Myenteric neuroprotective role of aspirin in acute and chronic experimental infections with Trypanosoma cruzi.

BACKGROUND: Experimental and clinical studies have shown that myenteric neuron cell death during infection with Trypanosoma cruzi mainly occurs in the esophagus and colon, resulting in megaesophagus and megacolon, respectively. Evidence suggests that the cyclooxygenase enzyme (COX) is involved in the T. cruzi invasion process. The use of low-dose aspirin (ASA), a COX-1/COX-2 inhibitor, has been shown to reduce infection with T. cruzi. Therefore, in this study, we evaluated the effects of treatment with low-dose ASA on myenteric colonic neurons during murine infection with T. cruzi. METHODS: Swiss mice were assigned into groups treated with either phosphate-buffered saline or low doses of ASA during the acute phase (20 mg/kg ASA) and chronic phase (50 mg/kg ASA) of infection with the Y strain of T. cruzi. Seventy-five days after infection, colon samples were collected to quantify inflammatory foci in histological sections and also general (myosin-V+ ), nitrergic, and VIPergic myenteric neurons in whole mounts. Gastrointestinal transit time was also measured. KEY RESULTS: Aspirin treatment during the acute phase of infection reduced parasitemia (P<.05). Aspirin treatment during the acute or chronic phase of the infection reduced the intensity of inflammatory foci in the colon, protected myenteric neurons from cell death and plastic changes, and recovered the gastrointestinal transit of mice infected with T. cruzi (P<.05). CONCLUSION & INFERENCES: Early and delayed treatment with low-dose ASA can reduce the morphofunctional damage of colonic myenteric neurons caused by murine T. cruzi infection.

Inhibitory effects of a standardized extract of Justicia pectoralis in an experimental rat model of airway hyper-responsiveness.

OBJECTIVE: Justicia pectoralis is a plant useful for the treatment of respiratory diseases. Here, we studied the antiasthmatic properties of a standardized extract of J. pectoralis (Jp). METHODS: Ovalbumin (OVA)-sensitized rats were actively challenged with saline or OVA to study airway hyper-responsiveness after oral treatment with saline or Jp. The ability of Jp to inhibit hyper-reactivity was evaluated in isolated trachea mounted in isolated organ bath chamber. KEY FINDINGS: Using KCl or carbachol as contractile agents, tracheal rings of OVA-challenged rats contracted with higher magnitude than trachea of rats challenged with saline. Such hyper-responsive phenotype of OVA-challenged tissues decreased with Jp administration. In Ca+ -free medium, Jp or its major constituent coumarin inhibited preferentially the contractions induced by Ca2+ addition in tissues of OVA-challenged rats stimulated with KCl or acetylcholine. In tissues depleted of their internal Ca+ stores in the presence of thapsigargin, Jp inhibited the contraction induced by capacitative Ca2+ entry. By gavage, Jp abolished the increase caused by challenge with OVA on the levels of IL-1ß and TNF-α in the bronchoalveolar fluid and also impaired the changes in gene expression of canonical transient receptor proteins. CONCLUSIONS: Jp has antiasthmatic properties in an experimental model that reproduces tracheal hyper-reactivity.

Viability of cryopreserved BM progenitor cells stored for more than a decade.

