Timothy M. Carlos

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.

Expression of endothelial adhesion molecules and recruitment of neutrophils after traumatic brain injury in rats.

Traumatic brain injury (TBI) is often accompanied by an acute inflammatory reaction mediated initially by neutrophils. Adhesion molecules expressed on vascular endothelium are requisite elements during recruitment of leukocytes at sites of inflammation. In a rat model of TBI the induction and persistent expression of E‐selectin (CD62E) on cerebrovascular endothelium ipsilateral, but not contralateral, to the site of contusion was demonstrated (P < 0.05 at 4 and 48 h posttrauma). In addition, these studies confirmed up‐regulation and prolonged expression of ICAM‐1 (CD54) on endothelium in the traumatized hemisphere (P < 0.05 at 4, 24, 48, and 72 h posttrauma). It is of interest that increased expression of CD54 was noted on blood vessels in the contralateral, non‐traumatized hemisphere 48 h posttrauma. Expression of a third endothelial adhesion molecule, PECAM‐1 (CD31), was unchanged following trauma. Administration of a murine monoclonal antibody (TM‐8) that inhibits the adhesive function of CD54 blocked a significant portion (37.9%) of neutrophil recruitment 24 h posttrauma (P = 0.04). Employing immunocytochemistry and a monoclonal antibody specific for rat neutrophils (RP‐3), peak infiltration of neutrophils was shown to occur 48 h after trauma. In contrast to emigration of neutrophils from blood vessels within the contusion, however, entry of neutrophils occurred from the surrounding leptomeninges and choroidal vessels. These studies demonstrate the relevance of CD54 (ICAM‐1) in recruitment of neutrophils following TBI. However, the majority of neutrophil influx relies on endothelial adhesion molecules other than CD54. Because emigration of neutrophils was shown to occur predominantly from vessels within the leptomeninges and choroid plexus, intrathecal delivery of agents that inhibit the adhesive interactions between neutrophils, endothelial CD54, and other endothelial adhesion molecules to be defined may offer a novel form of therapy to prevent the acute inflammatory response that follows TBI. J. Leukoc. Biol. 61: 279–285; 1997.

Inducible nitric oxide synthase expression in cerebrovascular smooth muscle and neutrophils after traumatic brain injury in immature rats

The inflammatory response after traumatic brain injury (TBI) includes cytokine production, leukocyte infiltration, and microglial activation. Production of nitric oxide by inducible nitric oxide synthase (iNOS) occurs during acute inflammation outside of the CNS and in models of cerebral ischemia, and therefore may contribute to the inflammatory response after TBI. The purpose of this study was to localize and define the time course of iNOS expression after TBI in the immature rat. Immature Wistar rats (age 3.5-4.5 wk) were anesthetized and subjected to percussive trauma to the right parietal cortex. Nontraumatized rats were used as controls (n = 7). At 2, 24, 48, or 168 h (n = 3/group) posttrauma rats were killed by perfusion fixation. Brains were removed, frozen, sectioned, immunostained with antibodies against iNOS and glial fibrillary acidic protein (GFAP, a marker specific for astrocytes), and imaged using fluorescent detection systems. There was no detectable expression of iNOS in control brains. At 2 h, minimal cerebrovascular iNOS expression was seen in the peritrauma area. At 24 and 48 h, there was marked peritrauma cerebrovascular iNOS expression that appeared to be restricted to vascular smooth muscle cells and infiltrated leukocytes. Further dual-immunolabeling showed that the leukocytes expressing iNOS were predominantly neutrophils. At 168 h, iNOS expression was no longer detectable. iNOS was not detectable in GFAP-positive cells. The prominent expression of iNOS protein after TBI in cerebrovascular smooth muscle cells and infiltrated neutrophils suggests that iNOS may play a role in cerebrovascular disturbances and secondary brain injury after trauma.

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 childrens hospital. Patients: Twenty‐seven children hospitalized with severe TBI (Glasgow Coma Scale score ≤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.

Complementary Adhesion Molecules Promote Neutrophil- Kupffer Cell Interaction and the Elimination of Bacteria Taken Up by the Liver

Most bacteria that enter the bloodstream are taken up by the liver. Previously, we reported that such organisms are initially bound extracellularly and subsequently killed by immigrating neutrophils, not Kupffer cells as widely presumed in the literature. Rather, the principal functions of Kupffer cells demonstrated herein are to clear bacteria from the peripheral blood and to promote accumulation of bactericidal neutrophils at the principal site of microbial deposition in the liver, i.e., the Kupffer cell surface. In a mouse model of listeriosis, uptake of bacteria by the liver at 10 min postinfection i.v. was reduced from approximately 60% of the inoculum in normal mice to ∼15% in mice rendered Kupffer cell deficient. Immunocytochemical analysis of liver sections derived from normal animals at 2 h postinfection revealed the massive immigration of neutrophils and their colocalization with Kupffer cells. Photomicrographs of the purified nonparenchymal liver cell population derived from these infected mice demonstrated listeriae inside neutrophils and neutrophils within Kupffer cells. Complementary adhesion molecules promoted the interaction between these two cell populations. Pretreatment of mice with mAbs specific for CD11b/CD18 (type 3 complement receptor) or its counter-receptor, CD54, inhibited the accumulation of neutrophils in the liver and the elimination of listeriae. Complement was not a factor; complement depletion affected neither the clearance of listeriae by Kupffer cells nor the antimicrobial activity expressed by infiltrating neutrophils.

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.

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.

Cost-effectiveness of hydroxyurea in sickle cell anemia

The Multicenter Study of Hydroxyurea in Sickle Cell Anemia (MSH) demonstrated the efficacy of hydroxyurea in reducing the rate of painful crises compared to placebo. We used resource utilization data collected in the MSH to determine the cost‐effectiveness of hydroxyurea. The MSH was a randomized, placebo‐controlled double‐blind clinical trial involving 299 patients at 21 sites. The primary outcome, visit to a medical facility, was one of the criteria to define occurrence of painful crisis. Cost estimates were applied to all outpatient and emergency department visits and inpatient hospital stays that were classified as a crisis. Other resources for which cost estimates were applied included hospitalization for chest syndrome, analgesics received, hydroxyurea dosing, laboratory testing, and clinic visits for management of patient care. Annualized differential costs were calculated between hydroxyurea‐ and placebo‐receiving patients. Hospitalization for painful crisis accounted for the majority of costs in both arms of the study, with an annual mean of

Leukocyte recruitment at sites of tumor: dissonant orchestration

12,160 (95% CI:

Acute systemic administration of interleukin-10 suppresses the beneficial effects of moderate hypothermia following traumatic brain injury in rats

9,440,