SLC22A17 as a Cell Death-Linked Regulator of Tight Junctions in Cerebral Ischemia.

BACKGROUND: Beyond neuronal injury, cell death pathways may also contribute to vascular injury after stroke. We examined protein networks linked to major cell death pathways and identified SLC22A17 (solute carrier family 22 member 17) as a novel mediator that regulates endothelial tight junctions after ischemia and inflammatory stress. METHODS: Protein-protein interactions and brain enrichment analyses were performed using STRING, Cytoscape, and a human tissue-specific expression RNA-seq database. In vivo experiments were performed using mouse models of transient focal cerebral ischemia. Human stroke brain tissues were used to detect SLC22A17 by immunostaining. In vitro experiments were performed using human brain endothelial cultures subjected to inflammatory stress. Immunostaining and Western blot were used to assess responses in SLC22A17 and endothelial tight junctional proteins. Water content, dextran permeability, and electrical resistance assays were used to assess edema and blood-brain barrier (BBB) integrity. Gain and loss-of-function studies were performed using lentiviral overexpression of SLC22A17 or short interfering RNA against SLC22A17, respectively. RESULTS: Protein-protein interaction analysis showed that core proteins from apoptosis, necroptosis, ferroptosis, and autophagy cell death pathways were closely linked. Among the 20 proteins identified in the network, the iron-handling solute carrier SLC22A17 emerged as the mediator enriched in the brain. After cerebral ischemia in vivo, endothelial expression of SLC22A17 increases in both human and mouse brains along with BBB leakage. In human brain endothelial cultures, short interfering RNA against SLC22A17 prevents TNF-α (tumor necrosis factor alpha)-induced ferroptosis and downregulation in tight junction proteins and disruption in transcellular permeability. Notably, SLC22A17 could repress the transcription of tight junctional genes. Finally, short interfering RNA against SLC22A17 ameliorates BBB leakage in a mouse model of focal cerebral ischemia. CONCLUSIONS: Using a combination of cell culture, human stroke samples, and mouse models, our data suggest that SLC22A17 may play a role in the control of BBB function after cerebral ischemia. These findings may offer a novel mechanism and target for ameliorating BBB injury and edema after stroke.

Double blind randomized controlled trial measuring the efficacy of nebulized steroid at half dose in comparison to high volume squeeze bottle steroid irrigation in management of patients with chronic rhinosinusitis.

KEY POINTS: Nebulized budesonide is effective at half dose compared to budesonide irrigation in CRS. Nasal nebulizers provide an alternative for delivery of topical steroids to the sinuses.

Circadian Biology and the Neurovascular Unit.

Mammalian physiology and cellular function are subject to significant oscillations over the course of every 24-hour day. It is likely that these daily rhythms will affect function as well as mechanisms of disease in the central nervous system. In this review, we attempt to survey and synthesize emerging studies that investigate how circadian biology may influence the neurovascular unit. We examine how circadian clocks may operate in neural, glial, and vascular compartments, review how circadian mechanisms regulate cell-cell signaling, assess interactions with aging and vascular comorbidities, and finally ask whether and how circadian effects and disruptions in rhythms may influence the risk and progression of pathophysiology in cerebrovascular disease. Overcoming identified challenges and leveraging opportunities for future research might support the development of novel circadian-based treatments for stroke.

Glymphatic and lymphatic communication with systemic responses during physiological and pathological conditions in the central nervous system.

Crosstalk between central nervous system (CNS) and systemic responses is important in many pathological conditions, including stroke, neurodegeneration, schizophrenia, epilepsy, etc. Accumulating evidence suggest that signals for central-systemic crosstalk may utilize glymphatic and lymphatic pathways. The glymphatic system is functionally connected to the meningeal lymphatic system, and together these pathways may be involved in the distribution of soluble proteins and clearance of metabolites and waste products from the CNS. Lymphatic vessels in the dura and meninges transport cerebrospinal fluid, in part collected from the glymphatic system, to the cervical lymph nodes, where solutes coming from the brain (i.e., VEGFC, oligomeric α-syn, ß-amyloid) might activate a systemic inflammatory response. There is also an element of time since the immune system is strongly regulated by circadian rhythms, and both glymphatic and lymphatic dynamics have been shown to change during the day and night. Understanding the mechanisms regulating the brain-cervical lymph node (CLN) signaling and how it might be affected by diurnal or circadian rhythms is fundamental to find specific targets and timing for therapeutic interventions.

