Supplemental Digital Content is available in the text. Secondary brain hypoxia portends significant mortality in ischemic brain diseases; yet, our understanding of hypoxic ischemic brain injury (HIBI) pathophysiology in humans remains rudimentary.To quantify the impact of secondary brain hypoxia on injury to the neurovascular unit in patients with HIBI.2 (PbtO2) and intracranial pressure were directly measured via intraparenchymal microcatheters. To isolate the cerebrovascular bed, we conducted paired sampling of arterial and jugular venous bulb blood and calculated the transcerebral release of biomarkers of neurovascular injury and inflammation in the patients with HIBI and 14 healthy volunteers for control comparisons. Ten patients with HIBI exhibited secondary brain hypoxia (PbtO2<20 mmHg), while 8 exhibited brain normoxia (PbtO2≥20 mmHg). In the patients with secondary brain hypoxia, we observed active cerebral release of glial fibrillary acidic protein , neurofilament light chain , total tau , neuron-specific enolase , and ubiquitin carboxy-terminal hydrolase L1  indicating de novo neuroglial injury. This injury was unrelated to the systemic global ischemic burden or cerebral endothelial injury but rather was associated with cerebral release of IL-6 . No cerebral release of the aforementioned biomarkers was observed in patients with HIBI with brain normoxia or the healthy volunteers. Hyperosmolar therapy in the patients with secondary brain hypoxia reduced the partial pressure of jugular venous O2-to-PbtO2 gradient  and increased PbtO2  suggesting improved cerebrovascular-to-parenchymal O2 transport.We conducted a prospective interventional study of invasive neuromonitoring in 18 post–cardiac arrest patients with HIBI. The partial pressures of brain tissue O2 transport is a promising therapeutic strategy for patients with HIBI with secondary brain hypoxia.Secondary brain hypoxia is associated with de novo neuroglial injury and cerebral release of IL-6. Mitigating cerebrovascular-to-parenchymal limitations to Ohttps://www.clinicaltrials.gov; Unique identifier: NCT03609333.URL:  Secondary injury takes place following resuscitation and restoration of innate cardiac output and CBF. During this phase, brief cerebral hyperemia is followed by a prolonged phase of depressed CBF, termed the no reflow period.7 During no-reflow, cerebral oligemia and a reduction in convective cerebral oxygen delivery (CDO2) may precipitate secondary brain hypoxia and cell death.The mortality and morbidity of hypoxic ischemic brain injury (HIBI) following return of spontaneous circulation (ROSC) after cardiac arrest is devastating,2 through augmentation of mean arterial pressure (MAP) along with maintenance of normocapnic ventilation10 and arterial oxygen content.12 Yet, recent randomized trials aimed at improving convective CDO2 through augmentation of MAP have failed to demonstrate biological or clinical efficacy.14 Further, despite goal directed resuscitation guided by neuromonitoring, patients with HIBI continue to experience a significant burden of brain hypoxia after ROSC15 and show physiological signatures consistent with increased diffusion barriers between the cerebral microvasculature and parenchyma.16 An improved understanding of the multiple mechanisms underlying secondary brain hypoxia in humans with HIBI is needed to facilitate the development of effective therapeutic strategies that extend beyond the augmentation of convective CDO2.A cornerstone in the critical care management of patients with HIBI post-resuscitation is the optimization of convective CDO18 perivascular edema,20 cellular metabolic perturbations,21 endothelial injury,22 oxidative stress,18 and inflammation23 may all be key factors in the pathophysiology of HIBI in humans. These factors all have the potential to intersect with—and impede oxygen transport at—multiple levels of the oxygen cascade to ultimately precipitate secondary brain hypoxia. If secondary brain hypoxia is of pathophysiologic significance in humans with HIBI and related ischemic brain disease states, injurious effects may be expected to arise within the neurovascular unit (NVU), which comprises the principal anatomic and functional interface between neuronal, astroglial, mural (smooth muscle cells and pericytes), and cerebrovascular endothelial cells.24 As function of the NVU determines the control of microvascular blood flow, nutrient exchange and cellular homeostasis of brain parenchyma,24 secondary hypoxic injury to the NVU  would be devastating.24 Therefore, it is imperative to characterize the injury pattern of the NVU, investigate the impact of secondary brain hypoxia on the