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Hippocampal astrocytes were acutely isolated by papain treatment and mechanical trituration. Astrocytes were identified by their distinctive stellate morphology and immunocytochemical staining for glial fibrillary acidic protein. The electrophysiological properties of these cells were investigated using whole-cell voltage-clamp techniques. Three kinetically and pharmacologically distinct voltage-activated K+ currents were identified in most cells; they resembled the neuronal A- current, delayed rectifier, and inward rectifier. The activation threshold of the A-current was -40 mV with a time to peak that ranged from 10 msec at -20 mV to 6 msec at 100 mV. Steady-state inactivation was observed when the holding potential was positive to -100 mV. The current was half-inactivated at -60 mV and totally inactivated at -20 mV. The A-current was suppressed by 4-aminopyridine (4-AP). The delayed rectifier was activated by depolarizing pulses more positive than -40 mV and had a half time of activation that ranged from 18 msec at -20 mV to 10 msec at potentials more positive than 40 mV. This current did not inactivate during a 100 msec pulse and was suppressed by extracellular tetraethylammonium (TEA). An inwardly rectifying current was elicited by hyperpolarizing pulses more negative than -80 mV. This current was not blocked by extracellular TEA or 4-AP and was never observed in the presence of external Ba2+. Voltage-activated inward Na+ currents were never observed. Voltage-activated K+ channels may enhance the local K+ spatial buffering capabilities of the astrocyte syncytium when extracellular [K+] increases during neuronal activity.

Vasopressin has been proposed as a potent vasoactive agent in the treatment of vasodilatory shock in adults and children. The objective of this trial was to evaluate the efficacy and safety of vasopressin as an adjunctive agent in pediatric vasodilatory shock.In this multicenter, double-blind trial, children with vasodilatory shock were randomized to receive low-dose vasopressin (0.0005-0.002 U/kg/min) or placebo in addition to open-label vasoactive agents. Vasoactive infusions were titrated to clinical endpoints of adequate perfusion. The primary outcome was time to vasoactive-free hemodynamic stability. Secondary outcomes included mortality, organ-failure-free days, length of critical care unit stay, and adverse events.Sixty-five of 69 children (94%) who were randomized received the study drug (33 vasopressin, 32 placebo) and were included in the analysis. There was no significant difference in the primary outcome between the vasopressin and placebo groups (49.7 vs. 47.1 hours; P = 0.85). There were 10 deaths (30%) in the vasopressin group and five (15.6%) in the placebo group (relative risk, 1.94; 95% confidence interval, 0.75-5.05; P = 0.24). There were no significant differences with respect to organ failure-free days (22 vs. 25.5 days; P = 0.11), ventilator-free days (16.5 23 days; P = 0.15), length of stay (8 vs. 8.5 days; P = 0.93), or adverse event rate ratios (12.0%; 95% confidence interval, -2.6 to 26.7; P = 0.15).Low-dose vasopressin did not demonstrate any beneficial effects in this pediatric trial. Although not statistically significant, there was a concerning trend toward increased mortality. Clinical trial registered with www.controlled-trials.com (ISRCTN11597444).

