Artificial Cerebrospinal Fluid Solution Research Articles

A Microfluidic, Multi-Antibody Cell Capture Method to Evaluate Tumor Cells in Cerebrospinal Fluid in Patients With Suspected Leptomeningeal Metastases.

Leptomeningeal disease (LMD) is a clinical sequela of central nervous system metastasis involving the cerebrospinal fluid (CSF), often seen in late-stage solid tumors. It has a grave prognosis without urgent treatment. Standard of care methodologies to diagnose LMD include CSF cytology, magnetic resonance imaging, and clinical evaluation. These methods offer limited sensitivity and specificity for the evaluation of LMD. Here, we describe the analytic performance characteristics of a microfluidic-based tumor cell enrichment and detection assay optimized to detect epithelial cells in CSF using both contrived samples as well as CSF from patients having suspected or confirmed LMD from carcinomas. To demonstrate the feasibility of using a microfluidic, multi-antibody cell capture assay to identify and quantify tumor cells in CSF. An artificial CSF solution was spiked with 34 different human carcinoma cell lines at different concentrations and assayed for the ability to detect tumor cells to assess analytic accuracy. Two cell lines were selected to assess linearity, intra-assay precision, interinstrument precision, and sample stability. Clinical verification was performed on 65 CSF specimens from patients. Parameters assessed included the number of tumor cells, coefficient of variation percentage, and percentage of tumor cell capture (TCC). Among contrived samples, average tumor cell capture ranged from 50% to 82% (261 of 522; 436 of 531), and coefficients of variation ranged from 7% to 67%. The cell capture assay demonstrated a sensitivity of 92% and a specificity of 95% among clinical samples. This assay demonstrated the ability to detect and enumerate epithelial cells in contrived and clinical specimens in an accurate and reproducible fashion. The use of cell capture assays in CSF may be useful as a sensitive test for the diagnosis and longitudinal monitoring of LMD from solid tumors.

Application of fiber loop ringdown spectroscopy technique for a new approach to beta-amyloid monitoring for Alzheimer Disease’s early detection

A novel fiber optic biosensor was purposed for a new approach to monitor amyloid beta protein fragment 1–42 (Aβ42) for Alzheimer’s Disease (AD) early detection. The sensor was fabricated by etching a part of fiber from single mode fiber loop in pure hydrofluoric acid solution and utilized as a Local Optical Refractometer (LOR) to monitor the change Aβ42 concentration in Artificial Cerebrospinal Fluid (ACSF). The Fiber Loop Ringdown Spectroscopy (FLRDS) technique is an ultra-sensitive measurement technique with low-cost, high sensitivity, real-time measurement, continuous measurement and portability features that was utilized with a fiber optic sensor for the first time for the detection of a biological signature in an ACSF environment. Here, the measurement is based on the total optical loss detection when specially fabricated sensor heads were immersed into ACSF solutions with and without different concentrations of Aβ42 biomarkers since the bulk refractive index change was performed. Baseline stability and the reference ring down times of the sensor head were measured in the air as 0.87% and 441.6 μs ± 3.9 μs, respectively. Afterward, the total optical loss of the system was measured when the sensor head was immersed in deionized water, ACSF solution, and ACSF solutions with Aβ42 in different concentrations. The lowest Aβ42 concentration of 2 ppm was detected by LOR. Results showed that LOR fabricated by single-mode fibers for FLRDS system design are promising candidates to be utilized as fiber optic biosensors after sensor head modification and have a high potential for early detection applications of not only AD but possibly also several fatal diseases such as diabetes and cancer.

Agar freezing solution for long-term cryopreservation of brain slices from non-hibernating animals

