RESUMEN

Necroptosis is a newly described form of regulated necrosis that contributes to neuronal death in experimental models of stroke and brain trauma. Although much work has been done elucidating initiating mechanisms, signaling events governing necroptosis remain largely unexplored. Akt is known to inhibit apoptotic neuronal cell death. Mechanistic target of rapamycin (mTOR) is a downstream effector of Akt that controls protein synthesis. We previously reported that dual inhibition of Akt and mTOR reduced acute cell death and improved long term cognitive deficits after controlled-cortical impact in mice. These findings raised the possibility that Akt/mTOR might regulate necroptosis. To test this hypothesis, we induced necroptosis in the hippocampal neuronal cell line HT22 using concomitant treatment with tumor necrosis factor α (TNFα) and the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. TNFα/zVAD treatment induced cell death within 4 h. Cell death was preceded by RIPK1-RIPK3-pAkt assembly, and phosphorylation of Thr-308 and Thr473 of AKT and its direct substrate glycogen synthase kinase-3ß, as well as mTOR and its direct substrate S6 ribosomal protein (S6), suggesting activation of Akt/mTOR pathways. Pretreatment with Akt inhibitor viii and rapamycin inhibited Akt and S6 phosphorylation events, mitochondrial reactive oxygen species production, and necroptosis by over 50% without affecting RIPK1-RIPK3 complex assembly. These data were confirmed using small inhibitory ribonucleic acid-mediated knockdown of AKT1/2 and mTOR. All of the aforementioned biochemical events were inhibited by necrostatin-1, including Akt and mTOR phosphorylation, generation of oxidative stress, and RIPK1-RIPK3-pAkt complex assembly. The data suggest a novel, heretofore unexpected role for Akt and mTOR downstream of RIPK1 activation in neuronal cell death.

Asunto(s)

Hipocampo/enzimología , Neuronas/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Inhibidores de Caspasas/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Activación Enzimática , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hipocampo/efectos de los fármacos , Hipocampo/patología , Imidazoles/farmacología , Indoles/farmacología , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Necrosis , Neuronas/efectos de los fármacos , Neuronas/patología , Oligopéptidos/farmacología , Estrés Oxidativo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína S6 Ribosómica/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Factores de Tiempo , Transfección , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

OBJECTIVE: Granulocyte colony stimulating factor (GCSF) has been used to increase systemic absolute neutrophil count (ANC) in patients with severe traumatic brain injury to reduce nosocomial infection risk. However, the effect of increasing systemic ANC on the pathogenesis of experimental traumatic brain injury has not been studied. Thus, we evaluated the effect of systemic ANC on blood-brain barrier (BBB) damage and brain edema after traumatic brain injury in rats. DESIGN: Experimental study. SETTING: Research laboratory at the University of Pittsburgh, PA. SUBJECTS: Forty-three adult male Sprague-Dawley rats. INTERVENTIONS: Protocol I: rats were randomized to receive either vinblastine sulfate to reduce ANC, GCSF to increase ANC, or saline before controlled cortical impact (CCI) of moderate overall severity. Evans blue was used to assess BBB damage at 4-24 hrs after CCI. Protocol II: rats received GCSF or saline before CCI. Brain edema was estimated at 24 hrs using wet - dry) / wet weight method. Protocol III: rats received GCSF or saline before CCI. Brain neutrophil accumulation was estimated at 24 hrs using a myeloperoxidase assay. MEASUREMENTS AND MAIN RESULTS: Physiologic variables were controlled before CCI was maintained at normal in all animals before traumatic brain injury. No rats were anemic, hypoglycemic, or hypotensive before CCI. Protocol I: compared with control, systemic ANC decreased in vinblastine-treated rats and increased in GCSF-treated rats. BBB damage correlated with systemic ANC. Protocol II: mean systemic ANC before traumatic brain injury increased 15-fold in rats given GCSF vs. control; however no difference in brain edema was observed at 24 hrs after injury between groups. Protocol III: median systemic ANC at the time of CCI was increased ten-fold in rats given GCSF vs. control. No difference in brain myeloperoxidase activity 24 hrs after CCI was observed in rats treated with GCSF vs. control. CONCLUSIONS: Systemic ANC influences BBB damage after traumatic brain injury produced by CCI. Because BBB damage and brain edema are discordant, mechanisms other than BBB damage likely predominate in the pathogenesis of brain edema after contusion. The implications of increased BBB permeability with the administration of GCSF in our model remains to be determined. Increasing systemic ANC before CCI with GCSF administration does not increase posttraumatic brain neutrophil accumulation or brain edema after CCI in rats. The finding that neutrophil infiltration is not enhanced by systemic neutrophilia suggests that the ability of GCSF-stimulated neutrophils to migrate into injured tissue may be impaired. Further studies are needed to evaluate the effects of GCSF administration on secondary injury and functional outcome in experimental models of traumatic brain injury.

