateral common carotid artery in mice. The activation of microglia and the related signal pathway p38MAPK/PKC in white matter, and working memory of mice were observed. We found that stenosis of common carotid arteries could induce MCP-1-mediated activation of microglia through p38MAPK/PKC pathway and white matter lesions. Taken together, our findings represent a novel mechanism of MCP-1 involved in activation of microglia and provide a novel therapeutical strategy for chronic cerebral hypoperfusion.     

A new model of white matter injury in neonatal rats with bilateral carotid artery occlusion

Periventricular leukomalacia is an important cause of cerebral palsy and characterized by cysts and coagulation necrosis in the periventricular white matter. Since no model of periventricular leukomalacia has been established in small animals, it is expected to establish a new model of white matter injury in immature rodents. Bilateral carotid arteries were occluded in neonatal rats at 5 days of age, and the brain neuropathologically examined at 7 days of age. Among 22 brains histologically examined, 20 (90.9%) had white matter changes including coagulation necrosis and cystic lesions in and around the internal capsule, while only two had small cerebral infarction and five showed some ischemic neurons in the cerebral cortex. Cerebral blood flow (CBF) decreased to about 25% of controls in the subcortical white matter in the animals with bilateral carotid artery occlusion (BCAO). Amyloid precursor protein (APP) immunohistochemistry demonstrated various APP-immunoreactive axonal profiles in the internal capsule and the subcortical white matter, and stronger expression of APP in pyramidal neurons in the cerebral cortex of BCAO brains. These results indicated that the white matter is more vulnerable than the cerebral cortex in 5-day-old rats when CBF decreases to about 25% and suggested that this model is useful for investigating the white matter changes induced by cerebral hypoperfusion in the neonatal brain, since previous models of hypoxic-ischemic brain injury in neonatal mice and rats revealed preferential susceptibility of the gray matter. It was also indicated that APP is a sensitive marker for mild axonal disruption in the white matter of the immature brain.     

Brain hypoperfusion: a critical factor in vascular dementia

Subcortical ischemic vascular dementia is a relatively common form of dementia. Anatomical changes of ageing in the brain arteries predispose the elderly to the effects of hypotension. Depending on their circulatory pattern, particular regions of the brain are susceptible to ischemic hypoperfusive lesions. These regions include the periventricular white matter, basal ganglia, and hippocampus. Interruption of prefrontal-basal ganglia circuits important for cognition and memory may result from these lesions. Hypotension and hypoperfusion explain the high risk for the development of cognitive impairment and vascular dementia in older patients affected by orthostatic hypotension, congestive heart failure, as well as in those undergoing surgical procedures such as hip and knee replacement and coronary artery bypass graft (CABG). Recognition of the susceptibility of elderly subjects to cerebral lesions induced by hypoperfusion should result in appropriate preventive measures and better treatment.     

Experimental model for repetitive ischemic attacks in the gerbil: the cumulative effect of repeated ischemic insults

An experimental model for repeated ischemic attacks, which allows easy induction of cerebral ischemia of any desired duration and frequency, has been developed in the gerbil. With this procedure, a pronounced cumulative effect on development of edema and tissue injury was observed using 3 separate, 5-min bilateral occlusions of the common carotid arteries spaced at various time intervals. This effect was most evident when the occlusions were carried out at 1-h intervals, i.e., during the period of marked postischemic hypoperfusion. Such animals, killed after 24 h of recirculation, showed significantly more severe edema and brain tissue injury in the areas exposed to ischemia than was observed in animals killed 24 h after single 5- or 15-min occlusions. The changes of regional CBF, assayed with a [3H]nicotine method, indicated a relatively rapid onset of hypoperfusion of similar degree after each release of arterial occlusion. The hypoperfusion recovered significantly within 6 h of recirculation following either single or multiple occlusions, and no residual hypoperfusion was observed in animals which, when killed at 24 h, showed severe edema and brain tissue injury. This model should prove useful in elucidating the pathophysiological mechanisms operative in repetitive cerebral ischemia.     