NVU, draw insights into the pathophysiology of secondary brain hypoxia, and determine the potential modifiability of brain hypoxia after ROSC in humans with HIBI.Preclinical animal models suggest blood-brain barrier (BBB) breakdown,22 as well as inflammation25 compared with patients without secondary brain hypoxia. We further hypothesized that secondary hypoxia would be related to impaired cerebrovascular-to-parenchymal diffusion of O2, and that this hypoxia would be reduced with hyperosmolar therapy. Utilizing arterial and jugular venous bulb blood sampling to isolate the cerebrovascular bed and determine transcerebral release of NVU injury markers, we demonstrate that persistent brain hypoxia following ROSC in humans is associated with de novo release of biomarkers related to neuronal and astroglial injury, namely, neurofilament-light (NF-L), NSE (neuron-specific enolase), total tau, and UCH-L1  as neuronal biomarkers and GFAP  as an astrocyte biomarker. Importantly, secondary brain hypoxia was related to a cerebral proinflammatory response but not concurrent cerebral endothelial injury. Our findings of elevated markers of neuronal and astroglial injury, BBB breakdown, and systemic endothelial injury were recapitulated at a physiological level by a reduction in cerebral oxygen gradients  and an improvement in the partial pressure of brain tissue oxygen (PbtO2) following hypertonic saline infusion.To investigate the pathophysiology of the secondary hypoxic injury that follows a global cerebral ischemic insult, we tested the hypothesis that post–cardiac arrest patients exhibiting persistent secondary brain hypoxia would demonstrate elevated biomarkers of neuronal, astroglial, and endothelial injuryData Supplement.The data that support the findings of this study are available from the corresponding author upon reasonable request. An expanded methods section is available in the 16; however, the study aims, hypotheses, data analysis, and results for this article are original. Patients were enrolled between November 2016 and October 2019 in the Vancouver General Hospital intensive care unit (ICU). Enrollment for this study is no longer ongoing and the study is now complete (NCT03609333). Inclusion/exclusion criteria and patient management are detailed in the expanded methods. Written informed consent was provided by a legally authorized representative.We conducted a prospective interventional study of invasive neuromonitoring in 18 patients with HIBI with paired sampling of arterial and jugular venous bulb blood to isolate the cerebrovascular bed and quantify transcerebral release of brain biomarkers . Fourteen patients in this cohort have been reported on in previous manuscriptsg for 10 minutes, with the serum supernatant aliquoted into cryovials and immediately frozen in a −80 °C freezer where they were stored for later analysis. All biomarker samples collected in the first 24 hours were collected before the administration of hypertonic saline (see below). Further, samples were collected from patients on day 2 and 3 if they remained alive and within the ICU.Simultaneous arterial and jugular venous bulb gases were taken on patients every 4 to 6 hours for measurement of pH, arterial carbon dioxide tension, arterial oxygen tension, and bicarbonate concentration. Further, arterial and jugular venous blood were simultaneously collected into serum separator tubes  within the first 24 hours of neuromonitoring (24 [13–40] hours post-arrest). These samples were set upright in the dark for 10 minutes and then centrifuged at 600Data Supplement). A continuous oximetric jugular venous bulb saturation catheter  was inserted retrograde in the dominant jugular vein, identified by bedside ultrasonography. Confirmation of the positioning of the catheter at the ipsilateral mastoid process was confirmed with anterior—postural and lateral skull base x-ray film . The median duration of neuromonitoring in our study was 43 hours . During this monitoring period, clinical directive was to maintain normoxemia (Pao2 80–100 mmHg) and normocapnia (Paco2 35–45 mmHg) for the duration of the study.Multimodal invasive neuromonitoring was undertaken via a dual lumen cranial access bolt  positioned at the frontal hair line on the side of the nondominant hemisphere. Intraparenchymal intracranial pressure  and brain tissue oxygenation  catheters were inserted via the dual lumen bolt and positioned in the subcortical white matter. Noncontrast head computed tomography  was undertaken upon admission and then again 24 hours thereafter to confirm adequate placement of the neuromonitoring catheters . A dose of 1 to 2 mL/kg of 5% hypertonic saline