Design of an adaptive randomized clinical trial of intravenous citrulline for sickle cell pain crisis in the emergency department.

Background: Vaso-occlusive pain crisis (VOC) is the most frequent cause for Emergency Department (ED) visits and hospital admissions for patients with sickle cell disease (SCD). Nitric oxide plays a critical role in the pathogenesis of vaso-occlusion. The amino acid, citrulline, is the main endothelial nitric oxide booster that offers the potential to ameliorate vaso-occlusion and decrease the risk of hospitalization. Objective: In this two-part study, the goal of the first part is to determine the pharmacokinetic profile of intravenous (IV) l-citrulline and optimal dose for the second part of the study, which is to determine the efficacy and tolerability of the intervention in patients with SCD. Design: A phase I/IIA open-label dose-finding study with subsequent double-blind, placebo-controlled, randomized Study of l-citrulline in children and adolescents with SCD presenting to the ED in VOC. Methods: Part 1: Subjects experiencing VOC are enrolled in an open-label, ascending dose of IV l-citrulline to identify the optimum dose with endpoints of pharmacokinetic parameters, pain scores, reduction of opioid use, quality of life, proportion admitted to the hospital for treatment of pain, readmission rates, and assessment of adverse events. Part 2 of the trial is a double-blind, placebo-controlled adaptive "pick-the-winner" design to evaluate the efficacy and tolerability of IV l-citrulline in patients with SCD while receiving standard of care therapy for VOC. Summary: This ED based sickle cell adaptive trial will determine the optimal dose for IV citrulline and whether the intervention improves outcome as a potential novel therapy for VOC in SCD.

Local influence of cell viability on stretch-induced permeability of alveolar epithelial cell monolayers.

Ventilator induced lung injury (VILI), often attributed to over-distension of the alveolar epithelial cell layer, can trigger loss of barrier function. Alveolar epithelial cell monolayers can be used as an idealized in vitro model of the pulmonary epithelium, with cell death and tight junction disruption and permeability employed to estimate stretch-induced changes in barrier function. We adapted a method published for vascular endothelial permeability, compare its sensitivity with our previously published method, and determine the relationship between breeches in barrier properties after stretch and regions of cell death After 4-5 days in culture, primary rat alveolar epithelial cells seeded on plasma treated polydimethylsiloxane membrane coated with biotin-labeled fibronectin, or fibronectin alone were stretched in the presence of FITC-tagged streptavidin (biotin-labeled membrane) or BODIPY-ouabain. We found that the FITC-labeling method was a more sensitive indicator of permeability disruption, with significantly larger positively stained areas visible in the presence of stretch and with ATP production inhibitor Antimycin-A. Triple-stained images with Hoescht (nuclei), Ethidium Homodimer (EthD, damaged cell nuclei) and FITC (permeable regions) were used to determine that within permeable regions intact cells were positioned closer to damaged cells than in non-permeable regions. We concluded that local cell death may be an important contributor to barrier integrity.

Encuentro y desencuentro de saberes: prácticas de salud en comunidades MBYA Guaraní de Alto Paraná, Misiones / Convergence and divergence of knowledge: a study of health practices in MBYA Guarani communities from Alto Paraná, Misiones