Transcriptomic Profiling Reveals Neuroinflammation in the Corpus Callosum of a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a widespread neurodegenerative disorder characterized by progressive cognitive decline, affecting a significant portion of the aging population. While the cerebral cortex and hippocampus have been the primary focus of AD research, accumulating evidence suggests that white matter lesions in the brain, particularly in the corpus callosum, play an important role in the pathogenesis of the disease. OBJECTIVE: This study aims to investigate the gene expression changes in the corpus callosum of 5xFAD transgenic mice, a widely used AD mouse model. METHODS: We conducted behavioral tests for spatial learning and memory in 5xFAD transgenic mice and performed RNA sequencing analyses on the corpus callosum to examine transcriptomic changes. RESULTS: Our results show cognitive decline and demyelination in the corpus callosum of 5xFAD transgenic mice. Transcriptomic analysis reveals a predominance of upregulated genes in AD mice, particularly those associated with immune cells, including microglia. Conversely, downregulation of genes related to chaperone function and clock genes such as Per1, Per2, and Cry1 is also observed. CONCLUSIONS: This study suggests that activation of neuroinflammation, disruption of chaperone function, and circadian dysfunction are involved in the pathogenesis of white matter lesions in AD. The findings provide insights into potential therapeutic targets and highlight the importance of addressing white matter pathology and circadian dysfunction in AD treatment strategies.

Sleep apnea multi-level surgery trial: long-term observational outcomes.

STUDY OBJECTIVES: The sleep apnea multi-level surgery (SAMS) randomized clinical trial showed surgery improved outcomes at 6 months compared to ongoing medical management in patients with moderate or severe obstructive sleep apnea (OSA) who failed continuous positive airway pressure therapy. This study reports the long-term outcomes of the multi-level surgery as a case series. METHODS: Surgical participants were reassessed >2 years postoperatively with the same outcomes reported in the main SAMS trial. Primary outcomes were apnea-hypopnea index (AHI) and Epworth sleepiness scale (ESS), with secondary outcomes including other polysomnography measures, symptoms, quality of life, and adverse events. Long-term effectiveness (baseline to long-term follow-up [LTFU]) and interval changes (6 month to LTFU) were assessed using mixed effects regression models. Control participants were also reassessed for rate of subsequent surgery and outcomes. RESULTS: 36/48 (75%) of surgical participants were reevaluated (mean (standard deviation)) 3.5 (1.0) years following surgery, with 29 undergoing polysomnography. AHI was 41/h (23) at preoperative baseline and 21/h (18) at follow-up, representing persistent improvement of -24/h (95% CI -32, -17; p < 0.001). ESS was 12.3 (3.5) at baseline and 5.5 (3.9) at follow-up, representing persistent improvement of -6.8 (95% CI -8.3, -5.4; p < 0.001). Secondary outcomes were improved long term, and adverse events were minor. Interval change analysis suggests stability of outcomes. 36/43 (84%) of the control participants were reevaluated, with 25 (69%) reporting subsequent surgery, with symptom and quality of life improvements. CONCLUSION: Multi-level upper airway surgery improves OSA burden with long-term maintenance of treatment effect in adults with moderate or severe OSA in whom conventional therapy failed. CLINICAL TRIAL: Multi-level airway surgery in patients with moderate-severe obstructive sleep apnea (OSA) who have failed medical management to assess change in OSA events and daytime sleepiness; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=366019&isReview=true; ACTRN12614000338662.

Sleep restoration by optogenetic targeting of GABAergic neurons reprograms microglia and ameliorates pathological phenotypes in an Alzheimer's disease model.

