John X. Wilson

Propofol neuroprotection in cerebral ischemia and its effects on low-molecular-weight antioxidants and skilled motor tasks.

Background: Propofol is neuroprotective when administered immediately after stroke. The therapeutic window, duration of administration, and antioxidant mechanisms of propofol in neuroprotection are not known. The effects of propofol after stroke were examined in the conscious animal. The authors have previously shown that light propofol anesthesia (25 mg · kg−1 · h−1) for a period of 4 h, even if delayed 1 h after the onset of ischemia, decreases infarct volume 3 days after the stroke. Methods: Cerebral ischemia was induced in awake Wistar rats by a local intracerebral injection of the potent vasoconstrictor, endothelin (6 pmol in 3 μl) into the striatum. Propofol treatment after ischemia was delayed up to 4 h, and the infusion period shortened from 4 h to 1 h. Infarct volume was assessed 3 or 21 days after the stroke. Neurologic outcome was evaluated on days 14–21 after ischemia. Tissue ascorbate and glutathione concentrations were evaluated at 4 h and 3 days after ischemia. Results: Infarct volumes were reduced 3 days after ischemia when propofol treatment (25 mg · kg−1 · h−1) was delayed for 2 h (0.5 ± 0.3 mm3) but not 4 h (2.0 ± 0.9 mm3), compared with intralipid controls (2.4 ± 0.7 mm3). The propofol infusion period of 3 h but not 1 h reduced infarct volume. Propofol treatment did not reduce infarct volume 21 days after the stroke, although motor function improvements (Montoya staircase test) were observed 14–21 days after the stroke. Propofol neuroprotection was independent of tissue ascorbate and glutathione concentrations. Conclusions: Concurrent or delayed administration of propofol is neuroprotective 3 days after ischemia. Although there were no differences in infarct volume 21 days after ischemia, propofol-treated animals had functional improvements at this time.

Mechanism of action of vitamin C in sepsis: Ascorbate modulates redox signaling in endothelium

Circulating levels of vitamin C (ascorbate) are low in patients with sepsis. Parenteral administration of ascorbate raises plasma and tissue concentrations of the vitamin and may decrease morbidity. In animal models of sepsis, intravenous ascorbate injection increases survival and protects several microvascular functions, namely, capillary blood flow, microvascular permeability barrier, and arteriolar responsiveness to vasoconstrictors and vasodilators. The effects of parenteral ascorbate on microvascular function are both rapid and persistent. Ascorbate quickly accumulates in microvascular endothelial cells, scavenges reactive oxygen species, and acts through tetrahydrobiopterin to stimulate nitric oxide production by endothelial nitric oxide synthase. A major reason for the long duration of the improvement in microvascular function is that cells retain high levels of ascorbate, which alter redox‐sensitive signaling pathways to diminish septic induction of NADPH oxidase and inducible nitric oxide synthase. These observations are consistent with the hypothesis that microvascular function in sepsis may be improved by parenteral administration of ascorbate as an adjuvant therapy.

iNOS expression requires NADPH oxidase-dependent redox signaling in microvascular endothelial cells

Redox regulation of inducible nitric oxide synthase (iNOS) expression was investigated in lipopolysaccharide and interferon‐γ (LPS + IFNγ)‐stimulated microvascular endothelial cells from mouse skeletal muscle. Unstimulated endothelial cells produced reactive oxygen species (ROS) sensitive to inhibition of NADPH oxidase (apocynin and DPI), mitochondrial respiration (rotenone) and NOS (L‐NAME). LPS + IFNγ caused a marked increase in ROS production; this increase was abolished by inhibition of NADPH oxidase (apocynin, DPI and p47phox deficiency). LPS + IFNγ induced substantial expression of iNOS protein. iNOS expression was prevented by the antioxidant ascorbate and by NADPH oxidase inhibition (apocynin, DPI and p47phox deficiency), but not by inhibition of mitochondrial respiration (rotenone) and xanthine oxidase (allopurinol). iNOS expression also was prevented by selective antagonists of ERK, JNK, Jak2, and NFκB activation. LPS + IFNγ stimulated activation/phosphorylation of ERK, JNK, and Jak2 and activation/degradation of IκB, but only the activation of JNK and Jak2 was sensitive to ascorbate, apocynin and p47phox deficiency. Ascorbate, apocynin and p47phox deficiency also inhibited the LPS + IFNγ‐induced DNA binding activity of transcription factors IRF1 and AP1 but not NFκB. In conclusion, LPS + IFNγ‐induced NFκB activation is necessary for iNOS induction but is not dependent on ROS signaling. LPS + IFNγ‐stimulated NADPH oxidase activity produces ROS that activate the JNK‐AP1 and Jak2‐IRF1 signaling pathways required for iNOS induction. Since blocking either NFκB activation or NADPH oxidase activity is sufficient to prevent iNOS expression, they are separate targets for therapeutic interventions that aim to modulate iNOS expression in sepsis. J. Cell. Physiol. 217: 207–214, 2008.