沙漠化地区缺血性脱髓鞘性脑白质病变危险因素研究

目的 回顾性研究武威市沙漠化地区缺血性脱髓鞘性脑白质病变的危险因素,为临床中预防沙漠地 区缺血性脱髓鞘性脑白质病变的发生提供理论依据。 方法 收集武威市人民医院神经内科2011年1月-2015年12月住院或门诊筛查出的缺血性脑血管病变 患者共500例。按不同居住地、不同危险因素对所筛选病例进行分析。采用多因素Logistic回归分析缺 血性脱髓鞘性脑白质病变患者的相关危险因素。 结果 沙漠组的缺血性脱髓鞘性脑白质病变发生率为62.4%,平原组的缺血性脱髓鞘性脑白质病变 发生率为49.6%;沙漠组中脑白质病变中重度病变占22.4%,平原组的脑白质病变中重度病变占8.4%。 多因素分析结果表示沙漠化天气、吸烟、饮酒、糖尿病、高血压、脂代谢异常、颈动脉硬化、高同型半 胱氨酸是加重缺血性脑白质病变的危险因素。 结论 沙漠化地区缺血性脱髓鞘脑白质病变的发生率较平原地区偏高,以重度病变为主。     

White matter lesion progression: a surrogate endpoint for trials in cerebral small-vessel disease

There is neuropathologic evidence that confluent MRI white matter lesions in the elderly reflect ischemic brain damage due to microangiopathy. The authors hypothesize that measuring changes in the progression of white matter lesions as shown by MRI may provide a surrogate marker in clinical trials on cerebral small-vessel disease in which the currently used primary outcomes are cognitive impairment and dementia. This hypothesis is based on evidence that confluent white matter lesions progress rapidly as shown in a recent follow-up study in community-dwelling subjects. The mean increase in lesion volume was 5.2 cm(3) after 3 years. Based on these data in a clinical trial, 195 subjects with confluent lesions would be required per treatment arm to demonstrate a 20% reduction in the rate of disease progression over a 3-year period. Like any other MRI metric, the change in white matter lesion volume cannot be considered preferable to clinical outcomes unless it has been demonstrated that it matters to the patient in terms of function.     

Experimental cerebral hypoperfusion induces white matter injury and microglial activation in the rat brain

Though cerebral white matter injury is a frequently described phenomenon in aging and dementia, the cause of white matter lesions has not been conclusively determined. Since the lesions are often associated with cerebrovascular risk factors, ischemia emerges as a potential condition for the development of white matter injury. In the present study, we induced experimental cerebral hypoperfusion by permanent, bilateral occlusion of the common carotid arteries of rats (n=6). A sham-operated group served as control (n=6). Thirteen weeks after the onset of occlusion, markers for astrocytes, microglia, and myelin were found to be labeled by means of immunocytochemistry in the corpus callosum, the internal capsule, and the optic tract. The ultrastructural integrity and oligodendrocyte density in the optic tract were investigated by electron microscopy. Quantitative analysis revealed that chronic cerebral hypoperfusion caused mild astrogliosis in the corpus callosum and the internal capsule, while astrocytic disintegration in the optic tract increased by 50%. Further, a ten-fold increase in microglial activation and a nearly doubled oligodendrocyte density were measured in the optic tract of the hypoperfused rats as compared with the controls. Finally, vacuolization and irregular myelin sheaths were observed at the ultrastructural level in the optic tract. In summary, the rat optic tract appears to be particularly vulnerable to ischemia, probably because of the rat brains angioarchitecture. Since the detected glial changes correspond with those reported in vascular and Alzheimer dementia, this model of cerebral hypoperfusion may serve to characterize the causal relationship between ischemia and white matter damage.     

慢性脑灌流不足老年大鼠脑组织Neurexin家族蛋白Caspr2的表达

目的 观察慢性脑灌流不足（chronic cerebral hypoperfusion，CCH）老年大鼠脑组织Caspr2表达的变化。方法 建立老年大鼠CCH模型，采用免疫荧光组织化学与Western blot法，对大鼠CCH后2周及1、3个月脑组织Caspr2的表达变化进行组织定位与半定量分析。结果 脑内Caspr2主要表达于皮质下白质，特别是胼胝体等神经纤维束密集区域，皮质表达较弱；CCH后Caspr2表达数量及强度与对照组相比显著下降，且其定位模式在白质纤维结构中出现分布紊乱；CCH后2周及1、3个月时其表达与对照组比较差异有统计学意义（P〈0．05）。结论 慢性缺血缺氧导致Caspr2表达显著降低与定位改变，可能是白质结构损害及相关功能减退的重要环节和分子基础。     

