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1.  Influences of Developmental Age on the Resolution of Diffuse Traumatic Intracranial Hemorrhage and Axonal Injury 
Journal of Neurotrauma  2014;31(2):206-214.
This study investigated the age-dependent injury response of diffuse traumatic axonal injury (TAI) and regional subdural and subarachnoid intracranial hemorrhage (ICH) in two pediatric age groups using a porcine head injury model. Fifty-five 5-day-old and 40 four-week-old piglets—which developmentally correspond to infants and toddlers, respectively—underwent either a sham injury or a single rapid non-impact rotational injury in the sagittal plane and were grouped by post-TBI survival time (sham, 3–8 h, one day, 3–4 days, and 5–6 days). Both age groups exhibited similar initial levels of ICH and a significant reduction of ICH over time (p<0.0001). However, ICH took longer to resolve in the five-day-old age group. At 5–6 days post-injury, ICH in the cerebrum had returned to sham levels in the four-week-old piglets, while the five-day-olds still had significantly elevated cerebral ICH (p=0.012). Both ages also exhibited similar resolution of axonal injury with a peak in TAI at one day post-injury (p<0.03) and significantly elevated levels even at 5–6 days after the injury (p<0.008), which suggests a window of vulnerability to a second insult at one day post-injury that may extend for a prolonged period of time. However, five-day-old piglets had significantly more TAI than four-week-olds overall (p=0.016), which presents some evidence for an increased vulnerability to brain injury in this age group. These results provide insight into an optimal window for clinical intervention, the period of increased susceptibility to a second injury, and an age dependency in brain injury tolerance within the pediatric population.
PMCID: PMC3901955  PMID: 23984914
axonal injury; hemorrhage; pediatric traumatic brain injury; swine, time course
2.  Improved Behavior, Motor, and Cognition Assessments in Neonatal Piglets 
Journal of Neurotrauma  2013;30(20):1770-1779.
The alterations of animal behavior after traumatic brain injury (TBI) can be subtle, and their quantitative characterization can present significant methodological challenges. Meeting these challenges is a critical need, because quantitative measures are required in studies that compare the efficacy of different clinical interventions. We developed a battery of assessments to quantify behavioral, motor, and cognitive changes in neonatal piglets with good sensitivity and specificity to the detection of persistent deficits that correlate with axonal injury severity after a rapid non-impact head rotation with a diffuse pattern of axonal injury. The battery of measures developed included open field behaviors of sniffing and moving a toy, locomotion measures of Lempel-Ziv complexity and the probability of remaining in the current location, and a novel metric for evaluating motor performance. Our composite porcine disability score was able to detect brain injury with a sensitivity of 100% and specificity of 85.7% at day +4 post-injury for n=8 injured and n=7 sham piglets and significantly correlated with the percent axonal injury in these animals (day +4: ρ=0.76, p=0.0011). A significant improvement over our previous assessments, this new porcine disability score has potential use in a wide variety of porcine disease and injury models.
PMCID: PMC3796335  PMID: 23758416
cognition; neurobehavioral assessment; pediatric brain injury; porcine; traumatic brain injury
3.  Cyclic Stretch–Induced Oxidative Stress Increases Pulmonary Alveolar Epithelial Permeability 
Mechanical ventilation with high tidal volumes has been associated with pulmonary alveolar flooding. Understanding the mechanisms underlying cyclic stretch–induced increases in alveolar epithelial permeability may be important in designing preventive measures for acute lung injury. In this work, we assessed whether cyclic stretch leads to the generation of reactive oxygen species in type I–like alveolar epithelial cells, which increase monolayer permeability via activation of NF-κB and extracellular signal–regulated kinase (ERK). We cyclically stretched type I–like rat primary alveolar epithelial cells at magnitudes of 12, 25, and 37% change in surface area (ΔSA) for 10 to 120 minutes. High levels of reactive oxygen species and of superoxide and NO specifically were detected in cells stretched at 37% ΔSA for 10 to 120 minutes. Exogenous superoxide and NO stimulation increased epithelial permeability in unstretched cells, which was preventable by the NF-κB inhibitor MG132. The cyclic stretch–induced increase in permeability was decreased by the superoxide scavenger tiron and by MG132. Furthermore, tiron had a dramatic protective effect on in vivo lung permeability under mechanical ventilation conditions. Cyclic stretch increased the activation of the NF-κB signaling pathway, which was significantly decreased with the ERK inhibitor U0126. Altogether, our in vitro and in vivo data demonstrate the sensitivity of permeability to stretch- and ventilation-induced superoxide production, suggesting that using antioxidants may be helpful in the prevention and treatment of ventilator-induced lung injury.