INTRODUCCIÓN El análisis de las prácticas de los equipos de salud en las comunidades mbya guaraní proporciona un panorama del alcance que ha tenido la implementación del sistema público de salud en dichos espacios. Además, permite visualizar las estrategias, los logros, las limitaciones y los conflictos que se dieron en el proceso. OBJETIVOS Describir y analizar las modalidades de institucionalización del sistema público de salud en las comunidades mbya guaraní de la región del Alto Paraná en la provincia de Misiones, así como su impacto en la población y las formas en que los saberes nativos y biomédicos se articulan para resolver las problemáticas de salud. MÉTODOS Se realizó un estudio exploratorio y descriptivo del proceso de contacto de salud pública con comunidades mbya guaraní. Se emplearon técnicas cuanticualitativas. Se efectuaron visitas a las salas de atención de las aldeas y los hospitales en el período 2014-2015. RESULTADOS Las comunidades mbya guaraní de Misiones articulan estrategias de resolución a sus problemas de salud. Las características de la cobertura de salud pública en las comunidades varían según su ubicación geográfica, accesibilidad, relación con dirigentes comunitarios y contacto con la población regional. La medicina tradicional tiene amplia demanda entre los mbya guaraní; igualmente, las estrategias que las familias desarrollan en el régimen de atención combinan tanto el saber tradicional como el biomédico. DISCUSIÓN Con el tiempo, las estrategias de atención pública a la salud de las comunidades mbya guaraní de Misiones se han modificado y adecuado a las necesidades de la población. El rechazo en la primera instancia de contacto se modificó gradualmente por una demanda del servicio de atención primaria a la salud. Los usuarios articulan actualmente los dos sistemas de atención que tienen a disposición tradicional y biomédico

Uses of Remnant Human Lung Tissue for Mechanical Stretch Studies.

Human lung tissue donated for research purposes is a precious resource which can enhance the exploration of mechanisms involved in ventilator-induced lung injury (VILI). The goal of this work was to establish methods and demonstrate the feasibility of obtaining viable primary human type I-like alveolar epithelial cells (AECs) from remnant tissue, even after a significant lapse in post-mortem time, as well as human precision-cut lung slices (PCLSs), and stretch them at magnitudes correlated with mechanical ventilation volumes. Although after 3 days in culture many of the isolated cells stained for the type II AEC marker pro-surfactant Protein C (pro-SPC), after 6 days in culture the monolayer stained only weakly and non-specifically for pro-SPC, and stained brightly for the type I AEC marker aquaporin-5. A strong zona-occludin 1 stain demonstrated the formation of tight junctions between the cells in the epithelial monolayer after only 3 days in culture. To demonstrate the utility of the preparations for the study of lung injury, we stretched the cells and the PCLSs cyclically, uniformly, and equibiaxially and quantified their viability. Our data show that the described methods allow the utilization of human tissue in in vitro stretch studies investigating VILI.

Reproducible uniform equibiaxial stretch of precision-cut lung slices.

Simulating ventilator-induced lung injury (VILI) in the laboratory requires stretching of lung alveolar tissue. Whereas precision-cut lung slices (PCLSs) are widely used for studying paracrine signaling pathways in the lungs, their use in stretch studies is very limited because of the technical challenge of fixing them to a stretchable substrate, stretching them uniformly, or holding them in a stretch device without causing rupture. We describe a novel method for attaching PCLSs to silicone membranes by stitching them together in a star-shaped pattern. Using a device that was previously designed in our laboratory for stretching primary alveolar epithelial cell monolayers, we demonstrate that in the central region of the PCLSs stretch is uniform, equibiaxial, and, after a short preconditioning period, also reproducible. The stitched and stretched PCLSs showed equal or better viability outcomes after 60 min of cyclic stretch at different magnitudes of physiological stretch compared with primary pulmonary alveolar epithelial cell monolayers. Preparing and stitching the PCLSs before stretch is relatively easy to perform, yields repeatable outcomes, and can be used with tissue from any species. Together with the ensuring uniform and equibiaxial stretch, the proposed methods provide an optimal model for VILI studies with PCLSs.

Premedication with meloxicam exacerbates intracranial haemorrhage in an immature swine model of non-impact inertial head injury.