was administered over 15 minutes via a central venous catheter. Serum sodium was measured before and after 5% hypertonic saline administration.2) were enrolled in a separate study, with unique research hypotheses, but are included here to provide normative control data for transcerebral A-v gradients of brain biomarkers (no data from this cohort has been previously published). This study was approved by the UBC Clinical Research Ethics Board (H18-01764) with the healthy volunteers providing written informed consent before participation.Fourteen healthy males  and oxygen (PETO2) was completed. Dynamic end-tidal forcing was utilized to hold PETO2 and PETCO2 steady26 for 10 minutes before and during blood sample collection. Arterial and jugular venous blood samples were simultaneously collected into serum separator tubes , set upright in the dark for 10 minutes and then centrifuged at 600g for 10 minutes (4 °C), with the serum supernatant aliquoted into cryovials and immediately snap-frozen in liquid N2 and then stored in a −80 °C freezer for later analysis.Participants were instrumented with an internal jugular venous and radial arterial catheter. Participants had ≥20 minutes to rest following cannulation while the remaining experimental set-up for monitoring minute ventilation, measuring the partial pressures of end-tidal carbon dioxide  discovery kit (102343), which reflects neuronal cell body injury27; total tau advantage kit (101552) and NF-L advantage kit (103186), which reveal axonal damage; NSE discovery kit (102475), an intracytosolic enzyme, which represents neuron cell body injury.27Serum samples from both healthy controls and patients with HIBI were analyzed for markers of neuronal and astroglial injury with the Quanterix platform using the Simoa HD-1 analyzer  by an investigator blinded to the patients’/controls’ physiological and outcome data. The following assays were preformed: GFAP discovery kit (102336), which reflects astroglial damage and BBB permeabilityInflammatory cytokines  were quantified using the Simoa HD-1 platform from Quanterix  using a cytokine 3-plex A advantage assay (101160).Markers of endothelial injury were measured as follows. E-selectin, P-selectin, sICAM-3 (soluble intracellular adhesion molecule 3), and thrombomodulin were analyzed using the MSD Human Vascular Injury 1 Kit . Syndecan-1 (LSEHSDC1) and von Willebrand Factor (LSEHVWF) were quantified using Invitrogen Human Elisa Kits . Details for all biomarker analyses are provided in the expanded methods.Data Supplement. Therefore, to make PbtO2 comparable relative to systemic hemodynamic conditions, we stratified by brain normoxia/hypoxia based upon PbtO2 values that were recorded when MAP was <100 mmHg. A PbtO2 value that occurred when MAP was >100 mmHg was not included in the 24-hour mean PbtO2 value utilized for determining patient stratification to account for removal of monitoring artifacts  and to adhere to current treatment paradigms that do not involve MAP augmentation above 100 mmHg14 rendering these values of limited physiological or clinical relevance as it pertains to hypoxic brain injury.All statistical analyses were performed in GraphPad Prism (Version 9.1.2). Systemic hemodynamic physiology was variable within and between patients as noted in Figure II in the This study included a sample of convenience. As this cohort of patients represents a first-in-human investigation of secondary brain hypoxia in the setting of HIBI post–cardiac arrest, no historical data were available for a priori power calculations. All data were assessed for normality using a Shapiro-Wilks test, with parametric and nonparametric tests used when appropriate (as described below). Data are presented as mean±SD or median (interquartile range) where appropriate.U test.For physiological data, the 6-hour binned neuromonitoring data  were assessed using linear mixed effects models (patient as a random effect) with both time (0–6 versus 7–12 versus 13–18 versus 19–24 hours) and brain oxygenation (normoxia versus hypoxia) as fixed factors. Twenty-four hour mean data were compared using a Mann-Whitney t test or Wilcoxin Signed Ranks test (population mean or median set at zero). Further, between group comparisons of A-v gradients were made using a Kruskal-Wallis test. Following the detection of a significant effect, post hoc testing was conducted and corrected for multiple comparisons with a Dunn multiple comparisons test.Differences in arterial biomarker levels  between patients with HIBI with brain normoxia and hypoxia as well as controls were made using a Kruskal-Wallis test. Following the detection of a significant effect, post hoc