Rodent Models of Cerebral Microinfarct and Microhemorrhage

HomeStrokeVol. 49, No. 3Rodent Models of Cerebral Microinfarct and Microhemorrhage Free AccessReview ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplementary MaterialsFree AccessReview ArticlePDF/EPUBRodent Models of Cerebral Microinfarct and Microhemorrhage Andy Y. Shih, PhD, Hyacinth I. Hyacinth, MD, PhD, MPH, David A. Hartmann, BA and Susanne J. van Veluw, PhD Andy Y. ShihAndy Y. Shih 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.). , Hyacinth I. HyacinthHyacinth I. Hyacinth 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.). , David A. HartmannDavid A. Hartmann 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.). and Susanne J. van VeluwSusanne J. van Veluw 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.). Originally published19 Feb 2018https://doi.org/10.1161/STROKEAHA.117.016995Stroke. 2018;49:803–810Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2018: Previous Version 1 Microinfarcts are prevalent but tiny ischemic lesions that may contribute to vascular cognitive impairment and dementia (Figure 1A and 1B).1 They are defined as areas of tissue infarction, often with gliosis or cavitation, visible only by examination of the autopsied brain at a microscopic level.2,3 Numerous autopsy studies have now shown that a greater microinfarct burden is correlated with increased likelihood of cognitive impairment.2,3 Cerebral microinfarcts are observed postmortem in the brains of ≈43% of patients with Alzheimer's disease, 62% of patients with vascular dementia, and 24% of nondemented elderly subjects.4 However, reported microinfarct numbers are a significant underestimation of total burden, as only a small portion of the brain is examined at routine autopsy.1 Indeed, they can number in the hundreds to thousands within a single brain. Microinfarcts can arise from a variety of etiologies, including cerebral small vessel disease, large vessel disease, cerebral hypoperfusion, and cardiac disease, but their role in the pathogenesis of vascular cognitive impairment and dementia remains poorly understood.3,5–7Download figureDownload PowerPointFigure 1. Human microinfarcts and microhemorrhages in cerebral amyloid angiopathy (CAA) cases. A, A cortical microinfarct on a hematoxylin & eosin–stained section. B, Microinfarct (black arrow) on a T2-weighted ex vivo magnetic resonance imaging (MRI) scan. C, A cortical microhemorrhage on a hematoxylin & eosin–stained section. D, Multiple lobar microbleeds (white arrows) on a gradient-repeat echo ex vivo MRI scan.Microhemorrhages are microscopic bleeds caused by rupture of cerebral microvessels, generating lesions on a similar scale as microinfarcts (Figure 1C and 1D).5 Pathologically, old microhemorrhages are defined as focal depositions of iron-positive hemosiderin-containing macrophages. Unlike microinfarcts, microhemorrhages easily escape detection on neuropathological examination, suggesting that they are not as widespread as microinfarcts. However, they can be detected with high sensitivity using in vivo magnetic resonance imaging (MRI).5,6 The 2 most common etiologies of age-related microhemorrhages are hypertensive arteriopathy and cerebral amyloid angiopathy (CAA). Microhemorrhages are associated with higher likelihood of dementia, and like microinfarcts, their role in vascular cognitive impairment and dementia remains incompletely understood.7Clinical studies have emphasized the need to better understand microinfarcts and microhemorrhages (microlesions) because their widespread nature and far-reaching effects could contribute to broad disruption of brain function in dementia. However, it is challenging to measure their functional impact in the human brain because their onset times and locations are unpredictable. Further, microlesions often coexist with other disease processes, making it difficult to isolate their specific contribution to brain dysfunction. Animal models that allow microlesions to be recreated in a more controlled environment are, therefore, valuable for understanding their impact on the brain. The purpose of this review is to collate existing rodent models of both microinfarcts and microhemorrhages that can be used to study microlesions at a preclinical level.Lesion Size CriterionMicroinfarcts and microhemorrhages are thought to arise from the occlusion or rupture of small parenchymal arterioles, such as penetrating arterioles and their smaller branches. We define microinfarcts in rodent models as lesions with sizes that could only arise from the occlusion of single penetrating arterioles or their downstream branches. Microinfarcts are