he external ventricular drain(EVD) is a catheter insertedpercutaneously into the ven-tricular system and connectedto an external strain gauge transducer.EVDs allow for both measurement of in-tracranial pressure and therapeuticdrainage of cerebrospinal fluid. EVD in-sertion is considered the most accurate,reliable, and cost-effective method for in-tracranial pressure monitoring in adults(1, 2). EVDs are also frequently used inthe pediatric population; however, thereare only a few published reports on theirindications (3–9) and specific complica-tions (6, 10–13). Total complicationswith EVD insertions in pediatric patientsare largely unknown, yet critical to un-derstand the risks associated with EVDinsertion, because EVD complications areoften interrelated (i.e., hemorrhage andinfection). Indeed, the recent pediatricguidelines for traumatic brain injury(TBI) management emphasize the needfor studies on EVD use in pediatric pa-tients (2).The adult literature regarding thesafety of intracranial pressure monitorsvaries widely when assessing rates ofcomplications, but generally intracranialpressure monitor placement is thoughtto be relatively safe (2, 14). The immedi-ate complications associated with EVDplacement include misplacement (10, 15)and hemorrhage (10, 11, 16). It is plau-sible that specific patient populations areat increased susceptibility to these imme-diate complications (i.e., TBI with slitventricles), but this has not been fullyaddressed in the literature. Delayed com-plications are primarily infection related.Studies examining the risk factors of pa-tients who develop secondary infections(17–25) and methods of antibiotic pro-phylaxis (22, 26–30) exist, but few studiesaddress these issues in children (11, 31).Furthermore, the rates of secondary in-fection with EVD insertion at the bedsidein the pediatric critical care unit (PCCU),compared with EVD insertion in the op-eratingroom(OR)undermorecontrolledenvironmental conditions, have not beenaddressed.Theaimsofthisstudyweretodescribethe indications and determine the com-plicationsassociatedwiththeuseofEVDsin the pediatric population. The relativerisk of EVD placement in patients withTBI with small ventricles was comparedwith hydrocephalus patients with dilatedventricles using the hypothesis that thesmaller ventricles found in patients withTBI would predispose to more EVD inser-tion complications. The relative risk be-tween EVD insertions occurring at thebedside in the PCCU vs. the OR was alsodetermined with the hypothesis thatthere would be a lower rate of EVD com-plications with EVDs inserted in the ORbecause of a more controlled surgical en-vironment.

We report a 13-month-old infant who developed acutely elevated intracranial pressure (ICP) as a result of a spontaneous intracerebral haemorrhage (ICH), secondary to idiopathic thrombocytopenic purpura (ITP). Her ICP remained severely elevated despite aggressive medical measures, with persistent obtundation, right hemiparesis and a dilated left pupil. Bilateral decompressive craniectomies (DCs) were performed, which resulted in a rapid decline in ICP. Ultimately, the patient regained consciousness and went on to complete neurological recovery. Tragically, she died of non-neurological, ITP-related complications 9 months later. In our review, we identified no other instances of bilateral DCs reported in the management of an infant with ITP and/or an ICH. We addressed three central questions: (1) Is there any value of DCs in children, and especially in infants, with elevated ICP? (2) Is there any value of DCs in the setting of non-traumatic ICH? And (3) is there any rationale for the use of bilateral versus unilateral DCs?

We report the successful management of a five-year-old child with severe diabetic ketoacidosis with dehydration, who received his initial resuscitative fluids and a continuous infusion of insulin via an intraosseous needle. The patient had presented to a remote community hospital and intravenous access could not be gained. The correction of hyperglycaemia and metabolic acidaemia was achieved at a rate comparable to intravenous therapy. No complications were observed. Although intraosseous access is well described in paediatric resuscitation guidelines, it is not mentioned in International Diabetes Society guidelines for the management of diabetic ketoacidosis. Alternatives to intravenous administration of insulin delivery recommended in such guidelines, such as the subcutaneous or intramuscular routes, may be less appropriate than the intraosseous route. This route can also allow resuscitation fluids and other drugs to be reliably administered in children with diabetic ketoacidosis and severe dehydration where intravenous access can not be attained. We suggest that the potential role of intraosseous access, when intravenous access can not be obtained, should be considered when management guidelines for paediatric diabetic ketoacidosis with dehydration are reviewed.

Cerebral edema is the most common neurologic complication of diabetic ketoacidosis in children. A minority of young patients with intracerebral crises in diabetic ketoacidosis present with cerebrovascular accidents. We report 2 adolescent patients with diabetic ketoacidosis who presented with coma and diffuse white matter hemorrhages in the absence of either cerebral edema or cerebrovascular accidents. These 2 cases illustrate a novel clinical and neuropathologic description of diffuse white matter hemorrhages, possibly related to a cytotoxic process as the underlying mechanism. These case descriptions emphasize that pediatric patients with diabetic ketoacidosis and coma can present with pathology not related to either cerebral edema or cerebrovascular accidents.

