Postnatal Exposure to Brief Hypoxia Alters Brain VEGF Expression and Capillary Density in Adult Mice.

Perinatal hypoxia leads to changes in cerebral angiogenesis and persistent structural and functional changes in the adult brain. It may also result in greater vulnerability to subsequent challenges. We investigated the effect of postnatal day 2 (P2) hypoxic preconditioning on adult brain capillary density and brain vascular endothelial growth factor (VEGF) expression in mice. P2 mice were exposed to hypoxia (5% O2) in a normobaric chamber for 2 h then returned to normoxia while their littermates remained in normoxia (P2 control). After 2-6 months, they were euthanised and their brains were removed for capillary density determination. Another set of animals (P2 hypoxic mice and P2 controls) were euthanised at 2, 10, 23, and 60 days after birth and brain VEGF expression was assessed by western blot. Adult brain capillary density was significantly increased in the P2 hypoxic mice when compared to the P2 control mice. Additionally, VEGF expression appeared to be elevated in the P2-hypoxia mice when compared to the P2-control mice at all time points, and VEGF levels in P2-hypoxia mice declined with age similarly to P2-control mice. These data demonstrate that transient early-postnatal hypoxic stress leads to an increase in capillary density that persists in the adult, possibly due to increased VEGF expression. These results might be explained by epigenetic factors in the VEGF gene.

Effects of Repeated Intravitreal Aflibercept Injection on the Corneal Endothelium in Patients With Age-Related Macular Degeneration: Outcomes From the RE-VIEW Study.

PURPOSE: The effects of repeated intravitreal aflibercept injection (IAI) on the corneal endothelium were studied in patients with unilateral neovascular age-related macular degeneration. METHODS: RE-VIEW was a phase 4, open-label, single-arm, multicenter study. Patients received IAI every 8 weeks after 3 monthly doses. Slit-lamp biomicroscopy was performed at all study visits. The central corneal endothelial health was evaluated by specular microscopy in the treated versus untreated fellow eyes at baseline and weeks 24 and 52. RESULTS: No slit-lamp abnormalities were noted in 154 enrolled patients (eyes). Baseline versus 52-week mean (±SD) endothelial morphometric values (n = 118) for the treated versus untreated fellow eyes were respectively as follows: endothelial cell density was 2410 ± 364 versus 2388 ± 384 cells/mm at baseline and remained unchanged at 2401 ± 353 versus 2376 ± 364 cells/mm at 52 weeks (P = 0.87); the coefficient of variation was 33.5 ± 4.4% versus 34.0 ± 5.0% at baseline and remained unchanged at 34.2 ± 4.7% versus 34.1 ± 4.9% at 52 weeks (P = 0.18); the percentage of hexagonal cells was 59.5 ± 5.8% versus 59.6 ± 6.4% at baseline and remained unchanged at 59.5 ± 6.0% versus 59.5 ± 5.8% at 52 weeks (P = 0.96). CONCLUSIONS: Repeated IAI for 52 weeks had no apparent corneal endothelial toxicity noted on specular microscopy in patients treated for neovascular age-related macular degeneration.

Environmental Enrichment Induces Increased Cerebral Capillary Density and Improved Cognitive Function in Mice.

Enrichment provides an environment that fosters increased physical activity and sensory stimulation as compared to standard housing. Promoting and sustaining stimulation increases neuronal activity and, consequently, brain oxygen demand. The mammalian brain modulates its microvascular network to accommodate tissue energy demand in a process referred to as angioplasticity. In this study we investigated the effect of an environmental enrichment on cerebral capillary density and cognitive performance in mice. Microvascular density (N/mm2) was determined by GLUT-1 immunohistochemistry in mice (3 months old) after 3 weeks of placement in a non-enriched or an enriched environment. The Y-maze test and a novel object recognition test were used to evaluate cognitive function in the aged mice (18 months old) after 4 weeks of environmental enrichment. Compared to the non-enriched control mice, the mice with environmental enrichment had significantly higher (~30%) capillary density in cortical brain. The enriched aged mice (n = 12) showed improved cognitive function, presented as a significantly higher alternation rate in the Y-Maze test compared to the non-enriched controls (n = 8). Our data suggest that environmental enrichment may result in increased brain capillary density and improved cognitive performance.

