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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) (Figure 1A and 1B).1 They are defined as areas because their widespread nature and far-reaching effects could of , often with 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 studies have now shown that a in the 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 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 -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 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 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 Bilateral (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 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 (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 , unique to the crossed cortex, hippocampus, and 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 ter- A second method allows reproducible targeting of microinfarct ritories of major cerebral .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 , includ- arterioles (or small pial arterioles that support flow to penetrat- ing intravascular clots, diffuse CAA, neuroinflammation, ing arterioles) are selectively occluded by inducing clots with and blood–brain barrier disruption. Microinfarcts in these precise laser irradiation. This is achieved most commonly by mice were postulated to result from small vessel 13 activating circulating photosensitizing agents with focused because of endothelial and dysfunction. green lasers, that is, focal photothrombosis.12,15,16 Occlusions Notch3 Mutant Mice can also be made without a photosensitizer by using ampli- Microinfarcts (and microhemorrhages) have been reported fied femtosecond laser ablation17 or targeted irradiation with in a mouse model of cerebral autosomal dominant arteri- higher laser powers from conventional two-photon imaging opathy with subcortical infarcts and leukoencephalopathy Shih et al Models of Microinfarct and Microhemorrhage 805

Figure 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

Downloaded from arteriole with focal photothrombosis (blue shade = region of ), 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.

(CADASIL).28 CADASIL is a hereditary form of vascular optically induced microinfarcts, this model requires implan- dementia caused by mutations on the gene for Notch3, a tation of a cranial window and a two-photon microscope to transmembrane receptor critical for mural cell–endothelial visualize and ablate the desired microvessel. An amplified http://stroke.ahajournals.org/ communication and vascular development. Mice with an femtosecond laser is used to damage the wall of target ves- Arg170Cys (R170C) mutation knocked into the endogenous sels, such as penetrating arterioles34 or capillaries.33 In vivo Notch3 gene (a prevalent substitution mutation seen in human two-photon imaging of laser-induced microhemorrhages CADASIL) developed cerebrovascular pathology akin to that shows rapid extravasation of blood cells forming a lesion core seen in human CADASIL. The authors reported microinfarcts roughly ≈100 μm in diameter and broad dissipation of blood in the motor cortex of 20-month-old mice, which appeared plasma over a region ≈5 times larger than the core.34 as small cystic cavities in deeper cortical layers. In contrast, another CADASIL mouse line (PAC-Notch3R169C) carrying Models With Spontaneous Microhemorrhages by guest on April 4, 2018 rat Notch3 with an Arg169Cys mutation exhibited cerebral CAA Models hypoperfusion and isolated white matter lesions but no micro- A variety of APP overexpressing mouse lines develop CAA. infarcts.29 For reasons still unclear, other mouse lines with var- These models include the PDAPP,37 Tg2576,38 double trans- ious Notch3 mutations develop arteriopathy but do not exhibit genic APP/PS1,39,40 triple transgenic Tg-SwDI,21 and APP2341 ischemic or hemorrhagic lesions.28,30 mouse lines. In these mice, Aβ plaque load, as well as CAA, Sickle Cell Mice increases gradually in an age-dependent fashion, with some is a well-established complication of model-specific variation. However, reports of spontaneous (SCD). About 40% of children with SCD microhemorrhages (or microinfarcts) have been sparse and develop small “silent” cerebral infarcts, with some falling in only described in lines that develop severe CAA during old the size range of microinfarcts.31 Recently, spontaneous corti- age or after a second hit. cal microinfarcts were reported in the Townes model of SCD The most commonly described observations of sponta- 42 (Figure 3D),14 a model that involves replacement of murine neous microhemorrhages occur in APP23 mice. Cerebral microbleeds could be observed with in vivo T2*-weighted β- gene with the human sickle β-globin and human MRI in APP23 mice starting around 16 months of age.35,43–45 γ-globin genes.32 Aged sickle cell mice (13 months old) exhib- Microbleed number and volume increased with animal age. ited faster capillary flow velocities and altered microvascular Postmortem analyses revealed that these MRI-observed topology, akin to that described in humans with SCD who were microbleeds were true microhemorrhages on corresponding at high risk for stroke.14 Spontaneous cortical microinfarcts in histopathologic Prussian blue–stained sections (Figure 4B). SCD mice were larger and more frequent than in controls, and Parenchymal microvessels in these mice show severe vascu- were associated with blood–brain barrier leakage and local tis- lar pathology, including smooth muscle cell degeneration and sue , suggesting vascular pathology as an origin. -like vasodilation.42 The APPDutch mouse model bears the mutation (E22Q- Models of Induced Microhemorrhage mutated Aβ) that causes hereditary cerebral hemorrhage with Laser-Induced Rupture of Parenchymal Vessels amyloidosis-Dutch type,46 a rare autosomal dominant disorder Microhemorrhages can be induced with high spatiotempo- in humans characterized by early-onset of severe CAA and ral precision in rodent cortex by directly rupturing cortical multiple recurrent lobar hemorrhages.47 Interestingly, when microvessels using focused lasers (Figure 4A).33,34 Much like APP23 mice are crossed with APPDutch mice, twice as many 806 Stroke March 2018 Downloaded from http://stroke.ahajournals.org/