Cerebral astrocytes transport ascorbic acid and dehydroascorbic acid through distinct mechanisms regulated by cyclic AMP.

Abstract: Cerebral ischemia and trauma lead to rapid increases in cerebral concentrations of cyclic AMP and dehydroascorbic acid (DHAA; oxidized vitamin C), depletion of intracellular ascorbic acid (AA; reduced vitamin C), and formation of reactive astrocytes. We investigated astrocytic transport of AA and DHAA and the effects of cyclic AMP on these transport systems. Primary cultures of astrocytes accumulated millimolar concentrations of intracellular AA when incubated in medium containing either AA or DHAA. AA uptake was Na+‐dependent and inhibited by 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS), whereas DHAA uptake was Na+‐independent and DIDS‐insensitive. DHAA uptake was inhibited by cytochalasin B, d‐glucose, and glucose analogues specific for facilitative hexose transporters. Once inside the cells, DHAA was reduced to AA. DHAA reduction greatly decreased astrocytic glutathione concentration. However, experiments with astrocytes that had been previously depleted of glutathione showed that DHAA reduction does not require physiological concentrations of glutathione. Astrocyte cultures were treated with a permeant analogue of cyclic AMP or forskolin, an activator of adenylyl cyclase, to induce cellular differentiation and thus provide in vitro models of reactive astrocytes. Cyclic AMP stimulated uptake of AA, DHAA, and 2‐deoxyglucose. The effects of cyclic AMP required at least 12 h and were inhibited by cycloheximide, consistent with a requirement for de novo protein synthesis. Uptake and reduction of DHAA by astrocytes may be a recycling pathway that contributes to brain AA homeostasis. These results also indicate a role for cyclic AMP in accelerating the clearance and detoxification of DHAA in the brain.

Septic impairment of capillary blood flow requires nicotinamide adenine dinucleotide phosphate oxidase but not nitric oxide synthase and is rapidly reversed by ascorbate through an endothelial nitric oxide synthase-dependent mechanism.

Objective:To determine the roles of nitric oxide synthase (NOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the impairment of capillary blood flow in sepsis and in the reversal of this impairment by ascorbate. Design:Prospective, controlled laboratory study. Setting:Animal laboratory in research institute. Subjects:Adult male wild type (WT), neuronal nitric oxide synthase (nNOS)−/−, inducible NOS (iNOS)−/−, endothelial NOS (eNOS)−/−, and gp91phox−/− mice. Interventions:Sepsis was induced by feces injection into peritoneum (FIP). A bolus of ascorbate or NADPH oxidase inhibitor apocynin was injected intravenously at 6 hrs post-FIP. Alternatively, NOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) or nitric oxide donor S-nitroso-N-acetylpenicillamine was superfused on the surface of the extensor digitorum longus muscle. Measurements and Main Results:Capillary blood flow impairment and NOS activity in the extensor digitorum longus muscle were measured by intravital microscopy and by enzymatic assay, respectively. Sepsis at 6 hrs impaired flow in WT mice. Apocynin, and knockout of gp91phox but not of any NOS isoforms, rescued this impairment. Constitutive NOS activity was unaffected by sepsis, but it was abolished by nNOS knockout (iNOS activity was negligible in all mice). Ascorbate rapidly (10 mins) rescued impaired flow in WT, nNOS−/−, iNOS−/− but not eNOS−/− mice. Ascorbate also improved survival of WT mice after FIP. BH4 and SNAP rescued flow in WT mice, while BH4 failed to rescue it in eNOS−/− mice. Conclusion:Capillary blood flow impairment in septic skeletal muscle requires NADPH oxidase but not NOS, and it is rapidly reversed by ascorbate and BH4 through an eNOS-dependent mechanism.