Our previous studies have shown that function of ionotropic glutamate receptors such as AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) and NMDA (N-methyl-D-aspartate) was impaired after long-term cryopreservation of brain slices at -10°C within 30-50 days. To elucidate the reasons for cryodamage to AMPA- and NMDA-dependent mechanisms, artificial cerebrospinal fluid (aCSF) solutions that contain agar at different concentrations (33, 44, and 50%) were used for cryopreservation of the rat olfactory cortex slices. After cryopreservation, the slices were warmed to 37°C and the amplitudes changes of AMPA and NMDA potentials, which reflected the activities of the AMPA and NMDA mechanisms, were evaluated; the results were compared with those obtained before cryopreservation. It was found that AMPA and NMDA potentials changed differently depending on the concentration of agar in artificial cerebrospinal fluid. In solutions with 33% agar, the amplitude of AMPA potentials increased by 60%, whereas, in contrast, the amplitude of NMDA potentials was equal to the values before cryopreservation. At agar concentration of 44% in the solution, the AMPA and NMDA amplitudes were increased by 70% and 80%, respectively. A complete recovery of the activities of AMPA and NMDA mechanisms was obtained after cryopreservation in a freezing solution with an agar concentration of 50%. Under these conditions, the amplitudes of the AMPA and NMDA potentials corresponded to those seen before cryopreservation. Thus, the results obtained indicate that agar added to the artificial cerebrospinal fluid solution is a cryoprotectant that protects AMPA- and NMDA-dependent mechanisms from cryoinjury. The freezing solution (artificial cerebrospinal fluid and agar) developed by us for cryopreservation of brain explants of non-hibernating animals will be used to create a cryobank of nervous tissue.

Cerebrospinal Fluid-philic and Biocompatibility-Enhanced Soft Cranial Window for Long-Term In Vivo Brain Imaging.

Soft, transparent poly(dimethyl siloxane) (PDMS)-based cranial windows in animal models have created many opportunities to investigate brain functions with multiple in vivo imaging modalities. However, due to the hydrophobic nature of PDMS, the wettability by cerebrospinal fluid (CSF) is poor, which may cause air bubble trapping beneath the window during implantation surgery, and favorable heterogeneous bubble nucleation at the interface between hydrophobic PDMS and CSF. This may result in excessive growth of the entrapped bubble under the soft cranial window. Herein, to yield biocompatibility-enhanced, trapped bubble-minimized, and soft cranial windows, this report introduces a CSF-philic PDMS window coated with hydroxyl-enriched poly(vinyl alcohol) (PVA) for long-term in vivo imaging. The PVA-coated PDMS (PVA/PDMS) film exhibits a low contact angle θACA (33.7 ± 1.9°) with artificial CSF solution and maintains sustained CSF-philicity. The presence of the PVA layer achieves air bubble-free implantation of the soft cranial window, as well as induces the formation of a thin wetting film that shows anti-biofouling performance through abundant water molecules on the surface, leading to long-term optical clarity. In vivo studies on the mice cortex verify that the soft and CSF-philic features of the PVA/PDMS film provide minimal damage to neuronal tissues and attenuate immune response. These advantages of the PVA/PDMS window are strongly correlated with the enhancement of cortical hemodynamic changes and the local field potential recorded through the PVA/PDMS film, respectively. This collection of results demonstrates the potential for future microfluidic platforms for minimally invasive CSF extraction utilizing a CSF-philic fluidic passage.

Motor behavioral abnormalities and histopathological findings in middle aged male Wistar rats inoculated with cerebrospinal fluid from patients with Amyotrophic Lateral Sclerosis

• Middle-aged male rats infused with CSF mimic pathogenic mechanisms observed in ALS. • Infusion of human CSF from patients of ALS causes motor impairment in middle-aged animals. • CSF from patients of ALS is a tool to investigate the characteristics of the disease. Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease associated with loss of upper and lower motor neurons in the primary motor cortex and spinal cord, respectively. Motor deficits are the main clinical features observed in patients with the disease. However, it has been suggested that the presence of neurotoxic factors in the cerebrospinal fluid (CSF) from ALS patients causes loss of motor neurons. The present study investigated the motor and histopathological changes induced by intracerebroventricular (ICV) infusion of CSF from ALS patients in middle aged male Wistar rats. Middle aged male rats were divided into three groups: (1) control group, animals injected with artificial CSF solution; (2) N-ALS group, animals injected with CSF from volunteers without neurological disease; and (3) ALS group, animals inoculated with CFS from a patient with definite ALS. After surgical and infusion procedures, animals were evaluated in different motor tests (grip strength; catalepsy and open field tests). Moreover, animals’ spinal cords were histologically investigated. We observed that ALS-CSF infusion reduced grip strength and led to motor changes and reduction in the number of motor neurons and glial cells in thoracic and lumbar regions of spinal cord. However, CSF N-ALS caused reduction of nerve and glial cells in the thoracic but not in the lumbar region. Our data suggest that ALS-CSF is associated with neurodegenerative mechanisms observed in ALS pathology.