PMCID: PMC3727879  PMID: 23526210
acute lung injury; MAPK; mechanical deformation; p65; ventilator-induced lung injury
4.  Folliculin Controls Lung Alveolar Enlargement and Epithelial Cell Survival through E-cadherin, LKB1 and AMPK 
Cell reports  2014;7(2):412-423.
Spontaneous pneumothoraces due to lung cyst rupture afflict patients with the rare disease Birt-Hogg-Dubé (BHD) syndrome caused by mutations of the tumor suppressor gene folliculin (FLCN) by unknown mechanism. BHD lungs exhibit increased alveolar epithelial cell apoptosis. We show that Flcn deletion in lung epithelium leads to cell apoptosis, alveolar enlargement and impaired lung function. FLCN loss also impairs alveolar epithelial barrier function. Flcn-null epithelial cell apoptosis is the result of impaired AMPK activation and increased cleaved caspase-3. AMPK activator LKB1 and E-cadherin are downregulated by Flcn loss and restored by its expression. Flcn-null cell survival is rescued by AICAR or constitutively active AMPK. AICAR also improves lung condition of Flcnf/f:SP-C-Cre mice. Our data show that Flcn regulates lung epithelial cell survival and alveolar size and suggest that lung cysts in BHD may result from an underlying defect in alveolar epithelial cell survival attributable to FLCN regulation of the E-cadherin-LKB1-AMPK axis.
PMCID: PMC4034569  PMID: 24726356
5.  The Anesthetic Effects on Vasopressor Modulation of Cerebral Blood Flow in an Immature Swine Model 
Anesthesia and analgesia  2013;116(4):838-844.
The effect of various sedatives and anesthetics on vasopressor modulation of cerebral blood flow (CBF) in children is unclear. In adults, isoflurane has been described to decrease CBF to a lesser extent than fentanyl and midazolam. Most large animal models of neurocritical care use inhaled anesthetics for anesthesia. Investigations involving modulations of CBF would have improved translatability within a model that more closely approximates the current practice in the pediatric intensive care unit.
Fifteen (15) four-week-old piglets were given one of 2 anesthetic protocols: total IV anesthesia (TIVA) (midazolam 1 mg/kg/hr and fentanyl 100 mcg/kg/hr, N=8) or ISO (isoflurane 1.5–2% and fentanyl 100 mcg/kg/hr, N=7). Mean arterial blood pressure, intracranial pressure (ICP), CBF, and brain tissue oxygen tension were measured continuously as piglets were exposed to escalating doses of arginine vasopressin, norepinephrine (NE) and phenylephrine (PE).
Baseline CBF was similar in two groups (ISO 38±10 vs. TIVA 35±26 ml/100gm/min) despite lower baseline cerebral perfusion pressure in the ISO group, 45±11 vs. 71±11 mmHg (p< 0.0005). Piglets in ISO group displayed increases in ICP with PE and NE (11±4 vs. 16±4 mmHg and 11±8 vs. 18±5 mmHg; p< 0.05), but in the TIVA group only exposure to PE resulted in increases in ICP when comparing maximal dose values to baseline data (11±4 vs. 15±5 mmHg; p < 0.05). Normalized CBF displayed statistically significant increases with regards to anesthetic group and vasopressor dose when piglets were exposed to NE and PE (p < 0.05), suggesting an impairment of autoregulation within ISO, but not TIVA.