Inflammatory Response in Microvascular Endothelium in Sepsis: Role of Oxidants

Sepsis, as a severe systemic inflammatory response to bacterial infection, represents a major clinical problem. It is characterized by the excessive production of reactive oxygen species (ROS) both in the circulation and in the affected organs. The excessive generation of ROS inevitably leads to oxidative stress in the microvasculature and has been implicated as a causative event in a number of pathologies including sepsis. In this review, we focus on the role of oxidative and nitrosative stress during the early onset of sepsis. Changes in microvascular endothelial cells, the cell type that occurs in all organs, are discussed. The mechanisms underlying septic induction of oxidative and nitrosative stresses, the functional consequences of these stresses, and potential adjunct therapies for microvascular dysfunction in sepsis are identified.

Delayed ascorbate bolus protects against maldistribution of microvascular blood flow in septic rat skeletal muscle.

Objective:Although early administration of ascorbate has been shown to protect against the microvascular dysfunction in sepsis, it is not clear if a delayed introduction of ascorbate also yields beneficial effects. The main objective was to determine the therapeutic window for treatment of an animal model of sepsis with bolus injection of ascorbate. We also determined if sepsis per se affects urinary excretion of ascorbate. Design:Prospective, controlled laboratory study. Setting:Animal laboratory in a university-affiliated research institute. Subjects:Male Sprague-Dawley rats, 300–400 g of body weight. Interventions:Rats were made septic by cecal ligation and perforation (CLP) and volume resuscitated by continuous saline infusion. Ascorbate bolus (7.6 mg/100 g of body weight) or saline vehicle was injected intravenously at 1, 6, or 24 hrs after CLP. Measurements and Main Results:At 24 hrs post-CLP, sepsis caused antidiuresis and decreased plasma ascorbate concentration, but it did not affect urinary excretion of ascorbate in rats that received only saline. Sepsis also caused maldistribution of capillary blood flow in skeletal muscle. This maldistribution of flow was prevented by ascorbate injected at 6 hrs post-CLP. At 48 hrs post-CLP, in addition to the flow maldistribution, sepsis caused systemic arterial hypotension and fever that were prevented by both immediate (1 hr post-CLP) and delayed injections of ascorbate (24 hrs post-CLP). Conclusion:Despite volume resuscitation, the present model of sepsis resulted in maldistribution of capillary blood flow within 24 hrs and hypotension within 48 hrs. Our finding that intravenous bolus of ascorbate can protect against these deficits even if delayed 6–24 hrs after the septic insult shows, for the first time, that ascorbate can reverse microcirculatory dysfunction after the onset of sepsis.

Propofol prevents peroxide-induced inhibition of glutamate transport in cultured astrocytes.

BACKGROUND Glutamate transporters located in the plasma membrane of cerebral astrocytes take up excitatory neurotransmitters from the synaptic cleft. In diseases characterized by oxidative stress, the extracellular glutamate concentration increases and contributes to neuronal death. The authors wanted to determine whether propofol defends brain cells against oxidant-induced changes in their transport of glutamate. METHODS Primary cultures of rat cerebral astrocytes were exposed to tert-butyl hydroperoxide (1 mM) to serve as an in vitro model of oxidative stress. Astrocytes were incubated with propofol for 2 h and tert-butyl hydroperoxide was added for the final hour. Alternatively, astrocytes were incubated with tert-butyl hydroperoxide for 30 min and then with propofol for another 30 min. Control cells received drug vehicle rather than propofol. The rate of uptake of glutamate, the efflux of the nonmetabolizable analog D-aspartate, and the intracellular concentration of the endogenous antioxidant glutathione were measured. RESULTS Tert-butyl hydroperoxide decreased the glutathione concentration and inhibited glutamate uptake but had a negligible effect on D-aspartate efflux. At clinically relevant concentrations, propofol did not affect the glutathione concentration but did prevent the effect of tert-butyl hydroperoxide on glutamate transport. Furthermore, the addition of propofol after tert-butyl hydroperoxide reversed the inhibition of glutamate uptake. CONCLUSIONS Propofol prevents and reverses the inhibition of excitatory amino acid uptake in astrocytes exposed to tert-butyl hydroperoxide. The ability of propofol to defend against peroxide-induced inhibition of glutamate clearance may prevent the pathologic increase in extracellular glutamate at synapses, and thus delay or prevent the onset of excitotoxic neuronal death.