Abstract P059: Exploiting mutant PPM1D-induced metabolic defects with nanoparticle-encapsulated NAMPT inhibitors

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is a leading cause of death in pediatric cancer, with an abysmal <1% 5-year survival rate due to lack of effective treatment options. A significant effort is needed to understand the genetic landscape of this disease to develop novel therapeutic strategies and modalities. Recently, the Bindra laboratory found that a truncation mutation in the gene PPM1D, found in ~30% of DIPG cases, causes global epigenetic alterations that lead to therapeutic vulnerabilities. They found that mutant PPM1D activity results in loss of the NAD+ biogenesis protein NAPRT that can be therapeutically targeted by inhibitors of another protein in the NAD+ biogenesis pathway NAMPT (NAMPTi), providing a viable therapeutic strategy for these cancers. Some NAMPTis have been FDA-approved for clinical usage, but present with systemic and retinal toxicity. Moreover, while there are few NAMPTis that can pass the blood brain barrier through systemic delivery, studies show diminished concentrations at the target site. Together, current studies show that the use of NAMPTis for precision targeting of CNS cancers such as DIPG with mutant PPM1D status is a promising therapeutic strategy, but impractical given the limitations of these drugs. However, recent developments in drug delivery systems offer a chance to overcome these issues. Tools such as nanoparticle (NP) drug delivery and unique injection set-ups such as convection-enhanced delivery (CED) allow for drugs such as NAMPTis to be reconsidered for clinical usage. To circumvent the challenges presented by these drugs, we have developed and characterized nanoparticles that encapsulate NAMPTis (NAMPTi-NP) and use CED for sustained intratumoral delivery. Thus far, we have fabricated and optimized PLA-PEG copolymeric nanoparticles capable of encapsulating the NAMPTi, GMX-1778. We characterized these nanoparticles based on (1) hydrodynamic diameter, (2) zeta potential, and (3) stability within an artificial cerebrospinal fluid solution. We have performed both in vitro and in vivo assays to determine the functionality of the NAMPTi-NPs through (1) cellular uptake studies via immunofluorescence, (2) functional analysis via NAD+ quantification, (3) short- and long-term cell viability assays to determine sensitivity to the NAMPTi-NP, and (4) in vivo biodistribution studies to assess sustained retention of NAMPTi-NP with CED intracranial injections. We find that NAMPTi-NPs have immediate and sustained cellular uptake, loss of NAD+ post-treatment indicating effective targeting, and enhanced sensitivity in long-term viability studies in mutant PPM1D models. Lastly, these NAMPTi-NP display prolonged retention in brain tissue compared to free drug injection over time. With further in vivo validation, this NP-based strategy will be a powerful tool for targeting mutant PPM1D DIPG and other cancers. Citation Format: Matthew A. Murray, Yazhe Wang, Ranjini Sundaram, Jason Beckta, Mark Saltzman, Ranjit Bindra. Exploiting mutant PPM1D-induced metabolic defects with nanoparticle-encapsulated NAMPT inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P059.

Cerebrospinal fluid replacement solutions promote neuroglia migratory behaviors and spinal explant outgrowth in microfluidic culture.

Disorders of the nervous system (NS) impact millions of adults, worldwide, as a consequence of traumatic injury, genetic illness, or chronic health conditions. Contemporary studies have begun to incorporate neuroglia into emerging NS therapies to harness the regenerative potential of glial-mediated synapses in the brain and spinal cord. However, the role of cerebrospinal fluid (CSF) that surrounds neuroglia and interfaces with their associated synapses remains only partially explored. The flow of CSF within subarachnoid spaces (SAS) circulates essential polypeptides, metabolites, and growth factors that directly impact neural response and recovery via signaling with healthy glia. Despite the availability of artificial CSF solutions used in neurosurgery and NS treatments, tissue engineering projects continue to use cell culture media, such as Neurobasal (NB) and Dulbecco's Modified Eagle Medium (DMEM), for development and characterization of many transplantable cells, matrixes, and integrated cellular systems. The current study examined in vitro behaviors of glial Schwann cells (ShC) and spinal cord explants (SCE) within a CSF replacement solution, Elliott's B Solution (EBS), used widely in the treatment of NS disorders. Our tests used EBS to create defined chemical microenvironments of extracellular factors within a glial line (gLL) microfluidic device, previously described by our group. The gLL is comparable in scale to the in vivo SAS that envelopes endogenous CSF and enables molecular transport via mechanisms of convective diffusion. Our results illustrate that EBS solutions facilitate ShC survival, morphology, and proliferation similar to those measured in traditional DMEM, and additionally support glial chemotactic behaviors in response to brain-derived growth factor (BDNF). Our data indicates that ShC undergo significant chemotaxis toward high and low concentration gradients of BDNF with statistical differences between gradients formed within diluents of EBS and DMEM solutions. Moreover, SCE cultured with EBS solutions facilitated measurement of neurite explant extension commensurate with reported in vivo measurements. This data highlights the translational significance and advantages of incorporating CSF replacement fluids to interrogate cellular behaviors and advance regenerative NS therapies.

