We quantified absolute myocardial blood flow (MBF) using a spin labeling magnetic resonance imaging (SL-MRI) method after transplantation of endothelial cells (ECs) into the infarcted heart. Our aims were to study the temporal changes in MBF in response to EC transplantation, and to compare regional MBF with contractile function (wall motion) and microvascular density.
Methods and Results
We first validated the SL-MRI method with the standard microsphere technique in normal rats. We then induced myocardial infarction (MI) in athymic rats and injected 5-million ECs (human umbilical vein endothelial cells) suspended in Matrigel or Matrigel alone (vehicle) along the border of the blanched infarcted area. At 2-weeks post-MI, MBF averaged over the entire slice (p = 0.038) and in the infarcted region (p = 0.0086) was significantly higher in EC vs. vehicle group; the greater MBF was accompanied by an increase of microvasculature density in the infarcted region (p = 0.0105 vs. vehicle). At 4-weeks post-MI, MBF in the remote region was significantly elevated in EC-treated hearts (p = 0.0277); this was accompanied by increased wall motion in this region assessed by circumferential strains. Intraclass correlation coefficients and Bland-Altman plot revealed a good reproducibility of the SL-MRI method.
Myocardial blood flow in free-breathing rats measured by SL-MRI is validated by the standard color microsphere technique. SL-MRI allows quantification of temporal changes of regional MBF in response to EC treatment. The proof-of-principle study indicates that MBF is a unique and sensitive index to evaluate EC-mediated therapy for the infarcted heart.
myocardial blood flow; spin labeling; magnetic resonance imaging; myocardial infarction; human umbilical vascular endothelial cells; microspheres; left ventricular ejection fraction
Small animal models have been used in traumatic brain injury (TBI) research to investigate the basic mechanisms and pathology of TBI. Unfortunately, successful TBI investigations in small animal models have not resulted in marked improvements in clinical outcomes of TBI patients.
To develop a clinically relevant immature large animal model of pediatric neurocritical care following TBI.
Eleven 4 week old piglets were randomized to either rapid axial head rotation without impact (N=6) or instrumented sham (N=5). All animals had an intracranial pressure monitor, brain tissue oxygen (PbtO2) probe, and cerebral microdialysis probe placed in the frontal lobe and data collected for 6 h following injury.
Injured animals had sustained elevations in intracranial pressure and lactate-pyruvate ratio (LPR), and decreased PbtO2 compared to sham. PbtO2 and LPR from separate frontal lobes had strong linear correlation in both sham and injured animals. Neuropathologic examination demonstrated significant axonal injury and infarct volumes in injured animals compared to sham at 6 hours post-injury. Averaged over time, PbtO2 in both injured and sham animals had a strong inverse correlation with total injury volume. Average LPR had a strong correlation with total injury volume.
LPR and PbtO2 can be utilized as serial non-terminal secondary markers in our injury model for neuropathology, and as evaluation metrics for novel interventions and therapeutics in the acute post-injury period. This translational model bridges a vital gap in knowledge between TBI studies in small animal models and clinical trials in the pediatric TBI population.
neurocritical care monitoring; pediatric head injury; swine; TBI model
Ventilation of septic patients often leads to the development of edema and impaired gas exchange. We hypothesized that septic alveolar epithelial monolayers would experience stretch-induced barrier dysfunction at a lower magnitude of stretch than healthy alveolar epithelial monolayers. Alveolar epithelial cells were isolated from rats 24 hours after cecal ligation and double puncture (2CLP) or sham surgery. Following a 5-day culture period, monolayers were cyclically stretched for 0, 10, or 60 minutes to a magnitude of 12% or 25% change in surface area (ΔSA). Barrier function, MAPk and myosin light chain (MLC) phosphorylation, tight junction (TJ) protein expression and actin cytoskeletal organization were examined after stretch. Significant increases in epithelial permeability were observed only in 2CLP monolayers at the 12% ΔSA stretch level, and in both 2CLP and sham monolayers at the 25% ΔSA stretch level. Increased permeability in 2CLP monolayers was not associated with MAPk signaling or alterations in expression of TJ proteins. 2CLP monolayers had fewer actin stress fibers before stretch, a more robust stretch-induced actin redistribution, and reduced phosphorylated MLCK than sham monolayers. Jasplakinolide stabilization of the actin cytoskeleton in 2CLP monolayers prevented significant increases in permeability following 60 minutes of stretch to 12% ΔSA. We concluded that septic alveolar epithelial monolayers are more susceptible to stretch-induced barrier dysfunction than healthy monolayers due to actin reorganization.
