Michael P. Flavin

Microglial tissue plasminogen activator (tPA) triggers neuronal apoptosis in vitro.

Several CNS disorders feature microglial activation. Microglia are known to have both restorative and cytotoxic capabilities. Neuronal apoptosis has been noted after an acute insult such as ischemia. Microglia may participate in this event. We previously showed that conditioned medium (CM) harvested from peritoneal macrophages or from activated microglia triggered apoptosis in rat hippocampal neurons in culture. We wished to characterize the factor responsible for triggering neuronal death. Quiescent microglia produced CM that did not disrupt hippocampal neurons. Lipopolysaccharide‐activated microglia produced CM which resulted in neuronal death. This effect was blocked by plasminogen activator inhibitor‐1, by tPA STOP, and by co‐incubation with tPA antibody. Recombinant human tPA exaggerated the neurotoxic effects of microglial CM, while tPA alone was toxic only at very high concentrations. This in vitro system, which probably excludes any significant impact of microglial free radicals, suggests that microglial tPA may contribute significantly to hippocampal neuronal death. GLIA 29:347–354, 2000.

Differential sensitivity of rat hippocampal and cortical astrocytes to oxygen-glucose deprivation injury.

Selective vulnerability of hippocampal neurons to ischemia is felt to relate to intense glutamatergic input and glutamate receptor expression. Since astrocytes are thought to have a neuroprotective role we speculated that hippocampal astrocyte sensitivity to insult could also contribute to this regional vulnerability. The purpose of the study was to determine if there is a differential sensitivity of cultured hippocampal and cortical astrocytes to oxygen-glucose deprivation (OGD). Hippocampal and cortical astrocytes were grown to confluence at matching cell density. Cultures were exposed to OGD for 2, 4, 6, 8 and 10 h. Progressive reduction in viability occurred in hippocampal astrocytes beginning at 2 h OGD. Reduction in cortical astrocyte viability was not observed until 4 h OGD. Death of hippocampal astrocytes was significantly greater than that of cortical astrocytes at each period of OGD. Based on acid phosphatase activity data the LD(50) for OGD duration in hippocampal astrocytes was 2 h compared to 8 h in cortical astrocytes. Regional differences in sensitivity of astrocytes to OGD implies that this may contribute to regional differences in neuronal vulnerability to ischemia.

Effect of inhaled hypertonic saline on hospital admission rate in children with viral bronchiolitis: a randomized trial

OBJECTIVE We sought to determine whether inhaled 3% hypertonic saline (HS) reduces admission to hospital in ambulatory children with moderately severe viral bronchiolitis. Secondary objectives compared changes in respiratory scores before and after treatment and assessed the need for unscheduled medical intervention within 7 days. METHODS Children under the age of 2 years presenting with moderately severe viral bronchiolitis to the emergency department of 4 general hospitals from November 2008 to March 2009 were randomly assigned to receive 3 consecutive 4-mL doses of nebulized 3% HS (treatment group) or 0.9% normal saline (NS; control group) in a double blind fashion, each coadministered with 1 mg salbutamol. Outcome measures included the difference in hospital admission rate and changes in respiratory distress scores. RESULTS A total of 81 children (mean age 8.9 mo, range 0.7-22 mo) were assessed over 88 visits on an intention-to-treat basis. No statistically significant differences were found between treatment groups. Children in the HS group had a nonsignificant trend toward greater improvement compared with NS controls with a same-day admission rate of 18% (95% confidence interval [CI] 9%-32%) versus 27% (95% CI 16%-42%), respectively. Respiratory Assessment Change Scores (RACS) favoured the HS group over NS controls (mean RACS 4.7 [95% CI 3.6-5.8] v. 3.7 [95% CI 2.5-4.9], respectively), although the CIs overlap and these differences were not statistically significant. CONCLUSION The short-term use of nebulized 3% HS did not result in any statistically significant benefits, although a nonsignificant trend toward a decrease in admission rate and improvement in respiratory distress was found. A larger study would be required to determine whether these trends arise from a clinically relevant treatment effect.

Tissue plasminogen activator protects hippocampal neurons from oxygen-glucose deprivation injury.