A 3-year-old girl was struck by a vehicle travelling at a high speed and was thrown approximately 6 m. Emergency medical services staff noted that the patient was seizing with left-deviated eyes. She was rushed to the closest community hospital for resuscitation and was subsequently transferred to

OBJECTIVES To describe the changes that have occurred in mechanical ventilation in children with acute lung injury in our institution over the last 10-15 yrs and to examine the impact of these changes, in particular of the delivered tidal volume on mortality. DESIGN Retrospective study. SETTING University-affiliated children's hospital. PATIENTS The management of mechanical ventilation between 1988 and 1992 (past group, n = 79) was compared with the management between 2000 and 2004 (recent group, n = 85). INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS The past group patients were ventilated with a significantly higher mean tidal volume (10.2 +/- 1.7 vs. 8.1 +/- 1.4 mL.kg actual body weight, p < .001), lower levels of positive end-expiratory pressure (6.1 +/- 2.7 vs. 7.1 +/- 2.4 cm H2O, p = .007), and higher mean peak inspiratory pressure (31.5 +/- 7.3 vs. 27.8 +/- 4.2 cm H2O, p < .001) than the recent group patients. The recent group had a lower mortality (21% vs. 35%, p = .04) and a greater number of ventilator-free days (16.0 +/- 9.0 vs. 12.6 +/- 9.9 days, p = .03) than the past group. A higher tidal volume was independently associated with increased mortality (odds ratio 1.59; 95% confidence interval 1.20, 2.10, p < .001) and reduction in ventilation-free days (95% confidence interval -1.24, -0.77, p < .001). CONCLUSIONS The changes in the clinical practice of mechanical ventilation in children in our institution reflect those reported for adults. In our experience, mortality among children with acute lung injury was reduced by 40%, and tidal volume was independently associated with reduced mortality and an increase in ventilation-free days.

Cerebral edema associated with diabetic ketoacidosis (DKA-CE) is a serious complication that occurs in 1–3% of children during DKA treatment. There is currently no consensus on the etiology of DKA-...

Objectives: The aims of this report are to 1) present a rare case of fatal cerebral edema associated with late-onset ornithine tran- scarbamylase (OTC) deficiency in a juvenile male patient receiving valproic acid and 2) review the neuropathologic changes associ- ated with the hyperammonemia. Design: Case report. Setting: A community hospital and a tertiary pediatric critical care unit. Interventions: Carbohydrate administration, intravenous nitro- gen excretion cocktail, and high-flux hemodialysis. Measurements and Main Results: Despite aggressive therapy for presumed late-onset OTC deficiency, the patient rapidly de- veloped fatal cerebral edema with tonsillar herniation. A liver biopsy confirmed OTC deficiency with 3% of residual hepatic enzyme activity. Chromosomal analysis showed a normal male karyotype. A thorough molecular analysis of the coding region in the OTC gene Xp21.1 was completed, but mutations were not identified, suggesting an upstream or downstream abnormality. Severe brain swelling was evident on neuropathology, and histo- pathology showed Alzheimer type II astrocytes, neuronal cyto- plasmic changes, and hypertrophy and eosinophilia of the small arterial walls. Conclusions: OTC deficiency is the most common urea cycle defect causing hyperammonemia. Late-onset presentations of OTC are infrequent, primarily affecting female patients. We present a rare case of a late-onset OTC deficiency in a juvenile male patient receiving valproic acid therapy who developed fatal cerebral edema. Valproic acid exacerbates acute elevations in ammonia and may contribute synergistically with ammonia to cerebral mitochondrial dysfunction. (Pediatr Crit Care Med 2006; 7:273-276)

The administration of drotrecogin alfa (activated) improves outcome in adult patients with severe sepsis. Since the published pediatric experience with this drug is limited, the role of drotrecogin alfa (activated) in children, and especially in newborns is not well established. We describe a 3-day-old neonate with septic shock and multiorgan system failure, including circulatory, respiratory, renal failure, and disseminated intravascular coagulation, refractory to intensive fluid resuscitation and inotrope support. Within hours of drotrecogin alfa (activated) administration, the neonate experienced dramatic improvement in hemodynamic parameters. The infusion was discontinued after 48 hours, without clinical deterioration. Aside from transient thrombocytopenia, no significant side effects were observed. A brain MRI performed on day 18 after discontinuation of treatment was normal. The positive hemodynamic effect and outcome of treatment in this patient, indicates that drotrecogin alfa (activated) may play a similar role in the treatment of sepsis in neonates as already established in adults.