Gelsolin is an actin-binding protein found in the cytoplasm and in extracellular fluids including blood plasma. Plasma gelsolin concentration decreases after a wide range of traumatic injuries. We hypothesized that the repletion of exogenous gelsolin would limit inflammation and tissue injury in a rat model of sepsis using cecal ligation and double puncture (2CLP).
Exogenous human plasma gelsolin (pGSN, 10 mg in 1 ml saline) was administered once immediately following surgery, and control 2CLP (2CLP Alb) and sham animals were injected with 1 ml saline containing equimolar albumin. Treatments were administered intraperitoneally (IP), intravenously (IV), or subcutaneously (SC).
Gelsolin levels in the 2CLP Alb group were lower than in sham animals compared to sham animals. Administration of pGSN increased levels when administered IV and SC, but not IP. Morbidity scores were significantly less severe in the 2CLP pGSN group than in the 2CLP Alb group when pGSN was administered IV and SC, but not IP. Furthermore, enzymatic activity indicative of tissue damage (lactate dehydrogenase and alanine transaminase) was significantly lower in 2CLP pGSN group when treated SC compared to 2CLP Alb group.
These data provide further evidence that exogenous gelsolin can reduce morbidity from sepsis.
Sepsis; tissue injury
Cyclosporin A (CsA) has been shown to be neuroprotective in mature animal models of traumatic brain injury (TBI), but its effects on immature animal models of TBI are unknown. In mature animal models, CsA inhibits the opening of the mitochondrial permeability transition pore (MPTP), thereby maintaining mitochondrial homeostasis following injury by inhibiting calcium influx and preserving mitochondrial membrane potential. The aim of the present study was to evaluate CsA's ability to preserve mitochondrial bioenergetic function following TBI (as measured by mitochondrial respiration and cerebral microdialysis), in two immature models (focal and diffuse), and in two different species (rat and piglet). Three groups were studied: injured+CsA, injured+saline vehicle, and uninjured shams. In addition, we evaluated CsA's effects on cerebral hemodynamics as measured by a novel thermal diffusion probe. The results demonstrate that post-injury administration of CsA ameliorates mitochondrial dysfunction, preserves cerebral blood flow (CBF), and limits neuropathology in immature animals 24 h post-TBI. Mitochondria were isolated 24 h after controlled cortical impact (CCI) in rats and rapid non-impact rotational injury (RNR) in piglets, and CsA ameliorated cerebral bioenergetic crisis with preservation of the respiratory control ratio (RCR) to sham levels. Results were more dramatic in RNR piglets than in CCI rats. In piglets, CsA also preserved lactate pyruvate ratios (LPR), as measured by cerebral microdialysis and CBF at sham levels 24 h after injury, in contrast to the significant alterations seen in injured piglets compared to shams (p<0.01). The administration of CsA to piglets following RNR promoted a 42% decrease in injured brain volume (p<0.01). We conclude that CsA exhibits significant neuroprotective activity in immature models of focal and diffuse TBI, and has exciting translational potential as a therapeutic agent for neuroprotection in children.
cerebral microdialysis; cyclosporin A; mitochondrial bioenergetics; neuroprotection; pediatric brain injury
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression implicated in multiple cellular processes. Cyclic stretch of alveoli is characteristic of mechanical ventilation, and is postulated to be partly responsible for the lung injury and inflammation in ventilator-induced lung injury. We propose that miRNAs may regulate some of the stretch response, and therefore hypothesized that miRNAs would be differentially expressed between cyclically stretched and unstretched rat alveolar epithelial cells (RAECs).