Figure 4. Microhemorrhages in mouse models. A, A cortical micro- hemorrhage observed in Prussian blue–stained mouse brain sec- Figure 3. Microinfarcts in rodent models. A, A cortical microin- tions after optically induced rupture of a single cortical penetrating by guest on April 4, 2018 farct observed in hematoxylin & eosin (H&E)–stained mouse brain arteriole. Adapted from Rosidi et al34 with permission. Copyright sections after injection of cholesterol crystals into the internal ©2011, Rosidi et al. B, Microbleeds detected by T2*-weighted carotid artery. Adapted from Wang et al10 with permission. Copy- magnetic resonance imaging (MRI; left) and corresponding micro- right ©2012, Society for Neurosience. B, A cortical microinfarct hemorrhages detected by Prussian blue (right) in an APP23 mouse. observed in NeuN and GFAP (glial fibrillary acidic protein) immu- Microbleeds seen by MRI appear larger than the actual lesion due nostained rat brain sections after occlusion of a single cortical to the blooming effect. Adapted from Reuter et al35 with permission. penetrating arteriole by focal photothrombosis. Adapted from Copyright ©2016, Reuter et al. C, Microhemorrhages detected by Shih et al12 with permission. Copyright ©2012, Springer Nature. Prussian blue staining in a mouse that received a specialized diet to C, Spontaneous microinfarcts observed in H&E-stained brain induce hyperhomocysteinemia. Reprinted from Sudduth et al36 with sections from an 18-month-old endothelial nitric oxide synthase permission. Copyright ©2013, SAGE Publications. (eNOS)–deficient mouse. Adapted from Tan et al13 with permis- sion, Copyright ©2012, Tan et al. D, Spontaneous microin- animals.52,53 Abnormal vascular remodeling may also generate farcts observed in NeuN-immunostained brain sections from a 50 13-month-old Townes sickle cell mouse. Adapted from Hyacinth weakened microvessels, leading to microhemorrhage. et al14 with permission. Copyright ©2017, SAGE Publications. Hypertension-induced microhemorrhages in mice require combining transgenic lines with treatments to chronically microhemorrhages arise compared with the APP23 geno- increase vascular tone. One study used a transgenic mouse line type alone.48 Further exacerbation of CAA was observed in expressing both the human renin and angiotensinogen genes (R+/ crossed mice, which may explain the increased incidence of A+), which develop chronic hypertension but are otherwise nor- microhemorrhage. mal.54 Challenging these hypertensive mice with a high salt diet Hypertension Models and L-NAME (an inhibitor of neuronal and endothelial NOS) A widely used model of severe hypertension is the inbred led to formation of microhemorrhages in multiple brain regions, strain stroke-prone spontaneously hypertensive rat.49 Early including brain stem, cerebellum, and basal ganglia. Another characterization studies showed that these rats developed study administered chronic angiotensin II and L-NAME to aged spontaneous ischemic lesions and hemorrhages around 9 to 12 Tg2576 mice and reported the development of more microhem- orrhages compared with mice receiving vehicle.55 months of age. Microhemorrhages (and some microinfarcts) coexisted with larger ischemic or hemorrhagic .50,51 Hyperhomocysteinemia Model Fibrinoid and thickening of the vascular walls were Hyperhomocysteinemia is a risk factor for stroke and regularly observed with cerebral penetrating arterioles, which Alzheimer’s disease. In diet-induced hyperhomocysteinemia, likely contributes to vascular occlusions and ruptures in these mice are placed on a diet deficient in folate, vitamin B6, and Shih et al Models of Microinfarct and Microhemorrhage 807

Table. Summary of Animal Models.