Gender differences in hypoxic acclimatization in cyclooxygenase-2-deficient mice.

The aim of this study was to determine the effect of cyclooxygenase-2 (COX-2) gene deletion on the adaptive responses during prolonged moderate hypobaric hypoxia. Wild-type (WT) and COX-2 knockout (KO) mice of both genders (3 months old) were exposed to hypobaric hypoxia (~0.4 ATM) or normoxia for 21 days and brain capillary densities were determined. Hematocrit was measured at different time intervals; brain hypoxia-inducible factor -1α (HIF-1α), angiopoietin 2 (Ang-2), brain erythropoietin (EPO), and kidney EPO were measured under normoxic and hypoxic conditions. There were no gender differences in hypoxic acclimatization in the WT mice and similar adaptive responses were observed in the female KO mice. However, the male KO mice exhibited progressive vulnerability to prolonged hypoxia. Compared to the WT and female KO mice, the male COX-2 KO mice had significantly lower survival rate and decreased erythropoietic and polycythemic responses, diminished cerebral angiogenesis, decreased brain accumulation of HIF-1α, and attenuated upregulation of VEGF, EPO, and Ang-2 during hypoxia. Our data suggest that there are physiologically important gender differences in hypoxic acclimatization in COX-2-deficient mice. The COX-2 signaling pathway appears to be required for acclimatization in oxygen-limiting environments only in males, whereas female COX-2-deficient mice may be able to access COX-2-independent mechanisms to achieve hypoxic acclimatization.

Hypoxia-induced angiogenesis and capillary density determination.

Chronic exposure to moderate hypoxia elicits structural and functional changes in the microvascular network of the mammalian CNS. Hypoxia-induced angiogenesis can be elicited and studied by a relatively simple experimental method. Rats or mice can be exposed to mild hypoxia in a hypobaric chamber, or alternatively in a normobaric hypoxia chamber. After 3 weeks, the animals are perfused and fixed, the brain removed, and paraffin embedded and sectioned at 5 µm. The sections are stained for the blood-brain barrier glucose transporter (GLUT-1) by immunohistochemistry, capillary profiles identified and counted as a measure of angiogenesis.

Defining the role of HIF and its downstream mediators in hypoxic-induced cerebral angiogenesis.

Now that some of the basic mechanisms that underlie hypoxia-induced cerebral angiogenesis have been described, it has become clear that the hypoxia-inducible transcription factors, HIF-1 and HIF-2, play an important role in the process by causing the upregulation of vascular endothelial growth factor (VEGF). The heterogeneity of the brain parenchyma means that further progress in understanding capillary pathophysiology requires techniques that allow determination of the roles of individual components of the neurovascular unit. Multi-stain fluorescence co-localization techniques provide one such approach.

Increased HIF-1α and HIF-2α accumulation, but decreased microvascular density, in chronic hyperoxia and hypercapnia in the mouse cerebral cortex.

The partial pressure of oxygen in the brain parenchyma is tightly controlled, and normal brain function is delicately sensitive to continuous and controlled oxygen delivery. The objective of this study was to determine brain angiogenic adaptive changes during chronic normobaric hyperoxia and hypercapnia in mice. Four-month-old C56BL/6 J mice were kept in a normobaric chamber at 50 % O2 and 2.5 % CO2 for up to 3 weeks. Normoxic littermates were kept adjacent to the chamber and maintained on the same schedule. Physiological variables were measured at time points throughout the 3 weeks or when the mice were sacrificed. Freshly collected or fixed brain specimens were analyzed by Western blot analysis and immunohistochemistry (IHC). We found significant accumulation of hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) and increased expression of erythropoietin (EPO), cyclooxygenase-2 (COX-2), and angiopoietin-2 (Ang-2) in hyperoxia and hypercapnia. Conversely, vascular endothelial growth factor (VEGF), and VEGF receptor-2 (KDR/Flk-1), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), and prolyl hydroxylase-2 (PHD-2) expressions were decreased in hyperoxia and hypercapnia. Capillary density was significantly diminished by the end of the 3rd week of hyperoxia and hypercapnia as compared to control. We conclude that HIF-independent mechanisms contribute to brain capillary density modulation that is continuously adjusted in accordance with tissue oxygen tension.