The vasopressor effect on CBF was limited when using a narcotic-benzodiazepine-based anesthetic protocol compared to volatile anesthetics, consistent with a preservation of autoregulation. Selection of anesthetic drugs is critical to investigate mechanisms of cerebrovascular hemodynamics, and in translating critical care investigations between the laboratory and bedside.
PMCID: PMC3606687  PMID: 23460561
6.  Behavioral Deficits and Axonal Injury Persistence after Rotational Head Injury Are Direction Dependent 
Journal of Neurotrauma  2013;30(7):538-545.
Pigs continue to grow in importance as a tool in neuroscience. However, behavioral tests that have been validated in the rodent model do not translate well to pigs because of their very different responses to behavioral stimuli. We refined metrics for assessing porcine open field behavior to detect a wide spectrum of clinically relevant behaviors in the piglet post-traumatic brain injury (TBI). Female neonatal piglets underwent a rapid non-impact head rotation in the sagittal plane (n=8 evaluable) or were instrumented shams (n=7 evaluable). Open field testing was conducted 1 day prior to injury (day −1) in order to establish an individual baseline for analysis, and at days +1 and +4 after injury. Animals were then killed on day +6 after injury for neuropathological assessment of axonal injury. Injured piglets were less interested in interacting with environmental stimuli and had a lower activity level than did shams. These data were compared with previously published data for axial rotational injuries in neonatal piglets. Acute behavioral outcomes post-TBI showed a dependence on the rotational plane of the brain injury, with animals with sagittal injuries demonstrating a greater level of inactivity and less random usage of the open field space than those with axial injuries. The persistence of axonal injury is also dependent on the rotational plane, with sagittal rotations causing more prolonged injuries than axial rotations. These results are consistent with animal studies, finite element models, and studies of concussions in football, which have all demonstrated differences in injury severity depending upon the direction of head impact rotation.
PMCID: PMC3636580  PMID: 23216054
behavioral assessments; cognitive function; pediatric brain injury; TBI
7.  Early Cerebral Perfusion Pressure Augmentation with Phenylephrine after Traumatic Brain Injury may be Neuroprotective in a Pediatric Swine Model 
Critical Care Medicine  2012;40(8):2400-2406.
Cerebral perfusion pressure (CPP) less than 40 mm Hg following pediatric traumatic brain injury (TBI) has been associated with increased mortality independent of age, and current guidelines recommend maintaining CPP between 40–60 mm Hg. Although adult TBI studies have observed an increased risk of complications associated with targeting a CPP > 70, we hypothesize that targeting a CPP of 70 mm Hg with the use of phenylephrine early after injury in the immature brain will be neuroprotective.
Animals were randomly assigned to injury with CPP = 70 mm Hg (CPP70) or CPP = 40 mm Hg (CPP40). Diffuse TBI was produced by a single rapid rotation of the head in the axial plane. Cerebral microdialysis, brain tissue oxygen, intracranial pressure, and cerebral blood flow (CBF) were measured 30 min – 6 h post-injury. One hour after injury, CPP was manipulated with the vasoconstrictor phenylephrine. Animals were euthanized 6 h post-TBI, brains fixed, and stained to assess regions of cell injury and axonal dysfunction.
University center.
21 four week-old female swine.
Measurements and Main Results
Augmentation of CPP to 70 mm Hg resulted in no change in axonal dysfunction, but significantly smaller cell injury volumes at 6 hours post injury compared to CPP40 (1.1% vs. 7.4%, p < 0.05). Microdialysis lactate/pyruvate ratios were improved at CPP70 compared to CPP40. CBF was higher in the CPP70 group but did not reach statistical significance. Phenylephrine was well tolerated and there were no observed increases in serum lactate or intracranial pressure in either group.
