Extravasation of biodegradable microspheres in the rat brain.

Drug development for neurological diseases is greatly impeded by the presence of the blood-brain barrier (BBB). We and others previously reported on extravasation of micrometer-sized particles from the cerebral microcirculation - across the BBB - into the brain tissue over the course of several weeks. This mechanism could potentially be used for sustained parenchymal drug delivery after extravasation of biodegradable microspheres. As a first step toward this goal, we set out to evaluate the extravasation potential in the rat brain of three classes of biodegradable microspheres with drug-carrying potential, having a median diameter of 13 µm (80% within 8-18 µm) and polyethylene glycol concentrations of 0%, 24% and 36%. Extravasation, capillary recanalization and tissue damage were determined in a rat cerebral microembolization model at day 14 after microsphere injection. Microspheres of all three classes had the potential to extravasate from the vessel into the brain parenchyma, with microspheres without polyethylene glycol extravasating the fastest. Microembolization with biodegradable microspheres led to impaired local capillary perfusion, which was substantially restored after bead extravasation. We did not observe overt tissue damage after microembolization with any microsphere: we found very limited BBB disruption (IgG extravasation), no microgliosis (Iba1 staining) and no large neuronal infarctions (NeuN staining). In conclusion, biodegradable microspheres with different polymer compositions can extravasate into the brain parenchyma while causing minimal tissue damage.

Perivascular clearance of blood proteins after blood-brain barrier disruption in a rat model of microinfarcts.

Microinfarcts result in a transient loss of the blood-brain barrier (BBB) in the ischemic territory. This leads to the extravasation of blood proteins into the brain parenchyma. It is not clear how these proteins are removed. Here we studied the role of perivascular spaces in brain clearance from extravasated blood proteins. Male and female Wistar rats were infused with microspheres of either 15, 25, or 50 µm in diameter (n = 6 rats per group) via the left carotid artery. We infused either 25,000 microspheres of 15 µm, 5500 of 25 µm, or 1000 of 50 µm. One day later, rats were infused with lectin and hypoxyprobe to label perfused blood vessels and hypoxic areas, respectively. Rats were then euthanized and perfusion-fixed. Brains were excised, sectioned, and analyzed using immunostaining and confocal imaging. Microspheres induced a size-dependent increase in ischemic volume per territory, but the cumulative ischemic volume was similar in all groups. The total volumes of ischemia, hypoxia and infarction affected 1-2 % of the left hemisphere. Immunoglobulins (IgG) were present in ischemic brain tissue surrounding lodged microspheres in all groups. In addition, staining for IgG was found in perivascular spaces of blood vessels nearby areas of BBB disruption. About 2/3 of these vessels were arteries, while the remaining 1/3 of these vessels were veins. The subarachnoid space (SAS) of the affected hemisphere stained stronger for IgG than the contralateral hemisphere in all groups: +27 %, +44 % and +27 % respectively. Microspheres of various sizes induce a local loss of BBB integrity, evidenced by parenchymal IgG staining. The presence of IgG in perivascular spaces of both arteries and veins distinct from the ischemic territories suggests that both contribute to the removal of blood proteins. The strong staining for IgG in the SAS of the affected hemisphere suggests that this perivascular route egresses via the CSF. Perivascular spaces therefore play a previously unrecognized role in tissue clearance of fluid and extravasated proteins after BBB disruption induced by microinfarcts.

Quantification of hypoxic regions distant from occlusions in cerebral penetrating arteriole trees.

The microvasculature plays a key role in oxygen transport in the mammalian brain. Despite the close coupling between cerebral vascular geometry and local oxygen demand, recent experiments have reported that microvascular occlusions can lead to unexpected distant tissue hypoxia and infarction. To better understand the spatial correlation between the hypoxic regions and the occlusion sites, we used both in vivo experiments and in silico simulations to investigate the effects of occlusions in cerebral penetrating arteriole trees on tissue hypoxia. In a rat model of microembolisation, 25 µm microspheres were injected through the carotid artery to occlude penetrating arterioles. In representative models of human cortical columns, the penetrating arterioles were occluded by simulating the transport of microspheres of the same size and the oxygen transport was simulated using a Green's function method. The locations of microspheres and hypoxic regions were segmented, and two novel distance analyses were implemented to study their spatial correlation. The distant hypoxic regions were found to be present in both experiments and simulations, and mainly due to the hypoperfusion in the region downstream of the occlusion site. Furthermore, a reasonable agreement for the spatial correlation between hypoxic regions and occlusion sites is shown between experiments and simulations, which indicates the good applicability of in silico models in understanding the response of cerebral blood flow and oxygen transport to microemboli.