Asunto(s)

Barrera Hematoencefálica/inmunología , Edema Encefálico/inmunología , Lesiones Encefálicas/inmunología , Factor Estimulante de Colonias de Granulocitos/fisiología , Leucocitosis/inmunología , Neutropenia/inmunología , Neutrófilos/inmunología , Animales , Permeabilidad Capilar/fisiología , Recuento de Leucocitos , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

OBJECTIVES: To determine whether bcl-2, a protein that inhibits apoptosis, would be increased in cerebrospinal fluid (CSF) in infants and children after traumatic brain injury (TBI) and to examine the association of bcl-2 concentration with clinical variables. STUDY DESIGN: Bcl-2 was measured in CSF from 23 children (aged 2 months-16 years) with severe TBI and from 19 children without TBI or meningitis (control subjects) by enzyme-linked immunosorbent assay. CSF oligonucleosome concentration was also determined as a marker of DNA degradation. Brain samples from 2 patients undergoing emergent decompressive craniectomies were analyzed for bcl-2 with Western blot and for DNA fragmentation with TUNEL (terminal deoxynucleotidyl-transferase mediated biotin-dUTP nick-end labeling). RESULTS: CSF bcl-2 concentrations were increased in patients with TBI versus control subjects (P =.01). Bcl-2 was increased in patients with TBI who survived versus those who died (P =.02). CSF oligonucleosome concentration tended to be increased after TBI (P =.07) and was not associated with bcl-2. Brain tissue samples showed an increase in bcl-2 in patients with TBI versus adult brain bank control samples and evidence of DNA fragmentation within cells with apoptotic morphology. CONCLUSIONS: Bcl-2 may participate in the regulation of cell death after TBI in infants and children. The increase in bcl-2 seen in patients who survived is consistent with a protective role for this anti-apoptotic protein after TBI.

Asunto(s)

Apoptosis , Lesiones Encefálicas/fisiopatología , Proteínas Proto-Oncogénicas c-bcl-2/líquido cefalorraquídeo , Adolescente , Factores de Edad , Análisis de Varianza , Lesiones Encefálicas/líquido cefalorraquídeo , Lesiones Encefálicas/etiología , Lesiones Encefálicas/mortalidad , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Escala de Coma de Glasgow , Humanos , Lactante , Modelos Lineales , Masculino , Análisis Multivariante , Nucleosomas/metabolismo , Pennsylvania/epidemiología , Análisis de Supervivencia , Lóbulo Temporal/metabolismo

RESUMEN

OBJECTIVES: To determine concentrations of circulating adhesion molecules endothelial (E)-selectin, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 in children with sepsis-induced multiple organ failure (MOF), and to determine associations among increased concentrations of these circulating adhesion molecules and important outcome measures. DESIGN: Prospective study. SETTING: University pediatric intensive care unit. PATIENTS: A total of 77 consecutive children with sepsis and 14 acutely ill children without sepsis. INTERVENTIONS: Plasma E-selectin, ICAM-1, and VCAM-1 concentrations and organ failure index (indicating number of failed organ systems) were determined in 77 children on days 1 and 3 of sepsis, and in 14 control children on pediatric intensive care unit day 1. Multivariate logistic regression analysis was used to determine associations between adhesion molecule concentrations and clinically relevant outcome measures. MEASUREMENTS AND RESULTS: Plasma concentrations of E-selectin, ICAM-1, and VCAM-1 were increased in children with sepsis vs. control on day 1 (p < .05). Plasma VCAM-1 (but not ICAM-1 or E-selectin) was increased in children with more than three organ failures vs. children with less than three organ failures (p < .05). Plasma ICAM-1 and VCAM-1 (but not E-selectin) concentrations independently predicted number of organs failed and development of more than three organ failures. Plasma ICAM-1 and VCAM-1 also predicted mortality and development of sequential (pulmonary/hepatic/renal) MOF (p < .05). CONCLUSIONS: The pronounced and persistent increase in plasma VCAM-1 and ICAM-1 that occurs in children with sepsis and persistent MOF may indicate a phenotypic change in endothelium toward a more proinflammatory state. Alternatively, the source for these adhesion molecules may be activated leukocytes and other cell types. Future studies are required to determine the role of ICAM-1 and VCAM-1 in the pathogenesis of sepsis-induced MOF.