Brain Interstitial Matrix Hydration Inversely Correlates with Neuronal Excytability

Interstitial flux of cerebrospinal fluid (CSF) mediates optimal transfer, clearance, and exchange of molecules in the brain. To determine whether disturbances during dehydration or edema lead to neuronal dysfunction, we quantified hydration in the brains of mice subjected to mild water deprivation and measured neuronal excitability in the dehydrated and rehydrated hippocampus. Specifically, we titrated water activity ex vivo using artificial CSF solutions of nonpenetrating inert polymers adjusted to give colloidosmotic pressures in the 0-200 mmHg range. Compared to nondeprived controls, the brains of water-deprived mice had hydration potentials of 94.5 ± 5.4 and 103.8 ± 3.5 mmHg after 12 and 24 hours, respectively, corresponding to 23 and 28 mmHg increases in potential, or 20% and 27% decreases in hydration. The brain-matrix hydraulic conductance, 0.051 ± 0.0019 µl/min/g/mmHg, did not change significantly (n = 6 hydrated/dehydrated pairs). The field excitatory postsynaptic potentials (fEPSP) in the hippocampus, adjusted to hydrations 25 and 125 mmHg below controls, increased 47% and 66%, respectively, but did not change at hydrations up to 75 mmHg above control. In addition, the level of phosphorylated CREB (cAMP response-binding protein), a key mediator of neuronal excitatory pathways, increased in dehydrated hippocampus after subthreshold stimulation.These results show that brain dehydration levels comparable to those measured following short-term water deprivation in mice increase neuronal excitability. Neurons appear very sensitive to water-activity changes, suggesting local dehydration in the interstitial matrix contributes to the cognitive deficits seen in dehydrated humans. As a plausible mechanism, we suggest that lower water activity in dehydrated brains increases the effective concentration of all solutes, modifying the timing, diffusivities, and probabilities of neurotransmitters binding to their cognate receptors.

Differential cholinergic and serotonergic neuromodulation of network activity in the mouse medial prefrontal cortex

Differential cholinergic and serotonergic neuromodulation of network activity in the mouse medial prefrontal cortex

Online 1 H-MRS measurements of time-varying lactate production in an animal model of glioma during administration of an anti-tumoral drug.

The aims of this study were to implement a magnetic resonance spectroscopy (MRS) protocol for the online profiling of subnanomolar quantities of metabolites sampled from the extracellular fluid using implanted microdialysis and to apply this protocol in glioma-bearing rats for the quantification of lactate concentration and the measurement of time-varying lactate concentration during drug administration. MRS acquisitions on the brain microdialysate were performed using a home-built, proton-tuned, microsolenoid with an active volume of 2μL. The microcoil was placed at the outlet of the microdialysis probe inside a preclinical magnetic resonance imaging (MRI) scanner. C6-bearing rats were implanted with microdialysis probes perfused with artificial cerebrospinal fluid solution and the lactate dehydrogenase (LDH) inhibitor oxamate. Microcoil magnetic resonance spectra were continuously updated using a single-pulse sequence. Localized in vivo spectra and high-resolution spectra on the dialysate were also acquired. The limit of detection and limit of quantification per unit time of the lactate methyl peak were determined as 0.37nmol/√min and 1.23nmol/√min, respectively. Signal-to-noise ratios (SNRs) of the lactate methyl peak above 120 were obtained from brain tumor microdialysate in an acquisition time of 4min. On average, the lactate methyl peak amplitude measured in vivo using the nuclear magnetic resonance (NMR) microcoil was 193 ± 46% higher in tumor dialysate relative to healthy brain dialysate. A similar ratio was obtained from high-resolution NMR spectra performed on the collected dialysate. Following oxamate addition in the perfusate, a monotonic decrease in the lactate peaks was observed in all animals with an average time constant of 4.6min. In the absence of overlapping NMR peaks, robust profiling of extracellular lactate can be obtained online using a dedicated sensitive NMR microcoil. MRS measurements of the dynamic changes in lactate production induced by anti-tumoral drugs can be assessed accurately with temporal resolutions on the order of minutes. The MRS protocol can be readily transferred to the clinical environment with the use of suitable clinical microdialysis probes.

