Normal brain function is dependent on continuous and controlled oxygen delivery. Chronic moderate hypoxia leads to angiogenesis, suggesting a modulatory role for oxygen in determining capillary density. The objective of this study was to determine physiologic and brain angiogenic adaptational changes during chronic moderate normobaric hyperoxia in mice. Four-month old C56BL/6J mice were kept in a normobaric chamber at 50% O2 for up to 3 weeks. Normoxic littermates were kept in the same room outside the chamber. Freshly collected or fixed brain specimens were analyzed by RT-PCR, Western blot analysis and immunohistochemistry. Results show accumulation of hypoxia inducible factors 1 and 2α (HIF-1 and 2α), and increased expression of erythropoietin (EPO), cyclooxygenase-2 (COX-2) and angiopoietin-2 (Ang-2). Conversely, vascular endothelial growth factor (VEGF), and VEGF receptor-2 (KDR/Flk-1), Peroxisome proliferator-activated receptor gamma coactivator 1-α(PGC-1α) and prolylhydroxylase-2 (PHD-2) expressions were decreased. VEGF mRNA level was diminished but there was no change in HIF-1α mRNA and von Hippel Lindau E3 ubiquitin ligase (VHL) protein expression. Microvascular density was significantly diminished by the end of the 3rd week of hyperoxia. Overall, our results are: 1) increased expression of the potent neuroprotective molecule, EPO; 2) diminished expression of the potent angiogenic factor, VEGF; and 3) decreased microvascular density. We can, therefore, conclude that brain microvascular density can be controlled by HIF-independent mechanisms, and that brain capillary density is a continuously adjusted variable with tissue oxygen availability as one of the controlling modulators.
COX-2; Ang-2; PGC-1α; PHD-2; brain angioplasticity
Neuroprotective properties of ketosis may be related to the up-regulation of hypoxia inducible factor 1 (HIF-1α), a primary constituent associated with hypoxic angiogenesis and a regulator of neuroprotective responses. The rationale that the utilization of ketones by brain results in elevation of intracellular succinate, a known inhibitor of prolyl-hydroxylase (the enzyme responsible for the degradation of HIF-1α) was deemed as a potential mechanism of ketosis on the up-regulation of HIF-1α. The neuroprotective effect of diet-induced ketosis (3 weeks of feeding a ketogenic diet), as pretreatment, on infarct volume, following reversible middle cerebral artery occlusion (MCAO) and the up-regulation of HIF-1α was investigated. The effect of beta-hydroxybutyrate (BHB), as a pretreatment via intraventricular infusion (4 days of infusion prior to stroke) was also investigated following MCAO. HIF-1α and Bcl-2 (anti-apoptotic protein) protein levels, and succinate content were measured. A 55–70% reduction in infarct volume was observed with BHB infusion or diet-induced ketosis, respectively. HIF-1α and Bcl-2 protein levels increased 3-fold with diet-induced ketosis; BHB infusions resulted in increases in these proteins. As hypothesized, succinate content increased by 55% with diet-induced ketosis and 4-fold with BHB infusion. We conclude, the biochemical link between ketosis and the stabilization of HIF-1α is through the elevation of succinate, and both HIF-1α stabilization and Bcl-2 up-regulation play a role in ketone induced neuroprotection in brain.
There is a high incidence of cardiac arrest and poorer post-resuscitation outcome in the elderly population. Cardiac arrest and resuscitation results in ischemia/reperfusion injury associated with oxidative stress, leading to post-resuscitation mortality and delayed selective neuronal cell loss. In this study we investigated recovery following cardiac arrest and resuscitation in the aged rat brain. Male Fischer 344 rats (6, 12 and 24 months old) underwent 7 minute cardiac arrest before resuscitation. Overall survival and hippocampal neuronal counts were determined at 4 days of recovery. Brainstem function was assessed by hypoxic ventilatory response (HVR). Mitochondria of brainstem, cortex and hippocampus were isolated and assessed for respiratory function. Effect of an antioxidant, alpha-phenyl-tert-butyl-nitrone (PBN) was used as a treatment strategy against oxidative stress in the 6 and 24-month old rats. The time course of mitochondrial function was established using 3-month old Wistar rats with 12-minute cardiac arrest. In the 24-month old rats, overall survival rate, hippocampal CA1 neuronal counts, HVR, and brain mitochondrial respiratory control ratio were significantly reduced following cardiac arrest and resuscitation compared to the younger rats, and PBN treatment improved outcome. The data suggest that (i) there was increased susceptibility to ischemia/reperfusion in aged rat brain; (ii) HVR was decreased in the aged rats; (iii) brain mitochondrial respiratory function related to coupled oxidation was decreased following cardiac arrest and resuscitation in rats, more so in the aged; and (iv) treatment with an antioxidant, such as PBN, reduced the oxidative damage following cardiac arrest and resuscitation.