testing was conducted and corrected for multiple comparisons with a Dunn multiple comparisons test. The presence of A-v gradients for each biomarker was assessed using a 2-tailed single sample 2 and related variables, the hypoxic patient data were compared pre-to-post hypertonic saline with a Wilcoxin Signed Ranks test. A P of <0.05 was considered statistically significant for all hypothesis testing.To assess the influence of hypertonic saline on PbtOIt is acknowledged that this study includes a relatively small sample of participants, which may increase the risk of Type 2 error, but also includes repeated comparisons that may lend themselves to increased risk of Type 1 error. Therefore, in addition to significance testing individual data is displayed for the sake of transparency and improved ease of data interpretation.2) admitted to Vancouver General Hospital  that met the inclusion criteria were consecutively enrolled in this study between November 2016 and November 2019. All patients had >10 minutes from the time of cardiac arrest to ROSC and were enrolled within 72 hours following their arrest. Patient details are included in Table I in the Data Supplement. This study conformed to the standards put forth in the Declaration of Helsinki, received institutional approvals , and was registered on www.clinicaltrials.gov (NCT03609333). Written informed consent was provided by a legally authorized representative.Eighteen post–cardiac arrest patients with HIBI ,15 through which 2 microcatheters were advanced into subcortical frontal lobe white matter to directly record PbtO2 and intracranial pressure. To investigate the pathophysiologic sequelae of secondary brain hypoxia in HIBI, patients were stratified into 2 groups based upon their mean PbtO2 over the first 24 hours of neuromonitoring: (1) those wherein clinical management achieved brain normoxia (mean PbtO2 ≥20 mm Hg) and (2) those with persistent brain hypoxia despite neuromonitoring-guided clinical management .29 Eight patients were classified as normoxic while 10 were classified as hypoxic.As part of clinical monitoring, a frontal dual access cranial bolt was placed in the mid-pupillary sagittal line on the side of the nondominant hemisphere . However, intracranial pressure was lower in the normoxic HIBI group  resulting in a comparatively higher CPP relative to the patients with hypoxic HIBI . Average core body temperature  over the first 24 hours of neuromonitoring was not different between the patients with brain normoxia (36.5 [34.25–37.0 °C]) compared with those with brain hypoxia . There were no statistical differences in hemoglobin concentration (P=0.408), or Pao2 (P=0.372) over the first 24 hours of neuromonitoring. There was, however, a trivial difference in Sao2 between patients with HIBI with brain normoxia (96 [96–97] %) and brain hypoxia . Paco2 was higher (and pH lower) in the patients with HIBI with brain hypoxia (39.5 [38.3–44.1] mm Hg) compared with brain normoxia .The differences in PbtOData Supplement. Specifically, loss of gray-white matter differentiation on head CT was noted in 1 patient in the normoxia group  and 7 patients  in the hypoxia group. Presence of restricted diffusion on diffusion weighted magnetic resonance imaging was noted in 2 patients in the normoxia group  and 4 patients in the hypoxia group . Clinical outcomes classified by the cerebral performance category in the normoxia and hypoxia groups are reported in Table I in the Data Supplement.Data pertaining to clinical and diagnostic markers of prognostication are reported in Table I in the P=0.65), as well as from a healthy volunteer control group. Arterial serum levels of biomarkers related to neuronal and astroglial injury, GFAP (both P<0.05), NF-L (both P<0.05), total tau (both P<0.05), and NSE (both P<0.05) were elevated in both HIBI groups compared with controls, while UCH-L1 was only elevated in the HIBI group with brain hypoxia (P=0.0054) compared with controls . We did not observe cerebral release of GFAP, NF-L, total tau, NSE or UCH-L1 in the control participants , NF-L (P=0.74), total tau (P=0.74), NSE (P=0.10), or UCH-L1 (P=0.64), nor were their A-v gradients significantly different compared with controls. However, there was active cerebral release of GFAP (P=0.0078), NF-L (P=0.010), total tau (P=0.0039), NSE (P=0.0039), and UCH-L1 (P=0.0059) in the patients with HIBI with brain hypoxia . The A-v gradients for GFAP, NF-L, total tau, NSE, and UCH-L1 were all greater by several orders of magnitude in the hypoxic HIBI group compared with the controls and the normoxic HIBI group , pH , HCO3− , and LDH  were not different between patients with HIBI with brain