Quantitative 3D analysis of tissue damage in a rat model of microembolization.

There is a discrepancy between successful recanalization and good clinical outcome after endovascular treatment (EVT) in acute ischemic stroke patients. During removal of a thrombus, a shower of microemboli may release and lodge to the distal circulation. The objective of this study was to determine the extent of damage on brain tissue caused by microemboli. In a rat model of microembolization, a mixture of microsphere (MS) sizes (15, 25 and 50 µm diameter) was injected via the left internal carotid artery. A 3D image of the left hemisphere was reconstructed and a point-pattern spatial analysis was applied based on G- and K-functions to unravel the spatial correlation between MS and the induced hypoxia or infarction. We show a spatial correlation between MS and hypoxia or infarction spreading up to a distance of 1000-1500 µm. These results imply that microemboli, which individually may not always be harmful, can interact and result in local areas of hypoxia or even infarction when lodged in large numbers.

Recovery of Hypoxic Regions in a Rat Model of Microembolism.

OBJECTIVES: Endovascular treatment (EVT) has become the standard of care for acute ischemic stroke. Despite successful recanalization, a limited subset of patients benefits from the new treatment. Human MRI studies have shown that during removal of the thrombus, a shower of microclots is released from the initial thrombus, possibly causing new ischemic lesions. The aim of the current study is to quantify tissue damage following microembolism. MATERIALS AND METHODS: In a rat model, microembolism was generated by injection of a mixture of polystyrene fluorescent microspheres (15, 25 and 50 µm in diameter). The animals were killed at three time-points: day 1, 3 or 7. AMIRA and IMARIS software was used for 3D reconstruction of brain structure and damage, respectively. CONCLUSIONS: Microembolism induces ischemia, hypoxia and infarction. Infarcted areas persist, but hypoxic regions recover over time suggesting that repair processes in the brain rescue the regions at risk.

Microembolus clearance through angiophagy is an auxiliary mechanism preserving tissue perfusion in the rat brain.

Considering its intolerance to ischemia, it is of critical importance for the brain to efficiently process microvascular occlusions and maintain tissue perfusion. In addition to collateral microvascular flow and enzymatic degradation of emboli, the endothelium has the potential to engulf microparticles and thereby recanalize the vessel, through a process called angiophagy. Here, we set out to study the dynamics of angiophagy in relation to cytoskeletal remodeling in vitro and reperfusion in vivo. We show that polystyrene microspheres and fibrin clots are actively taken up by (brain) endothelial cells in vitro, and chart the dynamics of the actin cytoskeleton during this process using live cell imaging. Whereas microspheres were taken up through the formation of a cup structure by the apical endothelial membrane, fibrin clots were completely engulfed by the cells, marked by dense F-actin accumulation surrounding the clot. Both microspheres and fibrin clots were retained in the endothelial cells. Notably, fibrin clots were not degraded intracellularly. Using an in vivo microembolization rat model, in which microparticles are injected into the common carotid artery, we found that microspheres are transported by the endothelium from the microvasculature into the brain parenchyma. Microembolization with microspheres caused temporal opening of the blood-brain barrier and vascular nonperfusion, followed by microsphere extravasation and restoration of vessel perfusion over time. Taken together, angiophagy is accompanied by active cytoskeletal remodeling of the endothelium, and is an effective mechanism to restore perfusion of the occluded microvasculature in vivo.

Extravasation of Microspheres in a Rat Model of Silent Brain Infarcts.