Asunto(s)

Selectina E/sangre , Molécula 1 de Adhesión Intercelular/sangre , Insuficiencia Multiorgánica/sangre , Sepsis/sangre , Molécula 1 de Adhesión Celular Vascular/sangre , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Unidades de Cuidado Intensivo Pediátrico , Modelos Logísticos , Masculino , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/mortalidad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Sepsis/complicaciones , Sepsis/mortalidad

RESUMEN

OBJECTIVE: To determine interleukin (IL)-8 concentrations in ventricular cerebrospinal fluid from children with severe traumatic brain injury (TBI). DESIGN: Prospective study. SETTING: University children's hospital. PATIENTS: Twenty-seven children hospitalized with severe TBI (Glasgow Coma Scale score < or =8), seven children with cerebrospinal fluid culture-positive bacterial meningitis, and twenty-four age-equivalent controls. INTERVENTIONS: Placement of an intraventricular catheter and continuous drainage of cerebrospinal fluid. MEASUREMENTS AND MAIN RESULTS: Median [range] cerebrospinal fluid IL-8 concentration in children with TBI (0-12 hrs) (4,452.5 [0-20,000] pg/mL) was markedly greater than that in controls (14.5 [0-250]) (p < .0001) and equivalent to concentrations in children with meningitis (5,300 [1,510-22,000] pg/mL) (p = .33). Cerebrospinal fluid IL-8 remained increased in children with severe TBI for up to 108 hrs after injury. Univariate logistic regression analysis demonstrated an association between cerebrospinal fluid IL-8 and child abuse (p = .07) and mortality (p = .01). Multivariate analysis demonstrated a strong, independent association between cerebrospinal fluid IL-8 and mortality (p = .01). CONCLUSIONS: The data are consistent with an acute inflammatory component of TBI in children and suggest an association between cerebrospinal fluid IL-8 and outcome after TBI. IL-8 may represent a potential target for anti-inflammatory therapy.

Asunto(s)

Traumatismos Craneocerebrales/líquido cefalorraquídeo , Interleucina-8/líquido cefalorraquídeo , Enfermedad Aguda , Adolescente , Biomarcadores/líquido cefalorraquídeo , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Lactante , Modelos Logísticos , Masculino , Meningitis Bacterianas/líquido cefalorraquídeo , Estudios Prospectivos , Factores de Tiempo

RESUMEN

Platelet (P-) selectin and intercellular adhesion molecule-1 (ICAM-1) mediate accumulation of neutrophils in brain. However, the mechanisms regulating neutrophil accumulation and damage after traumatic brain injury (TBI) are poorly defined. We hypothesized that mice deficient in both P-selectin and ICAM-1 (-/-) would have decreased brain neutrophil accumulation and edema, and improved functional and histopathological outcome after TBI compared with wild-type (+/+). In Protocol I, neutrophils and brain water content were quantified at 24 h after TBI. No difference in brain neutrophil accumulation was observed between groups; however, brain edema was decreased in dual P-selectin and ICAM-1 -/- (P < 0.05 vs. +/+ mice). In Protocol II, after TBI, tests of motor and memory function and histopathology were assessed over 21 days. No difference in motor or memory function or histopathological damage was observed between +/+ and -/- mice. A role for adhesion molecules in the pathogenesis of brain edema independent of leukocyte accumulation in brain is suggested.

Asunto(s)

Edema Encefálico/prevención & control , Lesiones Encefálicas/complicaciones , Molécula 1 de Adhesión Intercelular/fisiología , Selectina-P/fisiología , Animales , Barrera Hematoencefálica , Edema Encefálico/etiología , Edema Encefálico/metabolismo , Lesiones Encefálicas/metabolismo , Quimiotaxis de Leucocito , Contusiones/complicaciones , Contusiones/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Masculino , Trastornos de la Memoria/etiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trastornos del Movimiento/etiología , Selectina-P/genética , Peritonitis/inmunología , Heridas no Penetrantes/complicaciones , Heridas no Penetrantes/metabolismo

RESUMEN

Poly (ADP-ribose) polymerase (PARP) is a ubiquitous nuclear enzyme that, when activated by free-radical induced DNA damage, contributes to energy failure and cell death in models of central nervous system ischemia and reperfusion. PARP contributes to neuronal cell death in vivo after cerebral ischemia/reperfusion, however, the role of PARP in the pathogenesis of traumatic brain injury (TBI) is unknown. We hypothesized that, compared to wild type mice (+/+), mice deficient in PARP (-/-) would have reduced motor and cognitive deficits after TBI. Mice underwent controlled cortical impact (CCI) (6 m/s, 1.2 mm depth) and were tested for motor (d 1-5) and cognitive (d 14-18) function after CCI. PARP -/- mice demonstrated improved motor performance and improved cognitive function after CCI (both p < 0.05 compared to +/+). This is the first study to evaluate a role for PARP in functional outcome after TBI. The results suggest a detrimental role for PARP in the pathogenesis of TBI.