Meloxicam is a cyclo-oxygenase-2 (COX-2) preferential non-steroidal anti-inflammatory drug with very effective analgesic and anti-inflammatory effects in swine. Previous reports in piglets have demonstrated that meloxicam also inhibits COX-1 and reduces production of thromboxane significantly. We use preinjury analgesia in our immature swine (3-5-day-old piglets) model of brain injury using rapid head rotations without impact. In 23 consecutive subjects we found that premedication with meloxicam (n = 6) produced a significantly higher mortality rate (5/6 or 83%) than buprenorphine (n = 17, 1/17 or 6%, P < 0.02). On gross neuropathological examination of the meloxicam-treated swine, we observed massive subdural and subarachnoid bleeding which were not present in buprenorphine-premedicated animals. To our knowledge there are no previous reports in swine of increased bleeding or platelet inhibition associated with meloxicam administration and further research is needed to define mechanisms of action in piglets. We caution the use of meloxicam in swine when inhibition of platelet aggregation might adversely affect refinement of experimental research protocols, such as in stroke, trauma and cardiac arrest models.

Frequency and peak stretch magnitude affect alveolar epithelial permeability.

The present study measured stretch-induced changes in transepithelial permeability to uncharged tracers (1.5-5.5 A) using cultured monolayers of alveolar epithelial type-I like cells. Cultured alveolar epithelial cells were subjected to uniform cyclic (0, 0.25 and 1.0 Hz) biaxial stretch from 0% to 12, 25 or 37% change in surface area (DeltaSA) for 1 h. Significant changes in permeability of cell monolayers were observed when stretched from 0% to 37% DeltaSA at all frequencies, and from 0% to 25% DeltaSA only at high frequency (1 Hz), but not at all when stretched from 0% to 12% DeltaSA compared with unstretched controls. At stretch oscillation amplitudes of 25 and 37% DeltaSA, imposed at 1 Hz, tracer permeability increased compared with that at 0.25 Hz. Cells subjected to a single stretch cycle at 37% DeltaSA (0.25 Hz), to simulate a deep sigh, were not distinguishable from unstretched controls. Reducing stretch oscillation amplitude while maintaining a peak stretch of 37% DeltaSA (0.25 Hz) via the application of a simulated post-end-expiratory pressure did not protect barrier properties. In conclusion, peak stretch magnitude and stretch frequency were the primary determining factors for epithelial barrier dysfunction, as opposed to oscillation amplitude.

How do normals move during prolonged wheelchair-sitting?

The clinical practice for minimizing the risk of pressure sores (PS) is to relief pressures under bony prominences of immobilized patients by changing their postures frequently. The US Department of Health advises to relief sitting pressures at least every 1 hour, and every 15 minutes for individuals who are body-abled. Surprisingly, there is paucity of information in the literature concerning motion of healthy subjects during prolonged sitting, which can be compared with these recommendations. Considering that healthy individuals are able to sit for hours without suffering injuries, such information seems particularly important. Accordingly, our objective was to measure frequency of postural changes and extent of motion during postural changes among healthy subjects who sit in a wheelchair (N=10), in order to provide information that is missing in the literature of PS biomechanics. Subjects were asked to sit comfortably for 90 minutes, during which their trunk's frontal and sagittal motions and sitting pressures were measured. We found that normals change their posture every 9 +/- 6 minutes in the sagittal plane, and independently, every 6 +/- 2 minutes in the frontal plane. Shoulders, thoracic-spine and lumbar-spine frontal plane motions were 8 +/- 4 degrees , 14 +/- 7 degrees and 15 +/- 7 degrees , respectively, and sagittal trunk-thigh movement was 10.3 +/- 7 degrees . The frequency of postural changes in normals, measured herein, was higher than frequencies reported in the literature for patients who suffered PS. This small study population therefore supports the hypothesis that prolonged immobilization contributes to PS onset.

In vivo muscle stiffening under bone compression promotes deep pressure sores.

