Role of aquaporin-4 polarization in extracellular solute clearance.

Waste from the brain has been shown to be cleared via the perivascular spaces through the so-called glymphatic system. According to this model the cerebrospinal fluid (CSF) enters the brain in perivascular spaces of arteries, crosses the astrocyte endfoot layer, flows through the parenchyma collecting waste that is subsequently drained along veins. Glymphatic clearance is dependent on astrocytic aquaporin-4 (AQP4) water channels that are highly enriched in the endfeet. Even though the polarized expression of AQP4 in endfeet is thought to be of crucial importance for glymphatic CSF influx, its role in extracellular solute clearance has only been evaluated using non-quantitative fluorescence measurements. Here we have quantitatively evaluated clearance of intrastriatally infused small and large radioactively labeled solutes in mice lacking AQP4 (Aqp4-/-) or lacking the endfoot pool of AQP4 (Snta1-/-). We confirm that Aqp4-/- mice show reduced clearance of both small and large extracellular solutes. Moreover, we find that the Snta1-/- mice have reduced clearance only for the 500 kDa [3H]dextran, but not 0.18 kDa [3H]mannitol suggesting that polarization of AQP4 to the endfeet is primarily important for clearance of large, but not small molecules. Lastly, we observed that clearance of 500 kDa [3H]dextran increased with age in adult mice. Based on our quantitative measurements, we confirm that presence of AQP4 is important for clearance of extracellular solutes, while the perivascular AQP4 localization seems to have a greater impact on clearance of large versus small molecules.

MAIN POINTS: Solute clearance is reduced in mice lacking AQP4 Polarization of AQP4 to the endfeet may have a greater impact on clearance of large versus small molecules Clearance of large but not small solutes is correlated with age within adult age.

Rapid response team activation prior to in-hospital cardiac arrest: Areas for improvements based on a national cohort study.

INTRODUCTION: Rapid response teams (RRTs) are designed to improve the "chain of prevention" of in-hospital cardiac arrest (IHCA). We studied the 30-day survival of patients reviewed by RRTs within 24 hours prior to IHCA, as compared to patients not reviewed by RRTs. METHODS: A nationwide cohort study based on the Swedish Registry of Cardiopulmonary Resuscitation, between January 1st, 2014, and December 31st, 2021. An explorative, hypothesis-generating additional in-depth data collection from medical records was performed in a small subgroup of general ward patients reviewed by RRTs. RESULTS: In all, 12,915 IHCA patients were included. RRT-reviewed patients (n = 2,058) had a lower unadjusted 30-day survival (25% vs 33%, p < 0.001), a propensity score based Odds ratio for 30-day survival of 0.92 (95% Confidence interval 0.90-0.94, p < 0.001) and were more likely to have a respiratory cause of IHCA (22% vs 15%, p < 0.001). In the subgroup (n = 82), respiratory distress was the most common RRT trigger, and 24% of the RRT reviews were delayed. Patient transfer to a higher level of care was associated with a higher 30-day survival rate (20% vs 2%, p < 0.001). CONCLUSION: IHCA preceded by RRT review is associated with a lower 30-day survival rate and a greater likelihood of a respiratory cause of cardiac arrest. In the small explorative subgroup, respiratory distress was the most common RRT trigger and delayed RRT activation was frequent. Early detection of respiratory abnormalities and timely interventions may have a potential to improve outcomes in RRT-reviewed patients and prevent further progress into IHCA.

Deletion of aquaporin-4 improves capillary blood flow distribution in brain edema.

