Adam Sapirstein

Effect of duration of osmotherapy on blood-brain barrier disruption and regional cerebral edema after experimental stroke

Osmotherapy is the cornerstone of medical management for cerebral edema associated with large ischemic strokes. We determined the effect of duration of graded increases in serum osmolality with mannitol and hypertonic saline (HS) on blood-brain barrier (BBB) disruption and regional cerebral edema in a well-characterized rat model of large ischemic stroke. Halothane-anesthetized adult male Wistar rats were subjected to transient (2-h) middle cerebral artery occlusion (MCAO) by the intraluminal occlusion technique. Beginning at 6 h after MCAO, rats were treated with either no intravenous fluids or a continuous intravenous infusion (0.3 mL/h) of 0.9% saline, 20% mannitol, 3% HS, or 7.5% HS for 24, 48, 72, and 96 h. In the first series of experiments, BBB permeability was quantified by the Evans blue (EB) extravasation method. In the second series of experiments, water content was assessed by comparing wet-to-dry weight ratios in six predetermined brain regions. Blood-brain barrier disruption was maximal in rats treated with 0.9% saline for 48 h, but did not correlate with increases in serum osmolality or treatment duration with osmotic agents. Treatment with 7.5% HS attenuated water content in the periinfarct regions and all subregions of the contralateral nonischemic hemisphere to a greater extent than mannitol did with no adverse effect on survival rates. These data show that (1) BBB integrity is not affected by the duration and degree of serum osmolality with osmotic agents, and (2) attenuation of increases in brain water content with HS to target levels > 350 mOsm/L may have therapeutic implications in the treatment of cerebral edema associated with ischemic stroke.

Cytosolic phospholipase A2 alpha amplifies early cyclooxygenase-2 expression, oxidative stress and MAP kinase phosphorylation after cerebral ischemia in mice.

BackgroundThe enzyme cytosolic phospholipase A2 alpha (cPLA2α) has been implicated in the progression of cerebral injury following ischemia and reperfusion. Previous studies in rodents suggest that cPLA2α enhances delayed injury extension and disruption of the blood brain barrier many hours after reperfusion. In this study we investigated the role of cPLA2α in early ischemic cerebral injury.MethodsMiddle cerebral artery occlusion (MCAO) was performed on cPLA2α+/+ and cPLA2α-/- mice for 2 hours followed by 0, 2, or 6 hours of reperfusion. The levels of cPLA2α, cyclooxygenase-2, neuronal morphology and reactive oxygen species in the ischemic and contralateral hemispheres were evaluated by light and fluorescent microscopy. PGE2 content was compared between genotypes and hemispheres after MCAO and MCAO and 6 hours reperfusion. Regional cerebral blood flow was measured during MCAO and phosphorylation of relevant MAPKs in brain protein homogenates was measured by Western analysis after 6 hours of reperfusion.ResultsNeuronal cPLA2α protein increased by 2-fold immediately after MCAO and returned to pre-MCAO levels after 2 hours reperfusion. Neuronal cyclooxygenase-2 induction and PGE2 concentration were greater in cPLA2α+/+ compared to cPLA2α-/- ischemic cortex. Neuronal swelling in ischemic regions was significantly greater in the cPLA2α+/+ than in cPLA2α-/- brains (+/+: 2.2 ± 0.3 fold vs. -/-: 1.7 ± 0.4 fold increase; P < 0.01). The increase in reactive oxygen species following 2 hours of ischemia was also significantly greater in the cPLA2α+/+ ischemic core than in cPLA2α-/- (+/+: 7.12 ± 1.2 fold vs. -/-: 3.1 ± 1.4 fold; P < 0.01). After 6 hours of reperfusion ischemic cortex of cPLA2α+/+, but not cPLA2α-/-, had disruption of neuron morphology and decreased PGE2 content. Phosphorylation of the MAPKs-p38, ERK 1/2, and MEK 1/2-was significantly greater in cPLA2a+/+ than in cPLA2α-/- ischemic cortex 6 hours after reperfusion.ConclusionsThese results indicate that cPLA2α modulates the earliest molecular and injury responses after cerebral ischemia and have implications for the potential clinical use of cPLA2α inhibitors.