Targeting a CPP of 70 mm Hg resulted in a greater reduction in metabolic crisis and cell injury volumes compared to a CPP of 40 mm Hg in an immature swine model. Early aggressive CPP augmentation to a CPP of 70 mm Hg in pediatric TBI before severe intracranial hypertension has the potential to be neuroprotective, and further investigations are needed.
PMCID: PMC3400930  PMID: 22809910
pediatric head injury; cerebral perfusion pressure; phenylephrine; neuroprotection; swine; cerebral blood flow
8.  Influence of age and fall type on head injuries in infants and toddlers 
Age-based differences in fall type and neuroanatomy in infants and toddlers may affect clinical presentations and injury patterns.
Our goal is to understand the influence of fall type and age on injuries to help guide clinical evaluation.
Retrospectively, 285 children 0–48 months with accidental head injury from a fall and brain imaging between 2000–2006 were categorized by age (infant=<1 year and toddler=1–4 years) and fall type: low (≤3 ft), intermediate (>3 and <10 ft), high height falls (≥10 ft) and stair falls.
Outcome Measures
Clinical manifestations were noted and head injuries separated into primary (bleeding) and secondary (hypoxia, edema). The influence of age and fall type on head injuries sustained was evaluated.
Injury patterns in children <4 yrs varied with age. Despite similar injury severity scores, infants sustained more skull fractures than toddlers (71% v. 39%). Of children with skull fractures, 11% had no evidence of scalp/facial soft tissue swelling. Of the patients with primary intracranial injury, 30% had no skull fracture and 8% had neither skull fracture nor cranial soft tissue injury. Low height falls resulted in primary intracranial injury without soft tissue or skull injury in infants (6%) and toddlers (16%).
Within a given fall type, age-related differences in injuries exist between infants and toddlers. When interpreting a fall history, clinicians must consider the fall type and influence of age on resulting injury. For young children, intracranial injury is not always accompanied by external manifestations of their injury.
PMCID: PMC3288448  PMID: 22079853
falls; brain injury; mechanism; age
9.  Finite Element Model Predictions of Intracranial Hemorrhage from Non-Impact, Rapid Head Rotations in the Piglet 
Clinicians are charged with the significant task of distinguishing between accidental and inflicted head trauma. Oftentimes this distinction is straightforward, but many times probabilities of injuries from accidental scenarios are unknown making the differential diagnosis difficult. For example, it is unknown whether intracranial hemorrhage (IH) can occur at a location other than a focal contact site following a low height fall. To create a foundation for predicting regional IH in infants, we sought to identify the biomechanical response and injury threshold best able to predict IH in 3–5 day old piglets. First, finite element (FE) model simulations of in situ animal studies were performed to ascertain the optimal representation of the pia-arachnoid complex, cerebrospinal fluid and cortical vasculature (PCC) for predicting brain strain and brain/skull displacement. Second, rapid head rotations resulting in various degrees of IH were simulated (n=24) to determine the biomechanical predictor and injury threshold most closely correlated with IH. FE models representing the PCC with either spring connectors or solid elements between the brain and skull resulted in peak brain strain and brain/skull displacement similar to measured values in situ. However, when predicting IH, the spring connector representation of the PCC had the best predictive capability for IH with a sensitivity of 80% and a specificity of 85% when ≥ 1% of all spring connectors had at least a peak strain of 0.31 mm/mm. These findings and reported methodology will be used in the development of a human infant FE model to simulate real-world falls and identify injury thresholds for predicting IH in infants.
PMCID: PMC3322291  PMID: 22239917
subdural hemorrhage; traumatic brain injury; falls; brain-skull displacement; pediatric; finite element
10.  Spin Labeling-MRI Detects Increased Myocardial Blood Flow Post Endothelial Cell Transplantation in the Infarcted Heart 
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.
PMCID: PMC3322676  PMID: 22311739
myocardial blood flow; spin labeling; magnetic resonance imaging; myocardial infarction; human umbilical vascular endothelial cells; microspheres; left ventricular ejection fraction
11.  Neurocritical Care Monitoring Correlates with Neuropathology in a Swine Model of Pediatric Traumatic Brain Injury 
Neurosurgery  2011;69(5):1139-1147.