Nanoparticles based on gadolinium(iii) and europium(iii) complexes for biovisualization

Complexes Ln(TTA)3 and [Ln(TTA)3·1] (Ln = Eu, Gd; ТТА is thenoyltrifluoroacetyl-acetonate; 1 is 2-(5-chlorophenylene-2-hydroxy)-2-phenylethylene-bis(2-methoxy)phosphine oxide) in individual form, and as a part of a core of the polyelectrolyte stabilized colloids have been studied by Mossbauer spectroscopy and X-ray powder diffraction. The photophysical and colloidal characteristics of the solutions of polyelectrolyte nanoparticles were studied in water, artificial cerebrospinal fluid solution, solution of bovine serum albumin, and human blood serum. A stability of a luminescent response of the nanoparticles in solutions of bovine serum albumin and human blood serum at 37 °С for 2 hours has been revealed. This is a prerequisite for the potential application of studied nanoparticles for biovisualization.

Sodium Bicarbonate Facilitates Hemostasis in the Presence of Cerebrospinal Fluid Through Amplification of Platelet Aggregation.

Appropriate hemostasis is essential for clear visualization of the neural structures and cleavage planes. It is also essential for avoiding heat-induced injury, minimizing blood loss, and reducing operative time. To determine the role of cerebrospinal fluid (CSF) in platelet-dependent hemostasis during neurosurgery. The amplification of aggregation, activation of integrin αIIbβ3, intrinsic and extrinsic coagulation pathways, and activation of signaling cascades in platelets were evaluated. For comparison, various concentrations of a commercially available artificial CSF solution (aCSF), an artificial CSF solution prepared by the authors, and normal saline (NS) were used. Differences between aCSF and NS in obtaining in vivo hemostasis were assessed by measuring the tail vein bleeding time in C57BL/6N mice. Platelet aggregation was directly amplified by the addition of aCSF through increased activation of integrin αIIbβ3, phosphatidylserine exposure, and P-selectin expression. However, the prothrombin time and activated partial thromboplastin time were not primarily related to coagulation activity with the addition of aCSF. Activation of Src kinase was related to platelet activation by aCSF. The elimination of sodium bicarbonate from aCSF and the addition of the selective inhibitor of the HCO3/Cl exchanger, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt, significantly inhibited platelet aggregation. The bleeding time in aCSF-treated mice was significantly shorter than that in NS-treated mice. Sodium bicarbonate facilitates hemostasis through the amplification of platelet aggregation function. The existence of CSF and irrigation with aCSF provide better conditions for physiological hemostasis and they have the potential of improving hemostasis by bipolar coagulation or with irrigation during neuroendoscopic procedures.

Potential Risk of Artificial Cerebrospinal Fluid Solution Without Magnesium Ion for Cerebral Irrigation and Perfusion in Neurosurgical Practice

The effect of irrigation with artificial cerebrospinal fluid (CSF) containing various magnesium ion (Mg(2+)) concentrations on vasospastic arteries was investigated in the dog. Cerebral vasospasm was induced by the experimental subarachnoid hemorrhage model in 15 beagle dogs. Cisternal irrigation was performed for 1 hour via a microcatheter placed in the cisterna magna with commercially available artificial CSF (ARTCEREB®) with physiological concentration of Mg(2+) (2.2 mEq/l) (ACM group, n = 5), ARTCEREB solution without Mg(2+) (ACR group, n = 5), and ARTCEREB solution with higher Mg(2+) concentration (5 mEq/l) (ACMM group, n = 5). CSF electrolyte concentrations and the diameters of the basilar and vertebral arteries were measured. In the ACM group, no changes were detected in either CSF Mg(2+) concentration or arterial diameters. In the ACR group, the CSF Mg(2+) decreased significantly to 0.8 ± 0.07 mEq/l from the baseline value of 1.4 ± 0.03 mEq/l, and both basilar and vertebral artery diameters were significantly decreased to 0.61 ± 0.18 mm and 0.57 ± 0.23 mm from their baseline values of 0.74 ± 0.22 mm and 0.68 ± 0.17 mm, respectively. In the ACMM group, the CSF Mg(2+) significantly increased to 2.4 ± 0.15 mEq/l from the baseline value of 1.4 ± 0.05 mEq/l, and both basilar and vertebral artery diameters were significantly increased to 0.84 ± 0.19 mm and 0.90 ± 0.22 mm from their baseline values of 0.71 ± 0.21 mm and 0.69 ± 0.24 mm, respectively. Irrigation with artificial CSF solution without Mg(2+) causes vasoconstriction of the cerebral artery. Irrigation with artificial CSF with appropriate Mg(2+) concentration is essential, especially in patients with subarachnoid hemorrhage.

