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The influence of oxygen free radicals on the permeability of the monolayer of cultured brain endothelial cells
Abstract
Free radicals have been implicated in the pathogenesis of vasogenic brain edema caused by ischemic or traumatic injury. It has been reported that in transgenic mice overexpressing the human CuZn-superoxide dismutase, brain edema is decreased in many cerebral disorders. To investigate the effects of free radicals on the permeability of the blood brain barrier, we established an in vitro model system of the blood-brain barrier using brain endothelial cells cultivated from transgenic mice and non-transgenic mice. The blood-brain barrier model is originated by a monolayer of brain endothelial cells cultured on a membrane which has 0.45-micron pores. Electrical resistance across the cell monolayer, which reflects the paracellular flux of ionic molecules, was measured. The blood-brain barrier models were incubated with menadione (vitamin K3, an intracellular O2- producing agent), and segmental changes in the electrical resistance across the monolayer were compared between the transgenic and the non-transgenic mice. Superoxide dismutase activity of the cultured brain endothelial cells was 1.7 times higher in the transgenic than in the non-transgenic mice (n = 3, P < 0.001). The electrical resistance was reduced by menadione in the transgenic but not in the non-transgenic mice (n = 7, P < 0.05) in the early stage. Moreover, desferroxamine mesylate (Fe2+ chelating agent) inhibited the menadione-induced early decrease in electrical resistance in the transgenic mice (n = 7, P < 0.05). These results suggest that the permeability of the blood-brain barrier may be affected by hydroxyl radicals and/or peroxynitrite rather than the O2- itself.
... The overproduction of free radicals overpasses the antioxidant capacity of the body and produces damage to the nervous tissue [72]. Oxidative stress is also linked in MS to the impairment of regulatory T cells [73] and myeloid-derived suppressor cells (MDSCs) [74] that interfere in the adaptive immune response and in the loss of the BBB [68,75]. ...
Neurodegenerative diseases are characterized by gradually progressive, selective loss of anatomically or physiologically related neuronal systems that produce brain damage from which there is no recovery. Despite the differences in clinical manifestations and neuronal vulnerability, the pathological processes appear to be similar, suggesting common neurodegenerative pathways. It is well known that oxidative stress and the production of reactive oxygen radicals plays a key role in neuronal cell damage. It has been proposed that this stress, among other mechanisms, could contribute to neuronal degeneration and might be one of the factors triggering the development of these pathologies. Another common feature in most neurodegenerative diseases is neuron hyperexcitability, an aberrant electrical activity. This review, focusing mainly on primary motor cortex pyramidal neurons, critically evaluates the idea that oxidative stress and inflammation may be involved in neurodegeneration via their capacity to increase membrane excitability.
... Reactive oxygen species (ROS) weaken the blood-brain barrier [15] to facilitate CNS permeation by inflammatory cells. In the context of MS, DMF has principally been regarded as an activator of the NRF-2 pathway. ...
Dimethyl fumarate (DMF) has emerged as a first-line treatment for the relapsing-remitting multiple sclerosis (RRMS) subtype. It is hypothesized that DMF has anti-inflammatory and antioxidant effects although mechanisms are not fully understood. This study used RNA-seq to profile gene expression responses to DMF in cultured astrocytes. Responses were compared with those of isosorbide di-(methyl fumarate) (IDMF), a newly designed fumarate that may partially replicate DMF activity with fewer adverse effects. Both compounds altered the expression of MS-associated genes, including those near MS susceptibility loci and genes dysregulated in MS patient astrocytes. The shared DMF/IDMF transcriptome response involved altered expression of antioxidant genes (e.g., HMOX1) and genes linked to extracellular matrix integrity (TIMP3, MMP9) and migration of pro-inflammatory cells into CNS (CCL2). IDMF-specific transcriptome responses included down-regulation of mitotic genes associated with a proliferative reactive astrocyte phenotype (ICAM1) and repression of genes encoding NF-kappaB subunits (NFKB2, RELA, RELB) and NF-kappaB targets (NCAPG, CXCL1, OAS3). Overall, these results identify astrocyte-centered mechanisms that may contribute to the established efficacy of DMF as an RRMS treatment. Furthermore, our findings support a rationale for further studies of IDMF as a novel fumarate, which may have unique suppressive effects on astrocyte reactivity and glial scar formation. [200 words].
... Iron overload mediates endothelial cell damage and BBB opening after transient forebrain ischemia. This effect is attenuated by DFX [7,24]. Our study demonstrated that ferritin was upregulated after SAH in the cortex and was colocalized with endothelial cells, pericytes and astrocytes, which composing of BBB. ...
Blood brain barrier (BBB) disruption is a key mechanism of subarachnoid hemorrhage (SAH)-induced brain injury. This study examined the mechanism of iron-induced BBB disruption after SAH and investigated the potential therapeutic effect of iron chelation on SAH. Male adult Sprague-Dawley rats had an endovascular perforation of left internal carotid artery bifurcation or sham operation. The rats were treated with deferoxamine (DFX) or vehicle (100mg/kg) for a maximum of 7 days. Brain edema, BBB leakage, behavioral and cognitive impairment were examined. In SAH rat, the peak time of brain edema and BBB impairment in the cortex was at day 3 after SAH. SAH resulted in a significant increase in ferritin expression in the cortex. The ferritin positive cells were colocalized with endothelial cells, pericytes, astrocytes, microglia and neurons. Compared with vehicle, DFX caused less ferritin upregulation, brain water content, BBB impairment, behavioral and cognitive deficits in SAH rats. The results suggest iron overload could be a therapeutic target for SAH induced BBB damage.
... Endothelium cells, which are found at the interface between the CNS and periphery, are regulated in a MS-relevant manner by ROS. High ROS levels damage brain endothelium and affect blood-brain barrier (BBB) permeability (Imaizumi et al., 1996;Olesen, 1987). Thus, regulative effects of oxidative stress in MS probably include but are not limited to the CNS parenchyma. ...
Accumulating evidence suggests that oxidative stress plays a major role in the pathogenesis of multiple sclerosis (MS). Reactive oxygen species (ROS), which if produced in excess lead to oxidative stress, have been implicated as mediators of demyelination and axonal damage in both MS and its animal models. One of the most studied cell populations in the context of ROS-mediated tissue damage in MS are macrophages and their CNS companion, microglia cells. However, and this aspect is less well appreciated, the extracellular and intracellular redox milieu is integral to many processes underlying T cell activation, proliferation and apoptosis. In this review article we discuss how oxidative stress affects central as well as peripheral aspects of MS and how manipulation of ROS pathways can potentially affect the course of the disease. It is our strong belief that the well-directed shaping of ROS pathways has the potential to ameliorate disease progression in MS.
... Since the TEER value varies depending on type and source of cells, we evaluated TEER of our 1L-EC with other studies. TEER of 5L-AWMs was 17 Ωcm 2 , which was comparable to, but slightly lower than that of 1L-EC in this study and other studies [11][12][13][14]. This is probably because of the EC layer was cultured on layers of SMCs, unlike the 1L-EC which was grown on a polyester culture membrane and formed a compact monolayer. ...
Monolayers of endothelial cells (1L-ECs) have been generally used as in vitro vascular wall models to study the vascular mechanisms and transport of substances. However, these two-dimensional (2D-) system cannot represent the properties of native vascular wall which has 3D-structure and composed of not only ECs, but also smooth muscle cells (SMCs) and other surround tissues. Here in, five-layered (5L) 3D-arterial wall models (5L-AWMs) composed of EC monolayer and 4-layered SMCs were constructed by hierarchical cell manipulation. We applied the 5L-AWMs to evaluate their barrier function and permeability to nano-materials in order to analyze drug, or drug nanocarrier permeability to the blood vessel in vitro. Barrier property of the 3D-AWMs was confirm by Zonula occludens (ZO-1) staining and their transendothelial electrical resistance (TEER), which was comparable to 1L-ECs, while the SMCs showed close to zero. The effect of substance size to permeability across the 5L-AWMs was clearly observed from dextrans with various molecular weights, which agreed well with phenomena of the in vivo blood vessels. Importantly, transport of nano-materials could be observed across the depth of 5L-AWMs, suggesting the advantage of 3D-AWMs over general 2D-systems. By using this system, we evaluate the transport of 35 nm phenylalanine-modified poly(γ-Glutamic Acid) nanoparticles (γ-PGA-Phe NPs) as a candidate of biodegradable drug carrier. Interestingly, despite of having comparable size to dextran-2000k (28 nm), the γ-PGA-Phe NPs distinctly showed approximately 20 times faster transport across the 5L-AWMs, suggesting the effect of substance intrinsic properties to the transport. This in vitro evaluation system using the 3D-AWMs is therefore useful for the design and development of nano-drug carriers for treatment of vascular diseases, such as atherosclerosis.
Copyright © 2014. Published by Elsevier Inc.
... Overexpression of SOD1 protects striatal tissue from 3-NP-induced damage, whereas low levels of SOD1 activity exacerbate edema levels, suggesting that ROS production is an important component in this injury paradigm. There have also been reports that oxidative stress promotes BBB disruption in vivo (Chan et al., 1984(Chan et al., , 1991Kondo et al., 1997;Gasche et al., 2001;Aoki et al., 2002;Grzeschik et al., 2003) and in vitro ( Imaizumi et al., 1996;Schroeter et al., 1999;Mujumdar et al., 2001;Zaragoza et al., 2002). The present data show that oxidized HEt signals, which indicate ROS production, are spatially correlated with MMP-9 cellular expression 4 hr after 3-NP treatment (Fig. 5). ...
... Oxidative stress damages endothelial cells of the BBB and contributes to vasogenic edema Chan, 2001). Incubation of endothelial cells with ROS-generating systems increases the permeability of endothelial monolayers (Imaizumi et al., 1996;Lagrange et al., 1999;Fischer et al., 2005). The superoxide radical (O 2 -• ) has been identified as the primary ROS involved in increased vascular permeability and edema formation in global and focal cerebral ischemia, cold brain injury, and brain tumors (Heo et al., 2005). ...