normoxia and hypoxia  nor did markers of hepatic and renal injury, creatinine, AST (aspartate aminotransferase), and ALT . Moreover, the A-v gradients for markers of neuronal and astroglial injury were not correlated to time to ROSC (Table III in the Data Supplement). These results collectively suggest that the magnitude/severity of secondary injury in patients with HIBI is unrelated to the global ischemic burden of cardiac arrest.Next we determined if standard clinical laboratory measures were also significantly different in the patients with HIBI with brain normoxia and hypoxia. As a part of routine clinical care, clinical laboratory measures were taken daily over the first 72 hours, allowing for characterization of the temporal evolution of clinical changes in normoxic and hypoxic HIBI patients. Over the first 72 hours of monitoring, the admission, peak, and median values for general markers of global ischemic injury including: lactate (P=0.013), NF-L , total tau , NSE , and UCH-L1  levels were all able to accurately differentiate between brain normoxia and hypoxia, as were the peak and median levels of these biomarkers over the first 72 hours of neuromonitoring . Unsurprisingly, lactate, LDH, pH, troponin, and the other clinical laboratory results were not able to differentiate between these groups (Table IV in the Data Supplement).Next, we evaluated the ability of neurological and basic clinical blood biomarkers to differentiate between patients with HIBI with brain normoxia and hypoxia using receiver operator characteristic curve analysis. The initial serum GFAP , P-selectin , thrombomodulin , and von Willebrand Factor compared with controls . No statistical differences in arterial sICAM-3  of markers of endothelial injury and inflammation to provide cerebrovascular-specific mechanistic insight  into the pathophysiology of HIBI. The patients with HIBI with brain hypoxia had elevated levels of arterial serum E-selectin  and hypoxic (P<0.05) HIBI patients compared with controls , which was greater compared with patients with normoxic HIBI and controls, who did not exhibit any significant cerebral release .31 To test this hypothesis, we assessed PbtO2 and cerebrovascular-to-parenchymal oxygen gradients before and following osmotherapy with hypertonic saline in 9 hypoxic  Patients with HIBI. It is noteworthy that these sample sizes are reduced because hypertonic saline was only administered when clinically indicated and as such, statistical analyses were not performed in the normoxic patients receiving hypertonic saline. The hypertonic saline infusion increased PbtO2 in the Patients with HIBI with brain hypoxia  persistent secondary brain hypoxia following resuscitation is associated with de novo release of biomarkers that reflect injury to neuronal and astroglial components of the NVU; (2) the presence of secondary brain hypoxia and associated neuronal and astroglial injury is unrelated to the global systemic burden of ischemia or endothelial injury produced by cardiac arrest; (3) secondary brain hypoxia associated neuronal and astroglial injury coincided with cerebral release of IL-6; (4) secondary brain hypoxia post–cardiac arrest is attenuated by hypertonic saline infusion indicating a potential role for cerebral edema, specifically in the perivascular distribution, and consequent diffusion limitation of oxygen transport into the cerebral parenchyma as a key pathophysiologic characteristic of HIBI.We characterized injury to the NVU following cardiac arrest in human patients with HIBI with ongoing secondary brain hypoxia versus normoxia as well as in healthy controls. In these respective groups, we examined the effect of brain hypoxia on patterns of NVU injury by assessing transcerebral A-v gradients of neuronal, astroglial, and cerebrovascular endothelial injury biomarkers as well as inflammatory cytokines. Moreover, we demonstrated that persistent secondary brain hypoxia may be attenuated by resolving potential impairments in diffusive cerebrovascular-to-parenchymal O32 given that neurological injury is the primary cause of death in those that survive to hospital admission.2 Yet, measurement of one marker, as is typical, does not fully characterize the injury that may be taking place across the entire NVU. Herein, we demonstrate that de novo transcerebral release of NVU specific biomarkers reflecting astroglial injury (GFAP),33 neuronal cell body injury ,35 and axonal injury  occurs simultaneously in the context of post-ROSC brain hypoxia  were unrelated to the presence of secondary brain hypoxia or the magnitude of neuronal and astroglial injury. However, it has been well documented in other