Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue.

The extracellular space (ECS) of the brain has an extremely complex spatial organization, which has defied conventional light microscopy. Consequently, despite a marked interest in the physiological roles of brain ECS, its structure and dynamics remain largely inaccessible for experimenters. We combined 3D-STED microscopy and fluorescent labeling of the extracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living organotypic brain slices. SUSHI enables quantitative analysis of ECS structure and reveals dynamics on multiple scales in response to a variety of physiological stimuli. Because SUSHI produces sharp negative images of all cellular structures, it also enables unbiased imaging of unlabeled brain cells with respect to their anatomical context. Moreover, the extracellular labeling strategy greatly alleviates problems of photobleaching and phototoxicity associated with traditional imaging approaches. As a straightforward variant of STED microscopy, SUSHI provides unprecedented access to the structure and dynamics of live brain ECS and neuropil.

Glioblastoma Margin as a Diffusion Barrier Revealed by Photoactivation of Plasmonic Nanovesicles.

Glioblastoma (GBM) is the most complex and lethal primary brain cancer. Adequate drug diffusion and penetration are essential for treating GBM, but how the spatial heterogeneity in GBM impacts drug diffusion and transport is poorly understood. Herein, we report a new method, photoactivation of plasmonic nanovesicles (PANO), to measure molecular diffusion in the extracellular space of GBM. By examining three genetically engineered GBM mouse models that recapitulate key clinical features including the angiogenic core and diffuse infiltration, we found that the tumor margin has the lowest diffusion coefficient (highest tortuosity) compared with the tumor core and surrounding brain tissue. Analysis of the cellular composition shows that tortuosity in the GBM is strongly correlated with neuronal loss and astrocyte activation. Our all-optical measurement reveals the heterogeneous GBM microenvironment and highlights the tumor margin as a diffusion barrier for drug transport in the brain, with implications for therapeutic delivery.

Impedance Rhythms in Human Limbic System.

The impedance is a fundamental electrical property of brain tissue, playing a crucial role in shaping the characteristics of local field potentials, the extent of ephaptic coupling, and the volume of tissue activated by externally applied electrical brain stimulation. We tracked brain impedance, sleep-wake behavioral state, and epileptiform activity in five people with epilepsy living in their natural environment using an investigational device. The study identified impedance oscillations that span hours to weeks in the amygdala, hippocampus, and anterior nucleus thalamus. The impedance in these limbic brain regions exhibit multiscale cycles with ultradian (∼1.5-1.7 h), circadian (∼21.6-26.4 h), and infradian (∼20-33 d) periods. The ultradian and circadian period cycles are driven by sleep-wake state transitions between wakefulness, nonrapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. Limbic brain tissue impedance reaches a minimum value in NREM sleep, intermediate values in REM sleep, and rises through the day during wakefulness, reaching a maximum in the early evening before sleep onset. Infradian (∼20-33 d) impedance cycles were not associated with a distinct behavioral correlate. Brain tissue impedance is known to strongly depend on the extracellular space (ECS) volume, and the findings reported here are consistent with sleep-wake-dependent ECS volume changes recently observed in the rodent cortex related to the brain glymphatic system. We hypothesize that human limbic brain ECS changes during sleep-wake state transitions underlie the observed multiscale impedance cycles. Impedance is a simple electrophysiological biomarker that could prove useful for tracking ECS dynamics in human health, disease, and therapy.SIGNIFICANCE STATEMENT The electrical impedance in limbic brain structures (amygdala, hippocampus, anterior nucleus thalamus) is shown to exhibit oscillations over multiple timescales. We observe that impedance oscillations with ultradian and circadian periodicities are associated with transitions between wakefulness, NREM, and REM sleep states. There are also impedance oscillations spanning multiple weeks that do not have a clear behavioral correlate and whose origin remains unclear. These multiscale impedance oscillations will have an impact on extracellular ionic currents that give rise to local field potentials, ephaptic coupling, and the tissue activated by electrical brain stimulation. The approach for measuring tissue impedance using perturbational electrical currents is an established engineering technique that may be useful for tracking ECS volume.

Analysis of potassium ion diffusion from neurons to capillaries: Effects of astrocyte endfeet geometry.

