Cynthia Rovnaghi

Ketamine reduces the cell death following inflammatory pain in newborn rat brain.

Premature infants experience untreated repetitive pain that may alter their brain development. Effects of ketamine and repetitive pain on cellular death and subsequent behavior were studied in neonatal rats. Rat pups were randomized to undisturbed controls (C), 4% formalin injection (F), ketamine alone (K, 5 mg/kg) or formalin plus ketamine (KF) and were assessed for neuroactivation with Fos protein, cellular death with FluoroJade-B, cognition with the radial arm maze, and pain thresholds with the hot-plate. Greater Fos expression and cell death occurred in F vs. C groups in defined brain areas at 1 and 4 h in F compared with other groups. Cell death was accentuated 3.3-fold in cortical areas and 1.6-fold in subcortical areas in the F compared with the C group following repetitive pain and sacrifice 18–20 h later. These effects were ameliorated by ketamine. Compared with the F group, all other groups demonstrated greater exploratory and rearing behaviors and decreased time for bait consumption at 1-h and 3-h intervals. Significantly greater thermal pain latencies occurred in the KF and F groups. Repetitive neonatal pain accentuates neuronal excitation and cell death in developmentally regulated cortical and subcortical areas, which decreases the acquisition of visual-spatial clues, short-term and long-term memory, and increases pain latencies. Ketamine analgesia mitigates most of these effects.

Interactions of inflammatory pain and morphine in infant rats: Long-term behavioral effects

Neonatal rat pups exposed to repetitive acute pain show decreases in pain threshold and altered behavior during adulthood. A model using prolonged inflammatory pain in neonatal rats may have greater clinical relevance for investigating the long-term behavioral effects of neonatal pain in ex-preterm neonates. Neonatal rat pups were exposed to repeated formalin injections on postnatal (P) days 1-7 (P1-P7), with or without morphine pretreatment, and were compared with untreated controls. Behavioral testing during adulthood assessed pain thresholds using hot-plate (HP) and tail-flick (TF) tests, alcohol preference, and locomotor activity (baseline and postamphetamine). Adult rats exposed to neonatal inflammatory pain exhibited longer HP latencies than controls and male rats had longer HP thresholds compared to females. Male rats exposed to neonatal morphine alone exhibited longer TF latencies than controls. Both neonatal morphine treatment and neonatal inflammatory pain decreased ethanol preference, but their effects were not additive. During adulthood, male rats exposed to neonatal inflammatory pain exhibited less locomotor activity than untreated controls. We conclude that neonatal formalin and morphine treatment have specific patterns of long-term behavioral effects in adulthood, some of which are attenuated when the two treatments are combined.

Respiratory Syncytial Virus Disease Is Mediated by Age-Variable IL-33.

Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and severe RSV infections are a significant risk factor for childhood asthma. The pathogenic mechanisms responsible for RSV induced immunopathophysiology remain elusive. Using an age-appropriate mouse model of RSV, we show that IL-33 plays a critical role in the immunopathogenesis of severe RSV, which is associated with higher group 2 innate lymphoid cells (ILC2s) specifically in neonates. Infection with RSV induced rapid IL-33 expression and an increase in ILC2 numbers in the lungs of neonatal mice; this was not observed in adult mice. Blocking IL-33 with antibodies or using an IL-33 receptor knockout mouse during infection was sufficient to inhibit RSV immunopathogenesis (i.e., airway hyperresponsiveness, Th2 inflammation, eosinophilia, and mucus hyperproduction); whereas administration of IL-33 to adult mice during RSV infection was sufficient to induce RSV disease. Additionally, elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV; these cytokines declined during convalescence. In summary, IL-33 is necessary, either directly or indirectly, to induce ILC2s and the Th2 biased immunopathophysiology observed following neonatal RSV infection. This study provides a mechanism involving IL-33 and ILC2s in RSV mediated human asthma.

Ketamine alters the neurogenesis of rat cortical neural stem progenitor cells.