critical illnesses 37 and more recently in post–cardiac arrest patients38 that systemic circulating markers of inflammation and endothelial injury are related to poor outcomes. Indeed, post-ROSC ischemia-reperfusion injury incites a proinflammatory state thought to underlie endothelial injury and BBB breakdown in human patients with HIBI,25 an observation that is supported by preclinical experiments.22 Such endothelial damage and consequent BBB breakdown precipitates cerebral edema,39 which will impose a diffusional barrier to oxygen transport within the cerebral parenchyma.40The presence of secondary brain hypoxia and associated neuronal and astroglial injury following cardiac arrest was not related to measures of overall ischemic burden as indexes of global ischemic injury  and time to ROSC . Thus, while cerebral endothelial injury may be an important early factor in the pathophysiology of HIBI, it does not seem that ongoing cerebral endothelial injury is related to secondary brain hypoxia.Endothelial injury was evident in the Patients with HIBI. That some arterial markers of endothelial injury were elevated but no A-v gradients were present may be taken to reflect an initial ischemia-reperfusion injury to the endothelium43 We demonstrate increases in circulating systemic IL-625 but not TNF-α in agreement with previous work in mice.18 However, we also provide nuanced insight into cerebral-specific inflammation by demonstrating that persistent secondary brain hypoxia was related to cerebral release of IL-6. This is in agreement with preclinical work demonstrating increases in hippocampal IL-6 following cardiac arrest.44 IL-6 is produced by astrocytes (and other cells) in response to inflammation42 and may represent a key pathophysiological process of secondary hypoxic brain injury in humans with HIBI given the marked injury to astrocytes evidenced by the cerebral release of GFAP in the HIBI group with brain hypoxia. Our data therefore suggest that astrocyte IL-6 production may represent a potential therapeutic target46 as reducing cerebral IL-6 production is associated with reduced apoptotic signaling following cardiac arrest.44 To this effect, clinical trials assessing the influence of IL-6 receptor blockade with Tocilizumab on outcome following cardiac arrest are ongoing (NCT03863015). It is also possible that cerebral production of, and A-v gradient for, IL-6 may be in part explained by microglial activation and/or the expression of adhesion molecules  within the cerebral vasculature and consequent macrophage infiltration and cytokine release.23Cerebral inflammation leads to increased cerebral IL-6 production.31 which renders MAP augmentation less effective for ameliorating brain hypoxia in these vulnerable patients. The demonstration of cerebral release of GFAP in the hypoxic patients with HIBI, along with cerebral release of IL-6 and elevated arterial markers of endothelial injury, all suggest BBB disruption,17 fluid extravasation,17 and consequent perivascular edema40 as a potential mechanism for diffusion limitation and persistent secondary brain hypoxia in humans with HIBI. We extend this notion by experimentally demonstrating that hypertonic saline infusion improved PbtO2 and reduced the PvO2-PbtO2 gradient, which signifies improved oxygen transport into the cerebral parenchyma .40 Previous work has demonstrated an improvement in CBF with hypertonic saline infusion.49 However, while we cannot account for changes in cardiac output and any consequent influence on CBF50 following hypertonic saline infusion, the unaltered CPP in the current study lends support to the notion that the improvements in PbtO2 we observed were secondary to improved diffusion and not perfusion, likely a result of decreased perivascular edema.40 Therefore, these data suggest that therapeutic targeting of diffusion limitation may hold efficacy for mitigating secondary brain hypoxia in instances where MAP augmentation is not effective.An emerging hypothesis is that secondary brain hypoxia is related to a diffusion limitation that impedes cerebrovascular-to-parenchymal oxygen transport,6 However, such variability is integral to produce a range in the severity of brain hypoxia, a prerequisite to derive the important pathophysiologic relationships described herein. Previous efforts to elucidate the mechanisms of HIBI post–cardiac arrest and key contributors to mortality have not been capable of disentangling systemic physiology and ischemic injury from that of cerebral-specific injury. Our quantification of cerebral A-v gradients circumvents the potential for conflating systemic and cerebral injuries by isolating changes in multiple