Early life hypoxic or hypoxic/hypercapnic stress alters acute ventilatory sensitivity in adult mice.

In this study we investigated the effect of early life conditioning (hypoxia ± hypercapnia) on adult acute ventilatory sensitivity to hypoxia and hypercapnia. Mice were exposed to either hypoxia (5% O(2)) or hypoxia/hypercapnia (5% O(2)/8% CO(2)) in a normobaric chamber for 2 h at postnatal day 2 (P2), and then returned to normoxia. At 3 months of age, hypoxic ventilatory response (HVR) and hypercapnic ventilatory response (HCVR) were measured using a plethysmograph system. Results showed that HVR was significantly decreased in the P2-hypoxia mice but not in the P2 hypoxia/hypercapnia mice as compared to the P2-normoxic mice, respectively. However, HCVR was significantly decreased in the P2 hypoxia-hypercapnia group but not in the P2-hypoxia group. These data suggest early postnatal hypoxic stress vs. hypoxic/hypercapnic stress plays different roles in fetal programming of the respiratory control system as shown by altered adult acute ventilatory sensitivity.

Safety evaluation of a recombinant plasmin derivative lacking kringles 2-5 and rt-PA in a rat model of transient ischemic stroke.

BACKGROUND: Tissue type plasminogen activator is the only approved thrombolytic agent for the treatment of ischemic stroke. However, it carries the disadvantage of a 10-fold increase in symptomatic and asymptomatic intracranial hemorrhage. A safer thrombolytic agent may improve patient prognosis and increase patient participation in thrombolytic treatment. A novel direct-acting thrombolytic agent, Δ(K2-K5) plasmin, promising an improved safety profile was examined for safety in the snare ligature model of stroke in the rat. METHODS: Male spontaneously hypertensive rats were subjected to 6 hours middle cerebral artery occlusion followed by 18 hours reflow. Beginning 1 minute before reflow, they were dosed with saline, vehicle, Δ(K2-K5) plasmin (0.15, 0.5, 1.5, and 5 mg/kg) or recombinant tissue-type plasminogen activator (10 and 30 mg/kg) by local intra-arterial infusion lasting 10 to 60 minutes. The rats were assessed for bleeding score, infarct volume, modified Bederson score and general behavioral score. In a parallel study, temporal progression of infarct volume was determined. In an in vitro study, whole blood clots from humans, canines and rats were exposed to Δ(K2-K5). Clot lysis was monitored by absorbance at 280 nm. RESULTS: The main focus of this study was intracranial hemorrhage safety. Δ(K2-K5) plasmin treatment at the highest dose caused no more intracranial hemorrhage than the lowest dose of recombinant tissue type plasminogen activator, but showed at least a 5-fold superior safety margin. Secondary results include: temporal infarct volume progression shows that the greatest expansion of infarct volume occurs within 2-3 hours of middle cerebral artery occlusion in the spontaneously hypertensive rat. A spike in infarct volume was observed at 6 hours ischemia with reflow. Δ(K2-K5) plasmin tended to reduce infarct volume and improve behavior compared to controls. In vitro data suggests that Δ(K2-K5) plasmin is equally effective at lysing clots from humans, canines and rats. CONCLUSIONS: The superior intracranial hemorrhage safety profile of the direct-acting thrombolytic Δ(K2-K5) plasmin compared with recombinant tissue type plasminogen activator makes this agent a good candidate for clinical evaluation in the treatment of acute ischemic stroke.

Intra-arterial administration of recombinant tissue-type plasminogen activator (rt-PA) causes more intracranial bleeding than does intravenous rt-PA in a transient rat middle cerebral artery occlusion model.