Background and Purpose- We developed a rat model of silent brain infarcts based on microsphere infusion and investigated their impact on perfusion and tissue damage. Second, we studied the extent and mechanisms of perfusion recovery. Methods- At day 0, 15 µm fluorescent microspheres were injected into the right common carotid artery of F344 rats. At days 1, 7, or 28, the brain was removed, cut in 100-µm cryosections, and processed for immunofluorescent staining and analysis. Results- Injection of microspheres caused mild and transient damage to the treated hemisphere, with a decrease in perfused capillary volume at day 1, as compared with the untreated hemisphere. At day 1 but not at days 7 and 28, we observed IgG staining outside of the vessels, indicating vessel leakage. All microspheres were located inside the lumen of the vessels at day 1, whereas the vast majority (≈80%) of the microspheres were extravascular at day 7, and 100% at day 28. This was accompanied by restoration of perfused capillary volume. Conclusions- Microspheres cause mild and transient damage, and effective extravasation mechanisms exist in the brain to clear microsized emboli from the vessels.

Expression patterns of endothelial permeability pathways in the development of the blood-retinal barrier in mice.

Insight into the molecular and cellular processes in blood-retinal barrier (BRB) development, including the contribution of paracellular and transcellular pathways, is still incomplete but may help to understand the inverse process of BRB loss in pathologic eye conditions. In this comprehensive observational study, we describe in detail the formation of the BRB at the molecular level in physiologic conditions, using mice from postnatal day (P)3 to P25. Our data indicate that immature blood vessels already have tight junctions at P5, before the formation of a functional BRB. Expression of the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), which is known to be involved in transcellular transport and associated with BRB permeability, decreased during development and was absent when a functional barrier was formed. Moreover, we show that PLVAP deficiency causes a transient delay in retinal vascular development and changes in mRNA expression levels of endothelial permeability pathway proteins.-Van der Wijk, A.-E., Wisniewska-Kruk, J., Vogels, I. M. C., van Veen, H. A., Ip, W. F., van der Wel, N. N., van Noorden, C. J. F., Schlingemann, R. O., Klaassen, I. Expression patterns of endothelial permeability pathways in the development of the blood-retinal barrier in mice.

Glucocorticoids exert differential effects on the endothelium in an in vitro model of the blood-retinal barrier.

PURPOSE: Glucocorticoids (GCs) are used as treatment in diabetic macular oedema, a condition caused by blood-retinal barrier (BRB) disruption. The proposed mechanisms by which GCs reduce macular oedema are indirect anti-inflammatory effects and inhibition of VEGF production, but direct effects on the BRB endothelium may be equally important. Here, we investigated direct effects of GCs on the endothelium to understand the specific pathways of GC action, to enable development of novel therapeutics lacking the adverse side-effects of the presently used GCs. METHODS: Primary bovine retinal endothelial cells (BRECs) were grown on Transwell inserts and treated with hydrocortisone (HC), dexamethasone (Dex) or triamcinolone acetonide (TA). Molecular barrier integrity of the BRB was determined by mRNA and protein expression, and barrier function was assessed using permeability assays. In addition, we investigated whether TA was able to prevent barrier disruption after stimulation with VEGF or cytokines. RESULTS: Treatment of BRECs with GCs resulted in upregulation of tight junction mRNA (claudin-5, occludin, ZO-1) and protein (claudin-5 and ZO-1). In functional assays, only TA strengthened the barrier function by reducing endothelial permeability. Moreover, TA was able to prevent cytokine-induced permeability in human retinal endothelial cells and VEGF-induced expression of plasmalemma vesicle-associated protein (PLVAP), a key player in VEGF-induced retinal vascular leakage. CONCLUSION: Glucocorticoids have differential effects in an experimental in vitro BRB model. TA is the most potent in improving barrier function, both at the molecular and functional levels, and TA prevents VEGF-induced expression of PLVAP.

Spatial and temporal recruitment of the neurovascular unit during development of the mouse blood-retinal barrier.

The inner blood-retinal barrier (BRB) is made up by the neurovascular unit, consisting of endothelial cells, pericytes and glial cells. The BRB maintains homeostasis of the neural retina, but in pathological eye conditions the neurovascular unit is often disrupted, causing BRB loss. Here, we investigated in detail temporal and spatial recruitment of the neurovascular unit in the neonatal mouse retina from postnatal day (P)3 to P25 employing immunohistochemical staining of vascular endothelium (isolectin B4), pericytes (α-SMA and NG2) and astrocytes (GFAP). In addition, we investigated gene expression of polarized astrocytic end-feet markers aquaporin-4 and laminin α2 chain with qPCR. We observed GFAP-positive cells migrating ahead of the retinal vasculature during the first postnatal week, suggesting that the retinal vasculature follows an astrocytic meshwork. From P9 onwards, astrocytes acquired a mature phenotype, with a more stellate shape and increased expression of aquaporin-4. NG2-positive cells and tip cells co-localized at P5 and invaded the retina together as a vascular sprouting front. In summary, these data suggest that recruitment of the cell types of the neurovascular unit is a prerequisite for proper retinal vascularization and BRB formation.