RAECs were isolated and cultured to express type I epithelial characteristics. They were then equibiaxially stretched to 25% change in surface area at 15 cycles/minute for 1 hour or 6 hours, or served as unstretched controls, and miRNAs were extracted. Expression profiling of the miRNAs with at least 1.5-fold change over controls revealed 42 miRNAs were regulated (34 up and 8 down) with stretch. We validated 6 of the miRNAs using real-time PCR. Using a parallel mRNA array under identical conditions and publicly available databases, target genes for these 42 differentially regulated miRNAs were identified. Many of these genes had significant up- or down-regulation under the same stretch conditions. There were 362 down-regulated genes associated with up-regulated miRNAs, and 101 up-regulated genes associated with down-regulated miRNAs. Specific inhibition of two selected miRNAs demonstrated a reduction of the increased epithelial permeability seen with cyclic stretch.
We conclude that miRNA expression is differentially expressed between cyclically stretched and unstretched alveolar epithelial cells, and may offer opportunities for therapeutic intervention to ameliorate stretch-associated alveolar epithelial cell dysfunction.
For stroke and spinal cord injury, folic acid supplementation has been shown to enhance neurodevelopment and to provide neuroprotection. We hypothesized that folic acid would reduce brain injury and improve neurological outcome in a neonatal piglet model of traumatic brain injury (TBI), using 4 experimental groups of 3- to 5-day-old female piglets. Two groups were intubated, anesthetized and had moderate brain injury induced by rapid axial head rotation without impact. One group of injured (Inj) animals received folic acid (Fol; 80 μg/kg) by intraperitoneal (IP) injection 15 min following injury, and then daily for 6 days (Inj + Fol; n = 7). The second group of injured animals received an IP injection of saline (Sal) at the same time points (Inj + Sal; n = 8). Two uninjured (Uninj) control groups (Uninj + Fol, n = 8; Uninj + Sal, n = 7) were intubated, anesthetized and received folic acid (80 μg/kg) or saline by IP injection at the same time points as the injured animals following a sham procedure. Animals underwent neurobehavioral and cognitive testing on days 1 and 4 following injury to assess behavior, memory, learning and problem solving. Serum folic acid and homocysteine levels were collected prior to injury and again before euthanasia. The piglets were euthanized 6 days following injury, and their brains were perfusion fixed for histological analysis. Folic acid levels were significantly higher in both Fol groups on day 6. Homocysteine levels were not affected by treatment. On day 1 following injury, the Inj + Fol group showed significantly more exploratory interest, and better motor function, learning and problem solving compared to the Inj + Sal group. Inj + Fol animals had a significantly lower cognitive composite dysfunction score compared to all other groups on day 1. These functional improvements were not seen on day 4 following injury. Axonal injury measured by β-amyloid precursor protein staining 6 days after injury was not affected by treatment. These results suggest that folic acid may enhance early functional recovery in this piglet model of pediatric head injury. This is the first study to describe the application of complex functional testing to assess an intervention outcome in a swine model of TBI.
Neuroprotection; Folic acid; Traumatic brain injury; Neurobehavioral assessment; Pediatric brain injury; Axonal injury; Swine
Rotational inertial forces are thought to be the underlying mechanism for most severe brain injuries. However, little is known about the effect of head rotation direction on injury outcomes, particularly in the pediatric population. Neonatal piglets were subjected to a single non-impact head rotation in the horizontal, coronal, or sagittal direction, and physiological and histopathological responses were observed. Sagittal rotation produced the longest duration of unconsciousness, highest incidence of apnea, and largest intracranial pressure increase, while coronal rotation produced little change, and horizontal rotation produced intermediate and variable derangements. Significant cerebral blood flow reductions were observed following sagittal but not coronal or horizontal injury compared to sham. Subarachnoid hemorrhage, ischemia, and brainstem pathology were observed in the sagittal and horizontal groups but not in a single coronal animal. Significant axonal injury occurred following both horizontal and sagittal rotations. For both groups, the distribution of injury was greater in the frontal and parietotemporal lobes than in the occipital lobes, frequently occurred in the absence of ischemia, and did not correlate with regional cerebral blood flow reductions. We postulate that these direction-dependent differences in injury outcomes are due to differences in tissue mechanical loading produced during head rotation.