Lesion size Lesion number reported; Model Model Type Brain location reported/depicted Consistency Animal age Ref. Microinfarcts Injected Induced Cortex, hippocampus, ≈100–500 μm Numerous microinfarcts per Any age Silasi et al8; Wang et al10 microemboli striatum, thalamus, diameter (≈0.3 injection; Very consistent corpus callosum mm2 area) Laser-induced Induced Cortex ≈500 μm diameter One microinfarct per Any age Shih et al12; Taylor et al57 (≈0.2 mm3 volume) microvessel occlusion; Very consistent BCAS Spontaneous Cortex and thalamus Wide range of sizes <10 microinfarcts per brain; 3 mo start plus 6 Holland et al19 (C57Bl/6) (≈0.5 mm3 volume) ≈50% of mice examined mo of BCAS BCAS Spontaneous Cortex and ≈100–300 μm 1–10 microinfarcts per brain; 4–8 mo start Salvadores et al22; Okamoto (TgSwDI) hippocampus diameter (0.01– ≈25%–40% of mice examined plus 2–3 mo of et al23 0.03 mm2 area) BCAS BCAS (Apoe−/−) Spontaneous Hippocampus ≈200 μm diameter Multiple microinfarcts per 3 mo start plus Lee et al24 brain; ≈75% of mice examined 1.5 mo of BCAS Downloaded from APP/PS1×db Spontaneous Cortex ≈500 μm diameter Multiple microinfarcts per 7 mo Niedowicz et al25 brain; ≈75% of mice examined eNOS +/− Spontaneous Cortex, hippocampus, ≈100–500 μm Multiple microinfarcts per 12, 18 mo Tan et al13 thalamus diameter brain; Consistency unspecified Notch3 mutant Spontaneous Cortex (motor) ≈50–100 μm in Number per brain not specified; 20–22 mo Wallays et al28 http://stroke.ahajournals.org/ (R170C) diameter 12% of mice examined Townes sickle Spontaneous Cortex ≈100–500 μm ≈5 microinfarcts over 20 13 mo Hyacinth et al14 cell mouse diameter (≈0.05 sections per brain; 100% of mm2 area) mice examined Microhemorrhages Laser-induced Induced Cortex ≈100 μm diameter 1 microhemorrhage per Any age Nishimura et al33; Rosidi (hemorrhage core) rupture; Very consistent et al34

35 42

by guest on April 4, 2018 APP23 Spontaneous Cortex, thalamus ≈50–300 μm 15–30 microbleeds per brain; 20–28 mo Reuter et al ; Winkler et al ; diameter Very consistent Beckmann et al43; Marinescu et al44; Dudeffant et al45 BCAS Spontaneous Thalamus Not depicted Very few microhemorrhages 4–8 mo Salvadores et al22; Okamoto (TgSwDI) per brain; 30% of mice et al23 examined BCAS Spontaneous Thalamus ≈150 μm Multiple microhemorrhages per 6 mo Holland et al19 (C57Bl/6) diameter, but wide brain; ≈75% of mice examined range reported eNOS +/− Spontaneous Cortex ≈25 μm diameter ≈8 microhemorrhages over 18 mo Tan et al13 9–10 sections per brain; Consistency unspecified Notch3 mutant Spontaneous Cortex ≈25 μm diameter Number per brain not specified; 20–22 mo Wallays et al28 (R170C) 12% of mice examined SPSHR Spontaneous Cortex, hippocampus, ≈50–150 μm Multiple microhemorrhages per 8–12 mo Schreiber et al51 basal ganglia, corpus diameter brain; ≈60% of rats examined callosum R+/A+ Spontaneous Brain stem, ≈50–150 μm Multiple microhemorrhages per ≈11 mo start Iida et al54 (high salt and cerebellum, basal diameter brain; Very consistent plus 6 mo L-NAME) ganglia treatment Tg2576 Spontaneous Cortex and <100 μm in Multiple microhemorrhages per ≈15 mo start Passos et al55 (AngII and hippocampus diameter brain; Very consistent plus 1 wk L-NAME) treatment HHcy Spontaneous Cortex and ≈100 μm diameter ≈2–3 microhemorrhages 7 mo start plus 4 Sudduth et al36 (C57Bl/6) hippocampus per section; Consistency mo diet unspecified HHcy Spontaneous Cortex and ≈100 μm diameter ≈4 microhemorrhages 12 mo start plus Sudduth et al56 (APP/PS1) hippocampus per section; Consistency 6 mo diet unspecified BCAS indicates bilateral carotid artery stenosis; eNOS, Endothelial nitric oxide synthase; and HHcy, hyperhomocysteinemia. 808 Stroke March 2018