Pressure sores (PS) in deep muscles are potentially fatal and are considered one of the most costly complications in spinal cord injury patients. We hypothesize that continuous compression of the longissimus and gluteus muscles by the sacral and ischial bones during wheelchair sitting increases muscle stiffness around the bone-muscle interface over time, thereby causing muscles to bear intensified stresses in relentlessly widening regions, in a positive-feedback injury spiral. In this study, we measured long-term shear moduli of muscle tissue in vivo in rats after applying compression (35 KPa or 70 KPa for 1/4-2 h, N = 32), and evaluated tissue viability in matched groups (using phosphotungstic acid hematoxylin histology, N = 10). We found significant (1.8-fold to 3.3-fold, p < 0.05) stiffening of muscle tissue in vivo in muscles subjected to 35 KPa for 30 min or over, and in muscles subjected to 70 KPa for 15 min or over. By incorporating this effect into a finite element (FE) model of the buttocks of a wheelchair user we identified a mechanical stress wave which spreads from the bone-muscle interface outward through longissimus muscle tissue. After 4 h of FE simulated motionlessness, 50%-60% of the cross section of the longissimus was exposed to compressive stresses of 35 KPa or over (shown to induce cell death in rat muscle within 15 min). During these 4 h, the mean compressive stress across the transverse cross section of the longissimus increased by 30%-40%. The identification of the stiffening-stress-cell-death injury spiral developing during the initial 30 min of motionless sitting provides new mechanistic insight into deep PS formation and calls for reevaluation of the 1 h repositioning cycle recommended by the U.S. Department of Health.

Role of stretch on tight junction structure in alveolar epithelial cells.

Previous studies have demonstrated that high tidal volumes can cause interstitial and alveolar edema, with degradation of pulmonary epithelial barrier integrity. Separate studies have shown that F-actin disruption and decreased intracellular ATP (ATP(i)) levels in the nonpulmonary epithelium can increase tight junction (TJ) permeability. We hypothesized that large epithelial stretch perturbs ATP(i) and actin architecture, each of which adversely affects TJ structure, and thus increases TJ permeability. Primary alveolar epithelial cells were subjected to a uniform 25% or 37% change in surface area (DeltaSA), cyclic biaxial stretch (15 cycles/min) for 1 h, or treated with either glycolytic metabolic inhibitors or cytoskeletal disrupting agents. Unstretched, untreated cells served as controls. Changes in the TJ proteins occludin and ZO-1 were determined by immunocytochemical evaluation. A stretch amplitude of 25% DeltaSA did not produce any significant cytologic changes compared with controls, but an amplitude of 37% DeltaSA stretch resulted in significant decreases in the intensity of the peripheral occludin band, the degree of cell-cell attachment (CCA), and total cellular occludin content. ATP depletion significantly diminished the occludin band intensity and decreased CCA. Actin disruption did not affect TJ protein band intensities (although the occludin distribution became punctate) but altered CCA. Untreated cells stretched cyclically at 25% or 50% DeltaSA for 1 h had significantly decreased ATP(i) compared with unstretched controls. These results suggest that stretch-induced ATP(i) reduction and actin perturbation disrupt TJ structure and CCA, which may lead to the alveolar flooding associated with high tidal volumes.

Keratinocyte growth factor reduces alveolar epithelial susceptibility to in vitro mechanical deformation.

Keratinocyte growth factor (KGF) is a potent mitogen that prevents lung epithelial injury in vivo. We hypothesized that KGF treatment reduces ventilator-induced lung injury by increasing the alveolar epithelial tolerance to mechanical strain. We evaluated the effects of in vivo KGF treatment to rats on the response of alveolar type II (ATII) cells to in vitro controlled, uniform deformation. KGF (5 mg/kg) or saline (no-treatment control) was instilled intratracheally in rats, and ATII cells were isolated 48 h later. After 24 h in culture, both cell groups were exposed to 1 h of continuous cyclic strain (25% change in surface area); undeformed wells were included as controls. Cytotoxicity was evaluated quantitatively with fluorescent immunocytochemistry. There was >1% cell death in undeformed KGF-treated and control groups. KGF pretreatment significantly reduced deformation-related cell mortality to only 2.2 +/- 1.3% (SD) from 49 +/- 5.5% in control wells (P < 0.001). Effects of extracellular matrix, actin cytoskeleton, and phenotype of KGF-treated and control cells were examined. The large reduction in deformation-induced cell death demonstrates that KGF protects ATII cells by increasing their strain tolerance and supports KGF treatment as a potential preventative measure for ventilator-induced lung injury.