injury markers  to the cerebral vasculature/parenchyma  versus those with a PbtO2 >20 mmHg  post-resuscitation. Indeed, this provides evidence that 20 mmHg may be a meaningful cutoff for brain hypoxia in post–cardiac arrest patients, a notion that was previously extrapolated from traumatic brain injury literature.28 While the purpose of this study was not to assess differences in clinical outcomes between groups due to the small sample size, it is noteworthy that in the group with brain normoxia, 6 patients had a favorable outcome (CPC1-2), representing 75% of the normoxic patients, whereas in the patients with brain normoxia, only 2 patients (20%) had a favorable outcome (Table I in the Data Supplement).Assessment of human patients within the clinical setting precludes the experimental designs and uniformity that are typical of preclinical models of HIBI, where, for example, arrest times are highly controlled.a Figure . This ha51 and the potential contributions of microglia and vessel-associated macrophages.23 This study comprises a limited sample of human patients; yet, this limitation is offset by the state-of-the-art invasive and molecular methodology employed; and, for the first time, coupling of our HIBI patient data with that of a healthy control group that also underwent invasive catheterization to determine normative cerebral A-v gradients. We recognize that the generalizability of our findings may be limited due to the noncardiac causes of arrest in our cohort and our findings may not be directly relatable to patients with HIBI with sudden cardiac arrest resulting from either a primary arrhythmia or antecedent acute coronary syndrome. Given the nature of our exclusion criteria, we were unable to include patients with concomitant acute coronary syndromes because of contraindications to the insertion of invasive neuromonitoring in the setting of administration of anticoagulants or antiplatelet therapy. Additionally, the age of our cohort was younger than a typical post cardiac arrest HIBI population and although this may be viewed as a limitation, it should be noted that the strengths of our findings are enhanced as direct control comparisons may be made based upon the similar ages of our HIBI cohort and health controls.While we have assessed multiple aspects of the NVU as it relates to secondary hypoxic injury in humans, it is important to address that other factors not examined herein cannot be disregarded, such as the potential for pericyte injury52 Therefore, while we have used CPP as a surrogate for CBF, the regulation of CBF involves many factors in addition to perfusion pressure such as neuronal activity, arterial blood gases, and temperature. As these factors were not markedly altered during the stable periods of neuromonitoring  it is likely that CPP provided a reasonably accurate  proxy for CBF. Future work utilizing direct intraparenchymal measures of CBF are needed to delineate the importance of diffusion limitations to O2 transport in the pathophysiology of post–cardiac arrest HIBI. Nonetheless, small differences in CBF per group could not alone account for the marked differences in cerebral release of biomarkers we observed where the release of GFAP (230 fold greater), NF-L (577 fold greater), Tau (800 fold greater), NSE (81 fold greater), and UCH-L1 (10 fold greater) were much greater in magnitude than what could be accounted for by even large differences in CBF between patients with brain hypoxia compared with normoxia , Laerdal Foundation, Heart and Stroke Foundation of Canada and Vancouver General Hospital Foundation. M.S. Sekhon is funded through the Clinician Scientist Award from Vancouver Coastal Health Research Institute and a Health-Professional Investigator Award from the Michael Smith Foundation. D.E. Griesdale is funded through a Health-Professional Investigator Award from the Michael Smith Foundation for Health Research. R.L. Hoiland is funded by a Michael Smith Foundation for Health Research Trainee Fellowship, UBC Bluma Tischler Post-Doctoral Fellowship, and the Darin Daniel Green Memorial Scholarship.None.Expanded Materials and MethodsData Supplement Tables I–IVData Supplement Figures I–V