BACKGROUND: PBPC or BM is increasingly being harvested in remission for possible use in the event of relapse. Although the value of this approach has not been demonstrated, the long-term storage of progenitor cell components has become commonplace in many facilities. METHODS: We used multi-parameter flow cytometry to determine the viability of 11 long-term cryopreserved BM components (mean = 11.8 years) in liquid phase nitrogen. The components, prepared for autotransplantation but deaccessioned after confirming patient death, were carefully thawed, washed, and assayed immediately. The flow cytometry assay was performed according to the ISHAGE protocol, modified by the addition of 7AAD for analysis of progenitor viability (CD45+ CD34+ 7AAD-) and total leukocyte viability (CD45+ 7AAD-). In addition, total viability was assessed by fluorescence microscopy using acridine orange dye exclusion; granulocyte-monocyte colony-forming units (CFU-GM) were measured after 14 days culture. RESULTS: Leukocyte viability by flow cytometry and fluorescence microscopy agreed well (r2 = 0.55, slope = 0.83, P < 0.0005 by linear regression). CFU-GM did not correlate with CD34% or any of the viability parameters. Compared with short-term stored (mean = 33 days) PBPC assayed at infusion, long-term stored BM had a comparable percentage of CD34+ cells, comparable CFU-GM activity, increased CD34 viability, but decreased total cell viability, the latter most likely due to an increased proportion of differentiated myeloid cells. DISCUSSION: The results indicate that BM products can be cryopreserved for more than a decade without apparent loss of progenitor activity, as measured by these laboratory surrogates. This agrees with clinical anecdotes describing successful engraftment with long-term stored BM, and argues that expiration dates cannot be set for cryopreserved hematopoietic stem-cell components stored in liquid phase nitrogen.

Intraperitoneal, but not enteric, adenosine administration improves survival after volume-controlled hemorrhagic shock in rats.

OBJECTIVE: To circumvent the potential adverse systemic side effects of adenosine, this study explored the potential benefit of intraperitoneal or enteric adenosine on survival and inflammatory responses after volume-controlled hemorrhagic shock. DESIGN: Prospective, randomized, and blinded. A three-phase, volume-controlled hemorrhagic shock model was used: hemorrhagic shock phase (120 mins), resuscitation phase (60 mins), and observation phase (72 hrs). Three groups were compared: controls, intraperitoneal adenosine, and enteric adenosine. SETTING: Animal research facility. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Starting at 20 mins of hemorrhagic shock and continuing through the resuscitation phase, all three groups received both intraperitoneal lavage and repeated bolus injections into the ileum of vehicle (normal saline) or adenosine. In the intraperitoneal adenosine group (n = 10), adenosine solution (0.1 mM) was used for intraperitoneal lavage. In the enteric adenosine group (n = 10), adenosine (1.0 mM) was injected into the ileum. Blood cytokine concentrations and leukocyte infiltration in lungs and liver were studied in 12 separate rats (control and intraperitoneal adenosine, n = 6 each) with the same hemorrhagic shock model at resuscitation time 1 hr or 4 hrs. MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure and heart rate were similar between the three groups during hemorrhagic shock and resuscitation. Potassium, lactate, and blood urea nitrogen concentrations were lower and arterial pH was higher in the intraperitoneal and enteric adenosine groups compared with the control group (both p <.05). Survival time to 72 hrs was longer in the intraperitoneal adenosine group than in the control group(p <.05). Neither plasma interleukin-1beta, interleukin-6, interleukin-10, and tumor necrosis factor-alpha concentrations nor leukocyte infiltration in the lungs and liver was different between the control and intraperitoneal adenosine groups. CONCLUSIONS: The administration of adenosine via the intraperitoneal route improves survival time after severe volume-controlled hemorrhagic shock in rats without worsening hypotension or bradycardia. This beneficial effect may not be attributable to effects of adenosine on the inflammatory response.

Leukocyte recruitment at sites of tumor: dissonant orchestration.

Biopsies of tumors responding to interleukin 2 (IL-2) based immunotherapy have been reported to show a leukocytic infiltration. Clinical responses to IL-2-based immunotherapy, however, are limited, suggesting a failure of leukocyte localization at tumor sites. Leukocyte infiltration at inflammatory sites requires local activation of leukocytes and endothelial cells in a coordinated and defined temporal sequence. There is evidence supporting the theory that infiltration of leukocytes at tumor sites is suboptimal due to a failure of coordination of these localizing events. In this review, factors involved in leukocyte recruitment at sites of inflammation and the coordination of these factors in a successful model of inflammation, i.e., wound healing, are discussed. This example is contrasted with events at tumor sites where alterations in expression of cell adhesion molecules or in the production of activating agents may be present. Additionally, the systemic administration of an activating cytokine such as IL-2 may fail to duplicate events that normally occur within a local environment. These observations may facilitate the design of future immunotherapy trials.

