Ernesto R. Gonzales

Characterizing the Appearance and Growth of Amyloid Plaques in APP/PS1 Mice

Amyloid plaques are primarily composed of extracellular aggregates of amyloid-β (Aβ) peptide and are a pathological signature of Alzheimers disease. However, the factors that influence the dynamics of amyloid plaque formation and growth in vivo are largely unknown. Using serial intravital multiphoton microscopy through a thinned-skull cranial window in APP/PS1 transgenic mice, we found that amyloid plaques appear and grow over a period of weeks before reaching a mature size. Growth was more prominent early after initial plaque formation: plaques grew faster in 6-month-old compared with 10-month-old mice. Plaque growth rate was also size-related, as smaller plaques exhibited more rapid growth relative to larger plaques. Alterations in interstitial Aβ concentrations were associated with changes in plaque growth. Parallel studies using multiphoton microscopy and in vivo microdialysis revealed that pharmacological reduction of soluble extracellular Aβ by as little as 20–25% was associated with a dramatic decrease in plaque formation and growth. Furthermore, this small reduction in Aβ synthesis was sufficient to reduce amyloid plaque load in 6-month-old but not 10-month-old mice, suggesting that treatment early in disease pathogenesis may be more effective than later treatment. In contrast to thinned-skull windows, no significant plaque growth was observed under open-skull windows, which demonstrated extensive microglial and astrocytic activation. Together, these findings indicate that individual amyloid plaque growth in vivo occurs over a period of weeks and may be influenced by interstitial Aβ concentration as well as reactive gliosis.

Differences in vulnerability to permanent focal cerebral ischemia among 3 common mouse strains.

Background and Purpose Genetically engineered mice are used to study the role of single genes in cerebral ischemia, but inherent, strain-dependent differences in neuronal vulnerability may affect experimental end points. To examine this possibility, tissue injury resulting from focal ischemia and its relationship to cerebral hemodynamics were determined in 3 common mutant mouse strains. Methods Permanent middle cerebral artery ligation was performed in male C57BL/6J, Balb/C, and 129X1/SvJ mice. Mean arterial blood pressure, blood gases, basal and postischemic cortical blood flow ([14C]iodoantipyrine autoradiography and laser-Doppler flowmetry), posterior communicating artery patency, and infarct size were determined. Results Basal cortical blood flow did not differ among strains. Ten minutes after middle cerebral artery ligation, relative red cell flow in the ischemic cortex was 6% to 7% of preischemic flow in every strain. Despite similar hemodynamics, cortical infarcts in Balb/C mice were 3-fold larger than those in 129X1/SvJ and C57BL/6J mice; infarct size in the latter 2 strains was not significantly different. The posterior communicating artery was either poorly developed or absent in >90% of the Balb/C and C57BL/6J but in <50% of the 129X1/SvJ mice. Conclusions The extent of ischemic injury differed markedly between the 3 strains. The presence and patency of posterior communicating arteries, although variable among strains, did not affect preischemic or postischemic cortical blood flow or bear any relationship to ischemic injury. Therefore, intrinsic factors, other than hemodynamic variability, may contribute to the differences in ischemic vulnerability among strains. These findings underscore the importance of selecting genetically matched wild-type controls.

Cerebral protection by hypoxic preconditioning in a murine model of focal ischemia-reperfusion.

Sublethal periods of hypoxia or ischemia can induce adaptive mechanisms to protect against subsequent lethal ischemic insults in a process known as ischemic preconditioning. In the present study, we developed a murine model of cerebral preconditioning using several common strains of adult mice. Animals were exposed to sublethal hypoxia (11% oxygen for 2 h) 48 h prior to a 90 min period of transient focal middle cerebral artery occlusion, induced by an intraluminal filament; injury was assessed 24 h later by TTC staining. Infarct volume in hypoxia-preconditioned animals was reduced 46%, 58%, and 64% in C57Bl/6, 129SvEv, and Swiss-Webster ND4 mice relative to their respective untreated controls. This non-invasive murine model of ischemic tolerance should be useful for elucidating the molecular basis of this protection using transgenic and knockout mice.