Reperfusion injury induced by cardiac arrest and resuscitation leads to secondary challenges to the brainstem. A 12-minute cardiac arrest results in about 50% survival rate in the resuscitated rats over a 4-day recovery period. We investigated hypoxic ventilatory response (HVR) to mild hypoxia by measuring the minute volume before and during a brief exposure to 10% oxygen before and following cardiac arrest and resuscitation. Our results indicate that after cardiac arrest and resuscitation the baseline spontaneous ventilation was elevated significantly in all rats due to both increased frequency and tidal volume; HVR in the non-survivor group was essentially absent while the brainstem responsiveness to hypoxia is fully maintained in the survivor group. Thus, the HVR was shown in this study to be a reliable indicator of survival vs. non-survival during early days of recovery following cardiac arrest and resuscitation.
Brainstem; transient global ischemia; post-resuscitation survival; reperfusion injury
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 three 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 F344 rats is significantly attenuated during acute hypoxic exposure. In the present study, we show that cortical HIF-1α accumulation and HIF-1 activation remains absent during chronic hypoxic exposure in the aged rat brain (24 month 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 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.
Aging; Angiogenesis; Brain; Hypoxia; HIF-1; Microvessels; PGC-1α; VEGF
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.
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.
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.
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.
Ischemic stroke; Δ(K2-K5) plasmin; Intracranial hemorrhage; Spontaneously hypertensive rat model; Recombinant tissue-type plasminogen activator (rt-PA); Middle cerebral artery occlusion (MCAo)
Chronic moderate hypoxia results in systemic and central nervous system adaptations that allow acclimatization. Long-term responses to hypoxia involve systemic physiological changes, metabolic regulation, and vascular remodeling. To investigate whether aging affects systemic and cerebral angiogenic adaptational changes in response to prolonged hypoxia, the present study assessed the responses of 4 month old (“young”) C57BL/6 mice and 24 month old (“aged”) C57BL/6 mice to chronic hypobaric hypoxia of 0.4 ATM (290 torr). Compared to young mice, delayed body weight-loss recovery and a lag in polycythemic response were observed in aged mice. As previously shown, Hypoxia Inducible Factor-1α (HIF-1α) accumulation was attenuated and vascular endothelial growth factor (VEGF) expression was decreased in the cerebral cortex of aged mice. Conversely, Cyclooxygenase-2 (COX-2), angiopoietin-2 (Ang-2) and Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) protein upregulation were not affected in the aged mice. Despite an initial delay in cerebral angiogenic response in aged mice in the first week of hypoxia, no significant differences were observed in microvascular density between young and aged mice in normoxia and at 2 and 3 weeks of hypoxia. Taken together, these observations indicate that even though the HIF-1 response to hypoxia is greatly attenuated, HIF-1 independent compensatory pathways are eventually able to maintain baseline and cerebral angiogenic adaptational changes to chronic hypoxia in aged mice. The delayed adaptive response, however, may result in decreased survival in the aged cohort.
HIF-1; VEGF; Ang-2; COX-2; brain capillary density
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).
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.
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.
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.
The five known Na-coupled HCO3− transporters (NCBTs) of the solute carrier 4 (SLC4) family play important roles in pH regulation and transepithelial HCO3− transport. Nearly all of the NCBTs have multiple splice variants. One particular NCBT, the electroneutral Na/HCO3− cotransporter NBCn2 (SLC4A10), which is predominantly expressed in brain, has three known splice variants—NBCn2-A, -B, and -C—as well as a potential variant -D. It is important to know the tissue-specific expression of the splice variants for understanding the physiological roles of NBCn2 in central nervous system. In the present study, we developed three novel rabbit polyclonal antibodies against NBCn2: (1) anti-ABCD, which recognizes all four variants; (2) anti-BD, which recognizes NBCn2-B and -D; (3) anti-CD, which recognizes NBCn2-C and -D. By western blotting, we examined the expression and distribution of NBCn2 splice variants in five brain regions: cerebral cortex, subcortex, cerebellum, hippocampus, and medulla. The expression pattern revealed with anti-ABCD is distinct from those revealed with anti-BD and anti-CD. Moreover, by using immunoprecipitation in combination with western blotting, we demonstrate that NBCn2-D does indeed exist and that it is predominantly expressed in subcortex, to a lesser extent in medulla, but at very low levels in cortex, cerebellum, and hippocampus. NBCn2-A may be the dominant variant in mouse brain as a whole, and may also dominate in cerebral cortex, cerebellum, and hippocampus. Immunohistochemistry with anti-ABCD shows that NBCn2 is highly expressed in choroid plexus, cortex, molecular layer of cerebellum, hippocampus, and some specific regions of the brainstem.