animal models; brain ischemia; brain trauma; cerebral blood flow; neuropathology; subarachnoid hemorrhage
We used a nonimpact inertial rotational model of a closed head injury in neonatal piglets to simulate the conditions following traumatic brain injury in infants. Diffuse optical techniques, including diffuse reflectance spectroscopy and diffuse correlation spectroscopy (DCS), were used to measure cerebral blood oxygenation and blood flow continuously and noninvasively before injury and up to 6 h after the injury. The DCS measurements of relative cerebral blood flow were validated against the fluorescent microsphere method. A strong linear correlation was observed between the two techniques (R = 0.89, p < 0.00001). Injury-induced cerebral hemodynamic changes were quantified, and significant changes were found in oxy- and deoxy-hemoglobin concentrations, total hemoglobin concentration, blood oxygen saturation, and cerebral blood flow after the injury. The diffuse optical measurements were robust and also correlated well with recordings of vital physiological parameters over the 6-h monitoring period, such as mean arterial blood pressure, arterial oxygen saturation, and heart rate. Finally, the diffuse optical techniques demonstrated sensitivity to dynamic physiological events, such as apnea, cardiac arrest, and hypertonic saline infusion. In total, the investigation corraborates potential of the optical methods for bedside monitoring of pediatric and adult human patients in the neurointensive care unit.
diffuse correlation spectroscopy (DCS); diffuse reflectance spectroscopy (DRS); cerebral hemodynamics; cerebral blood flow; traumatic brain injury; near—infrared spectroscopy (NIRS)
Closed head injury is the leading cause of death in children less than 4 years of age, and is thought to be caused in part by rotational inertial motion of the brain. Injury patterns associated with inertial rotations are not well understood in the pediatric population. To characterize the physiological and pathological responses of the immature brain to inertial forces and their relationship to neurological development, toddler-age (4-week-old) piglets were subjected to a single non-impact head rotation at either low (31.6 ± 4.7 rad/sec2, n = 4) or moderate (61.0 ± 7.5 rad/sec2, n = 6) angular acceleration in the axial direction. Graded outcomes were observed for both physiological and histopathological responses such that increasing angular acceleration and velocity produced more severe responses. Unlike low-acceleration rotations, moderate-acceleration rotations produced marked EEG amplitude suppression immediately post-injury, which remained suppressed for the 6-h survival period. In addition, significantly more severe subarachnoid hemorrhage, ischemia, and axonal injury by β-amyloid precursor protein (β-APP) were observed in moderate-acceleration animals than low-acceleration animals. When compared to infant-age (5-day-old) animals subjected to similar (54.1 ± 9.6 rad/sec2) acceleration rotations, 4-week-old moderate-acceleration animals sustained similar severities of subarachnoid hemorrhage and axonal injury at 6 h post-injury, despite the larger, softer brain in the older piglets. We conclude that the traditional mechanical engineering approach of scaling by brain mass and stiffness cannot explain the vulnerability of the infant brain to acceleration-deceleration movements, compared with the toddler.
axonal injury; pediatric head injury; pig; toddler
Stretch is an essential mechanism for lung growth and development. Animal models in which fetal lungs have been chronically over- or under-distended demonstrate a disrupted mix of type II and type I cells, with static overdistention typically promoting a type I cell phenotype. The Rho GTPase family, key regulators of cytoskeletal signaling, are known to mediate cellular differentiation in response to stretch in other organs. Using a well-described model of alveolar epithelial cell differentiation and a validated stretch device, we investigated the effects of supraphysiologic stretch on human fetal lung (HFL) alveolar epithelial cell phenotype. Static stretch applied to epithelial cells suppressed type II cell markers (SP-B and Pepsinogen C, PGC), and induced type I cell markers (Caveolin-1, Claudin 7 and Plasminogen Activator Inhibitor-1, PAI-1) as predicted. Static stretch was also associated with Rho A activation. Furthermore, the Rho kinase (ROCK) inhibitor Y27632 decreased Rho A activation, and blunted the stretch-induced changes in alveolar epithelial cell marker expression. Together these data provide further evidence that mechanical stimulation of the cytoskeleton and Rho activation are key upstream events in mechanotransduction-associated alveolar epithelial cell differentiation.