Enhancing Astrocytic Lysosome Biogenesis Facilitates Aβ Clearance and Attenuates Amyloid Plaque Pathogenesis

In sporadic Alzheimers disease (AD), impaired Aβ removal contributes to elevated extracellular Aβ levels that drive amyloid plaque pathogenesis. Extracellular proteolysis, export across the blood–brain barrier, and cellular uptake facilitate physiologic Aβ clearance. Astrocytes can take up and degrade Aβ, but it remains unclear whether this function is insufficient in AD or can be enhanced to accelerate Aβ removal. Additionally, age-related dysfunction of lysosomes, the major degradative organelles wherein Aβ localizes after uptake, has been implicated in amyloid plaque pathogenesis. We tested the hypothesis that enhancing lysosomal function in astrocytes with transcription factor EB (TFEB), a master regulator of lysosome biogenesis, would promote Aβ uptake and catabolism and attenuate plaque pathogenesis. Exogenous TFEB localized to the nucleus with transcriptional induction of lysosomal biogenesis and function in vitro. This resulted in significantly accelerated uptake of exogenously applied Aβ42, with increased localization to and degradation within lysosomes in C17.2 cells and primary astrocytes, indicating that TFEB is sufficient to coordinately enhance uptake, trafficking, and degradation of Aβ. Stereotactic injection of adeno-associated viral particles carrying TFEB driven by a glial fibrillary acidic protein promoter was used to achieve astrocyte-specific expression in the hippocampus of APP/PS1 transgenic mice. Exogenous TFEB localized to astrocyte nuclei and enhanced lysosome function, resulting in reduced Aβ levels and shortened half-life in the brain interstitial fluid and reduced amyloid plaque load in the hippocampus compared with control virus-injected mice. Therefore, activation of TFEB in astrocytes is an effective strategy to restore adequate Aβ removal and counter amyloid plaque pathogenesis in AD.

Vascular Permeability Precedes Spontaneous Intracerebral Hemorrhage in Stroke-Prone Spontaneously Hypertensive Rats

Background and Purpose— Stroke-prone spontaneous hypertensive rats (SHRsp) fed a high-salt diet develop malignant hypertension, blood–brain barrier breakdown, and spontaneous intracerebral hemorrhage (ICH). The precise spatial and temporal relationship between these events has not been well-delineated. Methods— Ten SHRsp male rats, fed a high-salt diet, were imaged weekly using MRI, starting at 12 weeks of age. T1-weighted (with and without Gd), T2-weighted, and T2* sequences were acquired. Permeability maps were calculated. Results— Seven SHRsp rats had spontaneous ICH develop before death. Five of the 7 rats had focally increased vascular permeability at the site of the ICH; 3 of these rats had vascular permeability 1 to 2 weeks before spontaneous ICH. Conclusions— Salt-loaded SHRsp rats have increased vascular permeability up to 2 weeks before ICH, predicting hemorrhage both in space and time. These results suggest that hypertensive ICH is preceded by focal vasculopathy detectable by Gd leak.

Modulation of Basal and Postischemic Leukocyte-Endothelial Adherence by Nitric Oxide

BACKGROUND AND PURPOSE Recent studies indicate that leukocytes are important contributors to secondary vascular and parenchymal injury after cerebral ischemia. The present study was undertaken to define nitric oxide (NO)-based mechanisms that regulate leukocyte-endothelial interactions in the cerebral vasculature, how these mechanisms are affected by cerebral ischemia, and whether NO-based therapies can affect postischemic leukocyte dynamics. METHODS Leukocyte adherence to pial venules of anesthetized newborn piglets was quantified by in situ fluorescence videomicroscopy through closed cranial windows during basal conditions and during reperfusion after 9 minutes of asphyxia. Nitric oxide synthase (NOS) was inhibited by local window superfusion of L-nitroarginine; superfusion of sodium nitroprusside was used to donate NO. RESULTS Local inhibition of NOS under resting conditions increased leukocyte-endothelial adherence 2.2-fold and 3.9-fold over baseline values after 1 hour and 2 hours, respectively; this response was completely blocked by cosuperfusion with L-arginine. Cosuperfusion of superoxide dismutase reversed L-nitroarginine-induced leukocyte adherence by 89% and 63% at these respective time points. The extent of acute leukocyte adherence elicited by NOS inhibition was similar in magnitude to that observed during the initial 2 hours of reperfusion after asphyxia. Leukocyte adherence was not additionally increased in asphyxic animals treated with L-nitroarginine. Sodium nitroprusside robustly inhibited asphyxia-induced leukocyte adherence back to control levels. CONCLUSIONS NO exerts a tonic antiadherent effect in the cerebral microcirculation by inactivation of adherence-promoting superoxide radical formation. Cerebral ischemia is associated with an inhibition of NOS or lower levels of NO, which results in leukocyte-endothelial adherence that can be prevented by NO donors. The latter may be useful therapeutically to prevent the purported vascular and parenchymal dysfunction and injury caused by activated leukocytes in ischemic brain.

Neutrophil elastase and neurovascular injury following focal stroke and reperfusion.