Asunto(s)

Edema Encefálico/fisiopatología , Isquemia Encefálica/fisiopatología , Poli(ADP-Ribosa) Polimerasas/fisiología , Animales , Daño Encefálico Crónico/fisiopatología , Supervivencia Celular/fisiología , Lóbulo Frontal/irrigación sanguínea , Lóbulo Frontal/lesiones , Recuerdo Mental/fisiología , Ratones , Ratones Endogámicos , Destreza Motora/fisiología , Poli(ADP-Ribosa) Polimerasas/deficiencia , Flujo Sanguíneo Regional/fisiología , Daño por Reperfusión/fisiopatología

RESUMEN

Controlled cortical impact (CCI) produces blood-brain barrier (BBB) permeability and an acute inflammatory response in injured brain, associated with upregulation of cell adhesion molecules and accumulation of neutrophils. Nevertheless, the role of acute inflammation in the pathogenesis of BBB permeability after traumatic brain injury (TBI) is undefined. The purpose of this study was to examine the time course of acute inflammation and BBB permeability after CCI in rats and to determine the effect of neutrophil depletion on BBB permeability early after CCI. In the first protocol, four groups of rats (n = 4-7/group) were subjected to CCI. Expression of endothelial (E)-selectin on cerebrovascular endothelium, accumulation of neutrophils, and BBB permeability were measured in brain at 1, 4, 8, and 24 hours after injury by immunohistochemistry or spectrophotometric quantification of Evans blue. E-selectin upregulation and neutrophil accumulation in injured brain occurred at later times than maximal BBB permeability. In a second protocol, rats made neutropenic with a murine monoclonal IgM antibody (RP-3) specific for rat neutrophils were subjected to CCI, given Evans blue at 3.5 hours, and sacrificed at 4 hours after injury. Neutrophil depletion did not affect BBB permeability at 4 hours after CCI. We conclude that events other than those mediated by neutrophils initiate BBB permeability early after CCI.

Asunto(s)

Barrera Hematoencefálica/fisiología , Lesiones Encefálicas/fisiopatología , Corteza Cerebral/lesiones , Neutrófilos/fisiología , Animales , Lesiones Encefálicas/patología , Selectina E/biosíntesis , Endotelio Vascular/metabolismo , Inmunoglobulina M/toxicidad , Inmunohistoquímica , Inflamación/patología , Inflamación/fisiopatología , Masculino , Neutropenia/patología , Neutropenia/fisiopatología , Permeabilidad , Ratas , Ratas Sprague-Dawley , Espectrofotometría Ultravioleta , Factores de Tiempo , Regulación hacia Arriba

RESUMEN

Poly(ADP-ribose) polymerase (PARP), or poly-(ADP-ribose) synthetase, is a nuclear enzyme that consumes NAD when activated by DNA damage. The role of PARP in the pathogenesis of traumatic brain injury (TBI) is unknown. Using a controlled cortical impact (CCI) model of TBI and mice deficient in PARP, the authors studied the effect of PARP on functional and histologic outcome after CCI using two protocols. In protocol 1, naive mice (n = 7 +/+, n = 6 -/-) were evaluated for motor and memory acquisition before CCI. Mice were then subjected to severe CCI and killed at 24 hours for immunohistochemical detection of nitrated tyrosine, an indicator of peroxynitrite formation. Motor and memory performance did not differ between naive PARP +/+ and -/- mice. Both groups showed nitrotyrosine staining in the contusion, suggest ing that peroxynitrite is produced in contused brain. In protoco 2, mice (PARP +/+, n = 8; PARP -/-, n = 10) subjected to CCI were tested for motor and memory function, and contusion volume was determined by image analysis. PARP -/- mice demonstrated improved motor and memory function after CC versus PARP +/+ mice (P < 0.05). However, contusion volume was not different between groups. The results suggest a detri mental effect of PARP on functional outcome after TBI.

Asunto(s)

Lesiones Encefálicas/fisiopatología , Lesiones Encefálicas/psicología , Encéfalo/fisiopatología , Cognición/fisiología , Aprendizaje por Laberinto , Memoria/fisiología , Actividad Motora/fisiología , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Encéfalo/enzimología , Encéfalo/patología , Lesiones Encefálicas/genética , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/fisiopatología , Señales (Psicología) , Inmunohistoquímica , Ratones , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasas/deficiencia , Percepción Espacial

Asunto(s)

Lesiones Encefálicas/inmunología , Lesiones Encefálicas/terapia , Hemorragia Cerebral/inmunología , Hemorragia Cerebral/terapia , Infección Hospitalaria/prevención & control , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Lesiones Encefálicas/complicaciones , Hemorragia Cerebral/complicaciones , Infección Hospitalaria/etiología , Humanos , Inflamación/inducido químicamente , Neutrófilos/inmunología

