Frequency-Domain Low-Wavenumber Hyperspectral Stimulated Raman Scattering Microscopy.

In recent years, stimulated Raman scattering (SRS) microscopy has experienced rapid technological advancements and has found widespread applications in chemical analysis. Hyperspectral SRS (hSRS) microscopy further enhances the chemical selectivity in imaging by providing a Raman spectrum for each pixel. Time-domain hSRS techniques often require interferometry and ultrashort femtosecond laser pulses. They are especially suited to measuring low-wavenumber Raman transitions but are susceptible to scattering-induced distortions. Frequency-domain hSRS microscopy, on the other hand, offers a simpler optical configuration and demonstrates high tolerance to sample scattering but typically operates within the spectral range of 400-4000 cm-1. Conventional frequency-domain hSRS microscopy is widely employed in biological applications but falls short in detecting chemical bonds with a weaker vibrational energy. In this work, we extend the spectral coverage of picosecond spectral-focusing hSRS microscopy to below 100 cm-1. This frequency-domain low-wavenumber hSRS approach can measure the weaker vibrational energy from the sample and has a strong tolerance to sample scattering. By expanding spectral coverage to 100-4000 cm-1, this development enhances the capability of spectral-domain SRS microscopy for chemical imaging.

Hybrid Thrombectomy and Central Extracorporeal Membrane Oxygenation for Massive Pulmonary Embolism in a Child.

We describe a hybrid thrombectomy and central extracorporeal membrane oxygenation for a child in cardiogenic shock due to a massive pulmonary embolism.

Spatiotemporally Precise Optical Manipulation of Intracellular Molecular Activities.

Controlling chemical processes in live cells is a challenging task. The spatial heterogeneity of biochemical reactions in cells is often overlooked by conventional means of incubating cells with desired chemicals. A comprehensive understanding of spatially diverse biochemical processes requires precise control over molecular activities at the subcellular level. Herein, a closed-loop optoelectronic control system is developed that allows the manipulation of biomolecular activities in live cells at high spatiotemporal precision. Chemical-selective fluorescence signals are utilized to command lasers that trigger specific chemical processes or control the activation of photoswitchable inhibitors at desired targets. This technology is fully compatible with laser scanning confocal fluorescence microscopes. The authors demonstrate selective interactions of a 405 nm laser with targeted organelles and simultaneous monitoring of cell responses by fluorescent protein signals. Notably, blue laser interaction with the endoplasmic reticulum leads to a more pronounced reduction in cytosolic green fluorescent protein signals in comparison to that with nuclei and lipid droplets. Moreover, when combined with a photoswitchable inhibitor, microtubule polymerization is selectively inhibited within the subcellular compartments. This technology enables subcellular spatiotemporal optical manipulation over chemical processes and drug activities, exclusively at desired targets, while minimizing undesired effects on non-targeted locations.

Central Extracorporeal Membrane Oxygenation Support Following Calcium Channel Blocker Overdose in Children.

Refractory vasodilatory shock (RVS) following massive calcium channel blocker (CCB) overdose remains a challenging clinical entity. Peripheral venoarterial extracorporeal membrane oxygenation (ECMO) has proven useful in several cases of CCB intoxication, however, its use in the pediatric population poses unique challenges given the generally small size of pediatric peripheral vasculature in comparison to the high flow rates necessary for adequate mechanical circulatory support. As a result of these challenges, our group has adopted a "primary" central ECMO cannulation approach to the treatment of children and adolescents admitted to our center with profound RVS after CCB ingestion. We present four cases within the last year using this approach. All patients were successfully discharged from the hospital with no late morbidity at most recent follow-up. Central ECMO support in cases of massive vasodilatory shock following CCB overdose is safe and effective and should be considered early in the clinical course of these critically ill patients.

Amyloid ß Induces Lipid Droplet-Mediated Microglial Dysfunction in Alzheimer's Disease.

Several microglia-expressed genes have emerged as top risk variants for Alzheimer's disease (AD). Impaired microglial phagocytosis is one of the main proposed outcomes by which these AD-risk genes may contribute to neurodegeneration, but the mechanisms translating genetic association to cellular dysfunction remain unknown. Here we show that microglia form lipid droplets (LDs) upon exposure to amyloid-beta (Aß), and that their LD load increases with proximity to amyloid plaques in brains from human patients and the AD mouse model 5xFAD. LD formation is dependent upon age and disease progression and is more prominent in the hippocampus in mice and humans. Despite variability in LD load between microglia from male versus female animals and between cells from different brain regions, LD-laden microglia exhibited a deficit in Aß phagocytosis. Unbiased lipidomic analysis identified a substantial decrease in free fatty acids (FFAs) and a parallel increase in triacylglycerols (TAGs) as the key metabolic transition underlying LD formation. We demonstrate that DGAT2, a key enzyme for the conversion of FFAs to TAGs, promotes microglial LD formation, is increased in microglia from 5xFAD and human AD brains, and that inhibiting DGAT2 improved microglial uptake of Aß. These findings identify a new lipid-mediated mechanism underlying microglial dysfunction that could become a novel therapeutic target for AD.

Chemical-imaging-guided optical manipulation of biomolecules.

Chemical imaging via advanced optical microscopy technologies has revealed remarkable details of biomolecules in living specimens. However, the ways to control chemical processes in biological samples remain preliminary. The lack of appropriate methods to spatially regulate chemical reactions in live cells in real-time prevents investigation of site-specific molecular behaviors and biological functions. Chemical- and site-specific control of biomolecules requires the detection of chemicals with high specificity and spatially precise modulation of chemical reactions. Laser-scanning optical microscopes offer great platforms for high-speed chemical detection. A closed-loop feedback control system, when paired with a laser scanning microscope, allows real-time precision opto-control (RPOC) of chemical processes for dynamic molecular targets in live cells. In this perspective, we briefly review recent advancements in chemical imaging based on laser scanning microscopy, summarize methods developed for precise optical manipulation, and highlight a recently developed RPOC technology. Furthermore, we discuss future directions of precision opto-control of biomolecules.

Pulse-Picking Multimodal Nonlinear Optical Microscopy.

Nonlinear optical microscopy techniques can map chemical compositions in biological samples in a label-free manner. Commonly used nonlinear optical processes for imaging include multiphoton excitation fluorescence (MPEF), second harmonic generation (SHG), and coherent Raman scattering (CRS). Femtosecond lasers are typically used for MPEF and SHG due to the requirement of high peak power for excitation, while picosecond lasers are preferred for CRS due to the need for high spectral resolution. Therefore, it is challenging to integrate CRS with MPEF and SHG for chemical imaging. We develop a pulse-picking strategy based on an acousto-optic modulator that can program the duty cycle of the laser pulse train, significantly increasing the pulse peak power at low input average power. This approach offers strong enhancement of nonlinear optical signals and makes hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy compatible with MPEF and SHG for multimodal imaging at low laser average power. The pulse-picking method also enables the evaluation and comparison of phototoxicity of laser pulses at different average and peak power levels. The photo-perturbations to biological samples are evaluated using cellular dynamics and sample morphological changes, allowing the selection of optimal laser power for the best sensitivity and minimal phototoxicity.

Real-time precision opto-control of chemical processes in live cells.

Precision control of molecular activities and chemical reactions in live cells is a long-sought capability by life scientists. No existing technology can probe molecular targets in cells and simultaneously control the activities of only these targets at high spatial precision. We develop a real-time precision opto-control (RPOC) technology that detects a chemical-specific optical response from molecular targets during laser scanning and uses the optical signal to couple a separate laser to only interact with these molecules without affecting other sample locations. We demonstrate precision control of molecular states of a photochromic molecule in different regions of the cells. We also synthesize a photoswitchable compound and use it with RPOC to achieve site-specific inhibition of microtubule polymerization and control of organelle dynamics in live cells. RPOC can automatically detect and control biomolecular activities and chemical processes in dynamic living samples with submicron spatial accuracy, fast response time, and high chemical specificity.

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

Early Versus Late Sternal Closure in Infants-Perioperative Associations and Outcomes.

BACKGROUND: Delayed sternal closure (DSC) has been used for patients who develop bleeding, chest wall edema, and malignant arrhythmia following cardiopulmonary bypass. Multiple factors can influence the timing of when to perform DSC. We aimed to describe our DSC experience in neonates and infants by comparing outcomes between patients undergoing early (<48 hours) versus late DSC (> 48 hours). We explored the associations between specific clinical and laboratory variables and the timing of DSC. METHODS: Retrospective chart review of neonates and infants (

Pediatric Donor Management Goals in Use by US Organ Procurement Organizations.

INTRODUCTION: A recent study of pediatric organ donation after the neurologic determination of death (DNDD) demonstrated an association between the use of donor management goals (DMGs) by organ procurement organizations (OPOs) and organ yield. OBJECTIVE: To describe the pediatric DMGs used by OPOs and any association between specific DMGs and organ yield. DESIGN: Query of US OPOs who utilized DMGs in the care of pediatric DNDD organ donors from 2010 to 2013. RESULTS: All 23 OPOs using DMGs for pediatric DNDD organ donors during the study period participated (100%). The OPOs pursued an average 9.6 goals (standard deviation: 3.9; range: 5-22) with 113 unique definitions that targeted 33 aspects of donor hemodynamics, gas exchange/mechanical ventilation, electrolytes/renal function, blood products, thermoregulation, and infection control. The DMGs used by >50% of OPOs included blood pressure, oxygenation (partial pressure of arterial oxygen (PaO2), oxygen saturation of hemoglobin by pulse oximetry, or PaO2/fractional concentration of inspired oxygen [FiO2] ratio), pH, central venous pressure, serum sodium, urine output, limitations on inotropic support, and serum glucose. There was no significant correlation between the number of DMGs pursued by OPOs and organ yield. There was a difference in the observed/expected organs transplanted in the 0- to 10-year age-group for OPOs that included serum creatinine among their DMGs ( P = .046). CONCLUSIONS: The pediatric DMGs used by OPOs were generally measurable but diverse in definition and the number of goals pursued. There was no benefit in organ yield from larger DMG bundles. There may be a benefit in organ yield through the use of serum creatinine as a DMG in pediatric donors aged 0 to 10 years.

Variability of DKA Management Among Pediatric Emergency Room and Critical Care Providers: A Call for More Evidence-Based and Cost-Effective Care?

Management protocols have been shown to be effective in the pediatric emergency medicine (PEM) and pediatric critical care (PCC) settings. Treatment protocols define clear goals which are achieved with consistency in implementation. Over the last decade, many new recommendations have been proposed on managing diabetic ketoacidosis (DKA). Although no perfect set of guidelines exist, many institutions are developing DKA treatment protocols. We sought to determine the variability between institutions in implementation of these protocols.

Characterization of human serum butyrylcholinesterase in rhesus monkeys: behavioral and physiological effects.

The effects of a large dose of human serum butyrylcholinesterase (HuBChE) were evaluated in rhesus monkeys using a serial-probe recognition (SPR) task designed to assess attention and short-term memory. Each monkey received an intravenous injection of 150 mg (105,000 U or 30 mg/kg) of HuBChE 60 min prior to testing on the SPR task. Concurrent with the cognitive-behavioral assessment, blood was collected at various time points throughout the study and was analyzed for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities, anti-BChE antibody production and gross clinical pathology (i.e., complete blood count and clinical chemistry panel). HuBChE revealed a peak blood activity of 227 U/ml at 5 min after intravenous injection and a mean residence time of approximately 72 h. No cognitive-behavioral decrements of any kind in SPR performance and no toxic signs in clinical pathology were detected in any of the blood assays during the 5 weeks of observation. Anti-HuBChE antibodies peaked at about 14 days after injection, with no concomitant behavioral changes. These results demonstrate the behavioral and physiological safety of HuBChE in rhesus monkeys and support its development as a bioscavenger for the prophylaxis of chemical warfare agent toxicity in humans.

Systemic administration of the potential countermeasure huperzine reversibly inhibits central and peripheral acetylcholinesterase activity without adverse cognitive-behavioral effects.

Huperzine A is potentially superior to pyridostigmine bromide as a pretreatment for nerve agent intoxication because it inhibits acetylcholinesterase both peripherally and centrally, unlike pyridostigmine, which acts only peripherally. Using rhesus monkeys, we evaluated the time course of acetylcholinesterase and butyrylcholinesterase inhibition following four different doses of -(-)huperzine A: 5, 10, 20, and 40 microg/kg. Acetylcholinesterase inhibition peaked 30 min after intramuscular injection and varied dose dependently, ranging from about 30% to 75%. Subsequently, cognitive-behavioral functioning was also evaluated at each dose of huperzine A using a six-item serial-probe recognition task that assessed attention, motivation, and working memory. Huperzine did not impair performance, but physostigmine did. The results demonstrate that huperzine A can selectively and reversibly inhibit acetylcholinesterase without cognitive-behavioral side effects, thus warranting further study.

Galantamine is a novel post-exposure therapeutic against lethal VX challenge.

The ability of galantamine hydrobromide (GAL HBr) treatment to antagonize O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX)-induced lethality, impairment of muscle tension, and electroencephalographic (EEG) changes was assessed in guinea pigs. Guinea pigs were challenged with 16.8 microg/kg VX (2LD50). One min after challenge, animals were administered 0.5 mg/kg atropine sulfate (ATR) and 25 mg/kg pyridine-2-aldoxime methochloride (2-PAM). In addition, guinea pigs were given 0, 1, 2, 4, 8 or 10 mg/kg GAL as a post-exposure treatment immediately prior to ATR and 2-PAM. Animals were either monitored for 24-h survival, scheduled for electroencephalography (EEG) recording, or euthanized 60 min later for measurement of indirectly-elicited muscle tension in the hemidiaphragm. Post-exposure GAL therapy produced a dose-dependent increase in survival from lethal VX challenge. Optimal clinical benefits were observed in the presence of 10 mg/kg GAL, which led to 100% survival of VX-challenged guinea pigs. Based on muscle physiology studies, GAL post-exposure treatment protected the guinea pig diaphragm, the major effector muscle of respiration, from fatigue, tetanic fade, and muscular paralysis. Protection against the paralyzing effects of VX was dose-dependent. In EEG studies, GAL did not alter seizure onset for all doses tested. At the highest dose tested (10 mg/kg), GAL decreased seizure duration when administered as a post-exposure treatment 1 min after VX. GAL also reduced the high correlation associated between seizure activity and lethality after 2LD50 VX challenge. GAL may have additional benefits both centrally and peripherally that are unrelated to its established mechanism as a reversible acetylcholinesterase inhibitor (AChEI).

Serial-probe recognition in rhesus macaques: effects of midazolam.

A serial-probe recognition task was used to assess the effects of midazolam on visual attention and short-term memory in three rhesus monkeys. On each trial, six unique alphanumeric sample stimuli (list items) were presented sequentially followed by a choice period. Choosing the 'probe' stimulus was correct if the probe matched one of the list items; otherwise, choosing the 'default' stimulus (a white square) was correct. Behavior was examined under a range of doses of midazolam (0.065, 0.13, 0.26, and 0.52 mg/kg IM). Midazolam did not significantly reduce choice accuracy or change the shape of the serial position function and did not significantly reduce choice responding. However, choice reaction time was significantly increased by the two highest doses of midazolam. Responding directed at the sample stimuli was reduced at the two highest doses of midazolam. Furthermore, 0.52 mg/kg midazolam significantly increased sample-stimulus reaction time at all six serial positions. Overall, these data suggest that the two highest doses of midazolam tested increase reaction time, but do not directly impair short-term visual recognition memory. This is noteworthy because such doses appear capable of protecting against nerve agent-induced seizures.

Effects of physostigmine and human butyrylcholinesterase on acoustic startle reflex and prepulse inhibition in C57BL/6J mice.

The use of exogenously administered cholinesterases as bioscavengers of highly toxic organophosphorus nerve agents is a viable prophylactic against this threat. To use this strategy, cholinesterases must provide protection without disrupting behavior when administered alone. To assess behavioral safety, the acoustic startle reflex and prepulse inhibition (PPI) of C57BL/6J mice were investigated following administration of human plasma-derived butyrylcholinesterase (HuBChE). Two hours before testing, four groups of mice (n=10 per group) were pretreated with saline or HuBChE (2000 U, ip). Fifteen minutes before testing, subjects received either saline or the carbamate physostigmine (0.4 mg/kg, sc). Mice exposed to physostigmine exhibited a significant attenuation of the startle reflex, an increased time to peak startle amplitude, and significantly increased PPI. This effect was partially mitigated in mice pretreated with HuBChE. HuBChE alone did not change startle behavior or PPI significantly compared to saline controls. The circulatory time-course of butyrylcholinesterase was assessed in a separate group of mice and revealed levels approximately 600 times the physiological norm 2-4 h post administration. Thus, HuBChE does not appear to significantly alter startle or PPI behavior at a dose 30-fold higher than that estimated to be necessary for protection against 2LD50 of soman in humans.

Age and experience-related improvements in gap detection in the rat.

The ability to accurately process brief, successive acoustic signals rapidly presented to the central nervous system is believed to underlie successful language development. The limits of temporal resolution of the auditory system, often assessed using gap detection tasks, has been widely studied in relation to developing and decoding speech. In the present study, a reflex modification paradigm was used to investigate potential shifts in gap detection thresholds in rats across development, with test sessions beginning on postnatal day (P) 15, P35 and P64. We found that thresholds decreased over the course of development. These thresholds were determined to lie between 10 and 20 ms for the P15 and P35 groups, and between 5 and 10 ms for the P64 group. Moreover, we observed improvements in gap detection thresholds in all age groups over 5 days of testing, including the youngest age group (P15). These later results suggest that experience-dependent plasticity mechanisms at the level of sensory processing are operational and observable both very early in development, and also in adult animals. The present findings also demonstrate maturational improvements in silent gap detection using a pre-pulse inhibition paradigm.

Air and shock two-way shuttlebox avoidance in C57BL/6J and 129X1/SvJ mice.

Despite multiple advantages of the use of electric shock as an aversive stimulus, reasons exist for considering alternative aversive stimuli. In the present study, we examined and compared the acquisition of two-way shuttlebox avoidance with 275.8-kPa (40-psi) pulsed air and continuous 0.4-mA shock in two strains of mice commonly employed in targeted gene mutation research, C57BL/6J and 129X1/SvJ. Each trial consisted of a 5-s warning stimulus (WS, light) during which shuttling to the other side cancelled delivery of the aversive stimulus. Once initiated, the aversive stimulus remained active for 20 s or until an escape response occurred. For C57BL/6J mice, air and shock were equally and highly effective aversive stimuli. In contrast, air was less effective than shock for 129X1/SvJ mice. C57BL/6J mice outperformed 129X1/SvJ mice for both stimulus types. For 129X1/SvJ mice, longer escape latencies were observed initially for air, suggesting that shock is more effective. However, these differences in latency dissipated within the first seven sessions. Nevertheless, by the end of the 17-day study, asymptotic levels of avoidance proficiency were substantially lower for air than for shock in 129X1/SvJ mice. These results indicate that air is a suitable substitute for shock as an aversive stimulus in shuttlebox active avoidance; however, the relative efficacies of these aversive stimuli appear to depend upon the strain chosen for study.