The postconcussion syndrome after mild head trauma part II: is migraine underdiagnosed?

UNLABELLED: The evidence for post-traumatic migraine as the cause of the postconcussion syndrome in a proportion of patients is reviewed. CONCLUSION: patients suffering recurrent post-traumatic headaches or other elements of the postconcussion syndrome should be treated for migraine.

Infant skull and suture properties: measurements and implications for mechanisms of pediatric brain injury.

The mechanical properties of the adult human skull are well documented, but little information is available for the infant skull. To determine the age-dependent changes in skull properties, we tested human and porcine infant cranial bone in three-point bending. The measurement of elastic modulus in the human and porcine infant cranial bone agrees with and extends previous published data [McPherson, G. K., and Kriewall, T. J. (1980), J. Biomech., 13, pp. 9-16] for human infant cranial bone. After confirming that the porcine and human cranial bone properties were comparable, additional tensile and three-point bending studies were conducted on porcine cranial bone and suture. Comparisons of the porcine infant data with previously published adult human data demonstrate that the elastic modulus, ultimate stress, and energy absorbed to failure increase, and the ultimate strain decreases with age for cranial bone. Likewise, we conclude that the elastic modulus, ultimate stress, and energy absorbed to failure increase with age for sutures. We constructed two finite element models of an idealized one-month old infant head, one with pediatric and the other adult skull properties, and subjected them to impact loading to investigate the contribution of the cranial bone properties on the intracranial tissue deformation pattern. The computational simulations demonstrate that the comparatively compliant skull and membranous suture properties of the infant brain case are associated with large cranial shape changes, and a more diffuse pattern of brain distortion than when the skull takes on adult properties. These studies are a fundamental initial step in predicting the unique mechanical response of the pediatric skull to traumatic loads associated with head injury and, thus, for defining head injury thresholds for children.

Maturation-dependent response of the piglet brain to scaled cortical impact.

OBJECT: The goal of this study was to investigate the relationship between maturational stage and the brain's response to mechanical trauma in a gyrencephalic model of focal brain injury. Age-dependent differences in injury response might explain certain unique clinical syndromes seen in infants and young children and would determine whether specific therapies might be particularly effective or even counterproductive at different ages. METHODS: To deliver proportionally identical injury inputs to animals of different ages, the authors have developed a piglet model of focal contusion injury by using specific volumes of rapid cortical displacement that are precisely scaled to changes in size and dimensions of the growing brain. Using this model, the histological response to a scaled focal cortical impact was compared at 7 days after injury in piglets that were 5 days, 1 month, and 4 months of age at the time of trauma. Despite comparable injury inputs and stable physiological parameters, the percentage of hemisphere injured differed significantly among ages, with the youngest animals sustaining the smallest lesions (0.8%, 8.4%, and 21.5%, for 5-day-, 1-month-, and 4-month-old animals, respectively, p = 0.0018). CONCLUSIONS: These results demonstrate that, for this particular focal injury type and severity, vulnerability to mechanical trauma increases progressively during maturation. Because of its developmental and morphological similarity to the human brain, the piglet brain provides distinct advantages in modeling age-specific responses to mechanical trauma. Differences in pathways leading to cell death or repair may be relevant to designing therapies appropriate for patients of different ages.

Dynamic mechanical stretch of organotypic brain slice cultures induces differential genomic expression: relationship to mechanical parameters.

Although the material properties of biological tissues are reasonably well established, recent studies have suggested that the biological response of brain tissue and its constituent cells may also be viscoelastic and sensitive to both the magnitude and rate of a mechanical stimulus. Given the potential involvement of changes in gene expression in the pathogenic sequelae after head trauma, we analyzed the expression of 22 genes related to cell death and survival and found that a number of these genes were differentially regulated after mechanical stretch of an organotypic brain slice culture. Twenty-four hours after stretch, the expression of BDNF, NGF, and TrkA was significantly increased, whereas that of bcl-2, CREB, and GAD65 was significantly decreased (MANOVA followed by ANOVA, p < 0.05). Expression of CREB and GAD65 was negatively correlated with strain, whereas expression of APP695 was negatively correlated with strain rate (all p < 0.05). This study demonstrates that a subset of genes involved in cell death and survival are differentially regulated after dynamic stretch in vitro and that the expression of specific genes is correlated with mechanical parameters of that stretch.