RESUMEN

Intercellular adhesion molecule-1 (ICAM-1) is an adhesion molecule of the immunoglobulin family expressed on endothelial cells that is upregulated in brain as part of the acute inflammatory response to traumatic brain injury (TBI). ICAM-1 mediates neurologic injury in experimental meningitis and stroke; however, its role in the pathogenesis of TBI is unknown. We hypothesized that mutant mice deficient in ICAM-1 (-/-) would have decreased neutrophil accumulation, diminished histologic injury, and improved functional neurologic outcome versus ICAM-1 +/+ wild type control mice after TBI. Anesthetized ICAM-1 -/- mice and wild-type controls were subjected to controlled cortical impact (CCI, 6 m/sec, 1.2 mm depth). Neutrophils in brain parenchyma and ICAM-1 on vascular endothelium were assessed by immunohistochemistry in cryostat brain sections from the center of the contusion 24 h after TBI (n = 4/group). Separate groups of wild-type and ICAM-1-deficient mice (n = 9-10/group) underwent motor (wire grip test, days 1-5) and cognitive (Morris water maze [MWM], days 14-20) testing. Lesion volume was determined by image analysis 21 days following TBI. Robust expression of ICAM-1 was readily detected in choroid plexus and cerebral endothelium at 24 h in ICAM-1 +/+ mice but not in ICAM-1 -/- mice. No differences between groups were observed in brain neutrophil accumulation (9.4 +/- 2.2 versus 11.1 +/- 3.0 per x100 field, -/- versus +/+), wire grip score, MWM latency, or lesion volume (7.24 +/- 0.63 versus 7.21 +/- 0.45 mm3, -/- versus +/+). These studies fail to support a role for ICAM-1 in the pathogenesis of TBI.

Asunto(s)

Reacción de Fase Aguda/fisiopatología , Lesiones Encefálicas/fisiopatología , Molécula 1 de Adhesión Intercelular/fisiología , Reacción de Fase Aguda/metabolismo , Reacción de Fase Aguda/patología , Análisis de Varianza , Animales , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Corteza Cerebral/lesiones , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Quimiotaxis de Leucocito/fisiología , Trastornos del Conocimiento/fisiopatología , Progresión de la Enfermedad , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trastornos del Movimiento/fisiopatología , Neutrófilos/fisiología , Factores de Tiempo

RESUMEN

OBJECTIVE: To measure levels of defensins and lactoferrin in the cerebrospinal fluid (CSF) of children with meningitis. STUDY DESIGN. Prospective descriptive study involving children undergoing lumbar puncture during evaluation for meningitis. METHODS: CSF concentrations of defensins and lactoferrin were determined using enzyme-linked immunosorbent assays on 19 children with bacterial meningitis, 31 children with aseptic meningitis, and 32 control children found to have normal CSF during evaluation for meningitis. Pertinent clinical and laboratory data were gathered on all children. RESULTS: CSF concentrations of both defensins and lactoferrin were elevated markedly in children with bacterial and aseptic meningitis, compared with control children. No control subject had detectable levels of defensins in the CSF. Lactoferrin was undetectable in the CSF of 31 of 32 control subjects. Defensin and lactoferrin levels were significantly higher in the CSF of children with bacterial meningitis than in those with aseptic meningitis. Defensin levels in the CSF of children with bacterial meningitis ranged from 128 ng/mL to 99 430 ng/mL with a mean of 30 311 ng/mL (SD +/- 28 865) and a median of 23 042 ng/mL. Defensin levels in the CSF of children with aseptic meningitis ranged from 0 ng/mL to 1675 ng/mL with a mean of 227 ng/mL (SD +/- 433) and a median of 23 ng/mL. A significant correlation was found between defensin levels in the CSF and the total leukocyte count and the absolute neutrophil count in the CSF of children with bacterial meningitis. Lactoferrin levels in the CSF of children with bacterial meningitis ranged from 184 ng/mL to 31 412 ng/mL with a mean of 13 209 ng/mL (SD +/- 9644) and a median of 10 382 ng/mL. Lactoferrin levels in the CSF of children with aseptic meningitis ranged from 0 ng/mL to 2715 ng/mL with a mean of 1042 ng/mL (SD +/- 878) and a median of 852 ng/mL. No correlation was found between lactoferrin level in the CSF and the total leukocyte count or the absolute neutrophil count in the CSF of children with bacterial meningitis. In our study population, the sum total of CSF defensins and lactoferrin was found to be highly sensitive and specific in delineating bacterial from aseptic meningitis when compared with standard CSF studies. CONCLUSIONS: Significant elevations of defensins and lactoferrin, indicative of endogenous local antimicrobial peptide and polypeptide release, are found in the CSF of children with meningitis. We speculate that elevations in these antimicrobial molecules may reflect the intensity of the host response. Defensins seem to parallel neutrophil activation more closely than lactoferrin. Cumulative levels of CSF defensins and lactoferrin clearly distinguished bacterial meningitis from aseptic meningitis and control patients. Further investigation is warranted to determine the usefulness of measuring defensins and lactoferrin as a diagnostic tool and therapeutic monitor in the evaluation of children with meningitis.