The purpose of this study was to develop a more efficient fluorescent microsphere method to facilitate the rapid use of the histological technique and to enable its use in large tissue regions. Using fluorescent plate/slide imaging technology and automated detection and analysis software, we were able to rapidly image, detect, and count 3 separate microsphere colors in 200 μm-thick tissue sections from piglet brain. In resting newborn piglets (n = 6) on isoflurane anesthesia, we measured a median total cerebral blood flow (CBF) of 105 ml/min/100g (range 27–206 ml/min/100g). Compared with other FM analysis methods, our method reduces the time required to determine blood flow, improves accuracy in lipid-rich tissues and large tissue regions and, unlike the radiolabeled microsphere method, can be combined with histological analysis.
cerebral blood flow; fluorescent microspheres; pig
After a controlled, rapid, head rotation without impact, 73% of 51 neonatal pigs had ocular hemorrhage, primarily located in regions of strong vitreoretinal attachment. This model provides a platform for investigating traumatic ocular hemorrhage in children.
To characterize ocular hemorrhages from single, rapid head rotations in the neonatal pig.
Three- to 5-day-old anesthetized piglets (n = 51) underwent a single, rapid (117-266 rad/s) head rotation in the sagittal (n = 13), coronal (n = 7), or axial (n = 31) planes. Six hours after injury, the animals were euthanatized and perfusion fixed, and the brain and eyes were harvested for gross and histopathologic examination by masked neuro- and ocular pathologists.
Ocular hemorrhage was found in 73% of animals (51% bilateral). Intraocular hemorrhage was primarily located near the vitreous base (70% of injured animals had ciliary body hemorrhage, and 11% had peripheral retinal hemorrhage). Hemorrhages were also found in the anterior chamber (11%), vitreous (5%), and optic nerve (disc, 8%; nerve sheath, 57%). Rapid axial head rotations resulted in a higher incidence of intraocular hemorrhage than coronal or sagittal head rotations, but the difference did not reach statistical significance (P = 0.06). Control eyes had no injuries.
Optic nerve sheath and ciliary body hemorrhages were common in piglets that experienced a single, rapid head rotation. Retinal hemorrhage was present in a smaller number of animals. Most intraocular hemorrhages were located in regions of strong vitreous attachment, suggesting that this animal model will be useful in investigating the effect of vitreoretinal adhesion on ocular hemorrhage caused by inertial head rotations. Extrapolation of this model to the human infant should not be made until the effect of anatomic differences between the human and pig on the occurrence and patterns of ocular injuries is further investigated.
Mechanical ventilation with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses; but the mechanisms that regulate ventilator-associated lung injury and inflammation remain unclear. Analysis of the genomic response of the lung has been performed in intact lungs ventilated at large tidal volumes. This study is the first to study the genomic response of cultured primary alveolar epithelial cells undergoing large and moderate physiologic cyclic stretch. Responses were dependent on stretch magnitude and duration. Genomic expression was validated for 5 genes of interest: Amphiregulin, Glutamate-Cysteine Ligase Catalytic subunit, Matrix Metalloproteinase 7, Protein Phosphatase 1 regulatory inhibitor subunit 10, and Serpine-1, and protein expression mirrored genomic responses. Differences between results reported from homogenized intact lungs and monolayers of alveolar epithelial cells with type-I like phenotype provide provocative evidence that the whole lung preparation may mask the response of individual cell types.
Cell injury; Ventilator-associated lung injury; Microarray
We measured stretch-induced changes in transepithelial permeability in vitro to uncharged tracers 1.5–5.5 Å in radius to identify a critical stretch threshold associated with failure of the alveolar epithelial transport barrier. Cultured alveolar epithelial cells were subjected to a uniform cyclic (0.25 Hz) biaxial 12, 25, or 37% change in surface area (ΔSA) for 1 h. Additional cells served as unstretched controls. Only 37% ΔSA (100% total lung capacity) produced a significant increase in transepithelial tracer permeability, with the largest increases for bigger tracers. Using the permeability data, we modeled the epithelial permeability in each group as a population of small pores punctuated by occasional large pores. After 37% ΔSA, increases in paracellular transport were correlated with increases in the radii of both pore populations. Inhibition of protein kinase C and tyrosine kinase activity during stretch did not affect the permeability of stretched cells. In contrast, chelating intracellular calcium and/or stabilizing F-actin during 37% ΔSA stretch reduced but did not eliminate the stretch-induced increase in paracellular permeability. These results provide the first in vitro evidence that large magnitudes of stretch increase paracellular transport of micromolecules across the alveolar epithelium, partially mediated by intracellular signaling pathways. Our monolayer data are supported by whole lung permeability results, which also show an increase in alveolar permeability at high inflation volumes (20 ml/kg) at the same rate for both healthy and septic lungs.