TNFα-Induced Disruption of the Blood-Retinal Barrier In Vitro Is Regulated by Intracellular 3',5'-Cyclic Adenosine Monophosphate Levels.

Purpose: Proinflammatory cytokines such as tumor necrosis factor (TNFα) may have a causative role in blood-retinal barrier (BRB) disruption, which is an essential step in the development of diabetic macular edema. The purpose of our study was to determine whether TNFα increases permeability in an in vitro model of the BRB and to explore the mechanisms involved. Methods: Primary bovine retinal endothelial cells (BRECs) were grown on Transwell inserts and cells were stimulated with TNFα or a combination of TNFα, IL1ß, and VEGF. Molecular barrier integrity of the BRB was determined by gene and protein expression of BRB-specific components, and barrier function was assessed using permeability assays. Results: TNFα reduced the expression of tight and adherens junctions in BRECs. Permeability for a 376 Da molecular tracer was increased after TNFα stimulation, but not for larger tracers. We found that 3',5'-cyclic adenosine monophosphate (cAMP) stabilized the barrier properties of BRECs, and that TNFα significantly decreased intracellular cAMP levels. When BRECs were preincubated with a membrane-permeable cAMP analog, the effects of TNFα on claudin-5 expression and permeability were mitigated. The effects of TNFα on barrier function in BRECs were largely independent of the small Rho guanosine triphosphate (GTP)ases RhoA and Rac1, which is in contrast to TNFα effects on the nonbarrier endothelium. The combination of TNFα, IL1ß, and VEGF increased permeability for a 70 kDa-FITC tracer, also mediated by cAMP. Conclusions: TNFα alone, or in combination with IL1ß and VEGF, induces permeability of the BRB in vitro for differently sized molecular tracers mediated by cAMP, but independently of Rho/Rac signaling.

Is leukostasis a crucial step or epiphenomenon in the pathogenesis of diabetic retinopathy?

Leukostasis in the retinal microvasculature in animal model studies of diabetes is associated with the development of diabetes-like retinopathy. Therefore, it is generally assumed that adhesion of leukocytes is a central event inciting a chronic, low-grade form of inflammation that causes the vascular abnormalities that are specific for the early stages of diabetic retinopathy (DR), which culminate in diabetic macular edema, proliferative DR, and vision loss in humans. Here, we review the literature critically with respect to leukostasis and assess its pathologic consequences in the human diabetic retina. First, we review the pathologic processes that are known to be involved in the development of human DR. Then, we summarize experimental evidence for the role of leukostasis in the development of DR and the mechanisms involved in leukostasis in the retina. Based on our critical review, we conclude that leukostasis may be an epiphenomenon of the diabetic retinal milieu, rather than a crucial, specific step in the development of human DR.

Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Histatins are multifunctional histidine-rich peptides secreted by the salivary glands and exclusively present in the saliva of higher primates, where they play a fundamental role in the protection of the oral cavity. Our previously published results demonstrated that histatin-1 (Hst1) promotes cell-substrate adhesion in various cell types and hinted that it could also be involved in cell-cell adhesion, a process of fundamental importance to epithelial and endothelial barriers. Here we explore the effects of Hst1 on cellular barrier function. We show that Hst1 improved endothelial barrier integrity, decreased its permeability for large molecules, and prevented translocation of bacteria across epithelial cell layers. These effects are mediated by the adherens junction protein E-cadherin (E-cad) and by the tight junction protein zonula occludens 1, as Hst1 increases the levels of zonula occludens 1 and of active E-cad. Hst1 may also promote epithelial differentiation as Hst1 induced transcription of the epithelial cell differentiation marker apolipoprotein A-IV (a downstream E-cad target). In addition, Hst1 counteracted the effects of epithelial-mesenchymal transition inducers on the outgrowth of oral cancer cell spheroids, suggesting that Hst1 affects processes that are implicated in cancer progression.-Van Dijk, I. A., Ferrando, M. L., van der Wijk, A.-E., Hoebe, R. A., Nazmi, K., de Jonge, W. J., Krawczyk, P. M., Bolscher, J. G. M., Veerman, E. C. I., Stap, J. Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Plasmalemma Vesicle-Associated Protein Has a Key Role in Blood-Retinal Barrier Loss.