缺血性脑卒中患者脑微出血与脑白质病变的相关性研究

目的 探讨缺血性脑卒中患者脑微出血与脑白质病变的相关性。方法 选取2018年1月-2019年12月本院收治的缺血性脑卒中患者; 通过核磁共振成像扫描患者头部观察各脑区脑微出血、脑白质病变情况,并对脑微出血情况进行分级,对脑白质病情情况进行评分; 采用Logstic回归分析法分析各危险因素与脑微出血、脑白质病变的关系; 通过spearman相关分析法分析脑微出血分级与脑白质病变评分的相关性。结果 有脑微出血患者中皮质及皮质下出现脑微出血占比61.46%明显高于基底及丘脑23.96%及幕下区41.10%(P<0.05); 有脑白质病变患者中额区脑白质病变占比69.44%明显高于颞区14.81%、顶枕区6.48%、基底节3.70%(P<0.05); Logstic回归分析显示年龄>60岁、有高半胱氨酸血症及合并心房颤动是发生脑微出血的独立危险因素(P<0.05),年龄>60岁是发生脑白质病变的独立危险因素(P<0.05); 脑微出血各级与脑白质病变各评分均呈正相关(r=0.327,0.311,0.401,0.362,P<0.05)。结论 年龄>60岁是缺血性脑卒中患者发生脑微出血及脑白质病变的危险因素; 在缺血性脑卒中患者中脑微出血与脑白质病变呈正相关     

Chronic cerebral hypoperfusion induces white matter lesions and loss of oligodendroglia with DNA fragmentation in the rat

Cerebrovascular white matter lesions represent an age-related neurodegenerative condition that appears as a hyperintense signal on magnetic resonance images. These lesions are frequently observed in aging, hypertension and cerebrovascular disease, and are responsible for cognitive decline and gait disorders in the elderly population. In humans, cerebrovascular white matter lesions are accompanied by apoptosis of oligodendroglia, and have been thought to be caused by chronic cerebral ischemia. In the present study, we tested whether chronic cerebral hypoperfusion induces white matter lesions and apoptosis of oligodendroglia in the rat. Doppler flow meter analysis revealed an immediate reduction of cerebral blood flow ranging from 30% to 40% of that before operation; this remained at 52–64% between 7 and 30 days after operation. Transferrin-immunoreactive oligodendroglia decreased in number and the myelin became degenerated in the medial corpus callosum at 7 days and thereafter. Using the TUNEL method, the number of cells showing DNA fragmentation increased three- to eightfold between 3 and 30 days post-surgery compared to sham-operated animals. Double labeling with TUNEL and immunohistochemistry for markers of either astroglia or oligodendroglia showed that DNA fragmentation occurred in both of these glia. Messenger RNA for caspase-3 increased approximately twofold versus the sham-operated rats between 1 and 30 days post-surgery. Immunohistochemistry revealed up-regulation of caspase-3 in the oligodendroglia of the white matter, and also in the astroglia and neurons of the gray matter. Molecules involved in apoptotic signaling such as TNF- and Bax were also up-regulated in glial cells. These results indicate that chronic cerebral hypoperfusion induces white matter degeneration in association with DNA fragmentation in oligodendroglia.     

Multiple hemorrhagic cerebral amyloid angiopathy associated with leukoencephalopathy

The authors present a case of cerebral amyloid angiopathy (CAA) which caused multiple recurrent subcortical hemorrhages and leukoencephalopathy similar to Binswanger's disease. CT scan revealed bilateral hypodensity of the hemispheric white matter in addition to multiple subcortical hemorrhages. The difference between white and gray matter densities was 14 approximately 15 HU. T2-weighted MRI showed the hemispheric white matter as high intensity. These findings on CT and MRI are compatible with those observed in leukoencephalopathy like Binswanger's disease. A biopsy of the cortex was performed and histological examination revealed amyloid deposition in the arterial wall. In addition to amyloid deposition, obliterative intimal proliferation and hyaline degeneration of the vessel were observed. It is thought that the occlusive vascular change due to CAA may induce chronic hypoperfusion of the hemispheric white matter and consequently the leukoencephalopathy may develop. In the aged population, CAA is noteworthy as a cause of both hemorrhagic and ischemic lesions, because the incidence of CAA is increasing with age.