Neurovascular coupling (NVC) refers to a local increase in cerebral blood flow in response to increased neuronal activity. Mechanisms of communication between neurons and blood vessels remain unclear. Astrocyte endfeet almost completely cover cerebral capillaries, suggesting that astrocytes play a role in NVC by releasing vasoactive substances near capillaries. An alternative hypothesis is that direct diffusion through the extracellular space of potassium ions (K+ ) released by neurons contributes to NVC. Here, the goal is to determine whether astrocyte endfeet present a barrier to K+ diffusion from neurons to capillaries. Two simplified 2D geometries of extracellular space, clefts between endfeet, and perivascular space are used: (i) a source 1 µm from a capillary; (ii) a neuron 15 µm from a capillary. K+ release is modelled as a step increase in [K+ ] at the outer boundary of the extracellular space. The time-dependent diffusion equation is solved numerically. In the first geometry, perivascular [K+ ] approaches its final value within 0.05 s. Decreasing endfeet cleft width or increasing perivascular space width slows the rise in [K+ ]. In the second geometry, the increase in perivascular [K+ ] occurs within 0.5 s and is insensitive to changes in cleft width or perivascular space width. Predicted levels of perivascular [K+ ] are sufficient to cause vasodilation, and the rise time is within the time for flow increase in NVC. These results suggest that direct diffusion of K+ through the extracellular space is a possible NVC signalling mechanism.

MRI Extracellular Volume Fraction in Liver Fibrosis-A Comparison of Different Time Points and Blood Pool Measurements.

BACKGROUND: Extracellular volume (ECV) correlates with the degree of liver fibrosis. PURPOSE: To analyze the performance of liver MRI-based ECV evaluations with different blood pool measurements at different time points. STUDY TYPE: Prospective. SAMPLE: 73 consecutive patients (n = 31 females, mean age 56 years) with histopathology-proven liver fibrosis. FIELD STRENGTH/SEQUENCE: 3T acquisition within 90 days of biopsy, including shortened modified look-locker inversion recovery T1 mapping. ASSESSMENT: Polygonal regions of interest were manually drawn in the liver, aorta, vena cava, and in the main, left and right portal vein on four slices before and after Gd-DOTA administration at 5/10/15 minutes. ECV was calculated 1) on one single slice on portal bifurcation level, and 2) averaged over all four slices. STATISTICAL TESTS: Parameters were compared between patients with fibrosis grades F0-2 and F3-F4 with the Mann-Whitney U and fishers exact test. ROC analysis was used to assess the performance of the parameters to predict F3-4 fibrosis. A P-value <0.05 was considered statistically significant. RESULTS: ECV was significantly higher in F3-4 fibrosis (35.4% [33.1%-37.6%], 36.1% [34.2%-37.5%], and 37.0% [34.8%-39.2%] at 5/10/15 minutes) than in patients with F0-2 fibrosis (33.3% [30.8%-34.8%], 33.7% [31.6%-34.7%] and 34.9% [32.2%-36.0%]; AUC = 0.72-0.75). Blood pool T1 relaxation times in the aorta and vena cava were longer on the upper vs. lower slices at 5 minutes, but not at 10/15 minutes. AUC values were similar when measured on a single slice (AUC = 0.69-0.72) or based on blood pool measurements in the cava or portal vein (AUC = 0.63-0.67 and AUC = 0.65-0.70). DATA CONCLUSION: Liver ECV is significantly higher in F3-4 fibrosis compared to F0-2 fibrosis with blood pool measurements performed in the aorta, inferior vena cava, and portal vein at 5, 10, and 15 minutes. However, a smaller variability was observed for blood pool measurements between slices at 15 minutes. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 3.

Prediction of tumor regression grade in far-advanced gastric cancer after preoperative immuno-chemotherapy using dual-energy CT-derived extracellular volume fraction.