Systemic adrenocorticotropic hormone (ACTH) administration is a first-line therapy for the treatment of infantile spasms, an age-specific seizure disorder of infancy. It is proposed that exogenous ACTH acts via negative feedback to suppress the synthesis of corticotropin-releasing hormone (CRH), a possible endogenous convulsant in infant brain tissue. The aim of this study was to determine whether systemic ACTH treatment in infant rats down-regulates the hippocampal CRH system, including CRH, CRH-binding protein (CRH-BP), and CRH receptors (CRH-R1 and CRH-R2). Daily i.p. injection of ACTH for 7 consecutive days (postnatal days 3-9) elevated serum corticosterone levels 20-fold measured on postnatal day 10, indicating systemic absorption and circulation of the ACTH. Semiquantitative reverse transcriptase-PCR demonstrated that both CRH and CRH-BP mRNA obtained from the hippocampi of ACTH-injected infant rats was significantly depressed relative to saline-injected animals. Comparable reductions in both CRH and CRH-BP synthesis were further demonstrated with radioimmunoassay. In contrast, neither CRH-R1 nor CRH-R2 mRNA was altered by ACTH treatment, relative to saline-injected rats. This latter finding was confirmed electrophysiologically by measuring the enhancement of hippocampal population spikes by exogenous CRH, also showing no differences between ACTH- and saline-injected rats. The results of this study support the proposal that systemic ACTH treatment down-regulates CRH expression in infant brain, perhaps contributing to the therapeutic efficacy observed during treatment of infantile spasms.

The authors analyzed blood metabolites in nine children with epilepsy prior to starting the ketogenic diet (KD) and 3 to 4 weeks after KD therapy. Elevated beta-hydroxybutyrate and cortisol levels were observed in all children on the KD. Free fatty acids increased 2.2-fold on the KD, with significant elevations in most polyunsaturated fatty acids (PUFA; arachidonate increased 1.6- to 2.9-fold and docosahexaenoate increased 1.5- to 4.0-fold). The rise in total serum arachidonate correlated with improved seizure control. Elevated PUFA may represent a key anticonvulsant mechanism of the KD.

Epilepsy is a serious neurological disease that responds to two very different treatments involving lipids. Clinically, it responds to a state of ketosis induced by a very high-fat ‘ketogenic’ diet. Experimentally, in vitro and in vivo models demonstrate that injection or infusion of free (non-esterified) polyunsaturates such as arachidonate and docosahexaenoate also reduces seizure susceptibility. In our experience, rats on a very high-fat ketogenic diet not only have mild-to-moderate ketosis, but also have raised serum free fatty acids. Some polyunsaturates, particularly linoleate and α-linolenate, are relatively easily β-oxidized and are therefore ketogenic. We conclude that raised levels of free plasma polyunsaturates could contribute to the beneficial effect of the ketogenic diet in refractory epilepsy not only by helping sustain ketosis, but also by their own direct (though poorly defined) antiseizure effects.

Cholinergic stimulation of the hippocampal formation results in excitation and/or seizure. We report here, using whole-cell patch-clamp techniques in the hippocampal slice (34–35°C), a cholinergic-dependent slow afterdepolarization (sADP) and long-lasting plateau potential (PP). In the presence of 20 μmcarbachol, action potential firing evoked by weak intracellular current injection elicited an sADP that lasted several seconds. Increased spike firing evoked by stronger depolarizing stimuli resulted in long-duration PPs maintained close to −20 mV. Removal of either Na+or Ca2+from the external media, intracellular Ca2+([Ca2+]i) chelation with 10 mmbis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid, or the addition of 100 μmCd2+to the perfusate abolished both the sADP and PP. The sADP was depressed and the PP was abolished by either 10 μmnimodipine or 1 μmω-conotoxin, whereas 1.2 μmtetrodotoxin was ineffective. The involvement of a Na+/Ca2+exchanger was minimal because both the sADP and PP persisted after equimolar substitution of 50 mmLi+for Na+in the external media or reduction of the bath temperature to 25°C. Finally, in the absence of carbachol the sADP and PP could not be evoked when K+channels were suppressed, suggesting that depression of K+conductances alone was not sufficient to unmask the conductance. Based on these data, we propose that a Ca2+-activated nonselective cation conductance was directly enhanced by muscarinic stimulation. The sADP, therefore, represents activation of this conductance by residual [Ca2+]i, whereas the PP represents a novel regenerative event involving the interplay between high-voltage-activated Ca2+channels and the Ca2+-activated nonselective cation conductance. This latter mechanism may contribute significantly to ictal depolarizations observed during cholinergic-induced seizures.