ventilator-induced lung injury; acute lung injury; barrier properties
Cumulative effects of repetitive mild head injury in the pediatric population are unknown. We have developed a cognitive composite dysfunction score that correlates white matter injury severity in neonatal piglets with neurobehavioral assessments of executive function, memory, learning, and problem solving. Anesthetized 3- to 5-day-old piglets were subjected to single (n = 7), double one day apart (n = 7), and double one week apart (n = 7) moderate (190 rad/s) rapid non-impact axial rotations of the head and compared to instrumented shams (n = 7). Animals experiencing two head rotations one day apart had a significantly higher mortality rate (43%) compared to the other groups and had higher failures rates in visual-based problem solving compared to instrumented shams. White matter injury, assessed by β-APP staining, was significantly higher in the double one week apart group compared to that with single injury and sham. Worsening performance on cognitive composite score correlated well with increasing severity of white matter axonal injury. In our immature large animal model of TBI, two head rotations produced poorer outcome as assessed by neuropathology and neurobehavioral functional outcomes compared to that with single rotations. More importantly, we have observed an increase in injury severity and mortality when the head rotations occur 24 h apart compared to 7 days apart. These observations have important clinical translation to infants subjected to repeated inflicted head trauma.
axonal injury; neurobehavioral assessment; pediatric brain injury; traumatic brain injury
Sepsis results in the formation of pulmonary edema by increasing in epithelial permeability. Therefore we hypothesized that alveolar epithelial cells isolated from septic animals develop tight junctions with different protein composition and reduced barrier function relative to alveolar epithelial cells from healthy animals. Male rats (200–300g) were sacrificed 24 hours after cecal ligation and double puncture (2CLP) or sham surgery. Alveolar epithelial cells were isolated and plated on fibronectin-coated flexible membranes or permeable, non-flexible transwell substrates. After a 5 day culture period, cells were either lysed for western analysis of tight junction protein expressin (claudin 3, 4, 5, 7, 8, and 18, occludin, ZO-1, and JAM-A) and MAPk (JNK, ERK, an p38) signaling activation, or barrier function was examined by measuring transepithelial resistance (TER) or the flux of two molecular tracers (5 and 20 Å). Inhibitors of JNK (SP600125, 20 µM) and ERK (U0126, 10 µM) were used to determine the role of these pathways in sepsis induced epithelial barrier dysfunction. Expression of claudin 4, claudin 18, and occludin was significantly lower, and activation of JNK and ERK signaling pathways was significantly increased in 2CLP monolayers, relative to sham monolayers. Transepithelial resistance of the 2CLP monolayers was reduced significantly compared to sham (769 and 1234 ohm-cm2, respectively), however no significant difference in the flux of either tracer was observed. Inhibition of ERK, not JNK, significantly increased TER and expression of claudin 4 in 2CLP monolayers, and prevented significant differences in claudin 18 expression between 2CLP and sham monolayers. We conclude that alveolar epithelial cells isolated from septic animals form confluent monolayers with impaired barrier function compared to healthy monolayers, and inhibition of ERK signaling partially reverses differences between these monolayers. This model provides a unique preparation for probing the mechanisms by which sepsis alters alveolar epithelium.
Traumatic brain injury (TBI) initiates a cascade of numerous pathophysiological
events that evolve over time. Despite the complexity of TBI, research aimed at
therapy development has almost exclusively focused on single therapies, all of
which have failed in multicenter clinical trials. Therefore, in February 2008
the National Institute of Neurological Disorders and Stroke, with support from
the National Institute of Child Health and Development, the National Heart,
Lung, and Blood Institute, and the Department of Veterans Affairs, convened a
workshop to discuss the opportunities and challenges of testing combination
therapies for TBI. Workshop participants included clinicians and scientists from
a variety of disciplines, institutions, and agencies. The objectives of the
workshop were to: (1) identify the most promising combinations of therapies for
TBI; (2) identify challenges of testing combination therapies in clinical and
pre-clinical studies; and (3) propose research methodologies and study designs
to overcome these challenges. Several promising combination therapies were
discussed, but no one combination was identified as being the most promising.