In the brain, the transport of water and solute is precisely regulated. This maintains a stable and unique microenvironment
that is critical to brain function. Cerebral edema results from the excess of fluid in the brain’s intra- and extracellular
spaces. This occurs in response to a wide variety of insults, including cerebral ischemia, hypoxia, infection, brain tumors,
and neuroinflammation. Cytotoxic edema leads to intracellular swelling without alterations in vascular permeability. Vasogenic
edema is associated with damage to the blood–brain barrier. These types of edema rarely exist in isolation. In most neuropathological
conditions, one type of edema predominates, but both coexist. This chapter focuses on the major molecular mechanisms triggering
brain edema, including alterations in ion channels and transporters, matrix metalloproteinases, tight junction protein degradation,
free radicals, and products of the arachidonic acid metabolism. We review present knowledge of the contribution to brain edema
of molecules such as aquaporins, vasopressin, vascular endothelial growth factor, angiopoietins, and bradykinin. We further
examine brain imaging modalities that have revolutionized clinical diagnosis of cerebral edema. Finally, we provide a critical
evaluation of the current strategies for the treatment of brain edema.
KeywordsVasogenic edema-Neurovascular unit-Matrix metalloproteinases-Aquaporins-Blood–brain barrier-Imaging
... Abbreviations: WKY, Wistar Kyoto normotensive rat strain; SHR-SP, spontaneously hypertensive stroke-prone rat strain; 40, 40-week-old animals; 60-plac, 60-week-old animals fed by placebo diet for 20 weeks; 60-nimo, 60-week-old animals fed by nimodipine containing diet for 20 weeks; 60-nife, 60-week-old animals fed by nifedipine containing diet for 20 weeks. creased endothelial permeability (Wei et al., 1986;Imaizumi et al., 1996;Lagrange et al., 1999). The microvascular pathology in SHR-SP animals presented here can be associated with such a chain of events. ...
The aging of the central nervous system and the development of incapacitating neurological diseases like Alzheimer's disease (AD) are generally associated with a wide range of histological and pathophysiological changes eventually leading to a compromised cognitive status. Although the diverse triggers of the neurodegenerative processes and their interactions are still the topic of extensive debate, the possible contribution of cerebrovascular deficiencies has been vigorously promoted in recent years. Various forms of cerebrovascular insufficiency such as reduced blood supply to the brain or disrupted microvascular integrity in cortical regions may occupy an initiating or intermediate position in the chain of events ending with cognitive failure. When, for example, vasoconstriction takes over a dominating role in the cerebral vessels, the perfusion rate of the brain can considerably decrease causing directly or through structural vascular damage a drop in cerebral glucose utilization. Consequently, cerebral metabolism can suffer a setback leading to neuronal damage and a concomitant suboptimal cognitive capacity. The present review focuses on the microvascular aspects of neurodegenerative processes in aging and AD with special attention to cerebral blood flow, neural metabolic changes and the abnormalities in microvascular ultrastructure. In this context, a few of the specific triggers leading to the prominent cerebrovascular pathology, as well as the potential neurological outcome of the compromised cerebral microvascular system are also going to be touched upon to a certain extent, without aiming at total comprehensiveness. Finally, a set of animal models are going to be presented that are frequently used to uncover the functional relationship between cerebrovascular factors and the damage to neural networks.
... In the chronic phase of the disease, non-inflammatory mechanisms such as mitochondrial dysfunction support the formation of ROS and lead to oligodendrocyte damage and axonal degeneration [135]. However, high levels of free radicals can also damage brain endothelium and affect BBB permeability [136,137]. ROS can affect the CNS endothelium by disrupting the junctional proteins. In this regard, ONOO À is known to decrease the expression of claudin-5 [138], and H 2 O 2 induces aberrant expression of occludin and ZO1 associated with increase in BBB permeability [139]. ...
The presence of the blood-brain barrier (BBB) restricts the movement of soluble mediators and leukocytes from the periphery to the central nervous system (CNS). Leukocyte entry into the CNS is nonetheless an early event in multiple sclerosis (MS), an inflammatory disorder of the CNS. Whether BBB dysfunction precedes immune cell infiltration or is the consequence of perivascular leukocyte accumulation remains enigmatic, but leukocyte migration modifies BBB permeability. Immune cells of MS subjects express inflammatory cytokines, reactive oxygen species (ROS) and enzymes that can facilitate their migration to the CNS by influencing BBB function, either directly or indirectly. In this review, we describe how immune cells from the peripheral blood overcome the BBB and promote CNS inflammation in MS through BBB disruption.
... Stickstoffmonoxid und Arachidonsäuremetaboliten (Imaizumi et al., 1996;Yasuma et al., 1997;Schilling und Wahl, 1999;Unterberg et al., 1997). ...
Die konventionelle Therapie von malignen Gliomen (operative Entfernung, Bestrahlung, Chemotherapie) erzielt keine nachhaltigen Erfolge. Die Prognose von Patienten mit malignen Gliomen richtet sich nach wie vor nach der histologischen Wertigkeit des Tumors und dem Karnowski-Index des Patienten. Um die Ergebnisse der Therapie von malignen Gliomen zu verbessern, werden zusätzliche therapeutische Möglichkeiten benötigt. In früheren klinischen Studien konnte gezeigt werden, dass sich unter der Therapie mit H15® die perifokalen Ödeme bei Patienten mit malignen Gliomen signifikant zurückbilden und sich zudem ihre klinischen Symptome verbessern. Es war darüber hinaus eine Abnahme der Ausscheidung von LTE4, einem Metaboliten der Leukotriensynthese in Hirntumoren, zu beobachten. Zudem wurde in Zellkulturen eine proliferationshemmende Aktivität von H15® festgestellt. Mit der vorliegenden tierexperimentellen Studie wurden Einflüsse von H15® auf das Tumorwachstum untersucht. 40 weiblichen Wistar-Ratten mit einem Gewicht von 220g bis 250g wurden C6-Gliomzellen in den rechten Nucleus caudatus injiziert. Die Tiere wurden randomisiert und in vier Gruppen zu jeweils 10 Tieren unterteilt. Drei dieser Gruppen wurden mit verschiedenen Dosen von H15® behandelt; die vierte Gruppe diente als Kontrollgruppe. Nach 14 tägiger Verabreichung von H15® (3mal täglich) vom Tag nach der Tumorzellimplantation an wurden die Gehirne entnommen und mikroskopisch untersucht. Es wurde eine signifikante Stabilisierung des Vitalitätsindex in der Behandlungsgruppe mit der höchsten Medikamentendosis im Vergleich zur Kontrollgruppe aufgezeigt. Abhängig von der Dosierung wurde weiterhin ein signifikanter Unterschied im Tumorvolumen festgestellt. Der Anteil induzierter, apoptotischer Zellen war in Tieren, die mit hohen Dosen behandelt wurden, signifikant größer, als bei Tieren mit niedriger Dosierung. Bei den Tieren wurden keine bzw. geringe Nebenwirkungen auf das verabreichte Medikament beobachtet. Diese Daten belegen einen hemmenden Einfluss von H15® auf das Wachstum von C6-Gliomzellen in Rattenhirnen. The conventional malignant glioma therapy (surgery, radiation therapy and chemotherapy) does not yield satisfying results. The prognosis of the glioma patient depends more on the histological grading of the tumor and the patients age than on the therapy. Especially the adjuvant chemotherapy does not influence survival time in glioma patients significantly. To improve results in malignant glioma therapy additional therapeutic regimes are necessary. An earlier study showed a significant reduction of perifocal edema in patients with malignant glioma under therapy with H15®, accompanied with a clinical improvement. Also a decrease of urinary LTE4-excretion as a metabolite of leukotriene synthesis in brain tumors was observed. Furthermore a proliferation inhibiting activity of H15® was found in cell cultures. The purpose of this experimental study was to examine the effects of the boswellic acids, which are constituents of an extract from gum resin, on tumor growth in vivo. Fourty female wistar rats weighing 220250 g were inoculated C6 tumor cells into the right caudate nucleus and randomization into 4 groups each with 10 rats. The treatment groups received different dosages and were compared to a control group without any additional treatment. Drug treatment (3 times per day) was started immediately after implantation and stopped after 14 days. The animals were sacrificed and the brains were examined microscopically. Comparing low and high dosage of H15® treatment a significant difference in tumor volume was detected. The vitality index in animals with high dose treatment was significantly better than in the low dose treatment group. The proportion of apoptotic tumor cells in animals with high dose treatment was significantly larger than in the low dose treatment group. Only milde side effects were observed by the animal during the therapy with H15®. These data demonstrate an antiproliferative influence of H15® in rat glioma growth.
... Ein weiterer Kandidat der Mediatoren des Hirnödems ist VEGF, das nicht nur die Angiogenese beeinflusst, sondern zudem die Permeabilität des Kapillarendothels steigert (Bitzer et al., 1998). Eine zusätzliche Rolle spielen wahrscheinlich freie Radikale, einschließlich NO (Imaizumi et al., 1996; Yasuma et al., 1997; Schilling und Wahl, 1999). Auch der Arachidonsäure wurde eine permeabilitätssteigernde Wirkung zugeschrieben (Unterberg et al., 1987). ...
In der experimentellen Arbeit konnten Methoden zur Untersuchung von pharmakokinetischen Eigenschaften von 11-keto-ß-Boswelliasäure (KBA) und Acetyl-11-keto-beta-Boswelliasäure (AKBA) entwickelt werden. Durch Blutspiegelmessungen bei Probanden und Patienten sowie auch im Tierversuch konnte die orale Bioverfügbarkeit von KBA nachgewiesen werden. Die Detektion von AKBA im menschlichen Blut nach Einnahme von H15-Tabletten zeigte bei der eingesetzten Dosierung in den meisten Fällen nur kleinste Spuren. Im Tierexperiment wurde eine orale sowie intravenöse Bioverfügbarkeit von AKBA dokumentiert.