OBJECTIVES: This study examines the effectiveness of dual-energy CT (DECT) delayed-phase extracellular volume (ECV) fraction in predicting tumor regression grade (TRG) in far-advanced gastric cancer (FAGC) patients receiving preoperative immuno-chemotherapy. MATERIALS AND METHODS: A retrospective analysis was performed on far-advanced gastric adenocarcinoma patients treated with preoperative immuno-chemotherapy at our institution from August 2019 to March 2023. Patients were categorized based on their TRG into pathological complete response (pCR) and non-pCR groups. ECV was determined using the delayed-phase iodine maps. In addition, tumor iodine densities and standardized iodine ratios were meticulously analyzed using the triple-phase enhanced iodine maps. Univariate analysis with five-fold cross-validation and Spearman correlation determined DECT parameters and clinical indicators association with pCR. The predictive accuracy of these parameters for pCR was evaluated using a weighted logistic regression model with five-fold cross-validation. RESULTS: Of the 88 patients enrolled (mean age 60.8 ± 11.1 years, 63 males), 21 (23.9%) achieved pCR. Univariate analysis indicated ECV's significant role in differentiating between pCR and non-pCR groups (average p value = 0.021). In the logistic regression model, ECV independently predicted pCR with an average odds ratio of 0.911 (95% confidence interval, 0.798-0.994). The model, incorporating ECV, tumor area, and IDAV (the relative change rate of iodine density from venous phase to arterial phase), showed an average area under curves (AUCs) of 0.780 (0.770-0.791) and 0.766 (0.731-0.800) for the training and validation sets, respectively, in predicting pCR. CONCLUSION: DECT-derived ECV fraction is a valuable predictor of TRG in FAGC patients undergoing preoperative immuno-chemotherapy. CLINICAL RELEVANCE STATEMENT: This study demonstrates that DECT-derived extracellular volume fraction is a reliable predictor for pathological complete response in far-advanced gastric cancer patients receiving preoperative immuno-chemotherapy, offering a noninvasive tool for identifying potential treatment beneficiaries.

Efficacy of Citicoline Delivered via Brain Extracellular Space against Experimental Acute Ischemic Stroke in Rats.

Objective: Citicoline can be used to reduce acute ischemic stroke injury via venous infusion, however, its protective effects in the brain extracellular space remain largely unknown. Herein, we investigated the brain protective effects of citicoline administered via the brain extracellular space and sought precise effective dosage range that can protect against ischemic injury after experimental ischemic stroke in rats. Methods: Fifty-six Sprague-Dawley rats were randomly divided into control, intraperitoneal (IP), caudate-putamen (CPu)-25, CPu-40, CPu-50, CPu-60 and CPu-75 groups based on the infusion site and concentration of citicoline. Two hours after the administration of citicoline, the rats were subjected to a permanent middle cerebral artery occlusion to mimic acute ischemic stroke. Then, the brain infarct volume in rats after stroke was measured and their neurological deficiency was evaluated to explain the protective effects and effective dosage range of citicoline. Results: Compared to the control and IP groups, brain infarct volume of rats in CPu-40, CPu-50, and CPu-60 groups is significant smaller. Furthermore, the brain infarct volume of rats in CPu-50 is the least. Conclusions: Here, we showed that citicoline can decrease the brain infarct volume, thus protecting the brain from acute ischemic stroke injury. We also found that the appropriate effective citicoline dose delivered via the brain extracellular space is 50 mM. Our study provides novel insights into the precise treatment of acute ischemic stroke by citicoline via the brain extracellular space, further guiding the treatment of brain disease.

Local diffusion in the extracellular space of the brain.

The brain extracellular space (ECS) is a vast interstitial reticulum of extreme morphological complexity, composed of narrow gaps separated by local expansions, enabling interconnected highways between neural cells. Constituting on average 20% of brain volume, the ECS is key for intercellular communication, and understanding its diffusional properties is of paramount importance for understanding the brain. Within the ECS, neuroactive substances travel predominantly by diffusion, spreading through the interstitial fluid and the extracellular matrix scaffold after being focally released. The nanoscale dimensions of the ECS render it unresolvable by conventional live tissue compatible imaging methods, and historically diffusion of tracers has been used to indirectly infer its structure. Novel nanoscopic imaging techniques now show that the ECS is a highly dynamic compartment, and that diffusivity in the ECS is more heterogeneous than anticipated, with great variability across brain regions and physiological states. Diffusion is defined primarily by the local ECS geometry, and secondarily by the viscosity of the interstitial fluid, including the obstructive and binding properties of the extracellular matrix. ECS volume fraction and tortuosity both strongly determine diffusivity, and each can be independently regulated e.g. through alterations in glial morphology and the extracellular matrix composition. Here we aim to provide an overview of our current understanding of the ECS and its diffusional properties. We highlight emerging technological advances to respectively interrogate and model diffusion through the ECS, and point out how these may contribute in resolving the remaining enigmas of the ECS.