GABA receptors are distributed widely throughout the central nervous system on a variety of cell types. It has become increasingly clear that astrocytes, both in cell culture and tissue slices, express abundant GABAA receptors. In astrocytes, GABA activates Cl−-specific channels that are modulated by barbiturates and benzodiazepines; however, the neuronal inverse agonist methyl-4-ethyl-6, 7-dimethoxy-β-carboline-3-car-boxylate enhances the current in a subpopulation of astrocytes. The properties of astrocytic GABAA receptors, therefore, are remarkably similar to their neuronal counterparts, with only a few pharmacological exceptions. In stellate glial cells of the pituitary pars intermedia, GABA released from neuronal terminals activates postsynaptic potentials directly. The physiological significance of astrocytic GABAA-receptor activation remains unknown, but it may be involved in extracellular ion homeostasis and pH regulation. At present, there is considerably less evidence for the presence of GABAB receptors on astrocytes. The data that have emerged, however, indicate a prominent role for second-messenger regulation by this receptor. © 1994 Wiley-Liss, Inc.

In cultures of embryonic and adult mouse striatum, we previously demonstrated that EGF induces the proliferation of putative stem cells, which give rise to spheres of undifferentiated cells that can generate neurons and astrocytes. We report here that the spheres of undifferentiated cells contain mRNA and protein for the FGF receptor (FGFR1). Indirect immunocytochemistry demonstrated that many of the cells within the EGF-generated spheres were immunoreactive for FGFR1. Exogenous application of bFGF to the EGF-generated cells induced the proliferation of two progenitor cell types. The first, a bipotent progenitor cell, gave rise to cells with the antigenic and morphological properties of neurons and astrocytes; the other gave rise to cells with neuronal characteristics only. bFGF-generated cells with neuronal morphology exhibited electrophysiological properties indicative of immature central neurons. These results support the hypothesis that sequential actions of growth factors play a role in regulating the generation of neurons and astrocytes in the developing CNS.

In the striatum, dopamine generates arachidonic acid (AA) and induces synaptic depression. Here, we report that Na+ channels are a target for AA in both cultured and acutely isolated striatal neurons. AA depressed veratrine-induced Na+ influx and neurotransmitter release. Whole-cell voltage clamp revealed that peak Na+ currents are depressed, and steady-state inactivation shifts -15 mV in the presence of AA. Furthermore, inactivation was accelerated, and recovery from inactivation was delayed. These actions of AA were not produced by AA metabolites or protein kinase C activation. In addition, AA reduced both the amplitude and frequency of action potentials and depressed spontaneous inhibitory postsynaptic currents without affecting miniature inhibitory postsynaptic currents. These data suggest that AA modulates presynaptic, Na(+)-dependent action potentials, thereby contributing to striatal synaptic depression.

Background Identifying who is injured within a geographic region, by what mechanism, is essential for injury prevention (IP). Our objective was to define the injury and spatial epidemiology of severe adult traumas to prioritise and target initiatives. Methods Epidemiologic profiles were generated for severely injured (ISS>12) adult (≥18 years) patients treated at Lead Trauma Hospitals (LTH) in Southwestern Ontario, 2004–2009. Sub-analysis was undertaken by age groups (18–24; 25–64; 65+ years). Injury cases were mapped by patient residence and place of injury to examine spatial relationships. Results LTHs resuscitated 2804 severely injured adults (15% young adult, 55% adult, 30% senior; 72% male). Patient residences were dispersed throughout SWO, with clusters in cities and lower-income areas. MVCs accounted for 61% and 46% of injuries among young adults and adults, respectively. Only 60% of injured-occupants wore a seatbelt; 24% of drivers had a BAC above the legal limit. MVCs were overly concentrated on high-density urban areas with highly mixed land uses. Alcohol was involved with nearly one-third of non-senior severe injury (48% of assaults; 34% of crashes). Falls were the leading injury mechanism for seniors (68%); 67% occurred at home. Only 6% of patients were injured at work, half involved falls. Mortality was 15%, with 42% fall-related deaths. Conclusion Integrating injury epidemiology with geographic data on patients daily surroundings allowed for the identification of socio-spatial variations in injury patterns among vulnerable groups. This approach identified MVCs, falls and alcohol use as IP priorities to be targeted to the populations and regions of greatest need.