Im Tierversuch konnte die Passage der Blut-Hirn-Schranke durch beide Boswelliasäuren gezeigt werden, indem Gewebespiegel von KBA und AKBA im Hirn detektiert wurden. Dies ist in Hinblick auf eine mögliche antiproliferative Wirksamkeit bei malignen Tumorerkrankungen des zentralen Nervensystems von Bedeutung.
Anhand von Blutuntersuchungen bei freiwilligen Probanden konnte gezeigt werden, dass eine Erhebung pharmakokinetischer Parametern mit den vorgestellten Methoden möglich ist. Die Bestimmung von Bioverfügbarkeit, Halbwertszeit, Verteilungsvolumen und Clearance erfordert die Durchführung von weitergehenden, umfangreichen Versuchsreihen. Ausführliche pharmakokinetische und auch toxikologische Untersuchungen würden eine wichtige Grundlage für die Zulassung von Boswelliasäuren als Arzneimittel darstellen. This study developed approaches for the pharmacokinetic analysis of 11-keto-ß-boswellic acid (KBA) and Acetyl-11-keto-beta-boswellic acid (AKBA). An HPLC assay for the determination of the boswellic acids in blood was evaluated. KBA was quantitated in human blood of patients and healthy subjects after administration of a commercial Boswellia extract (H15). Only very small amounts of AKBA could be detected in humans. An animal trial demonstrated an oral and intravenous bioaviability of both KBA and AKBA.
The animal model showed concentrations of KBA and AKBA after administration of both boswellic acids in brain tissue. This finding is relevant to a possible therapeutical use of boswellic acids in malignant gliomas.
The developed analytical procedures were shown to be suitable for pharmacokinetic analyses of boswellic acids. Pharmacokinetic and toxicologic studies represent a prerequisite for the registration of boswellic acids as medicaments.
... Although excess NO may directly alter the BBB (Heo et al. 2005), a common mechanism that relates NO to BBB disruption involves oxidative/nitrosative stress. Indeed, peroxynitrite, the reaction product of NO and superoxide, was observed in free radical mediated-endothelial permeability changes in an in vitro study using endothelial cells obtained from CuZn-SOD-transgenic mice (Imaizumi et al. 1996) and in relation to BBB damage in animal models of stroke (Han et al. 2006), excitotoxicity (Parathath et al. 2006) and CNS inflammation (Phares et al. 2007). Oxidative/nitrosative stress has previ- ously been reported in brain in TD (Langlais et al. 1997;Calingasan et al. 1998;Kruse et al. 2004). ...
Wernicke's encephalopathy is a cerebral disorder caused by thiamine (vitamin B(1)) deficiency (TD). Neuropathologic consequences of TD include region-selective neuronal cell loss and blood-brain barrier (BBB) breakdown. Early increased expression of the endothelial isoform of nitric oxide synthase (eNOS) occurs selectively in vulnerable brain regions in TD. We hypothesize that region-selective eNOS induction in TD leads to altered expression of tight junction proteins and BBB breakdown. In order to address this issue, TD was induced in C57BL/6 wild-type (WT) and eNOS(-/-) mice by feeding a thiamine-deficient diet and treatment with the thiamine antagonist pyrithiamine. Pair-fed control mice were fed the same diet with additional thiamine. In medial thalamus of TD-WT mice (vulnerable area), increased heme oxygenase-1 and S-nitrosocysteine immunostaining was observed in vessel walls, compared to pair-fed control-WT mice. Concomitant increases in IgG extravasation, decreases in expression of the tight junction proteins occludin, zona occludens-1 and zona occludens-2, and up-regulation of matrix metalloproteinase-9 in endothelial cells were observed in the medial thalamus of TD-WT mice. eNOS gene deletion restored these BBB alterations, suggesting that eNOS-derived nitric oxide is a major factor leading to cerebrovascular alterations in TD. However, eNOS gene deletion only partially attenuated TD-related neuronal cell loss, suggesting the presence of mechanisms additional to BBB disruption in the pathogenesis of these changes.
... We did not measure superoxide dismutase activity in the present study, but this etiologic scenario remains plausible. Another possible mechanism for the involvement of superoxide radical is through the interaction with nitric oxide whereby the resultant highly reactive hydroxyl and peroxynitrite radicals alter the endothelial permeability (34,35). Elevated levels of both superoxide and nitric acid might affect capillary endothelial cell tight junction integrity (36,37), and other indirect mechanisms for BBB injury by oxidative stress may be involved. ...
Our previous studies implicated vitamin E deficiency as a risk factor for equine motor neuron disease, a possible model of human amyotrophic lateral sclerosis, and showed direct effects of this deficiency on brain vascular endothelium. To gain better understanding of the pathogenesis of equine motor neuron disease, we determined the effects of dietary antioxidant insufficiency and the resultant brain tissue oxidative stress on blood-brain barrier permeability. Rats (n = 40) were maintained on a diet deficient of vitamin E for 36 to 43 weeks; 40 controls were fed a normal diet. Permeability of the blood-brain barrier in the cerebral cortex was investigated using rhodamine B, and lipid peroxidation was measured as a marker for oxidative stress. Animals on the vitamin E-deficient diet showed less weight gain and had higher brain lipid peroxidation compared with the controls. Fluorometric studies demonstrated greater rhodamine B in the perivascular compartment and central nervous system parenchyma in rats on the deficient diet compared with controls. These results suggest that a deficiency in vitamin E increases brain tissue oxidative stress and impairs the integrity of the blood-brain barrier. These observations may have relevance to the pathogenesis of amyotrophic lateral sclerosis and other neurologic diseases.
... Our data are consistent with previous reports of increased peroxynitrite formation in pulmonary vessels of patients with acute inflammatory diseases and in atherosclerotic coronary arteries (3,26,31). Expression of these molecules by vascular endothelial and smooth muscle cells and by inflammatory cells may contribute to increased vascular permeability, mucus secretion, inflammatory cell recruitment, and reduction of eNOS in asthma (14,31,32,43). ...
Peroxynitrite is a potent oxidant formed by the rapid reaction of the free radicals nitric oxide (NO) and superoxide. It causes airway hyperresponsiveness and airway epithelial damage, enhances inflammatory cell recruitment, and inhibits pulmonary surfactant. Asthma is characterized by increased airway hyperresponsiveness, airway epithelial shedding, and inflammation. We examined the production of peroxynitrite and the expression of inducible nitric oxide synthase (iNOS) in airways of asthmatic patients compared to normal control subjects. We also performed a double-blind, crossover randomized-order, placebo-controlled study on 10 asthmatic patients to study the effects of inhaled glucocorticoid treatment (Budesonide) on the formation of peroxynitrite and NO. Fiberoptic bronchial biopsies were examined by immunohistochemistry with antiserum to nitrotyrosine, a marker of protein nitration by peroxynitrite. We also examined the expression of iNOS by immunohistochemistry and in situ hybridization, and measured exhaled NO by chemiluminescence. We correlated the airway production of peroxynitrite with pulmonary functions and airway responsiveness. In airway passages of control subjects, there was weak or no nitrotyrosine immunoreactivity. In contrast, there was strong immunoreactivity for nitrotyrosine in the airway epithelium and inflammatory cells in the airways of persons with asthma. Budesonide treatment resulted in a significant reduction in nitrotyrosine immunoreactivity. Expression of iNOS was evident in the airway pithelium of controls and asthmatic patients, but was significantly more abundant in asthmatic patients. The presence of nitrotyrosine in the airway epithelium (r=-0.841, P<0.0001; r=-0.771, P=0.0004) and inflammatory cells (r=-0.727, P=0014; r=-0.681, P=0.004) correlated inversely with methacholine PC20 and forced expiratory volume in 1 s, respectively. Asthma is associated with increased peroxynitrite formation in the airways, which is reduced after Budesonide treatment. The potent oxidant peroxynitrite may contribute to airway obstruction and hyperresponsiveness and epithelial damage in asthma.
... Furthermore, the elevated calcium concentration is known to activate detrimental metabolic cascades and is accompanied by free radical generation [47]. The calciumgenerated synthesis and accumulation of the easily translocating NO and its toxic products [2,34] can attack the integrity of the BBB: it may cause astrocytic dysfunction [43] and increased endothelial permeability [24,31,52]. The here presented microvascular pathology in SHR-SP animals can be associated with such a chain of events. ...
Chronic hypertension during aging is a serious threat to the cerebral vasculature. The larger brain arteries can react to hypertension with an abnormal wall thickening, a loss of elasticity and a narrowed lumen. However, little is known about the hypertension-induced alterations of cerebral capillaries. The present study describes ultrastructural alterations of the cerebrocortical capillary wall, such as thickening and collagen accumulation in the basement membrane of aging spontaneously hypertensive stroke-prone rats. The ratio of cortical capillaries with such vascular pathology occurred significantly more frequently in hypertensive animals. Nimodipine and nifedipine are potential drugs to decrease blood pressure in hypertension but their beneficial effects in experimental studies reach beyond the control of blood pressure. Nimodipine and nifedipine can alleviate ischemia-related symptoms and improve cognition. These drugs differ in that nifedipine, but not nimodipine reduces blood pressure at the here-used concentration while both drugs can penetrate the blood-brain barrier. Here we show that chronic treatment of aging hypertensive stroke-prone rats with nimodipine or nifedipine could preserve microvascular integrity in the cerebral cortex.
... Free radical generation plays a role in experimental BBB opening. Schilling et al. (Schilling and Wahl, 1997;Schilling and Wahl, 1999) point out that application of a free-radicalgenerating compound has BBB-opening effects in vitro (Imaizumi et al., 1996) and in vivo (Olesen and Crone, 1986). Moreover, intraparenchymal infusion of a mixture containing hypoxanthine and xanthine oxidase to generate oxygen-derived free radicals induced cerebral edema in rats (Chan et al., 1984). ...