Objective: High doses or prolonged exposure to ketamine increase neuronal apoptosis in the developing brain, although effects on neural stem progenitor cells remain unexplored. This study investigated dose- and time-dependent responses to ketamine on cell death and neurogenesis in cultured rat fetal cortical neural stem progenitor cells. Design: Laboratory-based study. Setting: University research laboratory. Subject: Sprague-Dawley rats. Interventions: Neural stem progenitor cells were isolated from the cortex of Sprague-Dawley rat fetuses on embryonic day 17. In dose-response experiments, cultured neural stem progenitor cells were exposed to different concentrations of ketamine (0–100 µM) for 24 hrs. In time-course experiments, neural stem progenitor cells cultures were exposed to 10 µM ketamine for different durations (0–48 hrs). Measurements and Main Results: Apoptosis and necrosis in neural stem progenitor cells were assessed using activated caspase-3 immunostaining and lactate dehydrogenase assays, respectively. Proliferative changes in neural stem progenitor cells were detected using bromo-deoxyuridine incorporation and Ki67 immunostaining. Neuronal differentiation was assessed using Tuj-1 immunostaining. Cultured neural stem progenitor cells were resistant to apoptosis and necrosis following all concentrations and durations of ketamine exposure tested. Ketamine inhibited proliferation with decreased numbers of bromo-deoxyuridine–positive cells following ketamine exposure to 100 µM for 24 hrs (p < .005) or 10 µM for 48 hrs (p < .01), and reduced numbers of Ki67-positive cells following exposure to ketamine concentration >10 µM for 24 hrs (p < .001) or at 10 µM for 48 hrs (p < .01). Ketamine enhanced neuronal differentiation, with all ketamine concentrations increasing Tuj-1-positive neurons (p < .001) after 24-hrs of exposure. This also occurred with all exposures to 10 µM ketamine for > 8 hrs (p < .001). Conclusions: Clinically relevant concentrations of ketamine do not induce cell death in neural stem progenitor cells via apoptosis or necrosis. Ketamine alters the proliferation and increases the neuronal differentiation of neural stem progenitor cells isolated from the rat neocortex. These studies imply that ketamine exposure during fetal or neonatal life may alter neurogenesis and subsequent brain development.

Ketamine analgesia for inflammatory pain in neonatal rats: a factorial randomized trial examining long-term effects

BackgroundNeonatal rats exposed to repetitive inflammatory pain have altered behaviors in young adulthood, partly ameliorated by Ketamine analgesia. We examined the relationships between protein expression, neuronal survival and plasticity in the neonatal rat brain, and correlated these changes with adult cognitive behavior.MethodsUsing Western immunoblot techniques, homogenates of cortical tissue were analyzed from neonatal rats 18–20 hours following repeated exposure to 4% formalin injections (F, N = 9), Ketamine (K, 2.5 mg/kg × 2, N = 9), Ketamine prior to formalin (KF, N = 9), or undisturbed controls (C, N = 9). Brain tissues from another cohort of rat pups (F = 11, K = 12, KF = 10, C = 15) were used for cellular staining with Fos immunohistochemistry or FluoroJade-B (FJB), followed by cell counting in eleven cortical and three hippocampal areas. Long-term cognitive testing using a delayed non-match to sample (DNMS) paradigm in the 8-arm radial maze was performed in adult rats receiving the same treatments (F = 20, K = 24, KF = 21, C = 27) in the neonatal period.ResultsGreater cell death occurred in F vs. C, K, KF in parietal and retrosplenial areas, vs. K, KF in piriform, temporal, and occipital areas, vs. C, K in frontal and hindlimb areas. In retrosplenial cortex, less Fos expression occurred in F vs. C, KF. Cell death correlated inversely with Fos expression in piriform, retrosplenial, and occipital areas, but only in F. Cortical expression of glial fibrillary acidic protein (GFAP) was elevated in F, K and KF vs. C. No significant differences occurred in Caspase-3, Bax, and Bcl-2 expression between groups, but cellular changes in cortical areas were significantly correlated with protein expression patterns. Cluster analysis of the frequencies and durations of behaviors grouped them as exploratory, learning, preparatory, consumptive, and foraging behaviors. Neonatal inflammatory pain exposure reduced exploratory behaviors in adult males, learning and preparatory behaviors in females, whereas Ketamine ameliorated these long-term effects.ConclusionNeuroprotective effects of Ketamine attenuate the impaired cognitive behaviors resulting from pain-induced cell death in the cortical and hippocampal fields of neonatal rats. This cell death was not dependent on the apoptosis associated proteins, but was correlated with glial activation.