We have previously shown that tissue plasminogen activator (tPA) participates in the neurotoxicity of microglial conditioned medium (MgCM). Killing of hippocampal neurons by MgCM was prevented by both plasminogen activator inhibitor‐1 (PAI‐1) and anti‐tPA antibody. An N‐methyl‐D‐aspartate (NMDA) receptor blocker protected neurons from MgCM, suggesting that this subtype of glutamate receptor is involved. Whereas glutamate receptor‐mediated events are important in cerebral ischemia and tPA has previously been shown to enhance excitotoxicity in hippocampus, we hypothesized that tPA would exaggerate oxygen glucose deprivation (OGD) injury in cultures of hippocampal neurons. Dissociated rat hippocampal cells were grown under conditions designed to optimize neuronal growth while minimizing glial replication. At 7–10 days, cultures were subjected to OGD for 2.5 hr. Recombinant human tPA (1,000 IU) was added immediately after OGD. Viability was assessed 24 hr later. Viable, apoptotic, and necrotic cells were classified and quantified based on staining patterns of acridine orange and ethidium bromide under fluorescence microscopy. tPA alone did not alter neuronal integrity. OGD produced significant neuronal death (viability reduced by 45%, P < 0.001). tPA completely protected OGD‐exposed cultures. Potential mechanisms of tPA protection were explored. Whereas tPA antibody abolished the protective effect of tPA, its proteolytic inhibitor PAI‐1 did not alter the effect. The effect of tPA was tested in separate free radical and excitatory amino acid insults. It did not protect neurons from hydrogen peroxide (1 μM), S‐nitro‐acetylpenicillamine (10 μM), glutamate (50 μM), or NMDA (10 μM) damage but significantly attenuated injury caused by 250 μM kainate. We conclude that tPA is capable of protecting hippocampal neurons from OGD by a nonproteolytic action. The mechanism of protection was not defined, although attenuation of AMPA/kainate glutamate receptors may play a role. J. Neurosci. Res. 63:388–394, 2001.

Influence of Dexamethasone on Neurotoxicity Caused by Oxygen and Glucose Deprivationin Vitro

There is conflicting evidence regarding the impact of glucocorticoid exposure on hypoxic ischemic brain injury. We examined the effects of timing, duration, and concentration of dexamethasone on neuronal injury following in vitro oxygen glucose deprivation (OGD). Dissociated embryonic rat basal forebrain cells were cultured and either preincubated with dexamethasone for 72 h or continuously exposed prior to, during, and after OGD. Injury was assessed by morphology rating and cholineacetyltransferase (ChAT) activity at Day 13 in vitro, 2 days after OGD. Preincubation with nanomolar concentrations of dexamethasone resulted in a dose-dependent exaggeration of injury. Combined glutamate receptor antagonist application negated this deleterious effect, suggesting that dexamethasone may increase glutamate release, decrease uptake, or upregulate glutamate receptor expression. Continuous application of a narrow concentration range of dexamethasone (100 nM and 1 microM) prior to, during, and after insult protected neurons. Dose, timing, and duration of glucocorticoid administration may each be critical variables influencing outcome of hypoxic ischemic brain insult.

Propentofylline protects neurons in culture from death triggered by macrophage or microglial secretory products

We recently demonstrated that conditioned medium (CM) from peritoneal macrophages or activated microglia triggers a predominantly apoptotic death in hippocampal neurons in culture. We tested the effects of propentofylline (ppf), an agent that is neuroprotective in focal ischemia and is also associated with reduced microglial antigen expression after insult. Ppf had no impact on the secretion of neurotoxin from microglia. However, ppf significantly attenuated the effects of macrophage and microglial conditioned medium on neurons. Ppf did not attenuate neuronal hypoxic injury but did reverse the exaggeration of hypoxic injury exerted by subsequent addition of macrophage CM. A1 and A2 adenosine receptor inhibitors and an inhibitor of adenosine uptake each mimicked the effect of ppf. Neither ATP nor a deaminase inhibitor blocked the effect of microglial CM. These findings may be relevant to the neuroprotective effects of ppf in ischemia and dementia. J. Neurosci. Res. 56:54–59, 1999. 

Neurotoxicity of Soluble Macrophage Productsin Vitro—Influence of Dexamethasone ☆