Traditionally, scientists and clinicians have explored peripheral physiological responses to acute hypoxia to explain the pathophysiological processes that lead to acute mountain sickness (AMS) and high-altitude cerebral edema (HACE). After more than 100 years of investigation, little is yet known about the fundamental causes of the headache and nausea that are the main symptoms of AMS. Thus, we review the evidence supporting a change in focus to the role of the central nervous system in AMS. Our justification is (i) that the symptoms of AMS and HACE are largely neurological, (ii) that HACE is considered to be the end-stage of severe AMS and was recently identified as a vasogenic edema, opening the door for a role for blood-brain barrier permeability in AMS, (iii) that new, non-invasive techniques make measurement of brain water levels and cerebral blood volume possible and (iv) that the available experimental evidence and theoretical arguments support a significant role for brain swelling in the pathophysiology of AMS. We believe that an examination of the responses of the central nervous system to acute hypoxia will reveal important new pathophysiological processes that may help explain AMS and HACE.
... Traumatic brain injury causes an early increase of the expression of all three isoforms of nitric oxide synthase (Gahm et al., 2000). The combination of NO and superoxide produces peroxynitrite (Beckman et al., 1996), a powerful oxidant that is more toxic than NO and superoxide themselves, which may increase microvascular permeability (Imaizumi et al., 1996). ...
In previous studies, the authors showed that the nitrone radical scavenger alpha-phenyl-N- tert -butyl nitrone (PBN) and its sulfo-derivative, 2-sulfo-phenyl-N- tert -butyl nitrone (S-PBN), attenuated cognitive disturbance and reduced tissue damage after traumatic brain injury (TBI) in rats. In the current study, the production of reactive oxygen species (ROS) after TBI was monitored with microdialysis and the 4-hydroxybenzoic acid (4-HBA) trapping method. A single dose of PBN (30 mg/kg) or an equimolar dose of S-PBN (47 mg/kg) was administered intravenously 30 minutes before a controlled cortical contusion injury in rats. Plasma and brain tissue drug concentrations were analyzed at the end of the microdialysis experiment (3 hours after injury) and, in a separate experiment with S-PBN, at 30 and 60 minutes after injury. Traumatic brain injury caused a significant increase in ROS formation that lasted for 60 minutes after the injury as evidenced by increased 3,4-dihydroxybenzoic acid (3,4-DHBA) concentrations in the dialysate. PBN and S-PBN equally and significantly attenuated the posttraumatic increase in 3,4-DHBA formation. High PBN concentrations were found bilaterally in brain tissue up to 3 hours after injury. In contrast, S-PBN was rapidly cleared from the circulation and was not detectable in brain at 30 minutes after injury or at any later time point. The results suggest that scavenging of ROS after TBI may contribute to the neuroprotective properties observed with nitrone spin-trapping agents. S-PBN, which remained undetectable even in traumatized brain tissue, reduced ROS production to the same extent as PBN that readily crossed the blood-brain barrier. This finding supports an important role for ROS production at the blood-endothelial interface in TBI.
The developmental profile of certain enzymatic antioxidants as well as the generation of reactive oxygen species was studied in the rat cerebral microvessels during first three weeks of life and the levels were compared to those present in adults. The data showed a higher generation of superoxide anion (+67%) and H2O2 (+200%) at postnatal day (PND) 21. Superoxide anion production was significantly lower (−24%) at PND 14 and almost comparable to adult values at PND 7. The activity of superoxide dismutase increased with development and attained an adult level at PND 21. Catalase was higher in neonates with a maximum activity at PND 7 and 14 (+68, 69%). The measurement of microvessel glutathione and glutathione‐related antioxidant enzymes showed that glutathione level was higher at PND 7, which declined to an adult level at PND 14. Se‐dependent GPx showed a marked increase between PND 14 and 21, however, it declined in adults. The activity of Se‐independent glutathione peroxidase was very low in cerebral microvessels. Glutathione reductase activity in 7‐day‐old, that was comparable to adult level, declined at PND 14 and 21. The level of glutathione S‐transferase was higher (+43%) at PND 21. The activity of microvessel marker enzyme γ‐glutatmyl transpeptidase increased with age, whereas, alkaline phosphatase showed a slight increase up to PND 14 and thereafter it declined. Lipid peroxidation was found to be significantly lower (−18%) at PND 21 as compared to adults. It may be concluded that developing cerebral microvessels contain high levels of several antioxidant enzymes that are more or equal to those present in adult brain microvessels.
Subarachnoid hemorrhage (SAH) results from the rupture of an intracranial aneurysm, and the first consequent events are increased intracranial pressure (ICP), reduced cerebral perfusion pressure (CPP), and decreased cerebral blood flow (CBF). The resultant hypoxic state alters autoregulation, ionic homeostasis, and excitotoxicity as well as initiates secondary injuries such as cytotoxic edema, blood-brain barrier (BBB) disruption, inflammation, and apoptotic cell death. Inflammation persists through hemorrhage degradation in the subarachnoid space. Several different aspects of the inflammatory response have been demonstrated in stroke pathogenesis, including cellular response (e.g., leukocyte adherence and microglia activation), expression of adhesion molecules (e.g., selectins, integrins, and immunoglobulin superfamily), production of inflammatory mediators (e.g., cytokines, nitric oxide/nitric oxide synthase (NO/NOS), and free radicals), and accumulation of platelet aggregates. Since all of these inflammatory aspects lead to brain edema and cell death, inflammation could be a particularly important target for designing therapeutic strategies against secondary injuries after SAH. Given these inflammatory contributions could be seen in large vessels, a plethora of research has been intended to reduce cerebral vasospasm (CVS) after SAH. The main research field, however, is moving toward studying early brain injury (EBI) because some human research demonstrated the morphological alleviation of CVS alone might not improve the functional recovery in patients after SAH. This chapter provides the current knowledge of the inflammatory response, translational research, and human clinical trials in SAH as well as discusses emerging opportunities for novel therapeutic strategies for clinical management of SAH.
The blood-brain barrier accounts for the high attrition rate of the treatments of most brain disorders, which therefore remain one of the greatest health-care challenges of the twenty first century. Against this background of hindrance to brain delivery, nanomedicine takes advantage of the assembly at the nanoscale of available biomaterials to provide a delivery platform with potential to raising brain levels of either imaging or therapeutic agents. Nevertheless, to prevent later failure due to ineffective drug levels at the target site, researchers have been endeavoring to develop a battery of in vitro screening procedures that can predict earlier in the drug discovery process the ability of these cutting-edge drug delivery platforms to cross the blood-brain barrier for biomedical purposes.
Before the discovery of insulin in 1921 the major cause of death in diabetic patients was diabetic ketoacidosis with coma. However, since the introduction of insulin diabetic patients more often die from vascular complications involving both large and small vessels.
Objective: To study the protective effects of the extract of camellia japonica L. on learning-memory dysfunction induced by ischemia-reperfusion in mice. Method: On day 7 after drug treatment, repeated cerebral ischemia -reperfusion was induced by fastening bilateral common carotid artery. The 7th day following surgery, learning-memory was measured using Morris water maze. The activity of MPO and the content of EB in brain were then measured by UV-photometer analysis system. Meanwhile, the content of water in brain were detected. Result: Ischemia-reperfusion impaired learning-memory (P < 0.05 and P < 0.01), and increased the activity of MPO and the content of EB and water in the brain of mice (P < 0.01). It was found that the extract of camellia japonica L. could remarkably decrease the content of EB (P < 0.01) and water (P < 0.05) in brain, and the activity of MPO (P < 0.05 and P < 0.01) when compared to the model group, finally dramatically improve the ability of learning-memory after ischemia-reperfusion (P < 0.05, P < 0.01). Conclusion: The extract of camellia japonica L. showed an improvement effect on the memory impairment in mice induced by ischemia/reperfusion. Improvement of the anti-blood-brain-barrier-damage and anti-inflammation in brain may be involved in the mechanism of this effect.
OBJECTIVE: To investigate the mechanism of bovine cerebral microvascular endothelial cells (BCMECs) damages induced by hydrogen peroxide and evaluate the protective effects of hydroxyethylpuerarin on the injured BCMECs. METHODS: Cells injury was determined by lactate dehydrogenase (LDH) activity in the extracellular medium. Malondialdelyde (MDA) and superoxide dismutase(SOD) activity both intracellular and in the extracellular medium were quantified by their reagents and the absorbance was assessed by automatic biochemistry analyser. Radioimmunoassay was applied to measure the amount of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α(6-keto-PGF1α) secreted by BCMECs. RESULTS: The exposure of cells to H2O2(200 μmol·L -1) for 2, 4 and 8 h caused a significant increase of LDH leakage, MDA level, and decrease of SOD activity compared with the normal group. Pre-incubation for 24 h with different concentrations of hydroxyethylpuerarin decreased the level of LDH and MDA, and increased the activity of SOD in a concentration-dependent manner in H2O2-induced cells. H2O2 also damaged the cells by increasing the content of TXB2 and decreasing the content of 6-keto-PGF1α secreted by BCMECs. Hydroxyethylpuerarin reduced the content of TXB2 and increased the content of 6-keto-PGF1α in a concentration-dependent manner. CONCLUSION: Hydroxyethylpuerarin protects the function of BCMEC s against hydrogen peroxide-induced injury contributing to its antioxidant effects.