Heterosynaptic plasticity induced by intracellular tetanization in layer 2/3 pyramidal neurons in rat auditory cortex

Associative Hebbian-type synaptic plasticity underlies the mechanisms of learning and memory; however, Hebbian learning rules lead to runaway dynamics of synaptic weights and lack mechanisms for synaptic competition.Heterosynaptic plasticity may solve these problems by complementing plasticity at synapses that were active during the induction, with opposite-sign changes at non-activated synapses. In visual cortex, a potential candidate mechanism for normalization is plasticity induced by a purely postsynaptic protocol, intracellular tetanization. Here we asked if intracellular tetanization can induce long-term plasticity in auditory cortex. We recorded excitatory postsynaptic potentials (EPSPs) of regular (n =76) and all-or-none (n =24) type in layer 2/3 pyramidal cells in slices from rat auditory cortex. After intracellular tetanization, 32 of 76 regular inputs (42%) showed long-term depression, 21 inputs (28%) showed potentiation and 23 inputs (30%) did not change. The direction of plasticity correlated with the initial release probability: inputs with initially low release probability tended to be potentiated, while inputs with high release probability tended to be depressed. Thus, intracellular tetanization had a normalizing effect on synaptic efficacy. Induction of plasticity by intracellular tetanization required a rise of intracellular [Ca(2+)], because it was impaired by chelating intracellular calcium with EGTA. The long-term changes induced by intracellular tetanization involved both pre and postsynaptic mechanisms. EPSP amplitude changes were correlated with changes of release indices: paired-pulse ratio and the inverse of the coefficient of variation (CV(-2)). Furthermore at some all-or-none synapses, changes of averaged response amplitude were correlated with a change of the failure rate, without a change of the synaptic potency, measured as averaged amplitude of successful responses. Presynaptic components of plastic changes were abolished in experiments with blockade of NO-synthesis and spread, indicating involvement of NO signalling. These results demonstrate that the ability of purely postsynaptic challenges to induce plasticity is a general property of pyramidal neurons of both auditory and visual cortices.

Testing neocortical slice viability in non-perfused no-magnesium artificial cerebrospinal fluid solutions

The acute in vitro brain slice model is a widely used neurophysiological research tool. When applying this method, most researchers continuously perfuse slices with carbogenated artificial cerebrospinal fluid (ACSF) to maintain pH balance and tissue oxygen delivery. Common wisdom suggests that static recordings are incompatible with submerged bath methodology because of deficiency in tissue oxygen supply. However, to our knowledge this has not been tested. In this study, we wanted to determine whether neocortical mouse slice viability could be maintained in the medium term (up to 2h) in a shallow, submerged recording bath under non-perfused, static conditions. Seizure-like events (SLEs) were generated in the slices utilizing no-magnesium ACSF and recorded for 2h under three conditions: (1) perfused ACSF condition (n=8), where slices were perfused continuously with carbogenated no-magnesium ACSF; (2) static ACSF condition (n=12), where slices were recorded in pre-carbogenated, but non-perfused (static) no-magnesium ACSF; and (3) static HEPES ACSF condition (n=12), where slices were recorded in non-perfused (static) no-magnesium ACSF with no pre-carbogenation but buffered with HEPES. SLE activity was stable for 2h across all three conditions. There was no statistically significant difference in SLE frequency, amplitude or length between static and perfused conditions. SLE frequency and amplitude were generally lower in the static HEPES buffer condition. The data indicate that robust and stable neocortical SLE activity can be generated for at least 2h in a submersion bath without ACSF perfusion if pH is adequately controlled.