B12 and supplemented with excess methionine.36 Treated In vivo calcium imaging revealed impaired neuronal responses mice developed microhemorrhages, visualized by in vivo ≤150 μm from the lesion core, but tissue function recovered MRI and Prussian blue staining of brain sections postmortem within 1 day.65 However, microhemorrhages induced persis- (Figure 4C).36 When hyperhomocysteinemia was induced in tent peri-lesional microgliosis and .34 Penetrating APP/PS1 mice, significantly more microhemorrhages were arterioles often remained flowing even after rupture,34 which observed in transgenics compared with their wild-type lit- suggests that local ischemia (as seen with microinfarcts) is termates.56 This increase was believed to be mediated by a necessary to induce neural deficits distant to the lesion core. heightened CAA and activation of matrix metalloproteinase-9 at the cerebrovascular wall. Summary and Future Directions Clinical efforts are now focused on understanding the causes, Model Selection: Advantages and risk factors, and functional effects of microinfarcts and micro- Disadvantages hemorrhages in vascular cognitive impairment and demen- The models discussed in this review have been collated in the tia.3,5 However, these microlesions can be difficult to study in Table. Inducible models allow one to ask how a microlesion humans because of their small size, unpredictable onset, and affects local brain activity or structure, independent of other coexistence with other disease factors. Preclinical studies can disease factors. Laser-induced microlesions are limited to cor- complement these clinical efforts by providing insight into tex but provide exquisite control over the location and timing the impact and of microlesions. This review has

Downloaded from of their onset. In a complementary fashion, intracarotid injec- shown that microlesions similar to those seen in humans can be tion of microemboli provides less control over lesion location reliably induced through microvascular occlusion and manipu- but produced distributed microinfarcts that cause measurable lation. Further, microlesions develop spontaneously in a variety deficits in common tests of cognitive function. of genetic and dietary-induced models of cerebrovascular dis- Models developing spontaneous microinfarct or microhe- ease. Future studies could use high-field MRI and white mat- morrhage provide an opportunity to understand the vascu- ter tractography to understand how microlesion accrual affects http://stroke.ahajournals.org/ lar deficiencies that lead to lesion formation and to identify white matter integrity and brain connectivity. This addresses potential targets for prevention. A wide variety of model types the possibility that individually small but broadly distributed develop spontaneous microinfarcts and microhemorrhages, microinfarcts can impair brain function on a global scale. and this supports the idea that diverse disease processes can Mechanistic studies can also be performed to understand the be contributing factors, including CAA, mural cell or endo- cellular/molecular changes occurring beyond the lesion core. thelial cell dysfunction, cerebral hypoperfusion, and vascular Technologies such as in vivo two-photon imaging­ allow direct . visualization of local neuronal, glial, vascular, and glymphatic