Anesthetic concentrations of propofol protect against oxidative stress in primary astrocyte cultures: comparison with hypothermia.

BackgroundThe extracellular concentration of glutamate in the brain increases after oxidative damage. This increase may be caused, in part, by changes in glutamate transport by astrocytes. The authors hypothesized that propofol and hypothermia mitigate the effects on astrocytes of oxidative stress. MethodsPrimary cultures of rat cerebral astrocytes were subjected to oxidative stress by incubation with tert-butyl hydroperoxide for 30 min, followed by a 30–90-min washout period. The effects of prophylactic (simultaneous with tert-butyl hydroperoxide application) and delayed (administered 30 min after the oxidant) propofol or hypothermia were determined by measuring the uptake of glutamate as well as the release of preloaded d-aspartate (a nonmetabolizable analog of glutamate) and endogenous lactate dehydrogenase (a cytosolic marker). ResultsDelayed administration of an anesthetic concentration of propofol (1–3 &mgr;m) prevented the inhibition of high-affinity glutamate uptake, stimulation of d-aspartate release, and increase in lactate dehydrogenase release caused by tert-butyl hydroperoxide (1 mm, 37°C). The protective effect of propofol (EC50 = 2 &mgr;m) on glutamate uptake was 20-fold more potent than that of &agr;-tocopherol (EC50 = 40 &mgr;m). Prophylactic hypothermia (28 and 33°C) also protected astrocytes from tert-butyl hydroperoxide. Delayed hypothermia was not protective but did not compromise rescue by propofol. ConclusionsClinical levels of propofol and hypothermia mitigate the effects of oxidative stress on astrocytic uptake and retention of glutamate, with propofol having a relatively larger therapeutic window. The ability of these treatments to normalize cell transport systems may attenuate the pathologic increase in extracellular glutamate at synapses and thus prevent excitotoxic neuronal death.

Osmotic swelling stimulates ascorbate efflux from cerebral astrocytes

Abstract: Ascorbate (reduced vitamin C) is an important enzyme cofactor, neuromodulator, and antioxidant that is stored at millimolar concentrations in the cytosol of cerebral astrocytes. Because these cells swell during hyponatremia, cerebral ischemia, and trauma, we investigated the effects of osmotic stress on astrocytic transport of ascorbate. Ascorbate efflux from primary cultures of rat astrocytes was rapidly (within 1 min) increased by incubation in hypotonic medium. Efflux also increased when astrocytes, which had been adapted to a hypertonic environment, were swollen by transfer to isotonic medium. Swelling‐induced ascorbate efflux was inhibited by the anion‐transport inhibitors 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS) and 4,4′‐dinitrostilbene‐2,2′‐disulfonic acid (DNDS). The pathway that mediates ascorbate efflux was found to be selective because a larger anion, 2′,7′‐bis(carboxyethyl)‐5‐(or ‐6)‐carboxyfluorescein (BCECF), was retained in the swollen astrocytes. Na+‐dependent ascorbate uptake into astrocytes was inhibited slightly during the first minute of hypotonic stress, indicating that the sodium ascorbate cotransporter does not mediate swelling‐induced efflux. Cell concentration of authentic ascorbate was measured by HPLC with electrochemical detection. When astrocytes were incubated in ascorbate‐free medium, hypotonicity decreased cell ascorbate concentration by 50% within 3 min. When astrocytes were incubated in ascorbate‐supplemented hypotonic medium, intracellular ascorbate concentration was restored within 10 min because uptake remained effective. Many pathological conditions cause brain cell swelling and formation of reactive oxygen species. Ascorbate release during astrocytic swelling may contribute to cellular osmoregulation in the short‐term and the scavenging of reactive oxygen species.