The aim of this work is the preclinical evaluation of three ferrocenyl complexes (FcOHTam, FcdiOH and ansa-FcdiOH) formulated into lipid nanocapsules (LNCs) for oncology purposes. Firstly, the anticancer activity of the ferrocifens is demonstrated on a triple negative breast cancer model with a significant delay of the tumor growth upon administration of LNCs loaded with FcOHTAM. In order to enhance the passive tumoral targeting, the second part is dedicated to the development of stealth LNCs through a one-step (OS) formulation process. These OS LNCs showed an extended blood circulation time and maintained their integrity several hours after intravenous (iv) injection, as observed by FRET imaging. Additionally, repeated injections of ansa-FcdiOH-LNCs led to the growth inhibition of subcutaneous glioma. The last part of this work is devoted to the treatment of orthotopic glioma through various strategy of LNC administration (local or peripheral). The intra-carotid injection of FcdiOH-LNCs coated with the NFL-TBS peptide resulted in an improved survival time for some animals (5/12). The intracerebral sustained infusion of ferrocifen-loaded LNCs provoked severe toxicity and mortality. Finally, the repeated iv injections turned out to be an encouraging approach with a slight increase in the mean survival time (7%). Taken together, these results demonstrate the promising activity of the ferrocenyl complexes associated to OS LNCs for an alternative anticancer therapy approach.
The drug discovery process for drugs that target the central nervous system suffers from a very high rate of failure due to the presence of the blood-brain barrier, which limits the entry of xenobiotics into the brain. To minimise drug failure at different stages of the drug development process, new methodologies have been developed to understand the absorption, distribution, metabolism, excretion and toxicity (ADMET) profile of drug candidates at early stages of drug development. Additionally, understanding the permeation of drug candidates is also important, particularly for drugs that target the central nervous system. During the first stages of the drug discovery process, in vitro methods that allow for the determination of permeability using high-throughput screening methods are advantageous. For example, performing the parallel artificial membrane permeability assay followed by cell-based models with interesting hits is a useful technique for identifying potential drugs. In silico models also provide interesting information but must be confirmed by in vitro models. Finally, in vivo models, such as in situ brain perfusion, should be studied to reduce a large number of drug candidates to a few lead compounds. This article reviews the different methodologies used in the drug discovery and drug development processes to determine the permeation of drug candidates through the blood-brain barrier.
Overexpression of Cu,Zn superoxide dismutase (SOD1) reduces ischemic injury in some stroke models but exacerbates injury in a neonatal stroke model and in other settings. The current study used a SOD1 transgenic (SOD1-Tg) murine cortical culture system, derived from the same mouse strain previously used for the stroke models, to identify conditions that determine whether SOD1 overexpression in neurons is protective or detrimental. The nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine, spermine-NONOate, and diethylamine-NONOate produced less death in SOD1-Tg neurons than in wild-type neurons (p < 0.01). Also, NO produced markedly less 3-nitrotyosine in SOD1-Tg cells. In contrast, the superoxide generator menadione produced significantly greater death and nearly twice as much 2′7′-dichlorofluorescein fluorescence in SOD1-Tg neurons than in wild-type neurons, suggesting increased peroxide formation in the SOD1-Tg cells. No significant difference was observed in the vulnerability of the two cell types to H2O2, the product of the SOD reaction. Over-expression of SOD1 also had no effect on neuronal vulnerability to glutamate, N-methyl-D-aspartate, or kainate. These observations suggest that SOD1 overexpression can reduce neuronal death under conditions where peroxynitrite formation is a significant factor, but may exacerbate neuronal death under conditions of rapid intracellular superoxide formation or impaired H2O2 disposal.Keywords: Superoxide dismutase; Transgenic; Neuronal death; Ischemia; Cell culture; Glutamate
Leukocyte trafficking serves a critical function in central nervous system (CNS) immune surveillance. However, in many disease states leukocyte entry into the CNS is increased, which can disrupt the blood-brain barrier (BBB) and propagate neuroinflammation. These pathologic processes result in BBB permeability, glial activation, and neuronal compromise, all of which contribute to CNS damage. The resulting neuronal injury and loss are characteristic of many neuroinflammatory conditions including Alzheimer disease, multiple sclerosis, HIV-1 encephalopathy, sepsis, ischemia and reperfusion, and CNS tumors. HIV-1 encephalopathy is unique among these processes in that viral activity exacerbates CNS immune dysregulation and promotes chronic neuroinflammation and neurodegeneration. Thus, a significant number of HIV-1-infected persons exhibit neurocognitive and/or motor impairment. This review discusses the mechanisms that regulate leukocyte recruitment into the CNS and how HIV-1 infection dysregulates this process and contributes to neuropathology. Experimental BBB models to study leukocyte transmigration and the potential of targeting this transmigration across the BBB as a therapeutic strategy are also discussed.
Münster (Westfalen), Univ., Diss., 2003 (Nicht für den Austausch).
Glutamate is a major excitatory neurotransmitter in the central nervous system and plays a significant role in the pathophysiology of ischemic stroke. During acute ischemic cerebrovascular disease, glutamate efflux in the CNS produces excitotoxicity in neurons and may mediate forms of stress in other tissues expressing glutamate ionotropic (N-methyl-D-aspartate (NMDA)) receptors, e.g., cerebral endothelial cells. While endothelial cell stress in response to glutamate has been reported (oxidant stress, loss of barrier function), changes in protein expression produced by glutamate (an agonist of metabotropic and NMDA receptors) have not been documented. Here, we have examined how exposure of human cerebral endothelial cells to glutamate, in the presence and absence of the NMDA receptor antagonist MK-801, can alter the proteomic profile of cerebral endothelial cells. We found several important changes in the proteins expressed by cerebral endothelial cells in response to glutamate. Interestingly, MK-801 itself had some direct effects on cerebral endothelial cells. Taken together, our findings demonstrate that cerebral endothelial cells respond to glutamate by altering their protein expression profile. We assume that protein alterations found in the cerebral endothelial proteome, in response to glutamate and which were blocked by MK-801, may be important vascular targets in better understanding the pathogenesis of ischemic stroke.
Since ischemic insults lead to a deregulation of nitric oxide production which contributes to delayed neuronal death, we investigated changes in the distribution and amount of nitric oxide synthases I and II and in the appearance of nitrotyrosine caused by small, well-defined photothrombic lesions (2 mm in diameter) in the somatosensory cortex of rats. Four hours after lesioning, cell loss was evident in the core of the lesion and no nitric oxide synthase was present within this area, indicating that neurons expressing nitric oxide synthase I were lost or that nitric oxide synthase I was degraded. No increase in the number of neurons expressing nitric oxide synthase I was visible in the area surrounding the lesion, nor in other parts of the brain. One day after lesioning, NADPH-diaphorase- and nitric oxide synthase II-positive leucocytes had invaded the perilesional cortex and were accumulated in injured blood vessels. By two to three days post-lesion, layer V and VI pyramidal neurons, microglia, astrocytes and invading leucocytes had become strongly immunoreactive for nitric oxide synthase II within a perilesional rim. The number of cells expressing nitric oxide synthase I remained stable. Nitric oxide synthase II immunoreactivity and related NADPH-diaphorase had decreased by seven days post-lesion in most animals. However, the number of activated microglia or macrophages and astrocytes, as revealed by other markers, remained elevated. In addition, nitrotyrosine immunoreactivity was evident in the blood vessels close to the lesion, as well as in the ipsilateral hippocampus and thalamus.
The activities of several enzymes involved in drug metabolism, NADPH-cytochrome P450 reductase, cytochrome P450 isoforms CYP1A and CYP2B, and uridine diphosphate glucuronosyltransferase (UGT) have been measured in primary cultures of rat cerebrovascular endothelial cells and in the immortalized rat brain endothelial cell line RBE4. These drug metabolizing activities were similar in the microsomes prepared from both cell types, even after 20 passages for RBE4 cells. These results were confirmed by Western immunoblotting analysis, using polyclonal antibodies raised against rat liver enzymes. The superoxide production observed during NADPH-cytochrome P450 reductase-dependent monoelectronic reduction of four xenobiotics, menadione, anthraquinone, nitrofurazone and diquat, was also investigated in these cultured cells at confluence. The rates of radical production were concentration-dependent. The superoxide formation induced by quinone metabolism was comparable in both cell cultures, and high amounts of superoxide radicals were produced even after 20 passages of RBE4 cells. On the other hand, nitrofurazone and diquat metabolism produced weak amounts of superoxide radicals in both cell types. Taken together, these results suggest that RBE4 cell line seems to constitute a valuable in vitro model for studies on the activity of some enzymatic systems involved in drug metabolism at the blood-brain barrier and the functional consequences of their activity.
In the present study, we investigated the changes in blood-brain barrier (BBB) permeability following brain endothelial cell exposure to different xenobiotics able to promote free radical generation during their metabolism. Our in vitro BBB model consisted of confluent monolayers of immortalized rat brain capillary endothelial cells (RBE4) grown on collagen-coated filters in the presence of C6 glioma cells grown in the lower compartment. We have recently shown that a range of xenobiotics, including menadione, nitrofurazone, and methylviologen (paraquat) may undergo monoelectronic redox cycling in isolated brain capillaries, giving rise to reactive oxygen species. In this study, addition of 100 microM menadione to the culture medium for 30 min significantly increased the permeability of endothelial cell monolayers to radiolabeled sucrose. The effect on endothelial permeability induced by menadione was dose-dependent and reversible. These permeability changes preceded the onset of cell death, as assessed by the Trypan blue exclusion method. Pre-incubation with superoxide dismutase and catalase blocked changes in sucrose permeability to control levels in a dose-dependent manner, suggesting the involvement of reactive oxygen species in menadione-induced BBB opening.
The developmental profile of certain enzymatic antioxidants as well as the generation of reactive oxygen species was studied in the rat cerebral microvessels during first three weeks of life and the levels were compared to those present in adults. The data showed a higher generation of superoxide anion (+67%) and H2O2 (+200%) at postnatal day (PND) 21. Superoxide anion production was significantly lower (-24%) at PND 14 and almost comparable to adult values at PND 7. The activity of superoxide dismutase increased with development and attained an adult level at PND 21. Catalase was higher in neonates with a maximum activity at PND 7 and 14 (+68, 69%). The measurement of microvessel glutathione and glutathione-related antioxidant enzymes showed that glutathione level was higher at PND 7, which declined to an adult level at PND 14. Se-dependent GPx showed a marked increase between PND 14 and 21, however, it declined in adults. The activity of Se-independent glutathione peroxidase was very low in cerebral microvessels. Glutathione reductase activity in 7-day-old, that was comparable to adult level, declined at PND 14 and 21. The level of glutathione S-transferase was higher (+43%) at PND 21. The activity of microvessel marker enzyme gamma-glutatmyl transpeptidase increased with age, whereas, alkaline phosphatase showed a slight increase up to PND 14 and thereafter it declined. Lipid peroxidation was found to be significantly lower (-18%) at PND 21 as compared to adults. It may be concluded that developing cerebral microvessels contain high levels of several antioxidant enzymes that are more or equal to those present in adult brain microvessels.