Neuroprotective effect of hydrogen peroxide on an in vitro model of brain ischaemia

Reactive oxygen species (ROS) have been postulated to play a crucial role in the pathogenesis of ischaemia-reperfusion injury. Among these, hydrogen peroxide (H(2)O(2)) is known to be a toxic compound responsible for free-radical-dependent neuronal damage. In recent years, however, the 'bad reputation' of H(2)O(2) and other ROS molecules has changed. The aim of this study was to assess the protective role of H(2)O(2) and modification in its endogenous production on the electrophysiological and morphological changes induced by oxygen/glucose deprivation (OGD) on CA1 hippocampal neurons. Neuroprotective effects of exogenous and endogenous H(2)O(2) were determined using extracellular electrophysiological recordings of field excitatory post synaptic potentials (fEPSPs) and morphological studies in a hippocampal slice preparation. In vitro OGD was delivered by switching to an artificial cerebrospinal fluid solution with no glucose and with oxygen replaced by nitrogen. Neuroprotection against in vitro OGD was observed in slices treated with H(2)O(2) (3 mM). The rescuing action of H(2)O(2) was mediated by catalase as pre-treatment with the catalase inhibitor 3-amino-1,2,4-triazole blocked this effect. More interestingly, we showed that an increase of the endogenous levels of H(2)O(2), due to a combination of an inhibitor of the glutathione peroxidase enzyme and addition of Cu,Zn-superoxide dismutase in the tissue bath, prevented the OGD-induced irreversible depression of fEPSPs. Taken together, our results suggest new possible strategies to lessen the damage produced by a transient brain ischaemia by increasing the endogenous tissue level of H(2)O(2).

Hypothermia Suppresses Excitatory Synaptic Transmission and Neuronal Death Induced by Experimental Ischemia in Spinal Ventral Horn Neurons

Whole-cell patch-clamp recordings were performed from the ventral horn neurons obtained from the rat spinal cord slices. This study investigated the effects of hypothermia on excitatory synaptic transmission and ischemia-induced neuronal death. Hypothermia has long been recognized as a promising physical strategy against both ischemic and traumatic spinal cord injuries. However, the mechanism of hypothermia-mediated neuroprotective action in the spinal cord is still not fully understood at the single cell level. Whole-cell patch-clamp recordings were performed from ventral horn neurons obtained from the spinal cord slices. Ischemia was simulated by superfusing an oxygen- and glucose-deprived medium [ischemia simulating medium (ISM)]. When the temperature of the superfusing artificial cerebrospinal fluid solution was changed from normothermia (36 degrees C) to hypothermia (32 degrees C, 28 degrees C, and 24 degrees C), the frequency of spontaneous excitatory postsynaptic currents was significantly decreased in a temperature-dependent manner. Surperfusing the ISM generated an agonal inward current which consisted of a slow and subsequent rapid inward current in all of the neurons tested. The latencies of the slow and rapid inward currents after the ISM exposures were significantly longer at hypothermia than at normothermia. Hypothermia decreased the slope of the ISM-induced slow inward current, although it did not affect the slope of the rapid inward current. Moreover, the glutamate receptor antagonists slightly prolonged the latencies of the slow and rapid inward currents that were induced by ISM and significantly decreased their slopes. These results suggest that hypothermia reduces the excitatory synaptic activities and ischemic neuronal death in the spinal ventral horn. This finding may help in achieving a better understanding of the mechanisms of hypothermia-mediated neuroprotection in the spinal cord.

Hydroxyamide Analogs of Propofol Exhibit State-Dependent Block of Sodium Channels in Hippocampal Neurons: Implications for Anticonvulsant Activity