Introduction Sepsis-related multiple organ dysfunction is a common cause of death in the intensive care unit. The effect of sepsis on markers of tissue repair is only partly understood. The aim of this study was to measure markers of collagen synthesis and degradation during sepsis and investigate the association with disease severity and outcome. Methods Forty-four patients with severe sepsis participated in the study and 15 volunteers acted as controls. Blood samples were collected for 10 days after the first sepsis-induced organ dysfunction and after three and six months. Procollagen type I and III aminoterminal propeptides (PINP and PIIINP) and cross-linked telopeptides of type I collagen (ICTP) were measured. Results The PIIINP concentration was elevated in the septic patients (8.8 ug/L, 25th to 75th percentile = 6.8 to 26.0) when compared with controls (3.0 ug/L, 25th to 75th percentile = 2.7 to 3.3; P < 0.001) on day one. Maximum serum PIIINP concentrations during sepsis were higher in non-survivors compared with survivors (26.1 ug/L, 25th to 75th percentile = 18.7 to 84.3; vs. 15.1 ug/L, 25th to 75th percentile = 9.6 to 25.5; P = 0.033) and in multiple organ failure (MOF) compared with multiple organ dysfunction syndrome (MODS) (24.2 ug/L, 25th to 75th percentile = 13.4 to 48.2; vs. 8.9 ug/L, 25th to 75th percentile = 7.4 to 19.4; P = 0.002). Although the PINP values of the septic patients remained within the laboratory reference values, patients with MOF had higher values than patients with MODS (79.8, 25th to 75th percentile = 44.1 to 150.0; vs.40.4, 25th to 75th percentile = 23.6 to 99.3; P = 0.007). Day one ICTP levels were elevated in septic patients compared with the controls (19.4 ug/L, 25th to 75th percentile = 12.0 to 29.8; vs. 4.1 ug/L, 25th to 75th percentile = 3.4 to 5.0; P < 0.001). Conclusions Markers of collagen metabolism are increased in patients with severe sepsis and can be investigated further as markers of disease severity and outcome.

Background: In some patients, SARS-CoV-2 infection induces cytokine storm, hypercoagulability and endothelial cell activation leading to worsening of COVID-19, intubation and death. Prompt identification of patients at risk of intubation or death is un unmet need. Objective: To derive a prognostic score for the risk of intubation or death in patients with critical COVID-19 by assessing biomarkers of hypercoagulability, endothelial cell activation and inflammation and a large panel of clinical analytes. Methods: We conducted a prospective, observational monocentric study enrolling 118 patients with COVID-19 admitted in the intensive care unit. At the 1st day of ICU admission all patients were assessed for the following biomarkers : protein C, protein S, antithrombin, D-Dimer, fibrin monomers, factors VIIa, V, XII, XII, VIII, von Willebrand antigen, fibrinogen, procoagulant phospholipid dependent clotting time, TFPI, thrombomodulin, P-selectin, heparinase, microparticles exposing tissue factor, IL-6, complement C3a, C5a, thrombin generation, prothrombin time, activated partial thromboplastin time, hemogram, platelet count) and clinical predictors. The clinical outcomes were intubation and mortality during hospitalization in ICU. Results: The intubation and mortality rate were 70 % and 18% respectively. Multivariate analysis led to the derivation of the COMPASS- COVID19-ICU score composed of P-Selectin, D-Dimer, free TFPI, TF activity, IL-6 and FXII, age and duration of hospitalization. The score predicted the risk of intubation or death with high sensitivity and specificity (0.90 and 0.92, respectively). Conclusions and Relevance: Critical COVID-19 is related with severe endothelial cell activation and hypercoagulability orchestrated in the context of inflammation. The COMPASS-COVID19-ICU score is an accurate predictive model for the evaluation of the risk of mechanical ventilation and death in patients with critical COVID-19. The assessment with the COMPASS- COVID-19-ICU score is feasible in tertiary hospitals. In this context it could be placed in the diagnostic procedure of personalized medical management and prompt therapeutic intervention. Disclosures Terpos: Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Takeda: Honoraria; Beigene: Honoraria; Janssen: Honoraria.