electroneutral bicarbonate transporter; immunohistochemistry; neuron; central nervous system
Cerebral hypoxia induces a profound angiogenic response in the central nervous system (CNS). Using a mouse model of chronic cerebral hypoxia, we previously demonstrated that angiogenic vessels in the hypoxic CNS show marked upregulation of the extracellular matrix (ECM) protein fibronectin, along with increased expression of its major receptor, α5β1integrin on brain endothelial cells (BEC). As cerebral hypoxia also leads to glial activation, the aim of the current study was to define the temporal relationship between BEC responses and glial cell activation in this model of cerebral hypoxia. This revealed that BEC fibronectin/α5β1 integrin expression and proliferation both reached maximal level after 4 days hypoxia. Interestingly, up to 4 days hypoxia, all dividing cells were BEC, but at later time-points proliferating astrocytes were also observed. GFAP staining revealed that hypoxia induced marked astrocyte activation that reached maximal level between 7–14 days hypoxia. As newly formed cerebral capillaries require ensheathment by astrocyte end-feet in order to acquire mature brain endothelium characteristics, we next examined how expression of astrocyte end-feet adhesion molecules is regulated by hypoxia. This showed that the astrocyte adhesion receptors α6β4 integrin and dystroglycan were both markedly upregulated, with a time-course that closely resembled astrocyte activation. Taken together, this evidence shows that cerebral hypoxia promotes first an endothelial response, in which fibronectin promotes BEC proliferation. This is then followed by an astrocyte response, involving astrocyte activation, proliferation and re-organization of astrocyte end-feet, which correlates with increased expression of astrocyte end-feet adhesion molecules.
Hypoxia; angiogenesis; endothelial; astrocyte; activation; integrin
Stem cells reside in specialized microenvironments or “niches” which regulate their function. In vitro studies employing hypoxic culture conditions (≤ 5% O2) have revealed strong regulatory links between O2 availability and stem/precursor cell functions1–6. Therefore, while some stem cells are perivascular, others may occupy hypoxic niches and be regulated by O2 gradients. However, the underlying mechanisms remain unclear. Here, we show that Hypoxia Inducible Factor-1α (HIF-1α), a principal mediator of hypoxic adaptations, modulates Wnt/β-catenin signalling in hypoxic embryonic stem (ES) cells by enhancing β-catenin activation and expression of downstream effectors LEF-1 and TCF-1. This regulation extends to primary cells, including isolated neural stem cells (NSCs), and is not observed in differentiated cells. In vivo, Wnt/β-catenin activity is closely associated with low O2 regions in the subgranular zone (SGZ) of the hippocampus, a key NSC niche7. Hif-1α deletion impairs hippocampal Wnt-dependent processes, including NSC proliferation, differentiation and neuronal maturation. This decline correlates with reduced Wnt/β-catenin signalling in the SGZ. Therefore, O2 availability may have a direct role in stem cell regulation via HIF-1α modulation of Wnt/β-catenin signalling.
Embryonic stem cells; hypoxic niche; HIF-1α/ARNT; Wnt/β-catenin signalling; neurogenesis
Aged rat brain is more sensitive to reperfusion injury induced by cardiac arrest and resuscitation. The mitochondrial respiratory chain, the major source of free radicals during reperfusion, is likely to be the target of lipid peroxidation. Previous work has shown a higher mortality and lower hippocampal neuronal survival in older rats. 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, was found to be elevated in cortex and brainstem after resuscitation. In this study we investigated the acute changes of mitochondrial function in aging rat brain following cardiac arrest and resuscitation; the effect of an antioxidant, alpha-phenyl-tert-butyl-nitrone (PBN) was also tested. Fischer 344 rats, 6 and 24-month old, were subjected to cardiac arrest (7-10 minutes) and allowed to recover 1 hour after resuscitation. Mitochondria of cortex and brainstem were isolated and assayed for respiratory function. Compared to their respective non-arrested control group, 1h untreated groups (both 6 month and 24 month) had similar state 3 (ADP-stimulated) but higher state 4 (resting state) respiratory rates. The respiratory control ratio (state 3/state 4) of cortex in the 1h untreated group was 26% lower than the non-arrested control group; similar results were found in brainstem. The decreased mitochondrial respiratory function was improved by PBN treatment. HNE–modified mitochondrial proteins were elevated 1h after resuscitation, with an evident change in the aged. Treatment with PBN reduced the elevated HNE production in mitochondria of cortex. The data suggest (i) there is increased sensitivity to lipid peroxidation with aging, (ii) mitochondrial respiratory function related to coupled oxidation decreases following cardiac arrest and resuscitation, and (iii) treatment with antioxidant, such as PBN, reduces the oxidative damage following cardiac arrest and resuscitation.
