Topical Review Section Editors: Steven M. Greenberg, MD, PhD, and Leonardo Pantoni, MD, PhD Rodent Models of Cerebral Microinfarct and Microhemorrhage Andy Y. Shih, PhD; Hyacinth I. Hyacinth, MD, PhD, MPH; David A. Hartmann, BA; Susanne J. van Veluw, PhD icroinfarcts are prevalent but tiny ischemic lesions that Clinical studies have emphasized the need to better under- Mmay contribute to vascular cognitive impairment and stand microinfarcts and microhemorrhages (microlesions) dementia (Figure 1A and 1B).1 They are defined as areas because their widespread nature and far-reaching effects could of tissue infarction, often with gliosis or cavitation, visible contribute to broad disruption of brain function in dementia. only by examination of the autopsied brain at a microscopic However, it is challenging to measure their functional impact level.2,3 Numerous autopsy studies have now shown that a in the human brain because their onset times and locations are Downloaded from greater microinfarct burden is correlated with increased like- unpredictable. Further, microlesions often coexist with other lihood of cognitive impairment.2,3 Cerebral microinfarcts are disease processes, making it difficult to isolate their specific observed postmortem in the brains of ≈43% of patients with contribution to brain dysfunction. Animal models that allow Alzheimer’s disease, 62% of patients with vascular demen- microlesions to be recreated in a more controlled environment tia, and 24% of nondemented elderly subjects.4 However, are, therefore, valuable for understanding their impact on the reported microinfarct numbers are a significant underestima- brain. The purpose of this review is to collate existing rodent http://stroke.ahajournals.org/ tion of total burden, as only a small portion of the brain is models of both microinfarcts and microhemorrhages that can examined at routine autopsy.1 Indeed, they can number in the be used to study microlesions at a preclinical level. hundreds to thousands within a single brain. Microinfarcts can arise from a variety of etiologies, including cerebral Lesion Size Criterion small vessel disease, large vessel disease, cerebral hypoper- Microinfarcts and microhemorrhages are thought to arise fusion, and cardiac disease, but their role in the pathogen- from the occlusion or rupture of small parenchymal arte- esis of vascular cognitive impairment and dementia remains rioles, such as penetrating arterioles and their smaller 3,5–7 by guest on April 4, 2018 poorly understood. branches. We define microinfarcts in rodent models as Microhemorrhages are microscopic bleeds caused lesions with sizes that could only arise from the occlusion of by rupture of cerebral microvessels, generating lesions single penetrating arterioles or their downstream branches. on a similar scale as microinfarcts (Figure 1C and 1D).5 Microinfarcts are typically no larger than 1 mm in diameter Pathologically, old microhemorrhages are defined as focal in the mouse and rat cortex. We also apply this size criteria depositions of iron-positive hemosiderin-containing mac- to microinfarcts in deeper brain structures, though the rela- rophages. Unlike microinfarcts, microhemorrhages easily tionship between vascular architecture and microinfarcts escape detection on neuropathological examination, sug- beyond cortex remains understudied. Microhemorrhages gesting that they are not as widespread as microinfarcts. induced in cortex, and occurring spontaneously in rodent However, they can be detected with high sensitivity using models, seem to be ≈200 to 300 μm or smaller at histopa- in vivo magnetic resonance imaging (MRI).5,6 The 2 most thology. We have adhered to this size range in our review common etiologies of age-related microhemorrhages are of the literature. We further note that the term microhemor- hypertensive arteriopathy and cerebral amyloid angiopa- rhage is used for histologically verified bleeds and micro- thy (CAA). Microhemorrhages are associated with higher bleeds for the MRI-visible correlate of microhemorrhages. likelihood of dementia, and like microinfarcts, their role Supplemental information for defining microinfarcts and in vascular cognitive impairment and dementia remains microhemorrhages in rodent tissues is provided in the incompletely understood.7 online-only Data Supplement. Received September 9, 2017; final revision received December 21, 2017; accepted January 9, 2018. From the Department of Neuroscience (A.Y.S., D.A.H.) and Center for Biomedical Imaging (A.Y.S.), Medical University of South Carolina, Charleston, SC; Aflac Cancer and Blood Disorder Center, Children’s Healthcare of Atlanta and Emory University Department of Pediatrics, GA (H.I.H.); and Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA (S.J.v.V.). The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA. 117.016995/-/DC1. Correspondence to Andy Y. Shih, PhD, Department of Neuroscience, Medical University of South Carolina, 173 Ashley Ave CRI 406, Charleston, SC 29425. E-mail [email protected] (Stroke. 