Consciousness, behavior, and clinical impact of the definition of pain

When we exposed the inapplicability of the current definition of pain to neonatal experiences [8], objections to our viewpoint hinged on the absence of consciousness in such nonlinguistic beings [37]. The evidence reviewed in this focus article demonstrates the occurrence and manifestations of consciousness during evolution and human development. Consciousness is described in the full-term newborn, the premature neonate, and the developing human fetus. The clinical implications of ignoring its existence excludes these patients from the benefit of pain relief, promotes more lenient criteria for their participation as research subjects, and produces marked changes in professional attitudes and analgesic practices. We conclude that the definition of pain imposes more stringent standards for acknowledging the presence of pain in nonlinguistic beings and should be changed to include sources of evidence other than verbal self-report.

Double-blind, placebo-controlled pilot randomized trial of methylprednisolone infusion in pediatric acute respiratory distress syndrome.

Objective: Low-dose methylprednisolone therapy in adults with early acute respiratory distress syndrome reduces systemic inflammation, duration of mechanical ventilation, and ICU length of stay. We report a pilot randomized trial of glucocorticoid treatment in early pediatric acute respiratory distress syndrome. Design: Double-blind, placebo-controlled randomized clinical trial. Setting: Le Bonheur Children’s Hospital, Memphis, TN. Patients: Children (0–18 yr) with acute respiratory distress syndrome undergoing mechanical ventilation. Interventions: Patients were randomly assigned to steroid or placebo groups within 72 hours of intubation. IV methylprednisolone administered as loading dose (2 mg/kg) and continuous infusions (1 mg/kg/d) on days 1–7 and then tapered over days 8–14. Both groups were ventilated according to the Acute Respiratory Distress Syndrome Network protocol modified for children. Daily surveillance was performed for adverse effects. Measurements and Main Results: Thirty-five patients were randomized to the steroid (n = 17, no death) and placebo groups (n = 18, two deaths). No differences occurred in length of mechanical ventilation, ICU stay, hospital stay, or mortality between the two groups. At baseline, higher plateau pressures (p = 0.006) and lower Pediatric Logistic Organ Dysfunction scores (p = 0.04) occurred in the steroid group; other characteristics were similar. Despite higher plateau pressures on days 1 (p = 0.006) and 2 (p = 0.025) due to poorer lung compliance in the steroid group, they had lower PaCO2 values on days 2 (p = 0.009) and 3 (p = 0.014), higher pH values on day 2 (p = 0.018), and higher PaO2/FIO2 ratios on days 8 (p = 0.047) and 9 (p = 0.002) compared with the placebo group. Fewer patients in the steroid group required treatment for postextubation stridor (p = 0.04) or supplemental oxygen at ICU transfer (p = 0.012). Steroid therapy was not associated with detectable adverse effects. Conclusion: This study demonstrates the feasibility of administering low-dose glucocorticoid therapy and measuring clinically relevant outcomes in pediatric acute respiratory distress syndrome. Changes in oxygenation and/or ventilation are consistent with early acute respiratory distress syndrome pathophysiology and results of similar clinical trials in adults. We propose and design a larger randomized trial to define the role of glucocorticoid therapy in pediatric acute respiratory distress syndrome.

Anaesthetic neurotoxicity in rodents: is the ketamine controversy real?

INTRODUCTION Different animal models have confirmed the neurotoxic effects of anaesthetics, such as ketamine, but these only occur following large doses or prolonged exposures in very young animals. These findings have questionable clinical relevance and should not preclude the clinician from using these agents. Basic scientists and clinicians need to collaborate actively to develop clinically relevant animal models. Ketamine and other N-methyl-D-aspartate (NMDA) receptor antagonists promote apoptosis in the newborn brain (1). Other commonly used anaesthetic agents (isoflurane, midazolam, nitrous oxide) also enhance neuroapoptosis leading to long-term learning deficits (2). These observations have sparked a widespread debate among paediatricians and anaesthesiologists about their clinical relevance (3,4). In this essay, we provide clinicians with an update on the most recent investigations on the neurotoxic effects of ketamine and suggest that high and prolonged exposure laboratory models are not relevant to decisions about the use of these anaesthetic agents in children.

Deficiency of the two-pore-domain potassium channel TREK-1 promotes hyperoxia-induced lung injury.