Rather, the general recommendation was to combine agents with complementary
targets and effects (e.g., mechanisms and time-points), rather than focusing on
a single target with multiple agents. In addition, it was recommended that
clinical management guidelines be carefully considered when designing
pre-clinical studies for therapeutic development. To overcome the challenges of
testing combination therapies it was recommended that statisticians and the U.S.
Food and Drug Administration be included in early discussions of experimental
design. Furthermore, it was agreed that an efficient and validated screening
platform for candidate therapeutics, sensitive and clinically relevant
biomarkers and outcome measures, and standardization and data sharing across
centers would greatly facilitate the development of successful combination
therapies for TBI. Overall there was great enthusiasm for working
collaboratively to act on these recommendations.
clinical trials; head injury; injury mechanisms; intervention; in-vitro and in-vivo models
We cultured (5 days) rat alveolar epithelial cells to investigate the role of mitogen-activated protein kinase (MAPk) signaling in ventilator induced epithelial barrier dysfunction. Cells were stretched to a magnitude of 12% or 37% change in surface area at a rate of 0.25 Hz with and without pretreatment with either the JNK inhibitor SP600125 or the ERK inhibitor U0126. Following stretch (0, 10, 30, or 60 min), MAPk phosphorylation was examined, monolayer permeability to the uncharged tracer carboxyfluorescein measured (0, 10, 60 min of stretch), and occludin expression determined (0 and 60 min of stretch). Stretch to 12%, previously shown not to increase monolayer permeability, did not alter phosphorylation of any MAPk or occludin expression at any time point. Following stretch to 37%, phosphorylation of JNK, ERK, and p38 was significantly higher by 10 minutes than in unstretched monolayers. Phosphorylation of JNK and p38 subsided as stretch continued, and by 30 minutes returned to unstretched levels. Phosphorylation of ERK remained significantly elevated compared to unstretched levels at all stretch durations. Epithelial permeability increased significantly by 10 minutes of stretch compared to unstretched controls, with further significant increases by 60 minutes. Inhibition with U0126 and SP600125 prevented stretch-induced phosphorylation increases of ERK and JNK, respectively, however neither prevented increases in permeability following 10 minutes. Separately, inhibition of JNK or ERK prevented subsequent additional permeability increases as stretch continued to 60 minute time points. Inhibition of JNK, not ERK, prevented loss of occludin, and minimized loss of cell-cell contact following 60 minutes of stretch. These data suggest that stretch-induced JNK signaling modulates epithelial permeability through regulation tight junction protein expression, and is a potential target for clinical treatments during mechanical ventilation.
Neurobehavioral deficits in higher cortical systems have not been described previously in a large animal model of diffuse brain injury. Anesthetized 3–5 day old piglets were subjected to either mild (142 rad/sec) or moderate (188 rad/sec) rapid non-impact axial rotations of the head. Multiple domains of cortical function were evaluated 5 times during the 12 day post-injury period using tests of neurobehavioral function devised for piglets. There were no observed differences in neurobehavioral outcomes between mild injury pigs (N = 8) and instrumented shams (N = 4). Moderately injured piglets (N = 7) had significantly lower interest in exploring their environment and had higher failure rates in visual-based problem solving compared to instrumented shams (N = 5) on Day 1 and 4 after injury. Neurobehavioral functional deficits correlated with neuropathologic damage in the neonatal pigs after inertial head injury. Injured axons detected by immunohistochemistry (β-APP) were absent in mild injury and sham piglets, but were observed in moderately injured piglet brains. In summary, we have developed a quantitative battery of neurobehavioral functional assessments for large animals that correlate with neuropathologic axonal damage and may have wide applications in the fields of cardiac resuscitation, stroke, and hypoxic-ischemic brain injury.
head injury; neurobehavioral assessment; axonal injury