A Highly Translatable Dual-arm Local Delivery Strategy To Achieve Widespread Therapeutic Coverage in Healthy and Tumor-bearing Brain Tissues.

Drug delivery nanoparticles (NPs) based entirely on materials generally recognized as safe that provide widespread parenchymal distribution following intracranial administration via convection-enhanced delivery (CED) are introduced. Poly(lactic-co-glycolic acid) (PLGA) NPs are coated with various poloxamers, including F68, F98, or F127, via physical adsorption to render particle surfaces non-adhesive, thereby resisting interactions with brain extracellular matrix. F127-coated PLGA (F127/PLGA) NPs provide markedly greater distribution in healthy rat brains compared to uncoated NPs and widespread coverage in orthotopically-established brain tumors. Distribution analysis of variously-sized F127/PLGA NPs determines the average rat brain tissue porosity to be between 135 and 170 nm while revealing unprecedented brain coverage of larger F127/PLGA NPs with an aid of hydraulic pressure provided by CED. Importantly, F127/PLGA NPs can be lyophilized for long-term storage without compromising their ability to penetrate the brain tissue. Further, 65- and 200-nm F127/PLGA NPs lyophilized-reconstituted and administered in a moderately hyperosmolar infusate solution show further enhance particle dissemination in the brain via osmotically-driven enlargement of the brain tissue porosity. Combination of F127/PLGA NPs and osmotic tissue modulation provides a means with a clear regulatory path to maximize the brain distribution of large NPs that enable greater drug loading and prolong drug release.

Anesthesia-related brain microstructure modulations detected by diffusion magnetic resonance imaging.

Recent studies have shown significant changes to brain microstructure during sleep and anesthesia. In vivo optical microscopy and magnetic resonance imaging (MRI) studies have attributed these changes to anesthesia and sleep-related modulation of the brain's extracellular space (ECS). Isoflurane anesthesia is widely used in preclinical diffusion MRI (dMRI) and it is therefore important to investigate if the brain's microstructure is affected by anesthesia to an extent detectable with dMRI. Here, we employ diffusion kurtosis imaging (DKI) to assess brain microstructure in the awake and anesthetized mouse brain (n = 22). We find both mean diffusivity (MD) and mean kurtosis (MK) to be significantly decreased in the anesthetized mouse brain compared with the awake state (p < 0.001 for both). This effect is observed in both gray matter and white matter. To further investigate the time course of these changes we introduce a method for time-resolved fast DKI. With this, we show the time course of the microstructural alterations in mice (n = 5) as they transition between states in an awake-anesthesia-awake paradigm. We find that the decrease in MD and MK occurs rapidly after delivery of gas isoflurane anesthesia and that values normalize only slowly when the animals return to the awake state. Finally, time-resolved fast DKI is employed in an experimental mouse model of brain edema (n = 4), where cell swelling causes the ECS volume to decrease. Our results show that isoflurane affects DKI parameters and metrics of brain microstructure and point to isoflurane causing a reduction in the ECS volume. The demonstrated DKI methods are suitable for in-bore perturbation studies, for example, for investigating microstructural modulations related to sleep/wake-dependent functions of the glymphatic system. Importantly, our study shows an effect of isoflurane anesthesia on rodent brain microstructure that has broad relevance to preclinical dMRI.

Recruitment of efferent lymph during infusion of 20 % albumin.