BACKGROUND: Alzheimer's disease (AD) patients exhibit memory disruptions and profound sleep disturbances, including disruption of deep non-rapid eye movement (NREM) sleep. Slow-wave activity (SWA) is a major restorative feature of NREM sleep and is important for memory consolidation. METHODS: We generated a mouse model where GABAergic interneurons could be targeted in the presence of APPswe/PS1dE9 (APP) amyloidosis, APP-GAD-Cre mice. An electroencephalography (EEG) / electromyography (EMG) telemetry system was used to monitor sleep disruptions in these animals. Optogenetic stimulation of GABAergic interneurons in the anterior cortex targeted with channelrhodopsin-2 (ChR2) allowed us to examine the role GABAergic interneurons play in sleep deficits. We also examined the effect of optogenetic stimulation on amyloid plaques, neuronal calcium as well as sleep-dependent memory consolidation. In addition, microglial morphological features and functions were assessed using confocal microscopy and flow cytometry. Finally, we performed sleep deprivation during optogenetic stimulation to investigate whether sleep restoration was necessary to slow AD progression. RESULTS: APP-GAD-Cre mice exhibited impairments in sleep architecture including decreased time spent in NREM sleep, decreased delta power, and increased sleep fragmentation compared to nontransgenic (NTG) NTG-GAD-Cre mice. Optogenetic stimulation of cortical GABAergic interneurons increased SWA and rescued sleep impairments in APP-GAD-Cre animals. Furthermore, it slowed AD progression by reducing amyloid deposition, normalizing neuronal calcium homeostasis, and improving memory function. These changes were accompanied by increased numbers and a morphological transformation of microglia, elevated phagocytic marker expression, and enhanced amyloid ß (Aß) phagocytic activity of microglia. Sleep was necessary for amelioration of pathophysiological phenotypes in APP-GAD-Cre mice. CONCLUSIONS: In summary, our study shows that optogenetic targeting of GABAergic interneurons rescues sleep, which then ameliorates neuropathological as well as behavioral deficits by increasing clearance of Aß by microglia in an AD mouse model.

O-GlcNAcylation is essential for therapeutic mitochondrial transplantation.

BACKGROUND: Transplantation of mitochondria is increasingly explored as a novel therapy in central nervous system (CNS) injury and disease. However, there are limitations in safety and efficacy because mitochondria are vulnerable in extracellular environments and damaged mitochondria can induce unfavorable danger signals. METHODS: Mitochondrial O-GlcNAc-modification was amplified by recombinant O-GlcNAc transferase (OGT) and UDP-GlcNAc. O-GlcNAcylated mitochondrial proteins were identified by mass spectrometry and the antiglycation ability of O-GlcNAcylated DJ1 was determined by loss-of-function via mutagenesis. Therapeutic efficacy of O-GlcNAcylated mitochondria was assessed in a mouse model of transient focal cerebral ischemia-reperfusion. To explore translational potential, we evaluated O-GlcNAcylated DJ1 in CSF collected from patients with subarachnoid hemorrhagic stroke (SAH). RESULTS: We show that isolated mitochondria are susceptible to advanced glycation end product (AGE) modification, and these glycated mitochondria induce the receptor for advanced glycation end product (RAGE)-mediated autophagy and oxidative stress when transferred into neurons. However, modifying mitochondria with O-GlcNAcylation counteracts glycation, diminishes RAGE-mediated effects, and improves viability of mitochondria recipient neurons. In a mouse model of stroke, treatment with extracellular mitochondria modified by O-GlcNAcylation reduces neuronal injury and improves neurologic deficits. In cerebrospinal fluid (CSF) samples from SAH patients, levels of O-GlcNAcylation in extracellular mitochondria correlate with better clinical outcomes. CONCLUSIONS: These findings suggest that AGE-modification in extracellular mitochondria may induce danger signals, but O-GlcNAcylation can prevent glycation and improve the therapeutic efficacy of transplanted mitochondria in the CNS.

Mitochondria are the part of a cell that generate most of its energy to perform its functions. In injury or disease, mitochondrial function can become disrupted. Transplantation of healthy mitochondria is being explored as a potential therapy to replace damaged mitochondria and restore normal cellular function. However, this approach is difficult to perform because mitochondria are not able to maintain their healthy state outside of cells. Here, we show that one of the reasons for this is due to a molecular process called advanced glycation end product modification. We show that simple modification of mitochondria with a sugar prevents this process and helps to improve the success of therapeutic mitochondrial transplantation in cells and in a mouse model of stroke. Our findings may help to guide future efforts to develop therapies based on mitochondrial transplantation.

Myelination- and migration-associated genes are downregulated after phagocytosis in cultured oligodendrocyte precursor cells.

In the central nervous system, microglia are responsible for removing infectious agents, damaged/dead cells, and amyloid plaques by phagocytosis. Other cell types, such as astrocytes, are also recently recognized to show phagocytotic activity under some conditions. Oligodendrocyte precursor cells (OPCs), which belong to the same glial cell family as microglia and astrocytes, may have similar functions. However, it remains largely unknown whether OPCs exhibit phagocytic activity against foreign materials like microglia. To answer this question, we examined the phagocytosis activity of OPCs using primary rat OPC cultures. Since innate phagocytosis activity could trigger cell death pathways, we also investigated whether participating in phagocytosis activity may lead to OPC cell death. Our data shows that cultured OPCs phagocytosed myelin-debris-rich lysates prepared from rat corpus callosum, without progressing to cell death. In contrast to OPCs, mature oligodendrocytes did not show phagocytotic activity against the bait. OPCs also exhibited phagocytosis towards lysates of rat brain cortex and cell membrane debris from cultured astrocytes, but the percentage of OPCs that phagocytosed beta-amyloid was much lower than the myelin debris. We then conducted RNA-seq experiments to examine the transcriptome profile of OPC cultures and found that myelination- and migration-associated genes were downregulated 24 h after phagocytosis. On the other hand, there were a few upregulated genes in OPCs 24 h after phagocytosis. These data confirm that OPCs play a role in debris removal and suggest that OPCs may remain in a quiescent state after phagocytosis.

Swallowing biomechanics in tracheostomised critically ill patients compared to age- and gender-matched healthy controls.

Objective: The mechanistic effects of a tracheostomy on swallowing are unclear. Pharyngeal high-resolution manometry with impedance (P-HRM-I) is a novel swallow assessment tool providing quantifiable metrics. This study aimed to characterise swallowing biomechanics in tracheostomised critically ill (non-neurological) patients. Design: Cohort study. Setting: Australian tertiary hospital intensive care unit. Participants: Tracheostomised adults, planned for decannulation. Main outcome measures: Swallowing assessment using P-HRM-I, compared to healthy age- and gender-matched controls. Results: In this tracheostomised cohort (n = 10), the Swallow Risk Index, a global measure of swallow function, was significantly elevated (p < 0.001). At the upper oesophageal sphincter (UOS), hypopharyngeal intrabolus pressure and UOS integrated relaxation pressure were significantly elevated (control 0.65 mmHg [-1.02, 2.33] v tracheostomy 13.7 mmHg [10.4, 16.9], P < 0.001; control -4.28 mmHg [-5.87, 2.69] v tracheostomy 12.2 mmHg [8.83, 15.6], P < 0.001, respectively). Furthermore, UOS opening extent and relaxation time were reduced (control 4.83 mS [4.60, 5.07] v tracheostomy 4.33 mS [3.97, 4.69], P = 0.002; control 0.52 s [0.49, 0.55] v tracheostomy 0.41 s [0.37, 0.45], P < 0.001, respectively). Total pharyngeal contractility (PhCI) measuring pharyngeal pressure generation was significantly elevated (control 199.5 mmHg cm.s [177.4, 221.6] v tracheostomy 326.5 mmHg cm.s [253.3, 399.7]; P = 0.001). Conclusion: In a critically ill tracheostomised cohort, UOS dysfunction was the prevalent biomechanical feature, with elevated pharyngeal pressures. Pharyngeal weakness is not contributing to dysphagia in this cohort. Instead, elevated pharyngeal pressures may represent a compensatory mechanism to overcome the UOS dysfunction. Further studies to extend these findings may inform the development of timely and targeted rehabilitation.

Postconditioning promotes recovery in the neurovascular unit after stroke.

Background and purpose: Experimental studies suggest that ischemic postconditioning interferes with cell death mechanisms and reduces infarction during the acute phase after focal cerebral ischemia. Postconditioning may be a practically feasible way to promote stroke recovery, but many drawbacks prevent its clinical translation. First, all existing studies are mostly on acute 24 h outcomes. Second, the mechanisms of protection and augmented long-term benefits remain unclear. Our study aims to define some of the mechanisms that explain long-term benefits of improved recovery. Methods: Male Sprague-Dawley rats were subjected to 100-min transient middle cerebral artery occlusion (MCAO) or postconditioning (100-min middle cerebral artery occlusion plus 10-min reperfusion plus 10-min reocclusion). After 3 days or 2 weeks, infarct volumes, western blot, and immunohistochemical markers of neurogenesis and angiogenesis were quantified. Fluorocitrate (FC) or saline were administrated ICV (intraventricular injection) every other day starting on day 5 after focal cerebral ischemia, animals were recovered for 2 weeks. Results: After postconditioning BDNF protein expression levels increased compared to animals subjected to MCAO. Immunostaining showed that BDNF increased specifically in astrocytes. Moreover, when astrocytes were metabolically inhibited by fluorocitrate the postconditioning neuroprotective effect together with the postconditioning-dependent new angiogenesis and neurogenesis, were no longer observed. Conclusion: These results suggest for the first time that therapeutic effects of postconditioning may involve the promotion of neurogenesis and angiogenic remodeling, via BDNF released by astrocytes, during the recovery phase after focal cerebral ischemia.

The impact of multifocal perineural invasion in predicting survival in patients with oral squamous cell carcinoma: A multicenter investigation.

BACKGROUND: Perineural invasion (PNI) in oral squamous cell carcinoma (OSCC) does not contribute to the current American Joint Committee on Cancer 8th edition (AJCC8) staging manual. This study seeks to validate the effect of multifocal PNI in a large cohort of patients. METHODS: Patients undergoing primary surgical treatment of OSCC with curative intent between 1995 and 2022 was retrieved from two Australian head and neck databases. PNI was categorized as a single focus or multiple foci. Study end points included disease-specific survival (DSS) and overall survival (OS). RESULTS: Complete data for survival analysis was available in 993 patients. Multifocal PNI was associated with a 61% increased risk of death due to OSCC (HR 1.61, 95% CI 1.11-2.33, p = 0.014) and a 32% increased risk of death from any cause (HR 1.32, 95% CI 1.01-1.73, p = 0.045). CONCLUSIONS: Multifocal PNI is a significant predictor of survival in OSCC.

Ultraflexible endovascular probes for brain recording through micrometer-scale vasculature.

Implantable neuroelectronic interfaces have enabled advances in both fundamental research and treatment of neurological diseases but traditional intracranial depth electrodes require invasive surgery to place and can disrupt neural networks during implantation. We developed an ultrasmall and flexible endovascular neural probe that can be implanted into sub-100-micrometer-scale blood vessels in the brains of rodents without damaging the brain or vasculature. In vivo electrophysiology recording of local field potentials and single-unit spikes have been selectively achieved in the cortex and olfactory bulb. Histology analysis of the tissue interface showed minimal immune response and long-term stability. This platform technology can be readily extended as both research tools and medical devices for the detection and intervention of neurological diseases.

Role of A-Kinase Anchoring Protein 12 in the Central Nervous System.

A-kinase anchoring protein (AKAP) 12 is a scaffolding protein that anchors various signaling proteins to the plasma membrane. These signaling proteins include protein kinase A, protein kinase C, protein phosphatase 2B, Src-family kinases, cyclins, and calmodulin, which regulate their respective signaling pathways. AKAP12 expression is observed in the neurons, astrocytes, endothelial cells, pericytes, and oligodendrocytes of the central nervous system (CNS). Its physiological roles include promoting the development of the blood-brain barrier, maintaining white-matter homeostasis, and even regulating complex cognitive functions such as long-term memory formation. Under pathological conditions, dysregulation of AKAP12 expression levels may be involved in the pathology of neurological diseases such as ischemic brain injury and Alzheimer's disease. This minireview aimed to summarize the current literature on the role of AKAP12 in the CNS.

Differential reductions in the capillary red-blood-cell flux between retina and brain under chronic global hypoperfusion.

Significance: It has been hypothesized that abnormal microcirculation in the retina might predict the risk of ischemic damages in the brain. Direct comparison between the retinal and the cerebral microcirculation using similar animal preparation and under similar experimental conditions would help test this hypothesis. Aim: We investigated capillary red-blood-cell (RBC) flux changes under controlled conditions and bilateral-carotid-artery-stenosis (BCAS)-induced hypoperfusion, and then compared them with our previous measurements performed in the brain. Approach: We measured capillary RBC flux in mouse retina with two-photon microscopy using a fluorescence-labeled RBC-passage approach. Key physiological parameters were monitored during experiments to ensure stable physiology. Results: We found that under the controlled conditions, capillary RBC flux in the retina was much higher than in the brain (i.e., cerebral cortical gray matter and subcortical white matter), and that BCAS induced a much larger decrease in capillary RBC flux in the retina than in the brain. Conclusions: We demonstrated a two-photon microscopy-based technique to efficiently measure capillary RBC flux in the retina. Since cerebral subcortical white matter often exhibits early pathological developments due to global hypoperfusion, our results suggest that retinal microcirculation may be utilized as an early marker of brain diseases involving global hypoperfusion.

BiZact™ tonsillectomy: Predictive factors for post-tonsillectomy haemorrhage from a 1717 case series.

OBJECTIVE: To determine primary and secondary post-tonsillectomy haemorrhage (PTH) rates and identify predictive factors in a cohort of consecutive adult and paediatric BiZact™ tonsillectomy cases. SETTING: Retrospective cohort study. Patients from Flinders Medical Centre, Noarlunga Hospital and private otolaryngology practices who underwent BiZact™ tonsillectomy from 2017 to 2020. DATA COLLECTED: patient age, indication for tonsillectomy, surgeon experience, time and severity of PTH, including return to theatre. Each secondary PTH was graded using the Stammberger classification. Logistic regression was utilised to identify predictors of secondary PTH. RESULTS: One thousand seven hundred and seventeen patient medical records were assessed (658 adults and 1059 children). The primary PTH rate was 0.1%, and secondary PTH rate was 5.9%. The majority of secondary PTH cases were Stammberger grade A (80/102, 78.4%) requiring observation only. Few secondary PTH required medical intervention (grade B; 9/102, 8.8%), return to theatre (grade C; 12/102, 11.8%), or blood transfusion (grade D; 1/102, 1.0%), with no death reported (grade E; 0/102, 0.0%). Recurrent secondary PTH occurred in 8 patients (0.5%). Predictive factors of secondary PTH in children were surgeon experience with trainees having greater chance of PTH (OR 2.502, 95% CI 1.345-4.654; p = .004) and age of child (OR 1.095, 95% CI 1.025-1.170; p = .007). Surgeon experience was a predictive factor for adults (OR 3.804, 95% CI 2.139-6.674; p < .001). CONCLUSIONS: BiZact™ tonsillectomy has a low primary PTH rate, with a secondary PTH rate comparable to other 'hot tonsillectomy' techniques. The majority of PTH events were minor and self-reported. There appears to be a learning curve for trainee surgeons.