Loss of blood-retinal barrier (BRB) properties induced by vascular endothelial growth factor (VEGF) and other factors is an important cause of diabetic macular edema. Previously, we found that the presence of plasmalemma vesicle-associated protein (PLVAP) in retinal capillaries associates with loss of BRB properties and correlates with increased vascular permeability in diabetic macular edema. In this study, we investigated whether absence of PLVAP protects the BRB from VEGF-induced permeability. We used lentiviral-delivered shRNA or siRNA to inhibit PLVAP expression. The barrier properties of in vitro BRB models were assessed by measuring transendothelial electrical resistance, permeability of differently sized tracers, and the presence of endothelial junction complexes. The effect of VEGF on caveolae formation was studied in human retinal explants. BRB loss in vivo was studied in the mouse oxygen-induced retinopathy model. The inhibition of PLVAP expression resulted in decreased VEGF-induced BRB permeability of fluorescent tracers, both in vivo and in vitro. PLVAP inhibition attenuated transendothelial electrical resistance reduction induced by VEGF in BRB models in vitro and significantly increased transendothelial electrical resistance of the nonbarrier human umbilical vein endothelial cells. Furthermore, PLVAP knockdown prevented VEGF-induced caveolae formation in retinal explants but did not rescue VEGF-induced alterations in endothelial junction complexes. In conclusion, PLVAP is an essential cofactor in VEGF-induced BRB permeability and may become an interesting novel target for diabetic macular edema therapy.

Immunological Adaptations to Pregnancy in Women with Type 1 Diabetes.

Despite adequate glycemic control, pregnancy outcome of women with type 1 diabetes (T1D) is still unfavorable as compared to healthy women. In a rat-model of T1D under normoglycemic conditions, adverse pregnancy outcome was also observed, which was associated with aberrant immunological adaptations to pregnancy. Because similar processes may occur in women with T1D we studied the systemic immune response in non-pregnant and pregnant women with and without T1D. The systemic immune response was assessed by using flow cytometry to evaluate the number and activational status of subpopulations of lymphocytes, Natural Killer cells and monocytes in peripheral blood of non-pregnant and pregnant women with and without T1D. An increased white blood cell count, an increased Th1/Th2 ratio, increased Natural Killer cell expression of CD335 and enhanced activation of intermediate and non-classical monocytes was observed in pregnant women with T1D vs. healthy pregnant women. Also, the pregnancy outcome (i.e. incidence of preterm delivery and macrosomia) of women with T1D was unfavorable as compared to healthy women. This study showed that in T1D, the immunological adaptations to pregnancy are disturbed. In addition to hyperglycemia, these different immunological adaptations may be responsible for the greater frequency of complications in pregnant women with T1D.

Pregnancy causes diminished myogenic tone and outward hypotrophic remodeling of the cerebral vein of Galen.

Pregnancy increases the risk of several complications associated with the cerebral veins, including thrombosis and hemorrhage. In contrast to the cerebral arteries and arterioles, few studies have focused on the effect of pregnancy on the cerebral venous side. Here, we investigated for the first time the effect of pregnancy on the function and structure of the cerebral vein of Galen in rats. Our major finding was that cerebral veins from late-pregnant (LP, n=11) rats had larger lumen diameters and thinner walls than veins from nonpregnant (NP, n=13) rats, indicating that pregnancy caused outward hypotrophic remodeling of the vein of Galen. Moreover, veins from NP animals had a small amount of myogenic tone at 10 mm Hg (3.9±1.0%) that was diminished in veins during pregnancy (0.8±0.3%; P<0.01). However, endothelium-dependent and -independent vasodilation of the veins was unchanged during pregnancy. Using immunohistochemistry, we show that the vein of Galen receives perivascular innervation, and that serotonergic innervation of cerebral veins is significantly higher in veins from LP animals. Outward hypotrophic remodeling and diminished tone of cerebral veins during pregnancy may contribute to the development of venous pathology through elevated wall tension and wall stress, and possibly by promoting venous blood stasis.