We measured age-dependent effects of the human ApoE4 on cerebral blood flow (CBF) using ApoE4 transgenic mice compared to age-matched wild-type (WT) mice by use of [14C] iodoantipyrene autoradiography. ApoE4 associated factors reduce CBF gradually to create brain hypoperfusion when compared to WT and the differences in CBF are greatest as animals age from 6-weeks to 12-months. Transmission electron microscopy with colloidal gold immunocytochemistry showed structural damage in young and aged microvessel endothelium of ApoE4 animals extended to the cytoplasm of perivascular cells, perivascular nerve terminals and hippocampal neurons and glial cells. These abnormalities coexist with mitochondrial structural alteration and mitochondrial DNA overproliferation and/or deletion in all brain cellular compartments. Spatial memory and temporal memory tests showed a trend in improving cognitive function in ApoE4 mice fed selective mitochondrial antioxidants acetyl-L-Carnitine and R-Lipoic acid. Our findings indicate that ApoE4 genotype-induced mitochondrial changes and associated structural damage may explain age-dependent pathology seen in AD, indicating potential for novel treatment strategies in the near future.
ApoE4; Alzheimer’s Disease; Cerebral Blood Flow; Electron Microscopy; Vascular and Mitochondrial Damage; Mitochondrial Antioxidant and Cognitive Performance
The brain is dependent on glucose as a primary energy substrate, but is capable of utilizing ketones such as β-hydroxybutyrate (βHB) and acetoacetate (AcAc), as occurs with fasting, prolonged starvation or chronic feeding of a high fat/low carbohydrate diet (ketogenic diet). In this study, the local cerebral metabolic rate of glucose consumption (CMRglu; μM/min/100g) was calculated in the cortex and cerebellum of control and ketotic rats using Patlak analysis. Rats were imaged on a rodent PET scanner and MRI was performed on a 7-Tesla Bruker scanner for registration with the PET images. Plasma glucose and βHB concentrations were measured and 90-minute dynamic PET scans were started simultaneously with bolus injection of 2-Deoxy-2[18F]Fluoro-D-Glucose (FDG). The blood radioactivity concentration was automatically sampled from the tail vein for 3 min following injection and manual periodic blood samples were taken. The calculated local CMRGlu decreased with increasing plasma BHB concentration in the cerebellum (CMRGlu = −4.07*[BHB] + 61.4, r² = 0.3) and in the frontal cortex (CMRGlu = −3.93*[BHB] + 42.7, r² = 0.5). These data indicate that, under conditions of ketosis, glucose consumption is decreased in the cortex and cerebellum by about 10% per each mM of plasma ketone bodies.
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-1α 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.
The extracellular matrix (ECM) is an important regulator of angiogenesis and vascular remodeling. We showed previously that angiogenic capillaries in the developing CNS express high levels of fibronectin and its receptor α5β1 integrin, and that this expression is developmentally downregulated. As cerebral hypoxia leads to an angiogenic response, we sought to determine whether angiogenic vessels in the adult CNS re-express fibronectin and the α5β1 integrin. Ten-week old mice were subject to hypobaric hypoxia for 0, 4, 7 and 14 days, and fibronectin/integrin expression examined. Fibronectin and the α5 integrin subunit were strongly upregulated on capillaries in the hypoxic CNS, with the effect maximal at the earliest time point examined (4 days). Immunofluorescent studies demonstrated that the α5 integrin was expressed by angiogenic endothelial cells. In light of the defined angiogenic role for fibronectin in other systems, this work suggests that induction of fibronectin-α5β1 integrin expression may be an important molecular switch driving angiogenesis in the hypoxic CNS.
CNS; Angiogenesis; Hypoxia; Capillary; Extracellular matrix (ECM); Fibronectin; α5β1 integrin; MECA-32
Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron- and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1α and HIF-2α. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1α and up-regulate HIF-dependent target genes (e.g. enolase, p21waf1/cip1, vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress.