Artificial Cerebral Spinal Fluid Research Articles

141 Polyvinylpyrrolidone-Coated Catheters Show Fewer Astrocytes and Choroid Plexus Epithelium Adhesion and Better Flow/Pressure Performance Than Other Commercial Catheters in a New Catheter Assay System

INTRODUCTION: The major cause of failure of cerebral spinal fluid (CSF) shunting devices in pediatric patients is proximal catheter occlusion. There are a number of shunt catheters commercially available, but their occlusion properties are unclear. METHODS: There were four catheters tested: 1) antibiotic-impregnated, 2) polyvinylpyrrolidone, 3) barium stripe, and 4) barium impregnated. Catheters were inoculated with cultured astrocytes or choroid cells and incubated in growth media for 7 days prior to testing. Sample catheters were placed into a 3D-printed ventricular replicating system to test occlusion properties. Artificial CSF was pumped through the system to mimic physiologic flow and pressure resistance sensors measured obstruction. Afterward testing, catheters were stained with anti-GFAP or anti-transthyretin to visualize cellular adhesion using a fluorescence microscope. RESULTS: Five catheters of each type were used. Polyvinylpyrrolidone catheters had the highest flow (e.g., least obstruction) in the ventricular replication system (0.12 mL/min) compared to the antibiotic coated (0.10 mL/min), barium stripe (0.02 mL/min) or barium impregnated (0.08 mL/min; p < 0.01). Polyvinylpyrrolidone catheters showed the lowest median cell attachment after cell marker staining (<1 cells per 0.07 mm2) compared to antibiotic coated (4.0 cells per 0.07 mm2), barium stripe (3.5 per 0.07 mm2) or barium impregnated (2.0 per 0.07 mm2). CONCLUSIONS: Polyvinylpyrrolidone catheters were least likely to be occluded by astrocytes or choroid cells during testing in a novel ventricular replicating system due to lower cellular adhesion. Our findings may have clinical significance when choosing proximal ventricular catheters that are least likely to occlude and prevent proximal shunt failure.

Responses of the in vitro turtle brain to visual and auditory stimuli during severe hypoxia.

North American pond turtles (Emydidae) are renowned for their ability to survive extreme hypoxia and anoxia, which enables several species to overwinter in ice-locked, anoxic freshwater ponds and bogs for months. Centrally important for surviving these conditions is a profound metabolic suppression, which enables ATP demands to be met entirely with glycolysis. To better understand whether anoxia limits special sensory functions, we recorded evoked potentials in a reduced brain preparation, in vitro, that was perfused with severely hypoxic artificial cerebral spinal fluid (aCSF). For recordings of visual responses, an LED was flashed onto retinal eyecups while evoked potentials were recorded from the retina or the optic tectum. For recordings of auditory responses, a piezomotor-controlled glass actuator displaced the tympanic membrane while evoked potentials were recorded from the cochlear nuclei. We found that visual responses decreased when perfused with hypoxic perfusate (aCSF PO2<4.0 kPa). In contrast, the evoked response within the cochlear nuclei was unattenuated. These data provide further support that pond turtles have a limited ability to sense visual information in their environment even while moderately hypoxic, but that auditory input may become a principal avenue of sensory perception during extreme diving in this species such as occurs during anoxic submergence.

Susceptibility of aged pericytes to Aβ1‐42 oligomers

AbstractBackgroundAging is associated with increased incidence of Alzheimer’s disease, blood brain barrier breakdown, and increased cellular senescence (Saez‐Atienzar et. al, 2020). Senescence is the permanent arrest of the proliferative state of the cell and is initiated as a result of aging and cellular stress (Martínez‐Cué et.al, 2020). Senescent cells induce production of proinflammatory cytokines. In the brain, chronic inflammation leads to synapse damage and cognitive decline (Saez‐Atienzar et. al, 2020). The objective of this study is to examine the impact of cellular senescence on brain vascular pericytes, which contribute to the regulation of cerebral blood flow.MethodFor this study, both in vitro as well as in vivo experiments were performed. To induce senescence by oxidative stress in vitro in a culture of mouse brain vascular pericytes, 100 uM of hydrogen peroxide was added to culture flasks. Senescence was confirmed in the treated cells via staining for β‐galactosidase, a known biomarker of senescent cells. The next steps for the in vitro aspects of this study include performing western blotting on senescent pericytes to determine if there is a differential expression of pericyte p75 neurotrophin receptor (p75NTR), which has a potential role in the impaired hemodynamic response and cell death in pericytes. Finally, cranial window procedures were performed on 18‐month‐old C57BL/6 mice followed by imaging the live brain tissue with a multiphoton microscope. While imaging, artificial cerebral spinal fluid (control animals) or 72 nM amyloid‐beta (Aβ)1‐42 oligomers were topically delivered to the brain surface to determine if Aβ1‐42 oligomers cause a significant reduction in erythrocyte flow through pericyte covered capillaries.ResultPreliminary data from the β‐galactosidase staining suggest that the hydrogen peroxide is an effective method for inducing senescence in mouse brain vascular pericytes. We have observed a decreased growth rate in cells treated with the hydrogen peroxide, further suggesting senescence in these cells.ConclusionThese findings characterize the impact of Aβ on pericyte contractility and could illuminate the role of senescence and aging on impaired hemodynamic responses because of dysfunctional pericytes.

Ultrasensitive dopamine detection with graphene aptasensor multitransistor arrays

Detecting physiological levels of neurotransmitters in biological samples can advance our understanding of brain disorders and lead to improved diagnostics and therapeutics. However, neurotransmitter sensors for real-world applications must reliably detect low concentrations of target analytes from small volume working samples. Herein, a platform for robust and ultrasensitive detection of dopamine, an essential neurotransmitter that underlies several brain disorders, based on graphene multitransistor arrays (gMTAs) functionalized with a selective DNA aptamer is presented. High-yield scalable methodologies optimized at the wafer level were employed to integrate multiple graphene transistors on small-size chips (4.5 × 4.5 mm). The multiple sensor array configuration permits independent and simultaneous replicate measurements of the same sample that produce robust average data, reducing sources of measurement variability. This procedure allowed sensitive and reproducible dopamine detection in ultra-low concentrations from small volume samples across physiological buffers and high ionic strength complex biological samples. The obtained limit-of-detection was 1 aM (10–18) with dynamic detection ranges spanning 10 orders of magnitude up to 100 µM (10–8), and a 22 mV/decade peak sensitivity in artificial cerebral spinal fluid. Dopamine detection in dopamine-depleted brain homogenates spiked with dopamine was also possible with a LOD of 1 aM, overcoming sensitivity losses typically observed in ion-sensitive sensors in complex biological samples. Furthermore, we show that our gMTAs platform can detect minimal changes in dopamine concentrations in small working volume samples (2 µL) of cerebral spinal fluid samples obtained from a mouse model of Parkinson’s Disease. The platform presented in this work can lead the way to graphene-based neurotransmitter sensors suitable for real-world academic and pre-clinical pharmaceutical research as well as clinical diagnosis.

In situ electrodeposition of bismuth oxide nanowires @MWNT on the carbon fiber microelectrode for the sensitively electrochemical detection of folic acid

A microminiaturized electrochemical device, BiO@CNW/CFE was fabricated based on the in situ co-electrodeposition of bismuth oxide nanowires (BiNWs) and multi-walled carbon nanotubes (MWNTs) on the surface of carbon fiber electrode (CFE). The nanostructure of BiNWs could bind MWNTs on the surface of CFE during the precipitation of bismuth at the potential of −1.1 V. The vimineous nanostructure of BiO@CNW improved the surface area and electrochemical activity of the microelectrode. With the low background noise, folic acid (FA) can be detected sensitively by BiO@CNW/CFE based on the electrochemical reduction via the method of square wave voltammetry. The linear range of FA in sodium acetate-acetic acid buffer was achieved in the range of 5.00 nM–200 nM, the detection limit was estimated to be 0.63 nM. The recoveries of FA in human serum and artificial cerebral spinal fluid were between 99% and 103%, which indicates BiO@CNW/CFE was a reliable sensor for the detection of FA in biological samples.

Abstract 2436: CIRBP is a functional mediator of leptomeningeal metastasis

Abstract Background: Cold-Inducible RNA Binding Protein (CIRBP) has been implicated in cancer initiation. Moreover, we have previously identified an association of CIRBP with the invasive growth of parenchymal brain metastases. Using a patient-derived xenograft (PDX) model, in which cells implanted in the mouse mammary fat pad spontaneously metastasize to the leptomeninges, we identified CIRBP as being elevated in leptomeningeal metastases (LM) compared to matched primary mammary tumors. LM grow in the subarachnoid space that harbors the cerebral spinal fluid (CSF). The leptomeninges represent an inhospitable environment for disseminated cancer cells since the CSF is mitogen and nutrient poor. To investigate the importance of CIRBP for metastatic breast cancer cells in the leptomeninges, we stably reduced CIRBP expression in MDA-MB-231 cells and performed mammary fat pad and cranial injections. CIRBP knockdown had no influence on primary tumor growth, but diminished growth within the brain. Hypothesis: We hypothesize that CIRBP functions as a stress response protein enabling cancer cells to grow in the harsh environment of the CSF. Results: In response to various cellular stressors, such as hypothermia, hypoxia or UV irradiation, CIRBP can translocate from the nucleus to the cytoplasm. We show that culturing MDA-MB-231 cells in artificial CSF (aCSF) also causes CIRBP relocation from nucleus to cytoplasm. Using RNA-IP approaches, we have identified mRNAs bound by CIRBP in breast cancer cells treated with aCSF. Ultimately, we will determine whether any of the identified CIRBP-regulated targets contribute to the formation of LM. Finally, we have generated CIRBP variants that are confined either to the nucleus or the cytoplasm. We are currently assessing the ability of these compartment-restricted CIRBP proteins to promote the formation of central nervous system metastases. Conclusions: We have identified CIRBP as a promoter of leptomeningeal metastasis. Our results have begun to reveal the mechanisms through which CIRBP mediates this process. Citation Format: Rima Ezzeddine, Matthew Dankner, Paul Savage, Steven Hebert, William Brothers, Matthew G. Annis, Sarah M. Maritan, Marc R. Fabian, Claudia Kleinman, Morag Park, Peter M. Siegel. CIRBP is a functional mediator of leptomeningeal metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2436.

Anti-inflammatory effect of P2Y1 receptor blocker MRS2179 in a rat model of traumatic brain injury

The aim of the current study was to determine the effects of cerebral contusion injury with purinergic adenosine triphosphate Y1 (P2Y1) receptor blockers on postinjury inflammatory responses. Adenosine triphosphate (ATP) is released into the extracellular space in several in vivo models, including traumatic brain injury. Released ATP triggers neuroinflammation via activation of microglial cells. P2Y1 receptor blockers were reported to suppress extracellular ATP elevation in several disease models through inhibition of cellular ATP release. In addition to the beneficial effects of inflammation, excess inflammatory reactions cause secondary damage and aggravate outcomes. Here, we assessed the effect of the selective P2Y1 receptor blocker MRS2179 on its potential to prevent posttraumatic inflammation in a rat cerebral contusion model. Cerebral contusion injury was induced in the rat cerebral cortex. Either MRS2179 or artificial cerebral spinal fluid as a control was administered in situ into the center of contused tissue via a subcutaneously implanted osmotic pump. Galectin 3, a marker of microglia and proinflammatory cytokines, was measured 1, 3 and 7 days following injury. Another group of rats was assessed for behavioral performance up to 28 days after injury, including the beam walk test, neurological response test and plus maze test. The Galectin 3 levels in the cortex around the contusion cavity and in the cortex far from the contusion cavity were significantly suppressed by MRS2179 administration on postinjury Days 1 and 3 (p < 0.05). However, administration of MRS2179 failed to improve behavioral outcome. Administration of MRS2179 successfully suppressed microglial activation in a traumatic brain injury model, which will be a potent treatment option in the future. Further study is required to conclude its therapeutic effects.

Effects of divalent cations on Schaffer collateral axon function.

Previously, we reported that distal Schaffer collaterals undergo biphasic changes in excitability during high-frequency stimulation (HFS), with an early hyper-excitability period followed by an excitability depression period. The extracellular divalent cations calcium and magnesium can regulate membrane excitability in neuronal tissue. Therefore, we hypothesized that altering the concentrations of extracellular calcium and magnesium would alter the biphasic excitability changes. We tested this hypothesis by recording distal Schaffer collateral fiber volleys in stratum radiatum of hippocampal area CA1 during 100Hz HFS in artificial cerebral spinal fluid (ACSF) containing normal and altered concentrations of extracellular divalent cations. Our normal ACSF contained 2.0mM calcium and 2.0mM magnesium. We examined four solutions with altered divalent cation concentrations: (1) high-calcium/low-magnesium (3.8mM/0.2mM), (2) low-calcium/high-magnesium (0.2mM/3.8mM), (3) high-calcium/normal-magnesium (3.8mM/2.0mM), or (4) normal-calcium/high-magnesium (2.0mM/10.0mM), and assessed the effects on Schaffer collateral responses. Increasing or decreasing extracellular calcium enhanced or reduced (respectively) the early hyper-excitable period whereas increasing extracellular magnesium reduced the later excitability depression. Because these results might be explained by altered calcium influx through voltage-gated calcium (CaV) channels, we tested CaV blockers (ω-agatoxin IVA, ω-conotoxin-GVIA, cadmium), but observed no effects on responses during HFS. Some of the effects of altered divalent cation concentration may be explained by altered membrane surface charge. Although this mechanism does not completely explain our findings, calcium influx through CaV channels is not required.

Conductive Polymers Modification for Carbon Fiber and Platinum Microelectrodes for the Measurement of Neuropeptide Y

Currently, one of the approaches in neuroscience research is the analysis and understanding of neurochemical behaviors. This is a great challenge since, although neurotransmitter measurements have been possible, these techniques have limitations, among which are the selectivity, sensitivity, and temporal resolution of the measurement. This work seeks to improve the Electrochemical Impedance Spectroscopy (EIS) technique using a Carbon Fiber Microelectrode and a Platinum Microelectrode for measurement with a conductive polymer modification of Pyrrole and Aniline. The EIS technique allows the measurement of the interaction of molecules in a solution and the surface of the electrode. The modification with conductive polymer is carried out since it is biocompatible, and having a load causes the selectivity to increase and, in turn, its sensitivity, in addition to being modifiable according to the application. Our objective is to find the optimal conditions for the modification of the surface of the microelectrode with the conductive polymers for use with EIS by measuring different neurotransmitters to study and understand its release mechanism and then be able to measure different concentrations. Our neuropeptide of interest is Neuropeptide Y (NPY) because it is related with many processes in the human body as regulation of appetite, stress, and diseases as Alzheimer and Parkinson disease. For conductive polymer modification we used 0.5M Pyrrole and Aniline in 0.5M H2SO4. Cyclic Voltammetry (CV) is applied to carry out the electropolimerization with 1 cycle per depositions 3 times. We used two solutions of ACSF (artificial cerebral-spinal fluid) one of them mixed with 10pg/ml of NPY and the other one without NPY to study the current behavior. SEM and EDS were used to study the morphology. As results for ACSF and ACSF-NPY we observed an increased in current at the first electropolymerization and a decreased after the second electropolymerization for CV.

Miniaturized probe on polymer SU-8 with array of individually addressable microelectrodes for electrochemical analysis in neural and other biological tissues.

An SU-8 probe with an array of nine, individually addressable gold microband electrodes (100μm long, 4μm wide, separated by 4-μm gaps) was photolithographically fabricated and characterized for detection of low concentrations of chemicals in confined spaces and in vivo studies of biological tissues. The probe's shank (6mm long, 100μm wide, 100μm thick) is flexible, but exhibits sufficient sharpness and rigidity to be inserted into soft tissue. Laser micromachining was used to define probe geometry by spatially revealing the underlying sacrificial aluminum layer, which was thenetched to free the probes from a silicon wafer. Perfusion with fluorescent nanobeads showed that, like a carbon fiber electrode, the probe produced no noticeable damage when inserted into rat brain, in contrast to damage from an inserted microdialysis probe. The individual addressability of the electrodes allows single and multiple electrode activation. Redox cycling is possible, where adjacent electrodes serve as generators (that oxidize or reduce molecules) and collectors (that do the opposite) to amplify signals of small concentrations without background subtraction. Information about electrochemical mechanisms and kinetics may also be obtained. Detection limits for potassium ferricyanide in potassium chloride electrolyte of 2.19, 1.25, and 2.08μM and for dopamine in artificial cerebral spinal fluid of 1.94, 1.08, and 5.66μM for generators alone and for generators and collectors during redox cycling, respectively, were obtained.

The Role of Arginine Vasopressin in Modulating Inspiratory Bursting Behavior in the Hypoglossal Motor Nucleus in Neonatal Mice

Arginine vasopressin (AVP) is classically known as a hormone, but it also acts as a neurotransmitter that binds V1a receptors in the central nervous system. V1a receptors are expressed at high levels early in postnatal life in cranial and spinal motoneurons. While AVP receptor expression levels decrease in some cranial motoneuron populations with increasing developmental age, AVP receptor mRNA levels are expressed at high levels in the hypoglossal (XII) motor nucleus in adult mice. Recordings from individual XII motoneurons demonstrated that AVP has excitatory effects on young XII motoneurons, although the signaling pathways and effector ion channels remain to be fully elucidated. Additionally, previous research demonstrated that GABAergic disinhibition within the paraventricular nucleus of adult male rats, which has the effect of promoting AVP release, led to stimulation of breathing and increased diaphragm and genioglossus (innervated by XII, primary tongue protruder) muscle activity. Whether AVP acts directly at XII motoneurons to potentiate inspiratory bursting is unknown. Our aim was to characterize the effects of AVP on inspiratory bursting behavior at XII motoneurons. To test our hypothesis that AVP will potentiate inspiratory bursting behavior, we utilized an in vitro medullary brainstem slice (600 µM) preparation of neonatal (postnatal day, P0-5) CD1 mouse brains of either sex that contains the nuclei for respiratory movement of the tongue. By superfusing the slice with warmed artificial cerebral spinal fluid, we were able to record inspiratory bursting activity using a suction electrode under baseline conditions and in response to local drug application. Local injections of AVP at 0.01 µM had little effect on inspiratory bursting amplitude (111% ± 3% of baseline, n=6), but at 0.1 µM or 1 µM, initial results suggest it increased inspiratory burst amplitude (134% ± 5% of baseline, n=13 and 130% ± 8% of baseline, n=7 respectively, p = 0.0889). The excitatory effect of AVP was significantly diminished using a V1a receptor antagonist, d(CH2)51Tyr(Me)2Arg8) Vasopressin (500nM) (Initial AVP increase in burst amplitude: 141% ± 8%; AVP after antagonist: 113% ± 4%; AVP after washout: 120% ± 3%, n=10, p = 0.0437). Vasopressin and oxytocin are closely related nonapeptides, and since the XII motor nucleus also expresses oxytocin receptors, we next tested whether the AVP effects could be attributed to activation of the oxytocin receptor. Our preliminary results suggest that the oxytocin receptor antagonist, L-371,257 (50nM), did attenuate the response of AVP (Initial AVP increase in burst amplitude: 144% ± 11%; AVP after antagonist: 114% ± 5%; AVP after Washout: 122% ± 6%, n=8, p = 0.0901). Lastly, we discovered that vasopressin-related potentiation of inspiratory burst amplitude has an age-dependent increase from P0-5 (regression fit: y=13.933x+12.401, R2=0.4411, p=0.000402). Our results indicate that vasopressin, acting at V1a, and possibly oxytocin, receptors, can potentiate XII inspiratory bursting behavior in neonatal mice. Whether vasopressin has a role in maintaining airway patency in vivo in adult animals will need to be addressed in future studies.

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 °C and positioned in a stereotactic frame with a cervical bend of about 30°. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 µL of blood was injected in the cisterna magna followed by a 30° downward tilt of the animal for 2 minutes. The second infusion of 30 µL of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics.

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Direct administration of ifenprodil and citalopram into the nucleus accumbens inhibits cue-induced nicotine seeking and associated glutamatergic plasticity.

Nicotine use disorder has been associated with glutamatergic alterations within the basal ganglia that might contribute to relapse. Specifically, initiation of cue-induced nicotine seeking produces rapid, transient synaptic potentiation (t-SP) in nucleus accumbens core (NAcore) medium spiny neurons (MSNs), defined as increases in spine head diameter and AMPA to NMDA current ratios (A/N). Ifenprodil, which inhibits nicotine reinstatement when administered systemically, antagonizes GluN2B-containing NMDA receptors, has affinity for serotonin receptors, and blocks serotonin transporters (SERT). The mechanisms underlying its therapeutic efficacy, however, remain unknown. Using pharmacological and genetic approaches, the current study examined the role of NAcore GluN2B receptors as well as SERT in mediating cue-induced nicotine seeking and associated MSN structure and physiology. Prior to reinstatement, rats received intra-NAcore injections of either ifenprodil, citalopram or artificial cerebral spinal fluid (15min prior), or GluN2B or control siRNAs (3 consecutive days prior). Rats were sacrificed after a 15-min cue-induced reinstatement session for dendritic spine analysis, western blotting or whole-cell electrophysiology. Intra-NAcore ifenprodil blocked nicotine-seeking behavior and promoted a higher frequency of shorter spines on MSN dendrites. However, a decrease in membrane-bound GluN2B receptor expression did not prevent cue-induced nicotine seeking or associated MSN cell physiology. Interestingly, intra-NAcore citalopram, an SSRI, prevented cue-induced nicotine seeking. Together, these results indicate that the therapeutic effects of ifenprodil on cue-induced nicotine seeking may, in part, be due to its actions at SERT rather than GluN2B, which may be specific to nicotine-seeking as opposed to other drugs of abuse.

Brainstem inflammation modulates the ventilatory pattern and its variability after acute lung injury in rodents.

Compared with sham rats, rats a week after acute lung injury (ALI) express more pro-inflammatory cytokines in their brainstem respiratory control nuclei, exhibit a higher respiratory frequency (fR) and breathe with a more predictable pattern. These characteristics of the respiratory pattern persist in insitu preparations even after minimizing pulmonary and chemo-afferent inputs. Interleukin (IL)-1β microinjected in the nucleus tractus solitarii increases fR and the predictability of the ventilatory pattern similar to rats with ALI. Intracerebroventricular infusion of indomethacin, an anti-inflammatory drug, mitigates the effect of ALI on fR and ventilatory pattern variability. We conclude that changes in the ventilatory pattern after ALI result not only from sensory input due to pulmonary damage and dysfunction but also from neuro-inflammation. Acute lung injury (ALI) increases respiratory rate (fR) and ventilatory pattern variability (VPV), but also evokes peripheral and central inflammation. We hypothesized that central inflammation has a role in determining the ventilatory pattern after ALI. In rat pups, we intratracheally injected either bleomycin to induce ALI or saline as a sham control. One week later, we recorded the ventilatory pattern of the rat pups using flow-through plethysmography, then formed insitu preparations from these pups and recorded their 'fictive' patterns from respiratory motor nerves. Compared with the ventilatory pattern of the sham rat pups, injured rat pups had increased fR and predictability. Surprisingly, the fictive patterns of the insitu preparations from ALI pups retained these characteristics despite removing their lungs to eliminate pulmonary sensory inputs and perfusing them with hyperoxic artificial cerebral spinal fluid to minimize peripheral chemoreceptor input. Histological processing revealed increased immunoreactivity of the pro-inflammatory cytokine Interleukin-1β (IL-1β) in the nucleus tractus solitarii (nTS) from ALI but not sham rats. In subsequent experiments, we microinjected IL-1β in the nTS bilaterally in anaesthetized naïve adult rats, which increased fR and predictability of ventilatory pattern variability (VPV) after 2h. Finally, we infused indomethacin intracerebroventricularly during the week of survival after ALI. This did not affect sham rats, but mitigated changes in fR and VPV in ALI rats. We conclude that neuro-inflammation has an essential role in determining the ventilatory pattern of ALI rats.

High-Throughput Liquid-Liquid Extractions with Nanoliter Volumes.

Current methods for liquid-liquid extractions generally require microliter to milliliter volumes of solvents and sample, long equilibration times, and manual procedures. Extraction methods for samples in microfluidic systems have been limited as this tool is difficult to implement on the nanoliter or smaller scale. We have developed slug-flow nanoextraction (SFNE), a method based on droplet microfluidics that allows multiple liquid-liquid extractions to be performed simultaneously in a capillary tube, using only 5 nL of sample and extraction solvent per extraction. Each two-phase slug is segmented from the others by immiscible carrier fluid. We found rapid extractions (<5 s), partition coefficients matching those achieved at larger scale extractions, and potential to preconcentrate samples through volume manipulation. This method was used to accurately and rapidly determine octanol-water partition coefficients (Kow), determining identical Kow as the shake-flask method for acetaminophen, Kow = 2.48 ± 0.02. The measurement, along with calibration and 12 replicates, was complete within 5 min, consuming under 150 nL of solvent and sample. The method was also applied to extract analytes from complex biological samples prior to electrospray ionization-tandem mass spectrometry (ESI-MS/MS) at a rate of 6 s per sample, allowing for simultaneous determination of five different drugs spiked into human plasma, synthetic urine (SU), and artificial cerebral spinal fluid (aCSF) using ethyl acetate as the extraction phase. The signal-to-noise (S/N) for analytes improved up to 19-fold compared to direct ESI-MS of single-phase droplets (aqueous plugs segmented by carrier fluid), with limits of detection down to 7 nM (35 amol).

Gadolinium-based MRI contrast agent for the detection of tyrosinase.

Tyrosinase is a key enzyme that has long been considered as a biomarker for melanoma as it catalyzes the oxidation of tyrosine and l-DOPA in melanogenesis. Recent studies also suggest a link between tyrosinase activity and Parkinson's disease; however, the mechanism of tyrosinase-mediated melanin formation in the brain is poorly understood. To better understand this connection, more advanced tools for the detection of tyrosinase in the brain are required. Herein, we successfully designed and synthesized a tyrosinase-targeting Gd(iii)-based MR contrast agent Tyr-GBCA 1. Tyr-GBCA 1 was synthesized by linking m-hydroxyphenyl to Gd-DOTA via a self-immolative linker. Tyr-GBCA 1 shows a 21% increase in the T1 relaxation rate (R1) in the presence of tyrosinase in artificial cerebral spinal fluid. Furthermore, Tyr-GBCA 1 is unreactive to hydrogen peroxide, which is a potential interferent in oxidation-based tyrosinase sensing systems. The reaction mechanism of the probe was studied by electrospray ionization (ESI) mass spectrometry and supports the cleavage of a reaction site.

Acute neurosteroid treatment with allopregnanolone enhances dorsal vagal motor neuron inhibition