Although propofol is most commonly known for its general anesthetic properties, at subanesthetic doses, propofol has been effectively used to suppress seizures during refractory status epilepticus, a mechanism, in part, attributed to the inhibition of neuronal sodium channels. In this study, we have designed and synthesized two novel analogs of propofol, HS245 [2-(3-ethyl-4-hydroxy-5-isopropyl-phenyl)-3,3,3-trifluoro-2-hydroxy-propionamide] and HS357 [2-hydroxy-8-(4-hydroxy-3,5-diisopropyl-phenyl)-2-trifluoromethyl-octanoic acid amide], and determined their effects on sodium currents recorded from cultured hippocampal neurons. HS357 had greater affinity for the inactivated state of the sodium channel than propofol and HS245 (0.22 versus 0.74 and 1.2 microM, respectively) and exhibited the greatest ratio of affinity for the resting over the inactivated state. HS357 also demonstrated greater use-dependent block and delayed recovery from inactivation in comparison with propofol and HS245. Under current-clamp conditions, action potentials from hippocampal CA1 neurons in slices were evoked by current injection, or following perfusion with a zero Mg(2+)/7 mM K(+) artificial cerebrospinal fluid solution. Propofol and HS357 reduced the number of current-induced action potentials; however, HS357 caused a greater reduction in the number of spontaneous action potentials. Consistent with these electrophysiology studies, propofol and HS357 protected mice against acute seizures in the 6-Hz (22-mA) partial psychomotor model. Efficacious doses of propofol were associated with an impairment of motor coordination as assessed in the rotorod toxicity assay. In contrast, HS357 demonstrated a 2-fold greater protective index than propofol. Thus, propofol analogs represent an important structural class from which not only effective, but also safer, anti-convulsants may be developed.

Are Primary Supratentorial Intracerebral Hemorrhages Surrounded by a Biochemical Penumbra? A Microdialysis Study

Opinions vary regarding the indications for surgical evacuation of spontaneous intracerebral hemorrhages (ICH) and whether or not penumbra zones surround them. We performed intracerebral microdialysis (mean duration, 3.5 d) after surgical evacuation of ICH in 22 patients. Probes were placed in the parenchyma within 1 to 2 cm of the evacuated hematoma; a postoperative computed tomographic scanning verified their positions. The catheters were perfused with an artificial cerebrospinal fluid solution at 0.3 microl/min. Biochemical variables (glucose, pyruvate, lactate, glutamate, and glycerol) were analyzed and displayed at the bedside. The levels obtained were compared with previous data from normal human brains and the pericontusional penumbra zones of patients with severe traumatic brain lesions. During 1 to 12 hours after surgery, interstitial levels of glucose (median level, 1.3 mmol/L; interquartile range, 0.6-2.2 mmol/L) were within normal variations, whereas the levels of lactate (median level, 6.4 mmol/L; interquartile range, 3.9-9.0 mmol/L), glutamate (median level, 14 micromol/L; interquartile range, 5-370 micromol/L), and glycerol (median level, 190 micromol/L; interquartile range, 74-380 micromol/L), as well as the lactate/pyruvate ratio (median ratio, 35; interquartile range, 23-50) were increased. A gradual normalization of the lactate/pyruvate ratio and glycerol level was observed within 48 hours. The area close to an evacuated ICH exhibits a biochemical pattern similar to that of the biochemical penumbra zone surrounding focal traumatic brain contusions. The presence of a penumbra zone around large ICH may be of importance for making surgical decisions.

Multiple Mechanisms Underlying the Neuroprotective Effects of Antiepileptic Drugs Against In Vitro Ischemia

The possible neuroprotective effects of classic and new antiepileptic drugs on the electrophysiological changes induced by in vitro ischemia on striatal neurons were investigated. In particular, the aim of the study was to correlate the putative neuroprotective effects with the action of these drugs on fast sodium (Na+) and high-voltage-activated (HVA) calcium (Ca2+) currents. Extracellular field potentials were recorded from rat corticostriatal brain-slice preparations. In vitro ischemia was delivered by switching to an artificial cerebrospinal fluid solution in which glucose and oxygen were omitted. Na+ and HVA Ca2+ currents were analyzed by whole-cell patch-clamp recordings from acutely isolated rat striatal neurons. Excitatory postsynaptic potential was measured following synaptic stimulation in corticostriatal slices by sharp intracellular microelectrodes. Neuroprotection against in vitro ischemia was observed in slices treated with carbamazepine (CBZ), valproic acid (VPA), and topiramate (TPM), whereas it was not achieved by using levetiracetam (LEV). Fast Na+ conductances were inhibited by CBZ and TPM, whereas VPA and LEV showed no effect. HVA Ca2+ conductances were reduced by CBZ, TPM, and LEV. VPA had no effect on this current. All antiepileptic drugs induced a small reduction of excitatory postsynaptic potential amplitude at concentrations higher than 100 microm without changes of paired-pulse facilitation. The concomitant inhibition of fast Na+ and HVA Ca2+ conductances is critically important for the neuroprotection, whereas the presynaptic inhibition on glutamate transmission does not seem to play a major role.