The blood-brain barrier (BBB) which is located in the continuous endothelial lining of cerebral blood vessels rigidly controls exchange of water soluble compounds under physiological conditions. Under pathological conditions such as trauma or ischemia, BBB permeability may increase thus allowing plasma constituents to escape into brain tissue. This "opening" of the BBB may, at least in part, be mediated by massive release of autacoids resulting in vasogenic brain edema. Five criteria have to be fulfilled by an individual autacoid to be considered a mediator candidate of cerebral edema: i) a permeability-enhancing action under physiological conditions, ii) a vasodilatory action, iii) the ability to induce vasogenic brain edema, iv) an increase of concentration in the tissue or interstitial fluid under pathological conditions, and v) a decrease of brain edema by specific interference with the release or action of a given autacoid. Among the mediator candidates considered, bradykinin is the only one to meet all criteria. Histamine, arachidonic acid and free radicals including nitric oxide may also be considered mediators of brain edema, but for each of these compounds evidence is less clear than for bradykinin. Although the concept of mediators inducing brain edema is well established by experimental studies, only a bradykinin receptor antagonist has so far gained entrance into clinical evaluation.
The lungs are a delicate interface between the atmosphere and our bodies across which oxygen diffuses from the air we breathe to the blood which carries oxygen to the cells and mitochondria. In healthy lungs at sea level where there is a surfeit of oxygen, this process occurs easily, whereas, in lungs with disease it becomes a task which may not be fully successful and hypoxemia may ensue or worsen. At high altitude where the barometric pressure (Pb) and thus the supply of oxygen is lower, the job of getting oxygen to the blood, even in the healthy lung is more difficult, and in the diseased lung it may be impossible. This presentation will review the lungs' responses to high altitude, with emphasis on the abnormal. Both acute and chronic responses of patients with pre-existing lung disease will be reviewed. Pulmonary diseases encountered at high altitude in previously healthy people, such as high altitude pulmonary edema and chronic mountain sickness will be touched on only as they pertain to other patients. Pre-existing lung disease (with and without hypoxemia at sea level) such as obstructive lung diseases (asthma, COPD, emphysema), and restrictive lung diseases (sarcoid, asbestosis, interstitial pulmonary fibrosis) will be discussed in terms of gas exchange, lung mechanics, and treatment at high altitude. Disorders of ventilatory control; e.g., obesity-hypoventilation syndrome and sleep apnea, may present formidable problems, and guidelines for their treatment will be discussed. Infectious lung diseases; e.g., pneumonia, cystic fibrosis, and pulmonary vascular disorders such as chronic mountain sickness, primary pulmonary hypertension, and congenital absence of the pulmonary artery are important disorders that require special attention because of the accentuated hypoxic pulmonary vascular response encountered at high altitude. The purpose therefore, is to provide the medical practitioner with the insight into prevention, recognition, and treatment of pulmonary problems encountered specifically at high altitude, as well as guidance on how best to advise patients with lung disease who want to fly in airplanes and/or ascend to high altitude for work or pleasure.
Previous works of our group demonstrated that xenobiotic metabolism by brain microsomes or cultured cerebral cells may promote the formation of reactive oxygen species. In order to characterise the risk of oxidative stress to both the central nervous system and the blood-brain barrier, we measured in the present work the release of superoxide in the culture medium of rat cerebrovascular endothelial cells during the metabolism of menadione, anthraquinone, diquat or nitrofurazone. Assays were run in the same experimental conditions on primary cultures of rat neurones and astrocytes. Quinone metabolism efficiently produced superoxide, but the production of radicals during the metabolism of diquat or nitrofurazone was very low, as a probable result of their reduced transport inside the cells. In all cell types assayed, superoxide production was time- and concentration-dependent, and cultured astrocytes always produced the highest amounts of radicals. Superoxide formation by microsomes prepared from the cultured cells was decreased by immunoinhibition of NADPH-cytochrome P450 reductase or by its irreversible inhibition by diphenyliodonium chloride, suggesting the involvement of this flavoprotein in radical production. Cerebrovascular endothelial cells cultured on collagen-coated filters produced equivalent amounts of superoxide both at their luminal side and through the artificial basement membrane, suggesting that in vivo, endothelial superoxide production may endanger adjacent astrocytes and neurones.
In human immunodeficiency virus (HIV)-1-associated dementia (HAD), consequences of interactions between infiltrating monocytes and brain endothelial cells are not yet fully understood. This study investigated whether the blood-brain barrier is affected in brain tissue of patients suffering from HAD and whether it was possible to find a correlation with the presence or absence of monocytic cells, which have been suggested to play a major role in HAD. Immunohistochemical analysis for zonula occludens 1, a tight junction protein, and CD68, a macrophage marker, revealed that loss of tight junction immunoreactivity was highly correlated with monocyte infiltration and with HAD. This suggests that the presence of perivascular macrophages cells is associated with breakdown of the blood-brain barrier thereby facilitating infiltration of more monocytic cells hence enhancing disease progression.
Overexpression of Cu,Zn SOD (SOD1) can increase survival of neurons under some pathological conditions. Prior studies have shown, however, that SOD1 overexpression can reduce neuronal survival during exposure to superoxide generators by a mechanism involving excess H(2)O(2) accumulation. Since astrocytes exhibit greater H(2)O(2) catabolism capacity than do neurons, the present study examined the effects of SOD1 overexpression on astrocyte survival under these conditions. Cultures were prepared from transgenic mice that overexpress human SOD1 and from nontransgenic littermate controls. Exposure to xanthine oxidase/hypoxanthine (XO/HPX) or menadione caused dose-dependent astrocyte death. In contrast to prior observations with neurons, astrocytes that overexpress SOD1 showed increased resistance to superoxide toxicity. Surprisingly, increased survival in SOD1 overexpressing cultures remained evident even when H(2)O(2) catabolism was inhibited by preincubation with aminotriazole (to block catalase) and buthionine sulfoximine (to deplete glutathione). These findings suggest differences in superoxide metabolism between neurons and astrocytes, and that the greater resistance of astrocytes to oxidative stress is due at least partly to factors other than greater glutathione peroxidase and catalase activity in astrocytes. GLIA 33:343-347, 2001. Published 2001 Wiley-Liss, Inc.
Three types of overlap occur among the disease states chronic fatigue syndrome (CFS), fibromyalgia (FM), multiple chemical sensitivity (MCS) and posttraumatic stress disorder (PTSD). They share common symptoms. Many patients meet the criteria for diagnosis for two or more of these disorders and each disorder appears to be often induced by a relatively short-term stress which is followed by a chronic pathology, suggesting that the stress may act by inducing a self-perpetuating vicious cycle. Such a vicious cycle mechanism has been proposed to explain the etiology of CFS and MCS, based on elevated levels of nitric oxide and its potent oxidant product, peroxynitrite. Six positive feedback loops were proposed to act such that when peroxynitrite levels are elevated, they may remain elevated. The biochemistry involved is not highly tissue-specific, so that variation in symptoms may be explained by a variation in nitric oxide/peroxynitrite tissue distribution. The evidence for the same biochemical mechanism in the etiology of PTSD and FM is discussed here, and while less extensive than in the case of CFS and MCS, it is nevertheless suggestive. Evidence supporting the role of elevated nitric oxide/peroxynitrite in these four disease states is summarized, including induction of nitric oxide by common apparent inducers of these disease states, markers of elevated nitric oxide/peroxynitrite in patients and evidence for an inductive role of elevated nitric oxide in animal models. This theory appears to be the first to provide a mechanistic explanation for the multiple overlaps of these disease states and it also explains the origin of many of their common symptoms and similarity to both Gulf War syndrome and chronic sequelae of carbon monoxide toxicity. This theory suggests multiple studies that should be performed to further test this proposed mechanism. If this mechanism proves central to the etiology of these four conditions, it may also be involved in other conditions of currently obscure etiology and criteria are suggested for identifying such conditions.