Neurons in the dorsal motor nucleus of the vagus (DMV) comprise the preganglionic parasympathetic motor output to much of the viscera and critically contribute to autonomic regulation of energy homeostasis. DMV neuron function is tightly regulated by gamma‐aminobutyric (GABA) input to GABAA receptors, which inhibits neuronal excitability. Extrasypnatically localized GABAA receptors, primarily containing δ‐subunits, respond to ambient GABA, setting the tonic inhibitory tone in DMV neurons. Allopregnanolone is a centrally and peripherally produced neuroactive steroid metabolite which readily crosses the blood brain barrier and is a potent allosteric activator of GABAA receptors. Outside of the DMV, allopregnanolone has been shown to increase inhibitory tonic current in a GABAA‐δ dependent manner. DMV neurons have significant tonic inhibition. These currents are largely mediated by the GABAA receptors containing the δ‐subunit. However, no studies to date have investigated the potential for allopregnanolone to induce changes in GABAergic signaling to DMV neurons. Therefore, we hypothesized that acute allopregnanolone exposure increases GABAA receptor mediated inhibition in DMV neurons through a GABAAR δ‐subunit dependent manner. To test this hypothesis, we incubated brainstem slices in allopregnanolone (100nM) or vehicle containing artificial cerebral spinal fluid (ACSF) for 30 minutes. We then analyzed GABAA receptor mediated inhibition in DMV neurons at 30, 60, and 120 minutes following the completion of this incubation. We examined both modalities of GABAA receptor inhibition, phasic and tonic using the specific GABAA antagonist bicuculline (BIC) and wholecell patch‐clamp electrophysiology. Allopregnanolone treatment increased most measures of inhibition in DMV neuron at 30 minutes, including spontaneous inhibitory postsynaptic current (sIPSC) decay time and BIC‐sensitive tonic current. DMV neurons from slices incubated in either allopregnanolone or control ACSF were probed for choline acetyltransferase and GABAA‐δ subunit expression using immunohistochemistry. Brief exposure to allopregnanolone transiently potentiates GABAA receptor response to ambient GABA. Allopregnanolone may play an important role in dynamically modulating vagal inhibition in models of disease.Support or Funding InformationAmerican Heart Association Grant# 16SDG26590000 and NIH Grant# 5T32HL007446‐37This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhanced responses to somatostatin interneuron activation in developmentally malformed cortex.

SummaryIntractable epilepsy is commonly associated with developmental cortical malformations. Using the rodent freeze lesion model, we have sought the underlying circuit abnormalities contributing to the epileptiform activity that occurs in association with the structural pathology of four‐layered microgyria. We showed previously that within the epileptogenic paramicrogyral region (PMR) surrounding the malformation, non–fast‐spiking neurons commonly containing somatostatin (SSt) exhibit alterations, including having a greater maximum firing rate. Here we examined the output of SSt interneurons with optogenetics, using SSt‐Cre mice mated to mice with floxed channelrhodopsin‐2. Voltage clamp recordings from layer V pyramidal neurons in ex vivo slices had significantly enhanced SSt‐evoked inhibitory postsynaptic currents in PMR cortex compared to control. In addition, under conditions of low‐Mg2+ artificial cerebral spinal fluid (aCSF), light activation of the SSt neurons evoked field potential epileptiform activity in the PMR cortex, but not in control. These data suggest that within the PMR cortex, SSts have a significantly larger effect on excitatory neurons. Surprisingly, the network effect of this enhanced inhibition is hyperexcitability with propagating epileptiform activity, perhaps due to disinhibition of other interneuron cell types or to enhanced synchrony of excitatory cortical elements. This identification creates a new locus for potential modulation of epileptiform activity associated with cortical malformation.

Proteomic analysis and ATP assay reveal a positive effect of artificial cerebral spinal fluid perfusion following microdialysis sampling on repair of probe-induced brain damage

BackgroundMicrodialysis (MD) is conventionally used to measure the in vivo levels of various substances and metabolites in extracellular and cerebrospinal fluid of brain. However, insertion of the MD probe and subsequent perfusion to obtain samples cause damage in the vicinity of the insertion site, raising questions regarding the validity of the measurements. New methodWe used fluorogenic derivatization liquid chromatography-tandem mass spectrometry, that quantifies both high and low abundance proteins, to differentiate the effects of perfusion from the effects of probe insertion on the proteomic profiles of expressed proteins in rat brain. ResultsWe found that the expression levels of five proteins were significantly lower in the perfusion group than in the non-perfusion group. Three of these proteins are directly involved in ATP synthesis. In contrast to decreased levels of the three proteins involved in ATP synthesis, ATP assays show that perfusion, following probe insertion, even for a short time (3 h) increased ATP level up to 148% that prior to perfusion, and returned it to normal state (before probe insertion). Comparison with existing methodThere is essentially no information regarding which observed changes are due to probe insertion and which to perfusion. ConclusionsOur findings partially demonstrate that the influence of whole MD sampling process may not significantly compromise brain function and subsequent analytical results may have physiological equivalence to normal, although energy production is transiently damaged by probe insertion.