BACKGROUND: Intra-arterial (IA) administration of rt-PA for ischemic stroke has the potential for greater thrombolytic efficacy, especially for a large thrombus in the M1 or M2 segment of the middle cerebral artery (MCA). Intracranial hemorrhage (ICH) is a concern with IA or intravenous (IV) administration especially as the therapeutic window is extended. However, because IA administration delivers a higher local concentration of agent, the incidence and severity of ICH may be greater than with similar doses IV. We investigated the safety of rt-PA administration by IA compared to IV infusion following 6 hours of MCA occlusion (MCAo) with reflow in the spontaneously hypertensive rat (SHR). METHODS: Male SHRs were subjected to 6 hours MCAo with 18 hours reflow using a snare ligature model. They were treated with IA saline, IA rt-PA (1, 5, 10, 30 mg/kg), or IV rt-PA (10 and 30 mg/kg) by a 10 to 60 minute infusion beginning approximately 1 minute before reflow. The rats were recovered for 24 hours after MCAo onset at which time Bleeding Score, infarct volume, and Modified Bederson Score were measured. RESULTS: Greater hemorrhagic transformation occurred with 10 and 30 mg/kg rt-PA administered IA than IV. The IV 10 mg/kg rt-PA dosage induced significantly less bleeding than did the 1 or 5 mg/kg IA groups. No significant increase in infarct volume was observed after IA or IV treatment. Rats treated with 30 mg/kg rt-PA by either the IA or IV route had greater neurological dysfunction compared to all other groups. CONCLUSIONS: Administration of rt-PA by the IA route following 6 hours of MCAo results in greater ICH and worse functional recovery than comparable dosages IV. Significantly greater bleeding was observed when the IA dose was a tenth of the IV dose. The increased bleeding did not translate in larger infarct volumes.

Hypoxia-inducible factor-1 (HIF-1)-independent microvascular angiogenesis in the aged rat brain.

Angiogenesis is a critical component of mammalian brain adaptation to prolonged hypoxia. Hypoxia-induced angiogenesis is mediated by hypoxia-inducible factor-1 (HIF-1)-dependent transcriptional activation of growth factors, such as vascular endothelial growth factor (VEGF). Microvascular angiogenesis occurs over a 3-week period in the rodent brain. We have recently reported that HIF-1α accumulation and transcriptional activation of HIF target genes in the aged cortex of 24-month-old F344 rats is significantly attenuated during acute hypoxic exposure. In the present study, we show that cortical HIF-1α accumulation and HIF-1 activation remain absent during chronic hypoxic exposure in the aged rat brain (24-month-old F344). Despite this lack of HIF-1 activation, there is no significant difference in baseline or post-hypoxic brain capillary density counts between the young (3-month-old F344) and old age groups. VEGF mRNA and protein levels are significantly elevated in the aged cortex despite the lack of HIF-1 activation. Other HIF-independent mediators of hypoxia-inducible genes could be involved during chronic hypoxia in the aged brain. PPAR-γ coactivator (PGC)-1α, a known regulator of VEGF gene transcription, is elevated in the young and aged cortex during the chronic hypoxic exposure. Overall, our results suggest a compensatory HIF-1-independent preservation of hypoxic-induced microvascular angiogenesis in the aged rat brain.

Diet-induced ketosis improves cognitive performance in aged rats.

Aging is associated with increased susceptibility to hypoxic/ischemic insult and declines in behavioral function which may be due to attenuated adaptive/defense responses. We investigated if diet-induced ketosis would improve behavioral performance in the aged rats. Fischer 344 rats (3- and 22-month-old) were fed standard (STD) or ketogenic (KG) diet for 3 weeks and then exposed to hypobaric hypoxia. Cognitive function was measured using the T-maze and object recognition tests. Motor function was measured using the inclined-screen test. Results showed that KG diet significantly increased blood ketone levels in both young and old rats. In the aged rats, the KG diet improved cognitive performance under normoxic and hypoxic conditions; while motor performance remained unchanged. Capillary density and HIF-1alpha levels were elevated in the aged ketotic group independent of hypoxic challenge. These data suggest that diet-induced ketosis may be beneficial in the treatment of neurodegenerative conditions.