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.
PMCID: PMC3188667  PMID: 21670716
neurocritical care monitoring; pediatric head injury; swine; TBI model
12.  Sepsis Enhances Epithelial Permeability with Stretch in an Actin Dependent Manner 
PLoS ONE  2012;7(6):e38748.
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.
PMCID: PMC3378620  PMID: 22723883
13.  Therapeutic potential of plasma gelsolin administration in a rat model of sepsis 
Cytokine  2011;54(3):235-238.
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.
PMCID: PMC3083472  PMID: 21420877
Sepsis; tissue injury
14.  Cyclosporin A Preserves Mitochondrial Function after Traumatic Brain Injury in the Immature Rat and Piglet 
Journal of Neurotrauma  2011;28(5):763-774.
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.
PMCID: PMC3125546  PMID: 21250918
cerebral microdialysis; cyclosporin A; mitochondrial bioenergetics; neuroprotection; pediatric brain injury
15.  MicroRNA modulate alveolar epithelial response to cyclic stretch 
BMC Genomics  2012;13:154.
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.
PMCID: PMC3425319  PMID: 22537220
16.  Folic Acid Enhances Early Functional Recovery in a Piglet Model of Pediatric Head Injury 
Developmental Neuroscience  2011;32(5-6):466-479.
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.
PMCID: PMC3073761  PMID: 21212637
Neuroprotection; Folic acid; Traumatic brain injury; Neurobehavioral assessment; Pediatric brain injury; Axonal injury; Swine
17.  Physiological and histopathological responses following closed rotational head injury depend on direction of head motion 
Experimental neurology  2010;227(1):79-88.
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.
PMCID: PMC3021173  PMID: 20875409
animal models; brain ischemia; brain trauma; cerebral blood flow; neuropathology; subarachnoid hemorrhage
18.  Diffuse optical monitoring of hemodynamic changes in piglet brain with closed head injury 
Journal of biomedical optics  2009;14(3):034015.
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.
PMCID: PMC3169814  PMID: 19566308
diffuse correlation spectroscopy (DCS); diffuse reflectance spectroscopy (DRS); cerebral hemodynamics; cerebral blood flow; traumatic brain injury; near—infrared spectroscopy (NIRS)
19.  Physiological and Pathological Responses to Head Rotations in Toddler Piglets 
Journal of Neurotrauma  2010;27(6):1021-1035.
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.
PMCID: PMC2943503  PMID: 20560753
axonal injury; pediatric head injury; pig; toddler
20.  The Rho pathway mediates transition to an alveolar type I cell phenotype during static stretch of alveolar type II cells 
Pediatric research  2010;67(6):585-590.
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.
PMCID: PMC3063400  PMID: 20220547
21.  Development of a fluorescent microsphere technique for rapid histological determination of cerebral blood flow 
Brain research  2010;1326:128-134.
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.
PMCID: PMC2855885  PMID: 20193669
cerebral blood flow; fluorescent microspheres; pig
22.  Ocular Hemorrhages in Neonatal Porcine Eyes from Single, Rapid Rotational Events 
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.
PMCID: PMC2941177  PMID: 20435592
23.  Cyclic Stretch Magnitude and Duration Affect Rat Alveolar Epithelial Gene Expression 
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.
PMCID: PMC3025888  PMID: 20054150
Cell injury; Ventilator-associated lung injury; Microarray
24.  Stretch increases alveolar epithelial permeability to uncharged micromolecules 
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.
PMCID: PMC2980812  PMID: 16282193
ventilator-induced lung injury; acute lung injury; barrier properties
25.  Repeated Traumatic Brain Injury Affects Composite Cognitive Function in Piglets 
Journal of Neurotrauma  2009;26(7):1111-1121.
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.
PMCID: PMC2848948  PMID: 19275468
axonal injury; neurobehavioral assessment; pediatric brain injury; traumatic brain injury

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