Reactive oxygen species (ROS) are involved in the pathogenesis of several inflammatory and degenerative diseases, including multiple sclerosis (MS), an inflammatory disease of the central nervous system. We investigated the potential involvement of ROS in the interaction of monocytes with cerebral endothelium, because this is likely to be an early event in the development of MS lesions. ROS are produced via two main pathways, one involving NADPH oxidase complex and the other involving xanthine oxidase (XO). We examined the effects of ROS, ROS scavengers, and ROS inhibitors of both pathways on the migration of monocytes across the blood-brain barrier in vitro. Scavengers and inhibitors of XO predominantly inhibited monocyte migration, whereas inhibitors and scavengers of the NADPH oxidase complex had no effect. Exposure of cerebral endothelial cells (CEC) to superoxide (O 2 - ) resulted in enhanced migration and adhesion of monocytes as well as disruption of the tight junctions, whereas hydroxyl radicals and hydrogen peroxide induced no significant effect on these parameters. Underlying mechanisms of the observed changes were found to reside in the phospholipase C- mediated signal transduction cascade, subsequent accumulation of inositol 1,4,5-trisphosphate, and mobilization of intracellular calcium. We conclude that O 2 - is a signaling molecule that is onocytes infiltrate the brain during various neurological disorders including HIV-1- associated dementia (HAD), Alzheimer's disease, and multiple sclerosis (MS). The presence of an excess of activated macrophages in brain tissue is accompanied by tissue damage, resulting in a loss of both neuronal function and viability. In HAD, infiltrated monocytes/macrophages fuse with HIV-infected macrophages to form nucleated giant cells,
Oxidative stress has been associated with the development of blood-brain barrier disruption and cellular injury after ischemia. The cytosolic antioxidant, copper/zinc superoxide dismutase, has been shown to protect against blood-brain barrier disruption and infarction after cerebral ischemia-reperfusion. However, it is not clear whether copper/zinc superoxide dismutase can protect against evolving ischemic lesions after thromboembolic cortical ischemia. In this study, the photothrombotic ischemia model, which is physiologically similar to thromboembolic stroke, was used to develop cortical ischemia. Blood-brain barrier disruption and oxidative cellular damage were investigated in transgenic mice that overexpress copper/zinc superoxide dismutase and in littermate wild-type mice after photothrombotic ischemia, which was induced by both injection of erythrosin B (30 mg/kg) and irradiation using a helium neon laser for 3 min. Free radical production, particularly superoxide, was increased in the lesioned cortex as early as 4 h after ischemia using hydroethidine in situ detection. The transgenic mice showed a prominent decrease in oxidative stress compared with the wild-type mice. Blood-brain barrier disruption, evidenced by quantitation of Evans Blue leakage, occurred 1 h after ischemia and gradually increased up to 24 h. Compared with the wild-type mice, the transgenic mice showed less blood-brain barrier disruption, a decrease in oxidative DNA damage using 8-hydroxyguanosine immunohistochemistry, a subsequent decrease in DNA fragmentation using the in situ nick-end labeling technique, and decreased infarct volume after ischemia. From these results we suggest that superoxide anion radical is an important factor in blood-brain barrier disruption and oxidative cellular injury, and that copper/zinc superoxide dismutase could protect against the evolving infarction after thromboembolic cortical ischemia.
We report a rare case with tetralogy of Fallot (TOF) and agenesis of the internal carotid artery (ICA) who presented serious intracerebral haematomas. In the literature, this is the first documented case having these complications simultaneously. Extreme hypoxic insults followed by recovery were detected by O2 saturation monitor before two bleeds. Chronic brain hypoxia could make the vasculature weak, which was shown in the histological examination.
A 2-year-old girl was transferred to us with a general convulsion due to intracerebral haematoma. She had been showing general cyanosis from birth due to TOF. Repeated intracerebral haemorrhages ended her life. Histological study showed dilated vascular channels in the subarachnoid space and necrotizing vasculature obstructed by fibrinous thrombi adjacent to the haematoma. Fibrosis of the vessel wall with infiltration of macrophages suggested subacute or chronic lesions rather than acute necrosis due to the multiple haemorrhages. The intracerebral haematomas and agenesis of the ICA were observed as unilateral hemispheric vascular complications of TOF. Chronic brain hypoxia could play an important role in weakening the vessel wall and erythrocytosis caused obstructing thrombi. We speculate these factors generated the intracerebral haematomas.
Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na+, K+-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO2 of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P2 membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na+, K+-ATPase activity was determined at 37 degrees C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl2, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na+, K+-ATPase activity. Following peroxynitrite exposure, the activity of Na+, K+-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na+, K+-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na+, K+-ATPase. The results demonstrate that peroxynitrite decreased activity of Na+, K+-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na+, K+-ATPase activity.
Various types of evidence implicate nitric oxide and an oxidant, possibly peroxynitrite, in MCS and chemical intolerance (CI). The positive feedback loops proposed earlier for CFS may explain the chronic nature of MCS (CI) as well as several of its other reported properties. These observations raise the possibility that this proposed elevated nitric oxide/peroxynitrite mechanism may be the mechanism of a new disease paradigm, answering the question raised by Miller earlier: "Are we on the threshold of a new theory of disease?"
Platelet-activating factor (PAF), released during inflammatory responses, increases microvascular permeability to fluid and macromolecules. Previous studies in the hamster cheek pouch microcirculation have shown that PAF-induced increases in permeability can be diminished by pretreatment with a nitric oxide synthase inhibitor indicating that nitric oxide is required for PAF to cause leakage, although nitric oxide itself does not cause leakage. We evaluated the hypothesis that PAF stimulates the production of reactive oxygen species (ROS) that then react with nitric oxide to form a new species that signals the increase in vascular permeability. The hamster cheek pouch microcirculation was used to quantify the leakage of FITC-dextran following topical application of PAF. PAF-induced leakage was markedly inhibited (70%) by prior superfusion of the cheek pouch with superoxide dismutase and catalase. Superfusing the cheek pouch with ROS generated by xanthine oxidase and hypoxanthine produced leakage similar to that observed with PAF. Pretreating the cheek pouch with a nitric oxide synthase inhibitor (N(omega)-nitro-L-arginine, L-NA) inhibited ROS-induced leakage by 59% and PAF-induced leakage by 64%. The effects of L-NA and superoxide dismutase plus catalase on PAF-induced leakage were not additive. Systemic administration of mercaptoethylguanidine, a peroxynitrite scavenger, inhibited PAF-induced leakage by 60%. These results suggest that PAF-induced leakage may be mediated by an interaction between ROS and NO, perhaps through the formation of peroxynitrite or one of its products.
Multiple chemical sensitivity (MCS) is a condition where previous exposure to hydrophobic organic solvents or pesticides appears to render people hypersensitive to a wide range of chemicals, including organic solvents. The hypersensitivity is often exquisite, with MCS individuals showing sensitivity that appears to be at least two orders of magnitude greater than that of normal individuals. This paper presents a plausible set of interacting mechanisms to explain such heightened sensitivity. It is based on two earlier theories of MCS: the elevated nitric oxide/peroxynitrite theory and the neural sensitization theory. It is also based on evidence implicating excessive NMDA activity in MCS. Four sensitization mechanisms are proposed to act synergistically, each based on known physiological mechanisms: Nitric oxide-mediated stimulation of neurotransmitter (glutamate) release; peroxynitrite-mediated ATP depletion and consequent hypersensitivity of NMDA receptors; peroxynitrite-mediated increased permeability of the blood-brain barrier, producing increased accessibility of organic chemicals to the central nervous system; and nitric oxide inhibition of cytochrome P450 metabolism. Evidence for each of these mechanisms, which may also be involved in Parkinson's disease, is reviewed. These interacting mechanisms provide explanations for diverse aspects of MCS and a framework for hypothesis-driven MCS research.
Gap junction channels enable the direct flow of signaling molecules and metabolites between cells. Alveolar epithelial cells show great variability in the expression of gap junction proteins (connexins) as a function of cell phenotype and cell state. Differential connexin expression and control by alveolar epithelial cells have the potential to enable these cells to regulate the extent of intercellular coupling in response to cell stress and to regulate surfactant secretion. However, defining the precise signals transmitted through gap junction channels and the cross talk between gap junctions and other signaling pathways has proven difficult. Insights from what is known about roles for gap junctions in other systems in the context of the connexin expression pattern by lung cells can be used to predict potential roles for gap junctional communication between alveolar epithelial cells.
To investigate the antioxidant and anti-endotoxin effects of propofol on endothelial cells and the possible mechanisms.
Cultured endothelial cells were treated with hydrogen peroxide (H(2)O(2)), propofol + H(2)O(2), lipopolysaccharide (LPS) and propofol + LPS, respectively. Endothelial cell damage was monitored for possible lactic dehydrogenase (LDH) release. The transcription and the protein expression levels of endothelial nitric oxide synthase (eNOS) were measured.
LDH release was higher in groups treated with H(2)O(2) or LPS than in the control group. After pretreatment with propofol, the effects induced by H(2)O(2) were attenuated, but propofol did not decrease the LDH release induced by LPS. Both H(2)O(2) and LPS significantly increased the eNOS transcript levels and the increases were significantly attenuated after pretreatment with propofol. Both H(2)O(2) and LPS significantly increased the eNOS protein expression and the increase was attenuated after pretreatment with propofol.
Propofol could protect endothelial cells against oxidative stress by inhibiting eNOS transcription and protein expression, but could not antagonise endotoxin induced cell injuries.
Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.
The rate constant for the reaction of NO with ·O2- was determined to be (6.7 ± 0.9) × 109 1 mol-1 s-1, considerably higher than previously reported. Rate measurements were made from pH 5.6 to 12.5 both by monitoring the loss of ·O2- and the formation of the product -OONO. The decay rate of -OONO, in the presence of 0.1 moll-1 formate, ranges from 1.2s-1 at pH 5 to about 0.2s-1 in strong base, the latter value probably reflecting catalysis by formate. © 1993 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
Nitric oxide (NO) released by vascular endothelial cells accounts for the relaxation of strips of vascular tissue1 and for the inhibition of platelet aggregation2 and platelet adhesion3 attributed to endothelium-derived relaxing factor4. We now demonstrate that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture. Nitric oxide was detected by bioassay5, chemiluminescence1 or by mass spectrometry. Release of NO from the endothelial cells induced by bradykinin and the calcium ionophore A23187 was reversibly enhanced by infusions of L-arginine and L-citrulline, but not D-arginine or other close structural analogues. Mass spectrometry studies using 15N-labelled L-arginine indicated that this enhancement was due to the formation of NO from the terminal guanidino nitrogen atom(s) of L-arginine. The strict substrate specificity of this reaction suggests that L-arginine is the precursor for NO synthesis in vascular endothelial cells.
Hypoxemia was induced in pigs by artificial ventilation with 7% oxygen. Death occurred after 21–99 min (mean 48 min). The plasma hypoxanthine and base deficit concentrations increased linearly during hypoxemia and were significantly elevated after 5 min compared to initial values. The correlation between hypoxanthine and base deficit was good (p < 0.001, r = 0.68). The rate of increase of plasma hypoxanthine concentration was correlated with survival time and a significant correlation was found between the negative slope of hypoxanthine increase and survival. Death occurred in all animals when the hypoxanthine concentrations exceeded about 125 μmol/l. These findings indicate that the plasma concentration and the rate of increase of hypoxanthine specifically and sensitively reflect tissue hypoxia.