We previously identified cholinergic-dependent plateau potentials (PPs) in CA1 pyramidal neurons that were intrinsically generated by interplay between voltage-gated calcium entry and a Ca2+-activated nonselective cation conductance. In the present study, we examined both the second-messenger pathway and the role of synaptic inhibition in the expression of PPs. The stimulation of m1/m3 cholinergic receptor subtypes and G-proteins were critical for activating PPs because selective receptor antagonists (pirenzepine, hexahydro-sila-difenidol hydrochloride, 4-diphenylacetoxy- N-methylpiperidine methiodide) and intracellular guanosine-5′- O-(2-thiodiphosphate) prevented PP generation in carbachol. Intense synaptic stimulation occasionally activated PPs in the presence of oxytremorine M, a cholinergic agonist with preference for m1/m3 receptors. PPs were consistently activated by synaptic stimulation only when oxytremorine M was combined with antagonists at both GABAA and GABAB receptors. These latter data indicate an important role for synaptic inhibition in preventing PP generation. Both intrinsically generated and synaptically activated PPs could not be elicited following inhibition of serine/threonine protein phosphatases by calyculin A, okadaic acid, or microcystin-L, suggesting that muscarinic-induced dephosphorylation is necessary for PP generation. PP genesis was also inhibited following irreversible thiophosphorylation by intracellular perfusion with ATP-γ-S. These data indicate that the expression of cholinergic-dependent PPs requires protein phosphatase-induced dephosphorylation via G-protein–linked m1/m3 receptor(s). Moreover, synaptic inhibition via both GABAA and GABAB receptors normally prevents the synaptic activation of PPs. Understanding the regulation of PPs should provide clues to the role of this regenerative potential in both normal activity and pathophysiological processes such as epilepsy.

Introduction Sepsis-associated encephalopathy (SAE) is a state of acute brain dysfunction in response to a systemic infection. We propose that systemic inflammation during sepsis causes increased adhesion of leukocytes to the brain microvasculature, resulting in blood-brain barrier dysfunction. Thus, our objectives were to measure inflammatory analytes in plasma of severe sepsis patients to create an experimental cytokine mixture (CM), and to use this CM to investigate the activation and interactions of polymorphonuclear leukocytes (PMN) and human cerebrovascular endothelial cells (hCMEC/D3) in vitro. Methods The concentrations of 41 inflammatory analytes were quantified in plasma obtained from 20 severe sepsis patients and 20 age- and sex-matched healthy controls employing an antibody microarray. Two CMs were prepared to mimic severe sepsis (SSCM) and control (CCM), and these CMs were then used for PMN and hCMEC/D3 stimulation in vitro. PMN adhesion to hCMEC/D3 was assessed under conditions of flow (shear stress 0.7 dyn/cm2). Results Eight inflammatory analytes elevated in plasma obtained from severe sepsis patients were used to prepare SSCM and CCM. Stimulation of PMN with SSCM led to a marked increase in PMN adhesion to hCMEC/D3, as compared to CCM. PMN adhesion was abolished with neutralizing antibodies to either β2 (CD18), αL/β2 (CD11α/CD18; LFA-1) or αM/β2 (CD11β/CD18; Mac-1) integrins. In addition, immune-neutralization of the endothelial (hCMEC/D3) cell adhesion molecule, ICAM-1 (CD54) also suppressed PMN adhesion. Conclusions Human SSCM up-regulates PMN pro-adhesive phenotype and promotes PMN adhesion to cerebrovascular endothelial cells through a β2-integrin-ICAM-1-dependent mechanism. PMN adhesion to the brain microvasculature may contribute to SAE.