1-HydroxyPGE1 reduces infarction volume in mouse transient cerebral ischemia

Differential neurological outcomes due to prostaglandin E2 activating G‐protein‐coupled prostaglandin E (EP) receptors have been observed. Here, we investigated the action of the EP4/EP3 agonist 1‐hydroxyPGE1 (1‐OHPGE1) in modulating transient ischemic brain damage. C57BL/6 mice were pretreated 50 min before transient occlusion of the middle cerebral artery with an intraventricular injection of 1‐OHPGE1 (0.1, 0.2, 2.0 nmol/0.2 µL). Brain damage 4 days after reperfusion, as estimated by infarct volume, was significantly reduced by more than 19% with 1‐OHPGE1 in the two higher‐dose groups (P < 0.05). To further address whether protection also was extended to neurons, primary mouse cultured neuronal cells were exposed to N‐methyl‐d‐aspartate. Co‐treatment with 1‐OHPGE1 resulted in significant neuroprotection (P < 0.05). To better understand potential mechanisms of action and to test whether changes in cyclic adenosine monophosphate (cAMP) levels and downstream signaling would be neuroprotective, we measured cAMP levels in primary neuronal cells. Brief exposure to 1‐OHPGE1 increased cAMP levels more than twofold and increased the phosphorylation of extracellular‐regulated kinases at positions Thr‐202/Tyr‐204. In a separate cohort of animals, 1‐OHPGE1 at all doses tested produced no significant effect on the physiological parameters of core body temperature, mean arterial pressure and relative cerebral blood flow observed following drug treatment. Together, these results suggest that modulation of PGE2 receptors that increase cAMP levels and activate extracellular‐regulated kinases 1/2 caused by treatment with 1‐OHPGE1 can be protective against neuronal injury induced by focal ischemia.

Stroke Outcomes in Mice Lacking the Genes for Neuronal Heme Oxygenase-2 and Nitric Oxide Synthase

Heme oxygenase-2 (HO-2) has been suggested to be a cytoprotective enzyme in a variety of in vivo experimental models. HO-2, the constitutive isozyme, is enriched in neurons and, under normal conditions, accounts for nearly all of brain HO activity. HO-2 deletion (HO-2-/-) leads to increased neurotoxicity in cultured brain cells and increased damage following transient cerebral ischemia in mice. Moreover, pharmacologic inhibition of HO activity significantly augments focal ischemic damage in wildtype (WT) mice, but does not further exacerbate it in HO-2-/- mice. The HO system shares some similarities with nitric oxide synthase (NOS), notably their syntheses of carbon monoxide (CO) and nitric oxide (NO), respectively, which are diffusible gases with numerous biological actions, including neurotransmission and vasodilation. While deletion of HO-2 results in greater stroke damage, the pharmacologic inhibition of neuronal nitric oxide synthase (nNOS), or its gene deletion, confers neuroprotection in animal models of transient cerebral ischemia. To investigate the interactions, the outcome of focal cerebral ischemia-reperfusion in double knockout (HO-2-/- X nNOS-/-) mice lacking both genes was compared to control WT mice. Wildtype and double knockout male mice underwent intraluminal middle cerebral occlusion for 2 hours, followed by reperfusion for 22 hours. Outcomes in neurologic deficits and infarct size were determined. No difference was observed between WT and double knockout mice in the volume of infarction, neurologic signs, decrease in relative cerebral blood flow during ischemia, or core body temperature. The results suggest that the deleterious action of nNOS would counteract the role of HO-2 in neuroprotection.

Perceived benefit of a telemedicine consultative service in a highly staffed intensive care unit.

PURPOSE The aim of this study was to evaluate whether a nocturnal telemedicine service improves culture, staff satisfaction, and perceptions of quality of care in a highly staffed university critical care system. METHODS We conducted an experiment to determine the effect of telemedicine on nursing-staff satisfaction and perceptions of the quality of care in an intensive care unit (ICU). We surveyed ICU nurses using a modified version of a previously validated tool before deployment and after a 2-month experimental program of tele-ICU. Nurses in another, similar ICU within the same hospital academic medical center served as concurrent controls for the survey responses. RESULTS Survey responses were measured using a 5-point Likert scale, and results were analyzed using paired t testing. Survey responses of the nurses in the intervention ICU (n = 27) improved significantly after implementation of the tele-ICU program in the relations and communication subscale (2.99 ± 1.13 pre vs 3.27 ± 1.27 post, P < .01), the psychological working conditions and burnout subscale (3.10 ± 1.10 pre vs 3.23 ± 1.11 post, P < .02), and the education subscale (3.52 ± 0.84 pre vs 3.76 ± 0.78 post, P < .03). In contrast, responses in the control ICU (n = 11) declined in the patient care and perceived effectiveness (3.94 ± 0.80 pre vs 3.48 ± 0.86 post, P < .01) and the education (3.95 ± 0.39 pre vs 3.50 ± 0.80 post, P < .05) subscales. CONCLUSION Telemedicine has the potential to improve staff satisfaction and communication in highly staffed ICUs.

24-hour intensivist staffing: balancing benefits and costs.

For the past three decades, quality of care has been evaluated using the “structure–process–outcome” paradigm of Donabedian (1). In this paradigm, how care is organized (structure) and how care is delivered (process) impacts on the results achieved (outcome). The relationship between structure and o

Cytosolic phospholipase A2 alpha modulates NMDA neurotoxicity in mouse hippocampal cultures