BACKGROUND: Intravenous infusion of hyper-oncotic 20 % albumin expands the plasma volume by approximately twice the infused volume. We investigated whether the recruited fluid stems from accelerated flow of efferent lymph, which would add protein to the plasma, or from reversed transcapillary solvent filtration, where the solvent is expected to be low in protein. METHODS: We analyzed data from 27 intravenous infusions of 20 % albumin (3 mL/kg; approximately 200 mL) over 30 min given to 27 volunteers and patients. Twelve of the volunteers were also given a 5 % solution and served as controls. The pattern of blood hemoglobin, colloid osmotic pressure, and the plasma concentrations of two immunoglobulins (IgG and IgM) were studied over 5 h. RESULTS: A decrease of the difference between the plasma colloid osmotic pressure and plasma albumin occurred during the infusions and was almost four times greater for 5 % albumin than for 20 % albumin at 40 min (P < 0.0036), which indicates that non-albumin protein enriched the plasma when 20 % was infused. Moreover, the difference between the infusion-derived dilution of the blood plasma based on hemoglobin and the two immunoglobulins amounted to -1.9 % (-6 to +0.2) for 20 % albumin and to -4.4 % (25th-75th percentile range - 8.5 to +0.2) during experiments with 5 % albumin (P < 0.001). This supports that the plasma was enriched by immunoglobulins, probably via the lymph, when 20 % was infused. CONCLUSIONS: Between half and two-thirds of the extravascular fluid that was recruited during infusion of 20 % albumin in humans consisted of protein-containing fluid consistent with efferent lymph.

Dietary nitrate supplementation increases nitrate and nitrite concentrations in human skin interstitial fluid.

Acute dietary nitrate (NO3-) supplementation can increase [NO3-], but not nitrite ([NO2-]), in human skeletal muscle, though its effect on [NO3-] and [NO2-] in skin remains unknown. In an independent group design, 11 young adults ingested 140 mL of NO3--rich beetroot juice (BR; 9.6 mmol NO3-), and 6 young adults ingested 140 mL of a NO3--depleted placebo (PL). Skin dialysate, acquired through intradermal microdialysis, and venous blood samples were collected at baseline and every hour post-ingestion up to 4 h to assess dialysate and plasma [NO3-] and [NO2-]. The relative recovery rate of NO3- and NO2- through the microdialysis probe (73.1% and 62.8%), determined in a separate experiment, was used to estimate skin interstitial [NO3-] and [NO2-]. Baseline [NO3-] was lower, whereas baseline [NO2-] was higher in the skin interstitial fluid relative to plasma (both P < 0.001). Acute BR ingestion increased [NO3-] and [NO2-] in the skin interstitial fluid and plasma (all P < 0.001), with the magnitude being smaller in the skin interstitial fluid (e.g., 183 ± 54 vs. 491 ± 62 µM for Δ[NO3-] from baseline and 155 ± 190 vs. 217 ± 204 nM for Δ[NO2-] from baseline at 3 h post BR ingestion, both P ≤ 0.037). However, due to the aforementioned baseline differences, skin interstitial fluid [NO2-] post BR ingestion was higher, whereas [NO3-] was lower relative to plasma (all P < 0.001). These findings extend our understanding of NO3- and NO2- distribution at rest and indicate that acute BR supplementation increases [NO3-] and [NO2-] in human skin interstitial fluid.

Energy optimisation predicts the capacity of ion buffering in the brain.

Neurons store energy in the ionic concentration gradients they build across their cell membrane. The amount of energy stored, and hence the work the ions can do by mixing, can be enhanced by the presence of ion buffers in extra- and intracellular space. Buffers act as sources and sinks of ions, however, and unless the buffering capacities for different ion species obey certain relationships, a complete mixing of the ions may be impeded by the physical conditions of charge neutrality and isotonicity. From these conditions, buffering capacities were calculated that enabled each ion species to mix completely. In all valid buffer distributions, the [Formula: see text] ions were buffered most, with a capacity exceeding that of [Formula: see text] and [Formula: see text] buffering by at least an order of magnitude. The similar magnitude of the (oppositely directed) [Formula: see text] and [Formula: see text] gradients made extracellular space behave as a [Formula: see text]-[Formula: see text] exchanger. Anions such as [Formula: see text] were buffered least. The great capacity of the extra- and intracellular [Formula: see text] buffers caused a large influx of [Formula: see text] ions as is typically observed during energy deprivation. These results explain many characteristics of the physiological buffer distributions but raise the question how the brain controls the capacity of its ion buffers. It is suggested that neurons and glial cells, by their great sensitivity to gradients of charge and osmolarity, respectively, sense deviations from electro-neutral and isotonic mixing, and use these signals to tune the chemical composition, and buffering capacity, of the extra- and intracellular matrices.

Extra-axonal contribution to double diffusion encoding-based pore size estimates in the corticospinal tract.

OBJECTIVE: To study the origin of compartment size overestimation in double diffusion encoding MRI (DDE) in vivo experiments in the human corticospinal tract. Here, the extracellular space is hypothesized to be the origin of the DDE signal. By exploiting the DDE sensitivity to pore shape, it could be possible to identify the origin of the measured signal. The signal difference between parallel and perpendicular diffusion gradient orientation can indicate if a compartment is regular or eccentric in shape. As extracellular space can be considered an eccentric compartment, a positive difference would mean a high contribution to the compartment size estimates. MATERIALS AND METHODS: Computer simulations using MISST and in vivo experiments in eight healthy volunteers were performed. DDE experiments using a double spin-echo preparation with eight perpendicular directions were measured in vivo. The difference between parallel and perpendicular gradient orientations was analyzed using a Wilcoxon signed-rank test and a Mann-Whitney U test. RESULTS: Simulations and MR experiments showed a statistically significant difference between parallel and perpendicular diffusion gradient orientation signals ([Formula: see text]). CONCLUSION: The results suggest that the DDE-based size estimate may be considerably influenced by the extra-axonal compartment. However, the experimental results are also consistent with purely intra-axonal contributions in combination with a large fiber orientation dispersion.

Near-Infrared Carbon Nanotube Tracking Reveals the Nanoscale Extracellular Space around Synapses.

We provide evidence of a local synaptic nanoenvironment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nanoenvironment might play a role in the regulation of brain activity.

A secretory system for extracellular production of spider neurotoxin huwentoxin-I in Escherichia coli.

Due to their advantages in structural stability and versatility, cysteine-rich peptides, which are secreted from the venom glands of venomous animals, constitute a naturally occurring pharmaceutical arsenal. However, the correct folding of disulfide bonds is a challenging task in the prokaryotic expression system like Escherichia coli due to the reducing environment. Here, a secretory expression plasmid pSE-G1M5-SUMO-HWTX-I for the spider neurotoxin huwentoxin-I (HWTX-I) with three disulfides as a model of cysteine-rich peptides was constructed. By utilizing the signal peptide G1M5, the fusion protein 6 × His-SUMO-HWTX-I was successfully secreted into extracellular medium of BL21(DE3). After enrichment using cation-exchange chromatography and purification utilizing the Ni-NTA column, 6 × His-SUMO-HWTX-I was digested via Ulp1 kinase to release recombinant HWTX-I (rHWTX-I), which was further purified utilizing RP-HPLC. Finally, both impurities with low and high molecular weights were completely removed. The molecular mass of rHWTX-I was identified as being 3750.8 Da, which was identical to natural HWTX-I with three disulfide bridges. Furthermore, by utilizing whole-cell patch clamp, the sodium currents of hNav1.7 could be inhibited by rHWTX-I and the IC50 value was 419 nmol/L.

Postmortem histological freeze-thaw artifacts: a case report of a frozen infant and literature review.

Freezing and thawing have the potential to alter the gross and histologic appearance of tissues, causing damage to individual cells and disrupting the overall architecture. In forensic investigations, freezing and thawing can play a crucial role in cases of unknown cause of death. Perpetrators may use freezing preservation to conceal the body or obscure the time of death. Freezing can also occur naturally when a body is exposed to the elements, sometimes even leading to death itself. We present a case report involving an autopsy performed on an infant, who died of natural causes, after undergoing freezing and thawing. The objective of this study was to identify and discuss the histological artifacts observed in different tissues as a result of the freeze-thaw process. Histologically, the infant's tissues exhibited the most common features described in the literature. Ice crystal artifacts, characterized by expansion of the extracellular space and tissue clefts, were found in the heart, brain, liver, lungs, and kidneys. On the contrary, adipose tissue was not affected, likely due to the scarcity of water. Freeze-thaw artifacts should be taken into account whether a body is known to have been frozen or to add further data if found already defrosted.

The kinetics of isotonic and hypertonic resuscitation fluids is dependent on the sizes of the body fluid volumes.