Asunto(s)

Antiinfecciosos/líquido cefalorraquídeo , Lactoferrina/líquido cefalorraquídeo , Meningitis Aséptica/líquido cefalorraquídeo , Meningitis Bacterianas/líquido cefalorraquídeo , Proteínas/análisis , Estudios de Casos y Controles , Niño , Preescolar , Defensinas , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Recuento de Leucocitos , Masculino , Meningitis Aséptica/inmunología , Meningitis Bacterianas/inmunología , Activación Neutrófila , Neutrófilos

RESUMEN

Leukocyte-endothelial adhesion molecules, critical to the development of acute inflammation, are expressed in brain as part of the acute inflammatory response to traumatic brain injury (TBI). We measured the concentrations of the adhesion molecules P-selectin, ICAM-1, E-selectin, L-selectin, and VCAM-1 in ventricular cerebrospinal fluid (CSF) from children with severe TBI (Glasgow coma score < 8) and compared these findings with those from children with bacterial meningitis. P-selectin, an adhesion molecule associated with ischemia/reperfusion, was increased in children with TBI versus meningitis and control. Univariate and multivariate regression analyses demonstrated associations between CSF P-selectin and child abuse and age of < 4 years, and a significant, independent association between CSF intercellular adhesion molecule-1 (ICAM-1) and child abuse. These results are consistent with a specific acute inflammatory component to TBI in children. Future studies of secondary injury mechanisms and therapy after TBI should assess on the roles of P-selectin and ICAM-1 in injury and repair processes in brain after TBI.

Asunto(s)

Lesiones Encefálicas/líquido cefalorraquídeo , Moléculas de Adhesión Celular/líquido cefalorraquídeo , Adolescente , Análisis de Varianza , Biomarcadores/líquido cefalorraquídeo , Lesiones Encefálicas/complicaciones , Estudios de Casos y Controles , Niño , Maltrato a los Niños , Preescolar , Femenino , Humanos , Lactante , Molécula 1 de Adhesión Intercelular/líquido cefalorraquídeo , Masculino , Estudios Prospectivos , Análisis de Regresión , Selectinas/líquido cefalorraquídeo , Molécula 1 de Adhesión Celular Vascular/líquido cefalorraquídeo

Asunto(s)

Electroencefalografía/efectos de los fármacos , Óxido Nítrico/efectos adversos , Vasodilatadores/efectos adversos , Administración por Inhalación , Animales , Preescolar , Humanos , Lactante , Óxido Nítrico/administración & dosificación , Vasodilatadores/administración & dosificación

RESUMEN

We sought to investigate the course and magnitude of blood brain barrier (BBB) permeability following focal and diffuse traumatic brain injury (TBI) in immature rats and examine the time course of markers of acute inflammation (neutrophil accumulation and E-selectin [E-sel] expression) following these two types of injury. We measured BBB permeability using i.v. injection Evans Blue (EB) and the extent of inflammation using immunohistochemical techniques identifying neutrophils (monoclonal antibody RP-3) and the endothelial adhesion molecule, E-selectin. Male Sprague-Dawley immature (17 day-old) rats (30-45 g, n = 80) were subjected to a controlled cortical impact (CCI: 2 mm, 4 m/s), a closed head diffuse injury (DI: 150 g/2m) or a corresponding sham procedure (with or without craniotomy). EB was injected i.v. at 30 min before sacrifice, which occurred at 1 h, 4 h, or 24 h after injury. BBB permeability was observed in both the CCI and DI rats at 1 h after injury which largely resolved by 24 h. In the CCI, EB extravasation was seen within and around the contusion. In DI, diffuse BBB permeability was seen. DI was not associated with acute inflammation since there was neither neutrophil accumulation nor E-selectin expression. The CCI rats though had 5.1 +/- 2.2 neutrophils/hpf and 3.0 +/- 0.4 endothelial cells/hpf expressing E-selectin (mean +/- SEM) (both p < 0.05 vs sham and DI). These data suggest that BBB breakdown occurs in the immature rat after both focal and diffuse TBI. This early BBB permeability was not associated with acute inflammation in DI but was in CCI. These data also suggest that contusion is a key factor in the development of a traditional acute inflammatory response after TBI in the immature rat.