Effect of neutropenia and granulocyte colony stimulating factor-induced neutrophilia on blood-brain barrier permeability and brain edema after traumatic brain injury in rats.

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.

Intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 are increased in the plasma of children with sepsis-induced multiple organ failure.

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.

Interleukin-8 is increased in cerebrospinal fluid of children with severe head injury.

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.

Reduced brain edema after traumatic brain injury in mice deficient in P-selectin and intercellular adhesion molecule-1.

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.

Inducible nitric oxide synthase is an endogenous neuroprotectant after traumatic brain injury in rats and mice.

Nitric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) is an inflammatory product implicated both in secondary damage and in recovery from brain injury. To address the role of iNOS in experimental traumatic brain injury (TBI), we used 2 paradigms in 2 species. In a model of controlled cortical impact (CCI) with secondary hypoxemia, rats were treated with vehicle or with 1 of 2 iNOS inhibitors (aminoguanidine and L-N-iminoethyl-lysine), administered by Alzet pump for 5 days and 1. 5 days after injury, respectively. In a model of CCI, knockout mice lacking the iNOS gene (iNOS(-/-)) were compared with wild-type (iNOS(+/+)) mice. Functional outcome (motor and cognitive) during the first 20 days after injury, and histopathology at 21 days, were assessed in both studies. Treatment of rats with either of the iNOS inhibitors after TBI significantly exacerbated deficits in cognitive performance, as assessed by Morris water maze (MWM) and increased neuron loss in vulnerable regions (CA3 and CA1) of hippocampus. Uninjured iNOS(+/+) and iNOS(-/-) mice performed equally well in both motor and cognitive tasks. However, after TBI, iNOS(-/-) mice showed markedly worse performance in the MWM task than iNOS(+/+) mice. A beneficial role for iNOS in TBI is supported.

Neutrophils do not mediate blood-brain barrier permeability early after controlled cortical impact in rats.

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.

Surgically induced leukocytic infiltrates within the rat intestinal muscularis mediate postoperative ileus.

BACKGROUND & AIMS: Postoperative ileus is a poorly understood and common problem. We previously demonstrated an association between a suppression in jejunal circular muscle activity and a massive extravasation of leukocytes into the muscularis after surgical manipulation of the small bowel. This study was pursued to establish a direct causal link between these events. METHODS: Reverse-transcription polymerase chain reaction and immunohistochemistry were used to detect and localize expression of adhesion molecules: P-selectin, intercellular adhesion molecule 1 (ICAM-1), and lymphocyte function-associated antigen 1 (LFA-1). Leukocyte infiltration and in vitro jejunal circular muscle function were quantified in controls and manipulated animals with and without antibody treatment (1A29, WT.1, and WT.3). RESULTS: Surgical manipulation caused a significant up-regulation within the muscularis of ICAM-1 and P-selectin messenger RNA. ICAM-1 and P-selectin protein expression was increased within the muscularis microvasculature, and ICAM-1 and LFA-1 were expressed on infiltrating cells. Administration of adhesion molecule antibodies prevented the recruitment of monocytes and neutrophils into the muscularis and also averted jejunal circular muscle dysfunction. CONCLUSIONS: The data demonstrate that adhesion molecule antibodies prevent surgically induced suppression of intestinal muscle contractions and therefore suggests that late postoperative ileus is mediated through a leukocytic inflammatory response within the intestinal muscularis externa.

Biology of marrow stromal cell lines derived from long-term bone marrow cultures of Trp53-deficient mice.