Brain edema is a feared complication to disorders and insults affecting the brain. It can be fatal if the increase in intracranial pressure is sufficiently large to cause brain herniation. Moreover, accruing evidence suggests that even slight elevations of intracranial pressure have adverse effects, for instance on brain perfusion. The water channel aquaporin-4 (AQP4), densely expressed in perivascular astrocytic endfeet, plays a key role in brain edema formation. Using two-photon microscopy, we have studied AQP4-mediated swelling of astrocytes affects capillary blood flow and intracranial pressure (ICP) in unanesthetized mice using a mild brain edema model. We found improved regulation of capillary blood flow in mice devoid of AQP4, independently of the severity of ICP increase. Furthermore, we found brisk AQP4-dependent astrocytic Ca2+ signals in perivascular endfeet during edema that may play a role in the perturbed capillary blood flow dynamics. The study suggests that astrocytic endfoot swelling and pathological signaling disrupts microvascular flow regulation during brain edema formation.

Selective Janus kinase 1 inhibition resolves inflammation and restores hair growth offering a viable treatment option for alopecia areata.

Background: Janus Kinase (JAK) inhibition has recently demonstrated therapeutic efficacy in both restoring hair growth and resolving inflammation in Alopecia Areata (AA). These effects are dose dependent and mainly efficacious at ranges close to a questionable risk profile. Objectives: We explored the possibility to separate the beneficial and adverse effects of JAK inhibition by selectively inhibiting JAK1 and thereby avoiding side effects associated with JAK2 blockade. Methods: The C3H/HeJ mouse model of AA was used to demonstrate therapeutic efficacy in vivo with different regimens of a selection of JAK inhibitors in regards to systemic versus local drug exposure. Human peripheral blood lymphocytes were stimulated in vitro to demonstrate translation to the human situation. Results: We demonstrate that selective inhibition of JAK1 produces fast resolution of inflammation and complete restoration of hair growth in the C3H/HeJ mouse model of AA. Furthermore, we show that topical treatment does not restore hair growth and that treatment needs to be extended well beyond that of restored hair growth in order to reach treatment-free remission. For translatability to human disease, we show that cytokines involved in AA pathogenesis are similarly inhibited by selective JAK1 and pan-JAK inhibition in stimulated human peripheral lymphocytes and specifically in CD8+ T cells. Conclusion: This study demonstrates that systemic exposure is required for efficacy in AA and we propose that a selective JAK1 inhibitor will offer a treatment option with a superior safety profile to pan-JAK inhibitors for these patients.

The predictive power of the National Early Warning Score (NEWS) 2, as compared to NEWS, among patients assessed by a Rapid response team: A prospective multi-centre trial.

AIM: Early identification of patients at risk of serious adverse events (SAEs) is of vital importance, yet it remains a challenging task. We investigated the predictive power of National Early Warning Score (NEWS) 2, as compared to NEWS, among patients assessed by a Rapid response team (RRT). METHODS: Prospective, observational cohort study on 898 consecutive patients assessed by the RRTs in 26 Swedish hospitals. For each patient, NEWS and NEWS 2 scores were uniformly calculated by the study team. The associations of NEWS and NEWS 2 scores with unanticipated admissions to Intensive care unit (ICU), mortality and in-hospital cardiac arrests (IHCA) within 24 h, and the composite of these three events were investigated using logistic regression. The predictive power of NEWS and NEWS 2 was assessed using the area under the receiver operating characteristic (AUROC) curves. RESULTS: The prognostic accuracy of NEWS/NEWS 2 in predicting mortality was acceptable (AUROC 0.69/0.67). In discriminating the composite outcome and unanticipated ICU admission, both NEWS and NEWS 2 were relatively weak (AUROC 0.62/0.62 and AUROC 0.59/0.60 respectively); for IHCA the performance was poor. There were no differences between NEWS and NEWS 2 as to the predictive power. CONCLUSION: The prognostic accuracy of NEWS 2 to predict mortality within 24 h was acceptable. However, the prognostic accuracy of NEWS 2 to predict IHCA was poor. NEWS and NEWS 2 performed similar in predicting the risk of SAEs but their performances were not sufficient for use as a risk stratification tool in patients assessed by a RRT.

Cardiac arrest in COVID-19: characteristics and outcomes of in- and out-of-hospital cardiac arrest. A report from the Swedish Registry for Cardiopulmonary Resuscitation.

AIM: To study the characteristics and outcome among cardiac arrest cases with COVID-19 and differences between the pre-pandemic and the pandemic period in out-of-hospital cardiac arrest (OHCA) and in-hospital cardiac arrest (IHCA). METHOD AND RESULTS: We included all patients reported to the Swedish Registry for Cardiopulmonary Resuscitation from 1 January to 20 July 2020. We defined 16 March 2020 as the start of the pandemic. We assessed overall and 30-day mortality using Cox regression and logistic regression, respectively. We studied 1946 cases of OHCA and 1080 cases of IHCA during the entire period. During the pandemic, 88 (10.0%) of OHCAs and 72 (16.1%) of IHCAs had ongoing COVID-19. With regards to OHCA during the pandemic, the odds ratio for 30-day mortality in COVID-19-positive cases, compared with COVID-19-negative cases, was 3.40 [95% confidence interval (CI) 1.31-11.64]; the corresponding hazard ratio was 1.45 (95% CI 1.13-1.85). Adjusted 30-day survival was 4.7% for patients with COVID-19, 9.8% for patients without COVID-19, and 7.6% in the pre-pandemic period. With regards to IHCA during the pandemic, the odds ratio for COVID-19-positive cases, compared with COVID-19-negative cases, was 2.27 (95% CI 1.27-4.24); the corresponding hazard ratio was 1.48 (95% CI 1.09-2.01). Adjusted 30-day survival was 23.1% in COVID-19-positive cases, 39.5% in patients without COVID-19, and 36.4% in the pre-pandemic period. CONCLUSION: During the pandemic phase, COVID-19 was involved in at least 10% of all OHCAs and 16% of IHCAs, and, among COVID-19 cases, 30-day mortality was increased 3.4-fold in OHCA and 2.3-fold in IHCA.

ECG-monitoring of in-hospital cardiac arrest and factors associated with survival.

BACKGROUND: ECG-monitoring is a strong predictor for 30-days survival after in-hospital cardiac arrest (IHCA). The aim of the study is to investigate factors influencing the effect of ECG-monitoring on 30-days survival after IHCA and elements of importance in everyday clinical practice regarding whether patients are ECG-monitored prior to IHCA. METHODS: In all, 19.225 adult IHCAs registered in the Swedish Registry for Cardiopulmonary Resuscitation (SRCR) were included. Cox-adjusted survival curves were computed to study survival post IHCA. Logistic regression was used to study the association between 15 predictors and 30-days survival. Using logistic regression we calculated propensity scores (PS) for ECG-monitoring; the PS was used as a covariate in a logistical regression estimating the association between ECG-monitoring and 30-days survival. Gradient boosting was used to study the relative importance of all predictors on ECG-monitoring. RESULTS: Overall 30-days survival was 30%. The ECG-monitored group (n = 10.133, 52%) had a 38% lower adjusted mortality (HR 0.62 95% CI 0.60-0.64). We observed tangible variations in ECG-monitoring ratio at different centres. The predictors of most relative influence on ECG-monitoring in IHCA were location in hospital and geographical localization. CONCLUSION: ECG-monitoring in IHCA was associated to a 38% lower adjusted mortality, despite this finding only every other IHCA patient was monitored. The significant variability in the frequency of ECG-monitoring in IHCA at different centres needs to be evaluated in future research. Guidelines for in-hospital ECG-monitoring could contribute to an improved identification and treatment of patients at risk, and possibly to an improved survival.

Astroglial endfeet exhibit distinct Ca2+ signals during hypoosmotic conditions.

Astrocytic endfeet cover the brain surface and form a sheath around the cerebral vasculature. An emerging concept is that endfeet control blood-brain water transport and drainage of interstitial fluid and waste along paravascular pathways. Little is known about the signaling mechanisms that regulate endfoot volume and hence the width of these drainage pathways. Here, we used the genetically encoded fluorescent Ca2+ indicator GCaMP6f to study Ca2+ signaling within astrocytic somata, processes, and endfeet in response to an osmotic challenge known to induce cell swelling. Acute cortical slices were subjected to artificial cerebrospinal fluid with 20% reduction in osmolarity while GCaMP6f fluorescence was imaged with two-photon microscopy. Ca2+ signals induced by hypoosmotic conditions were observed in all astrocytic compartments except the soma. The Ca2+ response was most prominent in subpial and perivascular endfeet and included spikes with single peaks, plateau-type elevations, and rapid oscillations, the latter restricted to subpial endfeet. Genetic removal of the type 2 inositol 1,4,5-triphosphate receptor (IP3R2) severely suppressed the Ca2+ responses in endfeet but failed to affect brain water accumulation in vivo after water intoxication. Furthermore, the increase in endfoot Ca2+ spike rate during hypoosmotic conditions was attenuated in mutant mice lacking the aquaporin-4 anchoring molecule dystrophin and after blockage of transient receptor potential vanilloid 4 channels. We conclude that the characteristics and underpinning of Ca2+ responses to hypoosmotic stress differ within the astrocytic territory and that IP3R2 is essential for the Ca2+ signals only in subpial and perivascular endfeet.

Removal of aquaporin-4 from glial and ependymal membranes causes brain water accumulation.

There is a constitutive production of water in brain. The efflux routes of this excess water remain to be identified. We used basal brain water content as a proxy for the capacity of water exit routes. Basal brain water content was increased in mice with a complete loss of aquaporin-4 (AQP4) water channels (global Aqp4-/- mice), but not in mice with a selective removal of perivascular AQP4 or in a novel mouse line with a selective deletion of ependymal AQP4 (Foxj1-Cre:Aqp4flox/flox mice). Unique for the global Aqp4-/- mice is the loss of the AQP4 pool subjacent to the pial membrane. Our data suggest that water accumulates in brain when subpial AQP4 is missing, pointing to a critical role of this pool of water channels in brain water exit.

Molecular scaffolds underpinning macroglial polarization: an analysis of retinal Müller cells and brain astrocytes in mouse.

Key roles of macroglia are inextricably coupled to specialized membrane domains. The perivascular endfoot membrane has drawn particular attention, as this domain contains a unique complement of aquaporin-4 (AQP4) and other channel proteins that distinguishes it from perisynaptic membranes. Recent studies indicate that the polarization of macroglia is lost in a number of diseases, including temporal lobe epilepsy and Alzheimer's disease. A better understanding is required of the molecular underpinning of astroglial polarization, particularly when it comes to the significance of the dystrophin associated protein complex (DAPC). Here, we employ immunofluorescence and immunogold cytochemistry to analyze the molecular scaffolding in perivascular endfeet in macroglia of retina and three regions of brain (cortex, dentate gyrus, and cerebellum), using AQP4 as a marker. Compared with brain astrocytes, Müller cells (a class of retinal macroglia) exhibit lower densities of the scaffold proteins dystrophin and α-syntrophin (a DAPC protein), but higher levels of AQP4. In agreement, depletion of dystrophin or α-syntrophin--while causing a dramatic loss of AQP4 from endfoot membranes of brain astrocytes--had only modest or insignificant effect, respectively, on the AQP4 pool in endfoot membranes of Müller cells. In addition, while polarization of brain macroglia was less affected by dystrophin depletion than by targeted deletion of α-syntrophin, the reverse was true for retinal macroglia. These data indicate that the molecular scaffolding in perivascular endfeet is more complex than previously assumed and that macroglia are heterogeneous with respect to the mechanisms that dictate their polarization.

Loss of perivascular Kir4.1 potassium channels in the sclerotic hippocampus of patients with mesial temporal lobe epilepsy.

Recent experimental data in mice have shown that the inwardly rectifying K channel Kir4.1 mediates K spatial buffering in the hippocampus. Here we used immunohistochemistry to examine the distribution of Kir4.1 in hippocampi from patients with medication-refractory temporal lobe epilepsy. The selectivity of the antibody was confirmed in mice with a glial conditional deletion of the gene encoding Kir4.1. These mice showed a complete loss of labeled cells, indicating that Kir4.1 is restricted to glia. In human cases, Kir4.1 immunoreactivity observed in cells morphologically consistent with astrocytes was significantly reduced in 12 patients with hippocampal sclerosis versus 11 patients without sclerosis and 4 normal autopsy controls. Loss of astrocytic Kir4.1 immunoreactivity was most pronounced around vessels and was restricted to gliotic areas. Loss of Kir4.1 expression was associated with loss of dystrophin and α-syntrophin, but not with loss of ß-dystroglycan, suggesting partial disruption of the dystrophin-associated protein complex. The changes identified in patients with hippocampal sclerosis likely interfere with K homeostasis and may contribute to the epileptogenicity of the sclerotic hippocampus.

Deletion of aquaporin-4 changes the perivascular glial protein scaffold without disrupting the brain endothelial barrier.

Expression of the water channel aquaporin-4 (AQP4) at the blood-brain interface is dependent upon the dystrophin associated protein complex. Here we investigated whether deletion of the Aqp4 gene affects the molecular composition of this protein scaffold and the integrity of the blood-brain barrier. High-resolution immunogold cytochemistry revealed that perivascular expression of α-syntrophin was reduced by 60% in Aqp4(-/-) mice. Additionally, perivascular AQP4 expression was reduced by 88% in α-syn(-/-) mice, in accordance with earlier reports. Immunofluorescence showed that Aqp4 deletion also caused a modest reduction in perivascular dystrophin, whereas ß-dystroglycan labeling was unaltered. Perivascular microglia were devoid of AQP4 immunoreactivity. Deletion of Aqp4 did not alter the ultrastructure of capillary endothelial cells, the expression of tight junction proteins (claudin-5, occludin, and zonula occludens 1), or the vascular permeability to horseradish peroxidase and Evans blue albumin dye. We conclude that Aqp4 deletion reduces the expression of perivascular glial scaffolding proteins without affecting the endothelial barrier. Our data also indicate that AQP4 and α-syntrophin are mutually dependent upon each other for proper perivascular expression.

Glial-conditional deletion of aquaporin-4 (Aqp4) reduces blood-brain water uptake and confers barrier function on perivascular astrocyte endfeet.

Tissue- and cell-specific deletion of the Aqp4 gene is required to differentiate between the numerous pools of aquaporin-4 (AQP4) water channels. A glial-conditional Aqp4 knockout mouse line was generated to resolve whether astroglial AQP4 controls water exchange across the blood-brain interface. The conditional knockout was driven by the glial fibrillary acidic protein promoter. Brains from conditional Aqp4 knockouts were devoid of AQP4 as assessed by Western blots, ruling out the presence of a significant endothelial pool of AQP4. In agreement, immunofluorescence analysis of cryostate sections and quantitative immunogold analysis of ultrathin sections revealed no AQP4 signals in capillary endothelia. Compared with litter controls, glial-conditional Aqp4 knockout mice showed a 31% reduction in brain water uptake after systemic hypoosmotic stress and a delayed postnatal resorption of brain water. Deletion of astroglial Aqp4 did not affect the barrier function to macromolecules. Our data suggest that the blood-brain barrier (BBB) is more complex than anticipated. Notably, under certain conditions, the astrocyte covering of brain microvessels is rate limiting to water movement.

The metabolism of C-glucose by neurons and astrocytes in brain subregions following focal cerebral ischemia in rats.

To provide insights into the effects of temporary focal ischemia on the function of neurons and astrocytes in vivo, we measured the incorporation of radiolabel from [U-14C]glucose into both glutamate and glutamine in brain subregions at 1 h of reperfusion following occlusion of the middle cerebral artery for 2 or 3 h. Under the experimental conditions used, 14C-glutamate is mainly produced in neurons whereas 14C-glutamine is generated in astrocytes from 14C-glutamate of both neuronal and astrocytic origin. Radiolabel incorporation into both amino acids was greatly decreased. The change in 14C-glutamate accumulation provides strong evidence for substantial reductions in neuronal glucose metabolism. The resulting decrease in delivery of 14C-glutamate from the neurons to astrocytes was probably also the major contributor to the change in 14C-glutamine content. These alterations probably result in part from a marked depression of glycolytic activity in the neurons, as suggested by previous studies assessing deoxyglucose utilization. Alterations in 14C-glucose metabolism were not restricted to tissue that would subsequently become infarcted. Thus, these changes did not inevitably lead to death of the affected cells. The ATP : ADP ratio and phosphocreatine content were essentially preserved during recirculation following 2 h of ischemia and showed at most only moderate losses in some subregions following 3 h of ischemia. This retention of energy reserves despite the decreases in 14C-glucose metabolism in neurons suggests that energy needs were substantially reduced in the post-ischemic brain. Marked increases in tissue lactate accumulation during recirculation, particularly following 3 h of ischemia, provided evidence that impaired pyruvate oxidation probably also contributed to the altered 14C-glucose metabolism. These findings indicate the presence of complex changes in energy metabolism that are likely to greatly influence the responses of neurons and astrocytes to temporary focal ischemia.

Astrocytic function assessed from 1-14C-acetate metabolism after temporary focal cerebral ischemia in rats.

Astrocytes play many roles essential for normal brain activity. The ability of these cells to recover after temporary focal cerebral ischemia is likely to be one important determinant of the extent of brain dysfunction and tissue damage. We have assessed astrocytic function based on the incorporation of radiolabel from 1-14C-acetate into glutamine at 1 hour of recirculation after middle cerebral artery occlusion for 2 or 3 hours in rats. There were marked differences in the response between subregions within the tissue subjected to ischemia, but the overall pattern of changes was similar after each ischemic period. The striatum, which forms part of the severely ischemic focal tissue during arterial occlusion, showed a large (44% to 68%) decrease in glutamine labeling compared with equivalent tissue from the contralateral hemisphere. In contrast, 14C-glutamine content was not significantly altered in perifocal tissue in the cerebral cortex, which was subjected to more moderate ischemia. Cortical focal tissue also was not significantly affected, but the response was much more variable between rats. In these brain subregions, the extent of recovery of the 14C-acetate metabolism after ischemia was not a good predictor of the likelihood of subsequent infarct development. Interestingly, a similar pattern of responses persisted when recirculation was extended to 4 hours. These results indicate that many astrocytes, particularly in the cortex, remain viable and capable of at least some complex oxidative metabolism during the first few hours of recirculation.

Fluorocitrate-mediated astroglial dysfunction causes seizures.

A role for astroglia in epileptogenesis has been hypothesised but is not established. Low doses of fluorocitrate specifically and reversibly disrupt astroglial metabolism by blocking aconitase, an enzyme integral to the tricarboxylic acid cycle. We used cerebral cortex injections of fluorocitrate, at a dose that we demonstrated to inhibit astroglial metabolism selectively, to determine whether astroglial disturbances lead to seizures. Rats were halothane-anesthetized, and 0.8 nmol of sodium fluorocitrate was injected into the cerebral cortex. Extradural electroencephalogram (EEG) electrodes were implanted, after which the anesthesia was ceased and the animals were observed. In all experiments, 14 of 15 fluorocitrate-treated animals exhibited epileptiform EEG discharges, with some animals exhibiting convulsive seizures. Discharges commenced as early as 30 min postfluorocitrate injection. Intraperitoneal octanol, but not halothane by inhalation, given to test the possible participation of gap junctions in EEG discharge generation, blocked or delayed the occurrence of discharges after fluorocitrate. These results indicate that focal cerebrocortical astroglial dysfunction leads to focal epileptiform discharges and sometimes to convulsive seizures and that the process possibly depends on effects mediated by gap junctions.