The involvement of oxygen radicals in the pathogenesis of Parkinson's disease has been suggested for some time. This article reviews the evidence supporting the involvement of oxygen radicals in the disease process in the brain. This includes a discussion of iron, lipid peroxidation, peroxidase, catalase, superoxide dismutase, and glutathione levels in the brain. In addition, various theories of induction of Parkinson's disease are discussed in relation to the possible involvement of oxygen radicals. These theories include the environmental toxin theory, the dopamine turnover theory, and the cerebral blood flow theory.
Recent advances in our knowledge of the blood-brain barrier (BBB) have in part been made by studying the properties and function of cerebral endothelial cells in vitro. After an era of working with a fraction, enriched in cerebral microvessels by centrifugation, the next generation of in vitro BBB model systems was introduced, when the conditions for routinely culturing the endothelial cells were established. This review summarizes the results obtained from this rapidly growing field. It can be stated with certainty that, in addition to providing a better insight into the chemical composition of cerebral endothelial cells, much has been learned from these studies about the characteristics of transport processes and cell-to-cell interactions during the last 12 years. With the application of new technologies, the approach offers a new means of investigation, applicable not only to biochemistry and physiology but also to the drug research, and may improve the transport of substances through the BBB. The in vitro approach has been and should remain an excellent model of the BBB to help unravel the complex molecular interactions underlying and regulating the permeability of the cerebral endothelium.
Oxygen-derived free radicals have been implicated in the pathogenesis of vasogenic edema and infarction caused by ischemia and reperfusion injury. In earlier studies, exogenously supplied liposome-entrapped CuZn superoxide dismutase (CuZn-SOD) ameliorated ischemic brain edema and infarction in rats following focal cerebral ischemia. To ascertain directly the role of SOD in the protection against superoxide radical-induced injury, we measured infarct size and water content 24 hr following focal cerebral ischemia in nontransgenic mice and in transgenic mice bearing the human SOD1 gene. These transgenic mice have 3.1-fold higher cellular CuZn-SOD activity in the brain than do their nontransgenic littermates. We also measured antioxidant levels (reduced glutathione and reduced ascorbate) of contralateral cortex, infarct cortex, surrounding cortex, and striatum. Infarct size and brain edema were significantly decreased in transgenic mice compared with nontransgenic mice. Reduced glutathione and reduced ascorbate levels decreased in the ischemic hemisphere, but levels in surrounding cortex and striatum were significantly higher in transgenic mice than in nontransgenic mice. These results indicate that increased endogenous SOD activity in brain reduces the level of ischemic damage and support the concept that superoxide radicals play an important role in the pathogenesis of infarction and edema following focal cerebral ischemia.
It has been proposed that oxygen-derived radicals, superoxide in particular, are involved in the alteration of blood-brain barrier permeability and the pathogenesis of brain edema following trauma, ischemia, and reperfusion injury. Using transgenic mice that overexpress the human gene for copper-zinc-superoxide dismutase, we studied the role of superoxide radicals in the blood-brain permeability changes, edema development, and delayed infarction resulting from cold-trauma brain injury. At 2 hours after a 30-second cold injury, cerebral water and Evans blue contents were reduced, respectively, from 80 +/- 0.2% and 132.7 +/- 12.9 micrograms/gm of dry weight for nontransgenic mice to 78.5 +/- 0.3% and 87.1 +/- 9.9 micrograms/gm of dry weight for transgenic mice. Infarction, as measured by 2,3,5-triphenyltetrazolium chloride staining, was reduced by 52% in transgenic brains. These data indicate that an increased level of superoxide dismutase activity in the brain reduces the development of vasogenic brain edema and infarction. Superoxide radicals play an important role in the pathogenesis of these lesions in cold-traumatized brain.
Endothelial cells grown on microcarriers are able to release H2O2 to the extracellular environment without any added stimulus. The extracellularly released H2O2 can be detected by luminol-amplified chemiluminescence (CL) if horseradish peroxidase is added. The CL response can be reduced by catalase and blocked by superoxide dismutase, indicating that O2- could be a precursor for H2O2. The CL kinetics, i.e., a long lag time followed by a rapid shift to a new level, indicate activation of an O2(-)-producing enzyme. The cells are also able to protect themselves from H2O2 stimulation by both catalase and the glutatione system. Bradykinin stimulates the H2O2 release, but if the effect is directly stimulatory or if it acts by reduction of the protective system is at present unclear. The extracellularly released H2O2 could be a cause of injury to the endothelial cells or to the subendothelial matrix.
The effects of quinones (benzoquinone, menadione, and doxorubicin) on the superoxide production in cell free systems (xanthine oxidase and rat liver microsomes) and of polycationic electrolyte- and latex-stimulated rat peritoneal macrophages have been studied. Contradictory results were obtained in cell free systems when two traditional assays for detection of superoxide ion, the cytochrome c reduction and the lucigenin-dependent chemiluminescence (CL), were used: all quinones inhibited the lucigenin-dependent CL at sufficiently large concentrations, but they did not inhibit at all the reduction of cytochrome c. It was proposed that the cytochrome c assay gave erroneous results due to the reversibility of the interaction of semiquinones with dioxygen. The effect of quinones on the superoxide production by peritoneal macrophages was biphasic: all quinones stimulated the O2-. formation at low concentrations and inhibited it at elevated concentrations. It was concluded that among the quinones studied, only menadione was capable of stimulating the superoxide production via a one-electron transfer mechanism in cell free systems, while the stimulatory effect of small concentrations of quinones on the O2-. production in macrophages was possibly due to their action on the activation of NADPH oxidase.
We studied the role of superoxide radicals in the pathogenesis of ischemic brain injury using a model of focal cerebral ischemia in 102 rats and liposome-entrapped CuZn-superoxide dismutase, which can penetrate the blood-brain barrier and cell membranes efficiently. The bolus intravenous administration of 25,000 units of liposome-entrapped CuZn-superoxide dismutase elevated superoxide dismutase activities in the blood and brain 1, 2, 8, and 24 hours later as well as in the ischemic hemisphere and contralateral cortex. Determined 24 hours after right middle cerebral and bilateral common carotid artery occlusion by the lack of staining for mitochondrial dehydrogenase activity with 2,3,5-triphenyltetrazolium chloride, infarct sizes were reduced by 33%, 25%, and 18% in the anterior, middle, and posterior brain slices, respectively, by treatment with liposome-entrapped CuZn-superoxide dismutase. Our data demonstrate that superoxide radicals are important determinants of infarct size following focal cerebral ischemia and that liposome-entrapped CuZn-superoxide dismutase may have pharmacologic value for the treatment of focal cerebral ischemic injury.
Down syndrome, the phenotypic expression of human trisomy 21, is presumed to result from a 1.5-fold increase in the expression of the genes on human chromosome 21. As an approach to the development of an animal model for Down syndrome, several strains of transgenic mice that carry the human Cu/Zn-superoxide dismutase gene have been prepared. These animals express the transgene in a manner similar to that of humans, with 0.9- and 0.7-kilobase transcripts in a 1:4 ratio, and synthesize the human enzyme in an active form capable of forming human-mouse enzyme heterodimers. Cu/Zn-superoxide superoxide dismutase activity is increased from 1.6- to 6.0-fold in the brains of four transgenic strains and to an equal or lesser extent in several other tissues. These animals provide a unique system for studying the consequences of increased dosage of the Cu/Zn-superoxide dismutase gene in Down syndrome and the role of this enzyme in a variety of other pathological processes.
Hypoxemia was induced in pigs by artificial ventilation with 7% oxygen. Death occurred after 21--99 min (mean 48 min). The plasma hypoxanthine and base deficit concentrations increased linearly during hypoxemia and were significantly elevated after 5 min compared to initial values. The correlation between hypoxanthine and base deficit was good (p < 0.001, r = 0.68). The rate of increase of plasma hypoxanthine concentration was correlated with survival time and a significant correlation was found between the negative slope of hypoxanthine increased and survival. Death occurred in all animals when the hypoxanthine concentrations exceeded about 125 mumol/l. These findings indicate that the plasma concentration and the rate of increase of hypoxanthine specifically and sensitively reflect tissue hypoxia.
Damage to the cerebral endothelium from ischemia could exacerbate brain injury by altering vascular integrity, but little is known concerning the response of cerebral endothelial cells to hypoxia. To address this issue, cerebral capillary endothelial cells were isolated from 14-day-old rats, grown to confluence, and placed in hypoxic chambers for up to 62 h. Cells were undamaged by 24 hours of hypoxia as assessed by lactate dehydrogenase release and ethidium bromide staining, but 48 h resulted in marked damage. Hypoxia was probably exacerbated by hypoglycemia because glucose levels fell to < 1 mM by 24 h, at which point ATP levels began to fall in hypoxic cultures (3.25 +/- 1.48 nmol/mg protein; mean +/- S.D.) relative to normoxic cultures (9.52 +/- 1.41 nmol/mg protein). Cells treated with 4 mM fructose-1,6-bisphosphate (FBP) had significantly less damage at 48 h of hypoxia than controls. FBP had little effect on rate of glucose depletion from the media, but ATP depletion due to hypoxia was significantly less. Thus, the protective effect of FBP may be mediated by the ability of treated cells to maintain higher ATP levels. Unlike FBP, glutamate receptor antagonists including MK-801, NBQX, DNQX, and kynurenic acid were ineffective in ameliorating hypoxia-induced endothelial cell injury.
Application of transgenic mice carrying the human CuZnSODgenein study of cerebral ischemia
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A procedure for the iso-lation of capillariesfrom rat brain. Cytobios. 8,4148. Joo F. and Klatzo I. (1989)Role of cerebral endotheliumin brain oedema
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Bovine aortic endothelial cells release hydrogenperoxide
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