To investigate the effect of Trp53 (formerly known as p53) on stromal cells of the hematopoietic microenvironment, long-term bone marrow cultures were established from mice in which the Trp53 gene had been inactivated by homologous recombination (Trp53(-/-)) or their wild-type littermates (Trp53(+/+)). Long-term bone marrow cultures from Trp53(-/-) mice continued to produce nonadherent cells for 22 weeks, while Trp53(+/+) cultures ceased production after 15 weeks. There was a significant increase in the number of nonadherent cells produced in Trp53(-/-) long-term bone marrow cultures beginning at week 9 and continuing to week 22 (P < 0.02). The Trp53(-/-) cultures also showed significantly increased cobblestone island formation indicative of early hematopoietic stem cell-containing colonies beginning at week 10 (P < 0.01). Cobblestone islands persisted until weeks 15 and 22 in Trp53(+/+) and Trp53(-/-) cultures, respectively. Co-cultivation experiments in which Trp53(+/+) Sca1(+)lin- enriched hematopoietic stem cells were plated on Trp53(-/-) stromal cells showed increased cobblestone island formation compared to Trp53(-/-) Scal+lin- cells plated on Trp53(+/+) or Trp53(-/-) stromal cells. Radiation survival curves for clonal bone marrow stromal cells revealed a similar D0 for the Trp53(+/+) and Trp53(-/-) cell lines (1.62 +/- 0.16 and 1.49 +/- 0. 08 Gy, respectively; P = 0.408), and similar n (8.60 +/- 3.23 and 10.71 +/- 0.78, respectively) (P = 0.491). Cell cycle analysis demonstrated a G2/M-phase arrest that occurred 6 h after irradiation for both Trp53(+/+) and Trp53(-/-) stromal cell lines. After 10 Gy irradiation, there was no significant increase in the frequency of apoptosis detected in Trp53(+/+) compared to Trp53(-/-) marrow stromal cell lines. In the stromal cell lines, ICAM-1 was constitutively expressed on Trp53(+/+) but not Trp53(-/-) cells; however, a 24-h exposure to TNF-alpha induced detectable ICAM-1 on Trp53(-/-) cells and increased expression on Trp53(+/+) cells. To test the effect of Trp53 on the radiation biology of hematopoietic progenitor cells, the 32D cl 3 cell line was compared with a subclone in which expression of an E6 inserted transgene accelerates ubiquitin-dependent degradation of Trp53, thus preventing accumulation of Trp53 after genotoxic stress. The radiation survival curves were similar with no significant difference in the D0 or n, or in the percentage of cells undergoing apoptosis after 10 Gy irradiation between the two cell lines. Cells of the 32D-E6 cell line displayed a G2/M-phase arrest 6 h after 10 Gy, while cells of the parent line exhibited both a G2/M-phase arrest and a G1-phase arrest at 24 and 48 h. The results suggest a complex mechanism of action of Trp53 on the interactions between stromal and hematopoietic cells in long-term bone marrow cultures.

Effect of traumatic brain injury in mice deficient in intercellular adhesion molecule-1: assessment of histopathologic and functional outcome.

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.

Soluble adhesion molecules in CSF are increased in children with severe head injury.

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.

Blood brain barrier permeability and acute inflammation in two models of traumatic brain injury in the immature rat: a preliminary report.

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.

Blood-brain barrier permeability, neutrophil accumulation and vascular adhesion molecule expression after controlled cortical impact in rats: a preliminary study.

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.

A role of neutrophils in the down-regulation of IL-6 and CD14 following hemorrhagic shock.

Hemorrhagic shock (HS) followed by resuscitation has been shown to initiate a series of events, including local cytokine production and PMN accumulation. To determine whether PMN are involved in the regulation of IL-6 expression in the liver or lungs, IL-6 mRNA levels were measured in rats made neutropenic by vinblastine pretreatment prior to HS. IL-6 mRNA levels were determined at 4 or 24 h following resuscitation from shock. Vinblastine alone in normal rats or sham-treated rats had no effect at 4 or 24 h. Vinblastine pretreatment had no effect on the HS-induced increase in IL-6 mRNA at 4 h but dramatically increased levels in both liver and lung at 24 h. Peripheral PMN counts were reduced by 95% in all vinblastine-treated animals. Similar changes seen in CD14 mRNA expression indicate that these effects are not limited to IL-6. These data show that normal PMN levels are not needed for induction of IL-6 and CD14 in HS, and suggest that PMN accumulation down-regulates the expression of these genes.

The effect of brain temperature on acute inflammation after traumatic brain injury in rats.

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.