typically no larger than 1 mm in diameter in the mouse and rat cortex. We also apply this size criteria to microinfarcts in deeper brain structures, though the relationship between vascular architecture and microinfarcts beyond cortex remains understudied. Microhemorrhages induced in cortex, and occurring spontaneously in rodent models, seem to be ≈200 to 300 μm or smaller at histopathology. We have adhered to this size range in our review of the literature. We further note that the term microhemorrhage is used for histologically verified bleeds and microbleeds for the MRI-visible correlate of microhemorrhages. Supplemental information for defining microinfarcts and microhemorrhages in rodent tissues is provided in the online-only Data Supplement.Models of Induced MicroinfarctsIntracarotid Injection of MicroemboliOne method of generating cerebral microinfarcts involves the injection of microemboli into the blood circulation, such as occlusive microbeads8,9 or cholesterol crystals10,11 (Figure 2, left). Injections are typically made through the internal carotid artery. This produces broadly distributed microinfarcts, with cortex, hippocampus, and thalamus being the major sites of accrual.8,10 A spectrum of microinfarct types are seen, including wedge or column-shaped lesions, in the cerebral cortex that are continuous with the pial surface (Figure 3A), as well as smaller circumscribed microinfarcts contained within the parenchyma. The choice of microembolus size, type, or number injected is important to achieve consistency of microinfarct formation.Download figureDownload PowerPointFigure 2. Models of induced microinfarct and microhemorrhage. The left hemisphere depicts the production of distributed microinfarcts by injecting microemboli through the internal carotid artery (ICA). The right hemisphere shows the selective occlusion of a penetrating arteriole with focal photothrombosis (blue shade = region of ischemia), or rupture of a penetrating arteriole with an amplified laser during in vivo optical imaging (red shade = region of blood leakage). CCA indicates common carotid artery; and ECA, external carotid artery.Download figureDownload PowerPointFigure 3. Microinfarcts in rodent models. A, A cortical microinfarct observed in hematoxylin & eosin (H&E)–stained mouse brain sections after injection of cholesterol crystals into the internal carotid artery. Adapted from Wang et al10 with permission. Copyright ©2012, Society for Neurosience. B, A cortical microinfarct observed in NeuN and GFAP (glial fibrillary acidic protein) immunostained rat brain sections after occlusion of a single cortical penetrating arteriole by focal photothrombosis. Adapted from Shih et al12 with permission. Copyright ©2012, Springer Nature. C, Spontaneous microinfarcts observed in H&E-stained brain sections from an 18-month-old endothelial nitric oxide synthase (eNOS)–deficient mouse. Adapted from Tan et al13 with permission, Copyright ©2012, Tan et al. D, Spontaneous microinfarcts observed in NeuN-immunostained brain sections from a 13-month-old Townes sickle cell mouse. Adapted from Hyacinth et al14 with permission. Copyright ©2017, SAGE Publications.Laser-Induced Occlusion of Penetrating ArteriolesA second method allows reproducible targeting of microinfarct location and size in rodent cortex (Figure 2, right).12 Typically coupled with in vivo two-photon imaging, single penetrating arterioles (or small pial arterioles that support flow to penetrating arterioles) are selectively occluded by inducing clots with precise laser irradiation. This is achieved most commonly by activating circulating photosensitizing agents with focused green lasers, that is, focal photothrombosis.12,15,16 Occlusions can also be made without a photosensitizer by using amplified femtosecond laser ablation17 or targeted irradiation with higher laser powers from conventional two-photon imaging lasers.18 Targeted penetrating arteriole occlusions primarily generate wedge or column-shaped cortical microinfarcts because clots are made in vessels at or near the pial surface (Figure 3B).Models With Spontaneous MicroinfarctsBilateral Carotid Artery StenosisBilateral carotid artery stenosis (BCAS) is a common manipulation 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 develops 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, 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 disease.Obesity and Diabetes MellitusSpontaneous microinfarcts were observed in a mouse model that crossed the Aβ overexpressing mouse line, APP/PS1, with the db/db mouse model for diabetes mellitus.25 The db/db line harbors a mutation of the diabetes mellitus (db) gene that leads to a leptin signaling defect, causing severe obesity, hypertension, and type 2 diabetes mellitus with hyperglycemia.26 The progeny of the APP/PS1-db/db cross-retained features of parental lines, but the combined risk factors led to cortical microinfarcts that were not observed in either parental strain. Microinfarcts appeared as small cystic cavities in various layers of cortex. The authors suspected that aberrant angiogenesis, unique to the crossed mice, led to immature and leaky microvessels that were prone to occlusion.Endothelial NOS-Deficient MiceEndothelial nitric oxide synthase is critical for regulation of vascular tone and blood pressure. A recent study showed that mice with partial deletion of endothelial nitric oxide synthase develop microinfarcts in cortex and, to a lesser extent, in the hippocampus and thalamus (Figure 3C).13 Cortical microinfarcts accrued in watershed regions between the perfusion territories of major cerebral arteries.27 They were noticeable by 6 months of age but were most prevalent at 12 to 18 months. This was accompanied by microvascular pathology, including intravascular clots, diffuse CAA, neuroinflammation, and blood–brain barrier disruption. Microinfarcts in these mice were postulated to result from small vessel thrombosis because of endothelial and platelet dysfunction.13Notch3 Mutant MiceMicroinfarcts (and microhemorrhages) have been reported in a mouse model of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).28 CADASIL is a hereditary form of vascular dementia caused by mutations on the gene for Notch3, a transmembrane receptor critical for mural cell–endothelial communication and vascular development. Mice with an Arg170Cys (R170C) mutation knocked into the endogenous Notch3 gene (a prevalent substitution mutation seen in human CADASIL) developed cerebrovascular pathology akin to that seen in human CADASIL. The authors reported microinfarcts in the motor cortex of 20-month-old mice, which appeared as small cystic cavities in deeper cortical layers. In contrast, another CADASIL mouse line (PAC-Notch3R169C) carrying rat Notch3 with an Arg169Cys mutation exhibited cerebral hypoperfusion and isolated white matter lesions but no microinfarcts.29 For reasons still unclear, other mouse lines with various Notch3 mutations develop arteriopathy but do not exhibit ischemic or hemorrhagic lesions.28,30Sickle Cell MiceCerebrovascular disease is a well-established complication of sickle cell disease (SCD). About 40% of children with SCD develop small "silent" cerebral infarcts, with some falling in the size range of microinfarcts.31 Recently, spontaneous cortical microinfarcts were reported in the Townes model of SCD (Figure 3D),14 a model that involves replacement of murine β-globin gene with the human sickle β-globin and human γ-globin genes.32 Aged sickle cell mice (13 months old) exhibited faster capillary flow velocities and altered microvascular topology, akin to that described in humans with SCD who were at high risk for stroke.14 Spontaneous cortical microinfarcts in SCD mice were larger and more frequent than in controls, and were associated with blood–brain barrier leakage and local tissue hypoxia, suggesting vascular pathology as an origin.Models of Induced MicrohemorrhageLaser-Induced Rupture of Parenchymal VesselsMicrohemorrhages can be induced with high spatiotemporal precision in rodent cortex by directly rupturing cortical microvessels using focused lasers (Figure 4A).33,34 Much like optically induced microinfarcts, this model requires implantation of a cranial window and a two-photon microscope to visualize and ablate the desired microvessel. An amplified femtosecond laser is used to damage the wall of target vessels, such as penetrating arterioles34 or capillaries.33 In vivo two-photon imaging of laser-induced microhemorrhages shows rapid extravasation of blood cells forming a lesion core roughly ≈100 μm in diameter and broad dissipation of blood plasma over a region ≈5 times larger than the core.34Download figureDownload PowerPointFigure 4. Microhemorrhages in mouse models. A, A cortical microhemorrhage observed in Prussian blue–stained mouse brain sections after optically induced rupture of a single cortical penetrating arteriole. Adapted from Rosidi et al34 with permission. Copyright ©2011, Rosidi et al. B, Microbleeds detected by T2*-weighted magnetic resonance imaging (MRI; left) and corresponding microhemorrhages detected by Prussian blue (right) in an APP23 mouse. Microbleeds seen by MRI appear larger than the actual lesion due to the blooming effect. Adapted from Reuter et al35 with permission. Copyright ©2016, Reuter et al. C, Microhemorrhages detected by Prussian blue staining in a mouse that received a specialized diet to induce hyperhomocysteinemia. Reprinted from Sudduth et al36 with permission. Copyright ©2013, SAGE Publications.Models With Spontaneous MicrohemorrhagesCAA ModelsA variety of APP overexpressing mouse lines develop CAA. These models include the PDAPP,37 Tg2576,38 double transgenic APP/PS1,39,40 triple transgenic Tg-SwDI,21 and APP2341 mouse lines. In these mice, Aβ plaque load, as well as CAA, increases gradually in an age-dependent fashion, with some model-specific variation. However, reports of spontaneous microhemorrhages (or microinfarcts) have been sparse and only described in lines that develop severe CAA during old age or after a second hit.The most commonly described observations of spontaneous microhemorrhages occur in APP23 mice.42 Cerebral microbleeds could be observed with in vivo T2*-weighted MRI in APP23 mice starting around 16 months of age.35,43–45 Microbleed number and volume increased with animal age. Postmortem analyses revealed that these MRI-observed microbleeds were true microhemorrhages on corresponding histopathologic Prussian blue–stained sections (Figure 4B). Parenchymal microvessels in these mice show severe vascular pathology, including smooth muscle cell degeneration and aneurysm-like vasodilation.42The APPDutch mouse model bears the mutation (E22Q-mutated Aβ) that causes hereditary cerebral hemorrhage with amyloidosis-Dutch type,46 a rare autosomal dominant disorder in humans characterized by early-onset of severe CAA and multiple recurrent lobar hemorrhages.47 Interestingly, when APP23 mice are crossed with APPDutch mice, twice as many microhemorrhages arise compared with the APP23 genotype alone.48 Further exacerbation of CAA was observed in crossed mice, which may explain the increased incidence of microhemorrhage.Hypertension ModelsA widely used model of severe hypertension is the inbred strain stroke-prone spontaneously hypertensive rat.49 Early characterization studies showed that these rats developed spontaneous ischemic lesions and hemorrhages around 9 to 12 months of age. Microhemorrhages (and some microinfarcts) coexisted with larger ischemic or hemorrhagic strokes.50,51 Fibrinoid necrosis and thickening of the vascular walls were regularly observed with cerebral penetrating arterioles, which likely contributes to vascular occlusions and ruptures in these animals.52,53 Abnormal vascular remodeling may also generate weakened microvessels, leading to microhemorrhage.50Hypertension-induced microhemorrhages in mice require combining transgenic lines with treatments to chronically increase vascular tone. One study used a transgenic mouse line expressing both the human renin and angiotensinogen genes (R+/A+), which develop chronic hypertension but are otherwise normal.54 Challenging these hypertensive mice with a high salt diet and L-NAME (an inhibitor of neuronal and endothelial NOS) led to formation of microhemorrhages in multiple brain regions, including brain stem, cerebellum, and basal ganglia. Another study administered chronic angiotensin II and L-NAME to aged Tg2576 mice and reported the development of more microhemorrhages compared with mice receiving vehicle.55Hyperhomocysteinemia ModelHyperhomocysteinemia is a risk factor for stroke and Alzheimer's disease. In diet-induced hyperhomocysteinemia, mice are placed on a diet deficient in folate, vitamin B6, and B12 and supplemented with excess methionine.36 Treated mice developed microhemorrhages, visualized by in vivo MRI and Prussian blue staining of brain sections postmortem (Figure 4C).36 When hyperhomocysteinemia was induced in APP/PS1 mice, significantly more microhemorrhages were observed in transgenics compared with their wild-type littermates.56 This increase was believed to be mediated by a heightened CAA and activation of matrix metalloproteinase-9 at the cerebrovascular wall.Model Selection: Advantages and DisadvantagesThe models discussed in this review have been collated in the Table. Inducible models allow one to ask how a microlesion affects local brain activity or structure, independent of other disease factors. Laser-induced microlesions are limited to cortex but provide exquisite control over the location and timing of their onset. In a complementary fashion, intracarotid injection of microemboli provides less control over lesion location but produced distributed microinfarcts that cause measurable deficits in common tests of cognitive function.Table. Summary of Animal Models.ModelModel TypeBrain locationLesion size reported/depictedLesion number reported; ConsistencyAnimal ageRef.Microinfarcts Injected microemboliInducedCortex, hippocampus, striatum, thalamus, corpus callosum≈100–500 μm diameter (≈0.3 mm2 area)Numerous microinfarcts per injection; Very consistentAny ageSilasi et al8; Wang et al10 Laser-inducedInducedCortex≈500 μm diameter (≈0.2 mm3 volume)One microinfarct per microvessel occlusion; Very consistentAny ageShih et al12; Taylor et al57 BCAS (C57Bl/6)SpontaneousCortex and thalamusWide range of sizes (≈0.5 mm3 volume)<10 microinfarcts per brain; ≈50% of mice examined3 mo start plus 6 mo of BCASHolland et al19 BCAS (TgSwDI)SpontaneousCortex and hippocampus≈100–300 μm diameter (0.01–0.03 mm2 area)1–10 microinfarcts per brain; ≈25%–40% of mice examined4–8 mo start plus 2–3 mo of BCASSalvadores et al22; et BCAS μm microinfarcts per brain; of mice examined3 mo start plus 1.5 mo of et μm microinfarcts per brain; of mice et hippocampus, μm microinfarcts per brain; 18 et al13 Notch3 μm in per brain not of mice et Townes sickle cell μm diameter mm2 microinfarcts over sections per brain; of mice et μm diameter microhemorrhage per Very consistentAny et Rosidi et al34 μm microbleeds per brain; Very et et et et et BCAS microhemorrhages per brain; of mice examined4–8 et al22; et BCAS μm but range microhemorrhages per brain; of mice et al19 μm microhemorrhages over sections per brain; et al13 Notch3 μm per brain not of mice et hippocampus, basal corpus μm microhemorrhages per brain; of rats et salt and stem, cerebellum, basal μm microhemorrhages per brain; Very mo start plus 6 mo et Tg2576 and and μm in microhemorrhages per brain; Very mo start plus 1 et (C57Bl/6)SpontaneousCortex and μm microhemorrhages per mo start plus mo et al36 and μm microhemorrhages per mo start plus 6 mo et indicates carotid artery nitric oxide and spontaneous microinfarct or microhemorrhage provide an to understand the vascular that to lesion formation and to for A variety of model types develop spontaneous microinfarcts and microhemorrhages, and this the that disease can be including CAA, mural cell or endothelial cell cerebral hypoperfusion, and vascular of Induced Microinfarcts and vivo optical imaging studies have revealed that microinfarcts induce and deficits in When microinfarcts were induced in the of APP/PS1 or Tg2576 mice, increase in Aβ plaque formation was seen in This effect was to of Indeed, 2 recent studies reported that distributed microinfarcts produced disruption of the Further, with a large effect on brain distributed microinfarcts also to deficits in cognitive total microinfarct volume being small compared with brain the microinfarct are but is of neuronal and This is with recent histopathologic from human of and blood–brain barrier disruption are also and altered these support the that microinfarcts the function of tissues well beyond their lesion existing on induced microhemorrhages that these lesions also deficits in tissues but more than microinfarcts. In vivo imaging revealed neuronal μm from the lesion but tissue function within 1 However, microhemorrhages induced and Penetrating arterioles often after which that local seen with microinfarcts) is necessary to induce deficits to the lesion and are now focused on understanding the risk and functional effects of microinfarcts and microhemorrhages in vascular cognitive impairment and However, these microlesions can be difficult to study in humans because of their small size, and with other disease factors. studies can these by into the impact and pathogenesis of This review shown that microlesions similar to seen in humans can be induced through microvascular occlusion and Further, microlesions develop spontaneously in a variety of and models of cerebrovascular disease. studies could MRI and white matter to understand how microlesion affects white matter and brain This the that small but broadly distributed microinfarcts can brain function on a studies can also be to understand the occurring beyond the lesion such as in vivo two-photon imaging allow of local and in tissues by ischemic Further, imaging of models that develop spontaneous microlesions can on disease pathogenesis by to the that could for or brain animal models as for A small number of studies have shown that microinfarct volume can be by suggesting a large and window for for large ischemic stroke may be as for smaller ischemic the was for et an occlusion of single cortical penetrating arterioles using focal of from a for critical of the and of Shih is by the the Alzheimer's and the on Alzheimer's Hyacinth is by and and an Emory University Hartmann is by from and and van Veluw is by a from the for online-only Data is with this at