When macrophage conditioned medium is added to neurons in vitro, there is a loss of cell membrane integrity, a loss of cell processes, and a large increase in apoptotic neurons. We tested the influence of a potent anti-inflammatory steroid on the interaction between macrophages and neurons. Dexamethasone was applied to macrophages in culture for 24 h while the culture was stimulated with lipopolysaccharide and hypoxia. Conditioned medium was collected after dexamethasone was removed. The dexamethasone pretreated medium was not toxic to hippocampal neurons in contrast to medium from stimulated macrophages not treated with steroid. The dexamethasone effect was concentration dependent. Pretreatment of macrophages with indomethacin and transforming growth factor beta had similar but less impressive effects when compared to dexamethasone. The effect of dexamethasone may have been mediated by inhibiting the synthesis or release of neurotoxic macrophage protein(s), as a combination of medium from steroid pretreated macrophages with medium from nontreated macrophages was not neuroprotective. The toxin(s) did not appear to be tumor necrosis factor alpha or arginase. A role for most neutral proteases was also excluded. We also assessed the consequence of stressing neurons with a mild hypoxic exposure immediately prior to conditioned medium application. Medium from dexamethasone-treated macrophages did not exaggerate hypoxic neuronal injury, unlike medium from non-dexamethasone-treated macrophages. It did not, however, block the exaggerating effect when coapplied in equal volume with medium from nontreated macrophages. Dexamethasone at 100 nM had no impact when applied directly to neurons while they were being exposed to conditioned medium. This in vitro protection by dexamethasone may be relevant to the demonstrated benefit of glucocorticoids in selected brain and spinal cord conditions. Suspicion of a potential link between this in vitro finding and in vivo CNS injury justifies an assessment of more specific agents acting on macrophage protein synthesis or secretion.

The effect of hypoxia on neurotransmitter phenotype of forebrain cholinergic neurons

The effect of hypoxia on the neurotransmitter phenotype of rat forebrain cholinergic neurons was analyzed using a dissociated fetal rat culture system. The aims of this study were to examine the feasibility of using choline acetyltransferase (ChAT) activity as a measure of cell injury and/or recovery, to measure the time course of hypoxic effects on ChAT activity, to determine how changes in ChAT activity at 48 h post-injury relate to microscopic changes and LDH release into the medium during that time, and finally to explore the possible mechanisms of hypoxic injury in this model. At exposure to 0.5-1.5% O2 there was a time-dependent decrease in ChAT activity when cells were harvested 48 h after exposure. Forty-eight hours after 8-9 h hypoxic exposure ChAT activity was 50-60% that of controls without any alteration in morphology of neurons. An 8 h exposure to hypoxic conditions caused a post-exposure time-dependent decrease in ChAT activity to 20% of control level at 72 h. Thereafter there was spontaneous recovery of phenotype to 60% of control which remained stable between 5 and 7 days post-exposure. Loss of neurotransmitter phenotype was not well correlated with other measures of cytotoxicity including morphological changes and LDH release. The loss of phenotype observed with hypoxia was mimicked by glutamate and kainate but not by NMDA. Consistent with these observations, neither APV nor AP3 significantly altered the effect of hypoxia on forebrain cholinergic neurons, while the addition of APV and CNQX in combination protected the phenotype of these neurons only if there was 50% or less loss of phenotype following hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of high and standard blood transfusion volumes in premature infants

UNLABELLED We compared high volume (20 ml/kg) with standard volume (15 ml/kg) packed red blood cell (PRBC) transfusions in a randomized trial. Ten high volume babies received a mean of 2.8 transfusions, while 10 standard volume babies received a mean of 3.4 (p=0.58). No adverse events were attributed to the use of the high volume transfusions. CONCLUSION Higher transfusion volumes appear to be well tolerated. The data can be used in the planning of larger controlled trials in VLBW infants.

Hyperthermia amplifies brain cytokine and reactive oxygen species response in a model of perinatal inflammation.

Chorioamnionitis, a perinatal infection of the fetal membranes, and maternal fever, which often accompanies infection are both risk factors for cerebral palsy (CP). Inflammation is a typical reaction to infection. Thus the aim of this study was to determine if hyperthermia alters newborn rat brain inflammatory response and oxidant stress after a maternal rat lipopolysaccaharide (LPS) injection. Since chorioamnionitis can predispose the fetus to perinatal hypoxia, we also explored the interaction with postnatal hypoxia. Exposure of newborn pups to brief hypoxia alone significantly increased brain tumor necrosis factor-alpha (TNF-alpha) and slightly increased levels of nitrite/nitrate. When maternal LPS was combined with postnatal hypoxia, the levels of TNF-alpha in were further increased when compared with hypoxia alone. Exposure of newborn pups to hyperthermia at 39 degrees C following maternal LPS and hypoxia caused yet more significant increases in brain TNF-alpha, nitrite/nitrate, and MDA/4-HAD compared to that under normal temperature conditions. This study supports the hypothesis that fever is a significant modifier of brain inflammatory response in developing brain particularly in a setting of hypoxia.