The arachidonic acid‐specific cytosolic phospholipase A2 alpha (cPLA2α) has been implicated in the generation of neurological injuries. cPLA2α‐dependent neurological injury has been postulated to be mediated through inflammatory and eicosanoid pathways. We determined if cPLA2α amplifies the injury of a non‐inflammatory, excitotoxic stimulus by modifying a well‐described toxicity assay to measure the toxicity of N‐methyl‐d‐aspartate (NMDA) in the CA1 region of organotypic, mouse hippocampal cultures. Hippocampal cultures from wild‐type and cPLA2α knockout mice were exposed to 5, 7.5 or 10 µm NMDA for 1 h. Toxicity was measured 23 h later. Cultures derived from cPLA2α–/– mice and cultures treated with the selective inhibitor AACOCF3 were significantly protected from NMDA toxicity, as compared with wild‐type cultures. To determine if cPLA2α‐dependent toxicity is cyclooxygenase (COX)‐2 dependent, COX‐2 and PGE2 levels were measured 7 and 25 h after NMDA treatment. NMDA treatment failed to induce COX‐2 protein or increase PGE2 in the culture media in either genotype at either time. In contrast, phorbol 12‐myristate 13‐acetate and ionophore treatment caused robust induction of COX‐2 and PGE2 in both genotypes. We conclude that cPLA2α may have a hitherto unrecognized direct effect on excitatory neurotoxicity, suggesting that cPLA2α inhibition is a therapeutic candidate for treatment of the early, excitotoxic injury observed in stroke.

Enhancing the Quality of Care in the Intensive Care Unit : A Systems Engineering Approach

This article presents an overview of systems engineering and describes common core principles found in systems engineering methodologies. The Patient Care Program Acute Care Initiative collaboration between the Armstrong Institute of the Johns Hopkins School of Medicine and the Gordon and Betty Moore Foundation, which will use systems engineering to reduce patient harm in the intensive care unit, is introduced. Specific examples of applying a systems engineering approach to the Patient Care Program Acute Care Initiative are presented.

Astrocyte Inositol Triphosphate Receptor Type 2 and Cytosolic Phospholipase A2 Alpha Regulate Arteriole Responses in Mouse Neocortical Brain Slices

Functional hyperemia of the cerebral vascular system matches regional blood flow to the metabolic demands of the brain. One current model of neurovascular control holds that glutamate released by neurons activates group I metabotropic glutamate receptors (mGluRs) on astrocytes, resulting in the production of diffusible messengers that act to regulate smooth muscle cells surrounding cerebral arterioles. The acute mouse brain slice is an experimental system in which changes in arteriole diameter can precisely measured with light microscopy. Stimulation of the brain slice triggers specific cellular responses that can be correlated to changes in arteriole diameter. Here we used inositol trisphosphate receptor type 2 (IP3R2) and cytosolic phospholipase A2 alpha (cPLA2α) deficient mice to determine if astrocyte mGluR activation coupled to IP3R2-mediated Ca2+ release and subsequent cPLA2α activation is required for arteriole regulation. We measured changes in astrocyte cytosolic free Ca2+ and arteriole diameters in response to mGluR agonist or electrical field stimulation in acute neocortical mouse brain slices maintained in 95% or 20% O2. Astrocyte Ca2+ and arteriole responses to mGluR activation were absent in IP3R2− /− slices. Astrocyte Ca2+ responses to mGluR activation were unchanged by deletion of cPLA2α but arteriole responses to either mGluR agonist or electrical stimulation were ablated. The valence of changes in arteriole diameter (dilation/constriction) was dependent upon both stimulus and O2 concentration. Neuron-derived NO and activation of the group I mGluRs are required for responses to electrical stimulation. These findings indicate that an mGluR/IP3R2/cPLA2α signaling cascade in astrocytes is required to transduce neuronal glutamate release into arteriole responses.

Inhibition of cytosolic phospholipase A2 alpha protects against focal ischemic brain damage in mice

It is postulated that inhibition of cytosolic phospholipase A(2) alpha (cPLA(2)α) can reduce severity of stroke injury. This is supported by the finding that cPLA(2)α-deficient mice are partially protected from transient, focal cerebral ischemia. The object of this study was to determine the effect of cPLA(2)α inhibition with arachidonyl trifluoromethyl ketone (ATK) on stroke injury in mice. Male C57BL/6 mice were subjected to 1h of focal cerebral ischemia followed by 24 or 72 h of reperfusion. Mice were treated with ATK or vehicle by intermittent intraperitoneal injection or continuous infusion via an implanted infusion pump. ATK injections 1h before and then 1 and 6h after the start of reperfusion significantly reduced infarction volumes in striatum and hemisphere after 24h of reperfusion. ATK did not reduce injury if it was not administered before onset of ischemia or was not administered after 6h of reperfusion. Intermittent doses of ATK failed to reduce infarct volume after 72 h of reperfusion. Continuous infusion with ATK throughout 72h of reperfusion significantly reduced cortical and whole hemispheric infarct volume compared to vehicle treatment. Following ischemia and reperfusion, ATK treatment significantly reduced brain PLA(2) activity. These results are the first to demonstrate a therapeutic effect of cPLA(2)α inhibition on ischemia and reperfusion injury and define a therapeutic time window. cPLA(2)α activity augments injury in the acute and delayed phases of cerebral ischemia and reperfusion injury. We conclude that cPLA(2)α inhibition may be clinically useful if started before initiation of cerebral ischemia.