Asunto(s)

Barrera Hematoencefálica/fisiología , Traumatismos Cerrados de la Cabeza/fisiopatología , Mediadores de Inflamación/sangre , Inflamación Neurogénica/fisiopatología , Animales , Animales Recién Nacidos , Conmoción Encefálica/fisiopatología , Edema Encefálico/fisiopatología , Permeabilidad Capilar/fisiología , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

UNLABELLED: Previous studies in our laboratory have shown that controlled cortical impact (CCI) produces an acute inflammatory response in rat brain, including neutrophil accumulation and upregulation of cell adhesion molecules. The purpose of this study was to compare the time course of acute inflammation to blood-brain barrier (BBB) breakdown after (CCI) in rats. METHODS: Male Wistar rats (n = 4-7/group) were subjected to CCI (2.5 mm depth, 4 m/s) and injected with Evans-blue dye (2%, 5 ml/kg) at 30 min, 3.5 h, 7.5 h, or 23.5 h after trauma. 30 min after dye injection rats were saline-perfused. BBB permeability was measured by spectrophotometric quantitation of Evans-blue in injured brain. Alternate cryostat sections from the anterior segment of the injured hemisphere were analyzed immunohistochemically for neutrophils (MoAb RP-3 vs rat neutrophils) or E-selectin (MoAb vs E-selectin). Neutrophils and E-selectin-positive blood vessels were quantitated by light microscopy in 100x cortical and hippocampal fields. RESULTS AND CONCLUSIONS: BBB breakdown was maximal early after CCI, whereas maximum E-selectin upregulation (8 h) and neutrophil accumulation (24 h) occurred later. Events other than acute inflammation initiate BBB permeability after CCI. Acute inflammation may contribute to BBB permeability at 4 h to 24 h after CCI.

Asunto(s)

Barrera Hematoencefálica/inmunología , Corteza Cerebral/lesiones , Selectina E/metabolismo , Traumatismos Cerrados de la Cabeza/inmunología , Inflamación Neurogénica/inmunología , Neutrófilos/inmunología , Animales , Permeabilidad de la Membrana Celular/inmunología , Corteza Cerebral/inmunología , Masculino , Ratas , Ratas Wistar

RESUMEN

Reduction in whole body cytochrome P450 (CYP 450) activity is evident in humans who develop trauma and sepsis-induced multiple organ failure (MOF). It is not known whether this has any deleterious or protective effect. Intraperitoneal injection of zymosan, the cell wall of Saccharomycoses A, induces dose-dependent inflammation with concomitant MOF in rats. High dose intraperitoneal zymosan (100 mg/100 g body weight) causes mortality and organomegaly in rats; low dose zymosan (20 mg/100 g body weight) does not. To study a role for CYP 450 in zymosan-induced toxicity, we examined the effect of the non-specific CYP 450 suicide inhibitor 1-aminobenzotriazole (1-ABT)(80 mg/kg/d), on rats treated with low dose zymosan. The 90% reduction in CYP 450 content achieved by this dose of 1-ABT was associated with 58% mortality in rats treated with low dose zymosan, in contrast to no mortality in rats treated with low dose zymosan alone (p < 0.01). In survivors, liver and lung organomegaly (p < 0.01), and polymorphonuclear leukocyte accumulation in the liver (p < 0.01) were increased after zymosan administration in rats treated with 1-ABT compared to those without 1-ABT. There was no effect of treatment with 1-ABT on the increased urinary excretion of nitric oxide byproducts observed after zymosan administration. These observations are consistent with the hypothesis that the CYP 450 enzyme system is an endogenous protectant in this experimental model of inflammation-induced MOF.

Asunto(s)

Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Triazoles/farmacología , Zimosan/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Sinergismo Farmacológico , Inflamación/inducido químicamente , Inflamación/enzimología , Recuento de Leucocitos , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Neutrófilos/efectos de los fármacos , Nitratos/orina , Nitritos/orina , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Análisis de Supervivencia