The postconcussion syndrome after mild head trauma: is brain damage overdiagnosed? Part 1.

UNLABELLED: Many investigators attribute the postconcussion syndrome following mild closed head injury to permanent brain damage. The evidence supporting this conclusion is reviewed, including the force necessary to cause permanent brain damage; the basis for determining whether the patient was exposed to sufficient force in the accident to permanently damage the brain; the basis for determining whether the patient actually has permanent brain damage (not just brain dysfunction) traceable to the accident; and whether the location and severity of brain damage is sufficient to account for the postconcussion syndrome. CONCLUSION: the evidence for permanent traumatic brain damage as the cause of the postconcussion syndrome following mild closed head injury is weak.

Defining brain mechanical properties: effects of region, direction, and species.

No regional or directional large-deformation constitutive data for brain exist in the current literature. To address this deficiency, the large strain (up to 50%) directional properties of gray and white matter were determined in the thalamus, corona radiata, and corpus callosum. The constitutive relationships of all regions and directions are well fit by an Ogden hyperelastic relationship, modified to include dissipation. The material parameter alpha, representing the non-linearity of the tissue, was not significantly sensitive to region, direction, or species. The average value of the material parameter mu, corresponding to the shear modulus of the tissue, was significantly different for each region, demonstrating that brain tissue is inhomogeneous. In each region, mu, obtained in 2 orthogonal directions, was compared. Consistent with local neuroarchitecture, gray matter showed the least amount of anisotropy and corpus callosum exhibited the greatest degree of anisotropy. Finally, human temporal lobe gray matter properties were determined and compared to porcine thalamic properties. The results show significant regional inhomogeneity at large strains and significant anisotropy in each region tested. The extent of regional anisotropy correlated with the degree of alignment in the local neuroarchitecture. These large strain, regional and directional data should enhance the biofidelity of computational models and provide important information regarding the mechanisms of traumatic brain injury.

Rectus sheath hematoma.

We describe 3 patients with rectus sheath hematoma presenting to the emergency department. Prompt consideration of this uncommon cause of abdominal pain may prevent more expensive and invasive diagnostic tests and, in some cases, unnecessary hospitalization and laparotomy.

A fiber-reinforced composite model of the viscoelastic behavior of the brainstem in shear.

Brainstem trauma occurs frequently in severe head injury, often resulting in fatal lesions due to importance of brainstem in crucial neural functions. Structurally, the brainstem is composed of bundles of axonal fibers distinctly oriented in a longitudinal direction surrounded by an extracellular matrix. We hypothesize that the oriented structure and architecture of the brainstem dictates this mechanical response and results in its selective vulnerability in rotational loading. In order to understand the relationship between the biologic architecture and the mechanical response and provide further insight into the high vulnerability of this region, a structural and mathematical model was created. A fiber-reinforced composite model composed of viscoelastic fibers surrounded by a viscoelastic matrix was used to relate the biological architecture of the brainstem to its anisotropic mechanical response. Relevant model parameters measured include the brainstem's composite complex moduli and relative fraction of matrix and fiber. The model predicted that the fiber component is three times stiffer and more viscous than the matrix. The fiber modulus predictions were compared with experimental tissue measurements. The optic nerve, a bundle of tightly packed longitudinally arranged myelinated fibers with little matrix, served as a surrogate for the brainstem fiber component. Model predictions agreed with experimental measures, offering a validation of the model. This approach provided an understanding of the relationship between the specific biologic architecture of the brainstem and the anisotropic mechanical response and allowed insight into reasons for the selective vulnerability of this region in rotational head injury.