Objectives: We previously reported the expression of the two-pore-domain K+ channel TREK-1 in lung epithelial cells and proposed a role for this channel in the regulation of alveolar epithelial cytokine secretion. In this study, we focused on investigating the role of TREK-1 in vivo in the development of hyperoxia-induced lung injury. Design: Laboratory animal experiments. Setting: University research laboratory. Subjects: Wild-type and TREK-1-deficient mice. Interventions: Mice were anesthetized and exposed to 1) room air, no mechanical ventilation, 2) 95% hyperoxia for 24 hours, and 3) 95% hyperoxia for 24 hours followed by mechanical ventilation for 4 hours. Measurements and Main Results: Hyperoxia exposure accentuated lung injury in TREK-1-deficient mice but not controls, resulting in increase in lung injury scores, bronchoalveolar lavage fluid cell numbers, and cellular apoptosis and a decrease in quasi-static lung compliance. Exposure to a combination of hyperoxia and injurious mechanical ventilation resulted in further morphological lung damage and increased lung injury scores and bronchoalveolar lavage fluid cell numbers in control but not TREK-1-deficient mice. At baseline and after hyperoxia exposure, bronchoalveolar lavage cytokine levels were unchanged in TREK-1-deficient mice compared with controls. Exposure to hyperoxia and mechanical ventilation resulted in an increase in bronchoalveolar lavage interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-&agr; levels in both mouse types, but the increase in interleukin-6 and monocyte chemotactic protein-1 levels was less prominent in TREK-1-deficient mice than in controls. Lung tissue macrophage inflammatory protein-2, keratinocyte-derived cytokine, and interleukin-1&bgr; gene expression was not altered by hyperoxia in TREK-1-deficient mice compared with controls. Furthermore, we show for the first time TREK-1 expression on alveolar macrophages and unimpaired tumor necrosis factor-&agr; secretion from TREK-1-deficient macrophages. Conclusions: TREK-1 deficiency resulted in increased sensitivity of lungs to hyperoxia, but this effect is less prominent if overwhelming injury is induced by the combination of hyperoxia and injurious mechanical ventilation. TREK-1 may constitute a new potential target for the development of novel treatment strategies against hyperoxia-induced lung injury.

Regulation of inflammatory biomarkers by intravenous methylprednisolone in pediatric ARDS patients: Results from a double-blind, placebo-controlled randomized pilot trial.

OBJECTIVE A double-blind, randomized controlled trial showed that low-dose glucocorticoid therapy in pediatric ARDS patients is feasible and may improve both ventilation and oxygenation indices in these patients. However, the molecular mechanisms underlying potential changes in outcomes remain unclear. Based on these clinical findings, this study was designed to examine the effects of intravenous methylprednisolone on circulating inflammatory biomarkers in pediatric ARDS patients. DESIGN Double-blind, placebo-controlled randomized trial with blood collection on study entry and day 7. SETTING Tertiary care childrens hospital. PATIENTS Children (0-18years) with ARDS undergoing mechanical ventilation. INTERVENTIONS 35 children were randomized within 72h of mechanical ventilation. The glucocorticoid group received methylprednisolone 2mg/kg loading dose followed by 1mg/kg/day continuous infusion from days 1 to 7. Both groups were ventilated following the ARDSnet recommendations. WBC and differential cell counts, plasma cytokines and CRP levels, and coagulation parameters were analyzed on days 0 and 7. RESULTS At study entry, the placebo group had higher IL-15 and basophil levels. On day 7, in comparison to study entry, the placebo group had lower IL-1α, IFN-γ and IL-10 levels. The glucocorticoid group had lower INF-α, IL-6, IL-10, MCP-1, G-CSF and GM-CSF levels, and higher IL-17α levels on day 7 in comparison to study entry. Total and differential cell counts remained unchanged within the placebo group between days 0 and 7, whereas in the glucocorticoid group total WBC and platelets counts were increased on day 7. Pearsons correlation studies within the placebo and glucocorticoid groups revealed positive and negative correlations between cytokine levels, cell counts, coagulation parameters and relevant clinical parameters of disease severity identified in our previous study. Multiple regression models identified several cytokines as predictors for alterations in clinical parameters of disease severity. CONCLUSION This pilot study shows the feasibility of simultaneously measuring multiple inflammatory cytokines, cell counts and coagulation parameters in pediatric ARDS patients. We report statistical models that may be useful for future, larger trials to predict ARDS severity and outcomes.