2018;49:803-810. DOI: 10.1161/STROKEAHA.117.016995.) © 2018 American Heart Association, Inc. Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.117.016995 803 804 Stroke March 2018 lasers.18 Targeted penetrating arteriole occlusions primar- ily generate wedge or column-shaped cortical microinfarcts because clots are made in vessels at or near the pial surface (Figure 3B). Models With Spontaneous Microinfarcts Bilateral Carotid Artery Stenosis Bilateral carotid artery stenosis (BCAS) is a common manip- ulation to induce chronic cerebral hypoperfusion in rodents. Normal C57Bl/6 mice develop subcortical microinfarcts after long periods of BCAS (6 months)19 but not after shorter periods (2–3 months).20 Two groups recently examined the effects of BCAS in the Tg-SwDI mouse model, which devel- ops early and pronounced CAA.21,22 Interestingly, only 2 to 3 months of BCAS was necessary to induce microinfarcts in these mice.23 Microinfarcts were observed in the cerebral cortex and hippocampus and were related to CAA severity, Downloaded from whereas no microinfarcts were seen in age-matched sham- operated Tg-SwDI mice. Further, a recent study showed that atherosclerotic Apoe knockout mice develop microinfarcts within 1.5 months after BCAS.24 Thus, prolonged cerebral hypoperfusion itself can cause microinfarcts, but this effect is exacerbated in transgenic mice with existing cerebrovascular http://stroke.ahajournals.org/ Figure 1. Human microinfarcts and microhemorrhages in cere- disease. bral amyloid angiopathy (CAA) cases. A, A cortical microinfarct on a hematoxylin & eosin–stained section. B, Microinfarct (black Obesity and Diabetes Mellitus arrow) on a T2-weighted ex vivo magnetic resonance imaging Spontaneous microinfarcts were observed in a mouse model (MRI) scan. C, A cortical microhemorrhage on a hematoxylin that crossed the Aβ overexpressing mouse line, APP/PS1, & eosin–stained section. D, Multiple lobar microbleeds (white 25 arrows) on a gradient-repeat echo ex vivo MRI scan. with the db/db mouse model for diabetes mellitus. The db/db line harbors a mutation of the diabetes mellitus (db) Models of Induced Microinfarcts gene that leads to a leptin signaling defect, causing severe by guest on April 4, 2018 obesity, hypertension, and type 2 diabetes mellitus with Intracarotid Injection of Microemboli hyperglycemia.26 The progeny of the APP/PS1-db/db cross- One method of generating cerebral microinfarcts involves the retained features of parental lines, but the combined risk injection of microemboli into the blood circulation, such as factors led to cortical microinfarcts that were not observed 8,9 10,11 occlusive microbeads or cholesterol crystals (Figure 2, in either parental strain. Microinfarcts appeared as small left). Injections are typically made through the internal carotid cystic cavities in various layers of cortex. The authors sus- artery. This produces broadly distributed microinfarcts, with pected that aberrant angiogenesis, unique to the crossed cortex, hippocampus, and thalamus being the major sites of mice, led to immature and leaky microvessels that were accrual.8,10 A spectrum of microinfarct types are seen, includ- prone to occlusion. ing wedge or column-shaped lesions, in the cerebral cortex Endothelial NOS-Deficient Mice that are continuous with the pial surface (Figure 3A), as well Endothelial nitric oxide synthase is critical for regulation of as smaller circumscribed microinfarcts contained within the vascular tone and blood pressure. A recent study showed that parenchyma. The choice of microembolus size, type, or num- mice with partial deletion of endothelial nitric oxide synthase ber injected is important to achieve consistency of microin- develop microinfarcts in cortex and, to a lesser extent, in the farct formation. hippocampus and thalamus (Figure 3C).13 Cortical microin- Laser-Induced Occlusion of Penetrating Arterioles farcts accrued in watershed regions between the perfusion ter- A second method allows reproducible targeting of microinfarct ritories of major cerebral arteries.27 They were noticeable by location and size in rodent cortex (Figure 2, right).12 Typically 6 months of age but were most prevalent at 12 to 18 months. coupled with in vivo two-photon imaging, single penetrating This was accompanied by microvascular pathology, includ- arterioles (or small pial arterioles that support flow