Neutrophil elastase (NE) degrades basal lamina and extracellular matrix molecules, and recruits leukocytes during inflammation; however, a basic understanding of the role of NE in stroke pathology is lacking. We measured an increased number of extravascular NE-positive cells, as well as increased levels of tissue elastase protein and activity, following transient middle cerebral artery occlusion (tMCAo). Both pharmacologic inhibition of NE with ZN200355 (ZN), and genetic deletion of NE, significantly reduced infarct volume, blood-brain barrier disruption, vasogenic edema, and leukocyte-endothelial adherence 24 h after tMCAo. ZN also reduced infarct volume in MMP9-null mice following tMCAo. There were, however, no reductions in infarct volume or vasogenic edema in NE-null mice in two models of permanent middle cerebral artery occlusion. Our findings confirm the involvement of NE in neurovascular stroke pathology, when reperfusion allows neutrophils access to vulnerable brain, with pharmacologic or genetic inhibition of NE being both neuro- and vasculo-protective in this setting.

Oligodendrocyte degeneration and recovery after focal cerebral ischemia

The vulnerability of oligodendrocytes to ischemic injury may contribute to functional loss in diseases of central white matter. Immunocytochemical methods to identify oligodendrocyte injury in experimental models rely on epitope availability, and fail to discriminate structural changes in oligodendrocyte morphology. We previously described the use of a lentiviral vector (LV) carrying enhanced green fluorescent protein (eGFP) under the myelin basic protein (MBP) promoter for selective visualization of oligodendrocyte cell bodies and processes. In this study, we used LV-MBP-eGFP to label oligodendrocytes in rat cerebral white matter prior to transient focal cerebral ischemia, and examined oligodendrocyte injury 24 h, 48 h and 1 week post-reperfusion by quantifying cell survival and assaying the integrity of myelin processes. There was progressive loss of GFP+ oligodendrocytes in ischemic white matter at 24 and 48 h. Surviving GFP+ cells had non-pyknotic nuclear morphology and were terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-negative, but there was marked fragmentation of myelin processes as early as 24 h after stroke. One week after stroke, we observed a restoration of GFP+ oligodendrocytes in ischemic white matter, reflected both by cell counts and by structural integrity of myelin processes. Proliferating cells were not the main source of GFP+ oligodendrocytes, as revealed by bromodeoxyuridine (BrdU) incorporation. These observations identify novel transient structural changes in oligodendrocyte cell bodies and myelinating processes, which may have consequences for white matter function after stroke.

Hydroxyethyl Starch Reduces Leukocyte Adherence and Vascular Injury in the Newborn Pig Cerebral Circulation After Asphyxia

Background and Purpose Hydroxyethyl starch (HES) has beneficial effects on ischemic brain injury; however, its mechanism of action remains unclear. The present study was undertaken to test the hypothesis that HES can attenuate increases in leukocyte adherence and vascular permeability in the cerebral vasculature after global cerebral ischemia induced by asphyxia. Methods Pial venular leukocyte adherence and permeability to sodium fluorescein were quantified in anesthetized newborn piglets by in situ fluorescence videomicroscopy through closed cranial windows during basal conditions and during 2 hours of reperfusion after global ischemia induced by 9 minutes of asphyxia. Experimental animals received HES after the asphyxial insult (10% HES 257/0.47, 600 mg/kg IV bolus 5 minutes after asphyxia, followed by 600 mg/kg per hour IV drip during reperfusion; n=9). Results A progressive and significant (P <0.05) increase in adherent leukocytes was observed during the initial 2 hours of reperfusion after asphyxia compared with nonasphyxial controls. In this model, vascular injury, as determined by significant (P <0.05) increases in fluorescein permeability at 2 hours of reperfusion, is largely dependent on adherent leukocytes. HES significantly reduced (P <0.05) leukocyte adherence at 1 hour and 2 hours of reperfusion and reduced fluorescein permeability at 2 hours. HES did not change hematocrit or alter pial arteriolar diameter. Conclusions These findings indicate that a vascular anti-inflammatory action may underlie the beneficial effects of HES in global cerebral ischemia secondary to asphyxia. Since this compound is well tolerated by patients, future preclinical and clinical studies may reveal improvements in functional outcome with the early introduction of this or similar agents after perinatal asphyxia or global ischemia.

Expression profiling identifies a molecular signature of reactive astrocytes stimulated by cyclic AMP or proinflammatory cytokines

Specialized glia, termed reactive astrocytes, accompany numerous pathologic conditions affecting the central nervous system, including stroke, multiple sclerosis, and neoplasia. To better define this important cell type, we employed high-density microarray gene expression profiling using two in vitro models of reactive gliosis (stimulation with dbcAMP or IL-1beta/IFNgamma). We identified 44 differentially expressed transcripts common to both in vitro models and demonstrated that a subset of these genes are also differentially expressed in response to experimental autoimmune encephalomyelitis and focal cerebral ischemia in vivo. Moreover, this pattern of differential gene expression is not observed in hyperproliferating or neoplastic glia.