RESUMEN

The effect of varying brain temperature on neutrophil accumulation in brain and the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) on cerebrovascular endothelium after controlled cortical impact (CCI) was studied in rats. Sprague Dawley rats were anesthetized and subjected to CCI to the left parietal cortex. Ten minutes after CCI, brain temperature was modulated and maintained at 32 degrees C, 37 degrees C, or 39 degrees C (n = 8 per group) for 4 h. Rats were then decapitated and immunohistochemistry on brain sections was performed using monoclonal antibodies (MoAb) that recognize neutrophils (RP-3), ICAM-1 (TM-8, Athena Neurosciences), or MoAb that react with E-selectin (La-Roche). Each of these markers was quantified in 100 x fields. Neutrophil accumulation was also quantified with myeloperoxidase (MPO) assay. Absolute neutrophil count (ANC) was measured in blood samples before and 1 h and 4 h after CCI. Neutrophil accumulation in injured brain was decreased in rats maintained at 32 degrees C vs 39 degrees C (4-fold difference as assessed by immunohistochemistry, p < 0.05; 8-fold difference as assessed by MPO assay, p < 0.05). Peripheral blood ANC was not affected by temperature. E-selectin was induced on cerebrovascular endothelium after CCI (p < 0.05), but was only decreased modestly at 32 degrees C versus 39 degrees C (p = 0.11). ICAM-1 was not upregulated on cerebrovascular endothelium at this early time following CCI. Neutrophil accumulation is directly dependent on brain temperature during the initial 4 h after CCI. This appears to be mediated by mechanisms other than effects of temperature on E-selectin or ICAM-1 expression or systemic ANC.

Asunto(s)

Temperatura Corporal/fisiología , Lesiones Encefálicas/fisiopatología , Encéfalo/fisiopatología , Encefalitis/fisiopatología , Animales , Encéfalo/patología , Química Encefálica/fisiología , Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/metabolismo , Encefalitis/etiología , Encefalitis/metabolismo , Endotelio Vascular/patología , Inmunohistoquímica , Masculino , Neutrófilos/fisiología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo

RESUMEN

Cytokines may play an important role in the pathophysiology of traumatic brain injury (TBI) in children. Interleukin-6 (IL-6) is a proinflammatory cyotkine that plays a role in regenerative processes within the central nervous system (CNS), whereas interleukin-10 (IL-10) is an antiinflammatory cytokine. Both have been measured in serum and cerebrospinal fluid (CSF) as an index of the degree of inflammation in diseases, including sepsis and meningitis. We hypothesized that both IL-6 and IL-10 would be increased in the CSF of children after severe TBI. Fifteen children who sustained severe TBI (Glascow Coma Score [GCS] < or = 7) were studied. Standard neurointensive care was provided. Ventricular CSF collected the first 3 days after TBI was analyzed for IL-6 and IL-10 concentrations by ELISA. Controls were 20 children who were evaluated for meningitis with diagnostic lumbar puncture subsequently found to have no CSF pleocytosis and negative cultures. IL-6 was increased in children after TBI versus controls on all days studied (day 1, 3158.2 +/- 621.8 pg/ml; day 2, 1111.6 +/- 337.0 pg/ml; day 3, 826.7 +/- 193.5 pg/ml vs. 20.6 +/- 5.8 pg/ml, p < 0.0001, Mann-Whitney Rank Sum). IL-10 was increased in children after TBI vs controls on all days studied (day 1, 47.2 +/- 12.9 pg/ml; day 2, 21.0 +/- 6.7 pg/ml; day 3, 15.5 +/- 5.9 pg/ml vs. 8.9 +/- 7.5 pg/ml, p < 0.01). Increased IL-10 concentrations were independently associated with age < 4 years and mortality (p = 0.004 and 0.04, respectively, multivariate linear model). This study demonstrates that IL-6 is increased after TBI in children to levels similar to those reported in adults and is the first to show that IL-10 is increased in CSF of humans after TBI. These data suggest that there may be an age-dependent production of IL-10 after TBI in children.

Asunto(s)

Lesiones Encefálicas/líquido cefalorraquídeo , Interleucina-10/líquido cefalorraquídeo , Interleucina-6/líquido cefalorraquídeo , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino

RESUMEN

Inflammation may play an important role in the evolution of damage after traumatic brain injury (TBI). IL-6 and IL-10 are markers of inflammation that are pro- and anti-inflammatory in nature, respectively. They have been used as an index of the degree of inflammation in diseases including sepsis and meningitis. We hypothesized that both IL-6 and IL-10 would be increased in the cerebrospinal fluid (CSF) of children after TBI. We measured ventricular CSF concentrations of these metabolites (ELISA) each of the first 3 days after TBI in 15 children. CSF IL-6 was increased on day 1 (p < 0.05 vs days 2 or 3). CSF IL-10 was similarly increased on day 1 (p < 0.05). CSF IL-6 after TBI is similar to serum IL-6 levels previously reported in children with septic shock. In contrast, the CSF IL-10 response was markedly attenuated following TBI compared to sepsis. These data suggest a unique balance between pro- and anti-inflammatory cytokines in brain after TBI.

Asunto(s)

Lesiones Encefálicas/líquido cefalorraquídeo , Ventrículos Cerebrales/metabolismo , Inflamación/líquido cefalorraquídeo , Interleucina-10/líquido cefalorraquídeo , Interleucina-6/líquido cefalorraquídeo , Choque Séptico/líquido cefalorraquídeo , Niño , Preescolar , Humanos , Lactante