Background and Aims: The extracellular and intracellular fluid volumes (ECV and ICV) vary not only with age, gender, and body weight but also with the habitual intake of water. The present study examines whether the baseline variations in the ECV and ICV change the distribution and elimination of subsequently given infusion fluids. Material and Methods: Twenty healthy male volunteers underwent 50 infusion experiments with crystalloid fluid for which the fluid volume kinetics was calculated based on frequent measurements of the hemodilution using mixed-effects modeling software. The results were compared with the ECV and ICV measured with multifrequency bioimpedance analysis before each infusion started. The fluids were given over 30 minutes and comprised 25 mL/kg Ringer's acetate (N = 20), Ringer's lactate, 5 mL/kg 7.5% saline, and 3 mL/kg 7.5% saline in 6% dextran 70 (these fluids, N = 10). Results: A large ICV was associated with a small extravascular accumulation of infused fluid, which increased the plasma volume expansion and the urinary excretion. With hypertonic fluid, a large ECV greatly accelerated urinary excretion. The body weight did not serve as a covariate in the kinetic models. Albumin was recruited to the plasma during infusion of both types of fluid. The hypertonic fluids served as diuretics. The infused excess sodium and osmolality were distributed over a 35% larger space than the sum of the ECV and ICV. Conclusion: A large ICV reduced the rate of distribution of Ringer's solution, whereas a large ECV accelerated the excretion of hypertonic saline.

Deciphering the functional nano-anatomy of the tripartite synapse using stimulated emission depletion microscopy.

A major challenge for studying neuron-astrocyte communication lies in visualizing the tripartite synapse, which is the physical site where astrocytic processes contact and interact with neuronal synapses. While conventional light microscopy cannot resolve the anatomical details of the tripartite synapse, electron microscopy only provides ultrastructural snapshots that tell us little about its living state and dynamics. Stimulated emission depletion (STED) microscopy is a super-resolution fluorescence imaging technique that can provide live images of tripartite synapses with nanoscale spatial resolution. It is compatible with physiology experiments and imaging in the intact brain in vivo, opening up new opportunities to link the nanoscale structure of the tripartite system with functional readouts of neurons and astrocytes or even behavior. In this review, we first summarize the findings and insights from previous studies addressing the structure-function relationship of the tripartite synapse using conventional imaging techniques. We then explain the basic principle of STED microscopy and the main challenges facing its application to live-tissue imaging of fine astrocytic processes. We summarize insights from our recent STED studies, which revealed new aspects of the structure and physiology of the tripartite synapse and the surrounding extracellular space. Finally, we discuss how the STED approach and other advanced optical techniques can illuminate the role of astrocytes for brain physiology and animal behavior.

Cellular mechanisms mediating activity-dependent extracellular space shrinkage in the retina.

Volume transmission plays an essential role in CNS function, with neurotransmitters released from synapses diffusing through the extracellular space (ECS) to distant sites. Changes in the ECS volume fraction (α) will influence the diffusion and the concentration of transmitters within the ECS. We have recently shown that neuronal activity evoked by physiological photic stimuli results in rapid decreases in ECS α as large as 10% in the retina. We now characterize the cellular mechanisms responsible for this ECS shrinkage. We find that block of inwardly rectifying K+ channels with Ba2+ , inhibition of the Na+ /K+ /2Cl- cotransporter with bumetanide, or block of AQP4 water channels with TGN-020 do not diminish the light-evoked ECS decrease. Inhibition of the Na+ /HCO3 - cotransporter by removing HCO3 - from the superfusate, in contrast, reduces the light-evoked ECS decrease by 95.6%. Inhibition of the monocarboxylate transporter with alpha-cyano-4-hydroxycinnamate (4-CIN) also reduces the ECS shrinkage, but only by 32.5%. We tested whether the swelling of Müller cells, the principal glial cells of the retina, is responsible for the light-evoked ECS shrinkage. Light stimulation evoked a 6.3% increase in the volume of the fine processes of Müller cells. This volume increase was reduced by 97.1% when HCO3 - was removed from the superfusate. We conclude that a large fraction of the activity-dependent decrease in ECS α is generated by the activation of the Na+ /HCO3 - cotransporter in Müller cells. The monocarboxylate transporter may also contribute to the response.