by guest on April 4, 2018 changes in tissues affected by ischemic . Further, longitu- Physiological Impact of Induced Microinfarcts dinal imaging of models that develop spontaneous microlesions and Microhemorrhages can shed light on disease pathogenesis by identifying changes In vivo optical imaging studies have revealed that microinfarcts to the neurovascular unit that could account for narrowing or induce lasting neural and hemodynamic deficits in surround- weakening brain microvessels. Finally, animal models serve as ing tissues.58,59 When microinfarcts were photothrombotically test beds for therapeutics. A small number of past studies have induced in the cortices of APP/PS1 or Tg2576 mice, increase shown that microinfarct volume can be extensively reduced in Aβ plaque formation was seen in surrounding tissues.60,61 by neuroprotectants,12,18 suggesting a relatively large penum- This effect was attributed to impaired drainage of interstitial bra and window for therapeutic intervention.10 Thus neuro- fluid. Indeed, 2 recent studies reported that distributed micro- protective strategies previously considered for large ischemic infarcts produced bi-hemispheric disruption of the brain’s stroke may be worth re-evaluating as preventative therapies for glymphatic system.62,63 Further, consistent with having a large smaller ischemic events. effect on brain function, distributed microinfarcts also lead to deficits in cognitive tasks, despite total microinfarct volume Addendum being small compared with overall brain volume.10,11 After the manuscript was accepted for publication, Dönmez-Demir et Surrounding the microinfarct core, neurons are viable, al published an article describing occlusion of single cortical penetrat- ing arterioles using focal application of FeCl from a micropipette.66 but there is of neuronal dendrites, reduced dendritic 3 spine density,58 axonal damage, and myelin loss.8,10,63 This is consistent with recent histopathologic findings from human Acknowledgments microinfarcts.64 Extensive peri-lesional astrogliosis, mislocal- We thank Brian Bacskai for critical reading of the article and helpful ization of aquaporin 4, and blood–brain barrier disruption are discussions. also observed, indicating neuroinflammation and altered neu- ronal–glial signaling.10,12,58 Collectively, these findings support Sources of Funding the idea that microinfarcts impair the function of tissues well Dr Shih is supported by NIH-NINDS (NS085402, NS096997, beyond their restricted lesion cores. NS097775), NIH-NIGMS (P20GM109040), National Science Foundation (1539034), the Dana Foundation, the American Heart The existing data on experimentally induced microhemor- Association (14GRNT20480366), Alzheimer’s Association, and the rhages suggest that these lesions also produce neural deficits Charleston Conference on Alzheimer’s Disease. Dr Hyacinth is sup- in surrounding tissues but more transiently than microinfarcts. ported by NIH-NHLBI (U01HL117721 and R01HL138423) and Shih et al Models of Microinfarct and Microhemorrhage 809

an Emory University Pilot/HeRO Award. D.A. Hartmann is sup- 18. Zhang Q, Lan Y, He XF, Luo CM, Wang QM, Liang FY, et al. ported by awards from NIH (T32 GM08716), NIH-NCATS (UL1 Allopurinol protects against ischemic insults in a mouse model of cor- TR001450 and TL1 TR001451), and NIH-NINDS (F30NS096868). tical microinfarction. Brain Res. 2015;1622:361–367. doi: 10.1016/j. Dr van Veluw is supported by a Rubicon grant from the Netherlands brainres.2015.07.010. Organization for Scientific Research (019.153LW.014). 19. Holland PR, Searcy JL, Salvadores N, Scullion G, Chen G, Lawson G, et al. Gliovascular disruption and cognitive deficits in a mouse model with features of small vessel disease. J Cereb Blood Flow Metab. Disclosures 2015;35:1005–1014. doi: 10.1038/jcbfm.2015.12. None. 20. Shibata M, Yamasaki N, Miyakawa T, Kalaria RN, Fujita Y, Ohtani R, et al. Selective impairment of working memory in a mouse model of chronic cerebral hypoperfusion. Stroke. 2007;38:2826–2832. doi: References 10.1161/STROKEAHA.107.490151. 1. Westover MB, Bianchi MT, Yang C, Schneider JA, Greenberg SM. 21. Davis J, Xu F, Deane R, Romanov G, Previti ML, Zeigler K, et al. Estimating cerebral microinfarct burden from autopsy samples. Early-onset and robust cerebral microvascular accumulation of amyloid Neurology. 2013;80:1365–1369. doi: 10.1212/WNL.0b013e31828c2f52. beta-protein in transgenic mice expressing low levels of a vasculotropic 2. Smith EE, Schneider JA, Wardlaw JM, Greenberg SM. Cerebral micro- Dutch/Iowa mutant form of amyloid beta-protein precursor. J Biol Chem. infarcts: the invisible lesions. Lancet Neurol. 2012;11:272–282. doi: 2004;279:20296–20306. doi: 10.1074/jbc.M312946200. 10.1016/S1474-4422(11)70307-6. 22. Salvadores N, Searcy JL, Holland PR, Horsburgh K. Chronic cerebral 3. van Veluw SJ, Shih AY, Smith EE, Chen C, Schneider JA, Wardlaw hypoperfusion alters amyloid-β peptide pools leading to cerebral amy- loid angiopathy, microinfarcts and haemorrhages in Tg-SwDI mice. Clin JM, et al. Detection, risk factors, and functional consequences of cere- Sci (Lond). 2017;131:2109–2123. doi: 10.1042/CS20170962. bral microinfarcts. Lancet Neurol. 2017;16:730–740. doi: 10.1016/ 23. Okamoto Y, Yamamoto T, Kalaria RN, Senzaki H, Maki T, Hase Y, et S1474-4422(17)30196-5. al. Cerebral hypoperfusion accelerates cerebral amyloid angiopathy and Downloaded from 4. Brundel M, de Bresser J, van Dillen JJ, Kappelle LJ, Biessels GJ. Cerebral promotes cortical microinfarcts. Acta Neuropathol. 2012;123:381–394. microinfarcts: a systematic review of neuropathological studies. J Cereb doi: 10.1007/s00401-011-0925-9. Blood Flow Metab. 2012;32:425–436. doi: 10.1038/jcbfm.2011.200. 24. Lee ES, Yoon JH, Choi J, Andika FR, Lee T, Jeong Y. A mouse model of 5. Greenberg SM, Vernooij MW, Cordonnier C, Viswanathan A, Al-Shahi subcortical reflecting degeneration of cerebral white Salman R, Warach S, et al; Microbleed Study Group. Cerebral micro- matter and microcirculation [published online ahead of print October 20, bleeds: a guide to detection and interpretation. Lancet Neurol. 2017]. J Cereb Blood Flow Metabo. doi: 10.1177/0271678X17736963. 2009;8:165–174. doi: 10.1016/S1474-4422(09)70013-4.

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SUPPLEMENTAL MATERIALS

Criterion for defining microinfarcts and microhemorrhages in rodent studies.

Recent reviews have established guidelines for identifying human cerebral microinfarcts1 and microhemorrhages.2 However, no criteria yet exist for defining spontaneously occurring microlesions in rodent models. Microinfarct size is the primary means to distinguish these lesions from larger ischemic strokes caused by blockage or rupture of major cerebral arterioles. We suggest that an upper size threshold of 1 mm lesion diameter be used for microinfarcts. This would encompass most microinfarcts observed across the preclinical studies reviewed here, and is also consistent with the size of penetrating arteriole perfusion domains observed in rats and mice. A caveat, however, is that microinfarct shapes can be irregular and individual tissue slices may not transect the widest region of the lesion. It is therefore best to report microinfarct volume, if possible, by measuring lesion area over multiple adjacent brain slices. In addition to defining a region of infarction with loss of NeuN or Hematoxylin & Eosin , it is recommended to also confirm the presence of neuroinflammation by staining for CD68, Iba1 or GFAP, which should be upregulated following tissue ischemia. For old microhemorrhages, stains such as Perl’s Prussian blue should be used to detect the presence of iron. We suggest that lesions should consist of at least 5 iron-positive depositions as a lower threshold and the overall region of staining be less than approximately 0.5 mm in diameter. We noticed that regions with only a single iron deposit were counted as microhemorrhages in some studies, and these may be too small to represent true microhemorrhages. By standardizing the reporting of microinfarcts and microhemorrhages with lesion size criterion, and data on lesion size range, brain location, and prevalence, consistency among research groups and comparisons between different models can be evaluated more rigorously.

References

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