Closer reading of computed tomography pulmonary angiography (CTPA) scans of patients presenting with acute pulmonary embolism (PE) may identify those at high risk of developing chronic thromboembolic pulmonary hypertension (CTEPH). We aimed to validate the predictive value of six radiological predictors that were previously proposed.Three hundred forty-one patients with acute PE were prospectively followed for development of CTEPH in six European hospitals. Index CTPAs were analysed post hoc by expert chest radiologists blinded to the final diagnosis. The accuracy of the predictors using a predefined threshold for ‘high risk’ (≥ 3 predictors) and the expert overall judgment on the presence of CTEPH were assessed.CTEPH was confirmed in nine patients (2.6%) during 2-year follow-up. Any sign of chronic thrombi was already present in 74/341 patients (22%) on the index CTPA, which was associated with CTEPH ; 37 patients (11%) had ≥ 3 of 6 radiological predictors, of whom 4 (11%) were diagnosed with CTEPH . Expert judgment raised suspicion of CTEPH in 27 patients, which was confirmed in 8 .The presence of ≥ 3 of 6 predefined radiological predictors was highly specific for a future CTEPH diagnosis, comparable to overall expert judgment, while the latter was associated with higher sensitivity. Dedicated CTPA reading for signs of CTEPH may therefore help in early detection of CTEPH after PE, although in our cohort this strategy would not have detected all cases.Three expert chest radiologists re-assessed CTPA scans performed at the moment of acute pulmonary embolism diagnosis and observed a high prevalence of chronic thrombi and signs of pulmonary hypertension.• On these index scans, the presence of ≥ 3 of 6 predefined radiological predictors was highly specific for a future diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH), comparable to overall expert judgment.• Dedicated CTPA reading for signs of CTEPH may help in early detection of CTEPH after acute pulmonary embolism.• The online version contains supplementary material available at 10.1007/s00330-021-08364-0. The potentially life-threatening disease chronic thromboembolic pulmonary hypertension (CTEPH) is preceded by a diagnosis of acute pulmonary embolism (PE) in 75% , 2. EstaThe recent InShape III study has investigated the radiological differentiation of acute PE from CTEPH on CTPAs performed for suspected PE . For thiEven though a more detailed reading of index CTPA scans may help in early detection of CTEPH, several important questions need to be answered before recommending implementation in routine care . Since tThis is a predefined post hoc analysis based on results of the InShape II study, which was a prospective, multicentre management study assessing the accuracy of a non-invasive follow-up strategy for early identification of CTEPH in consecutive patients treated for acute PE. Criteria for study inclusion have been described previously and included (1) a CTPA proven diagnosis of first or recurrent symptomatic acute PE (2) and treatment with therapeutically dosed anticoagulant therapy for at least three months according to current guidelines . Main exAll study participants were managed according to the predefined InShape II algorithm, which is a screening strategy for excluding CTEPH early after acute PE  to examine the prevalence of the six predefined radiological predictors established in the InShape III study, as well as the overall judgment on the presence of CTEPH by expert radiologists among the InShape II study population; (2) to investigate the association between the presence of six predictors and the overall expert judgment on the presence of CTEPH with a final CTEPH diagnosis; (3) to evaluate the prognostic value of CTPA reading for a future CTEPH diagnosis in several subgroups based on pre-test probability of CTEPH and sex.Of all InShape II study patients, available CTPA scans at the time of index PE diagnosis were collected post hoc at each of the six study sites. All scans had been performed using a CT scanner with at least 64 slices and a slice thickness of 1 to 3 mm. After full anonymisation and removal of meta-data, only the original axial images were available for local expert chest radiologists to re-assess the images. These radiologists all had over 15 years of experience in pulmonary CTPA reading.All involved radiologists were unaware of the results of the InShape II screening algorithm and of the results of a 2-year follow-up, i.e. whether CTEPH was ultimately diagnosed or not. Standardised re-assessment of CTPA scans was done according to an identical scoring form as previously used in the InShape III study (Patient characteristics were described as mean with standard deviation (SD), median with interquartile range (IQR), or numbers with proportions if appropriate. Descriptive analyses were used to show the results of the CTPA reading. The number of patients judged to have chronic thrombi or PH were assessed, as well as the prevalence of the six previously mentioned predictors on the index CTPA scan. Using logistic regression analysis, both the presence of ≥ 3 of these predictors (a cut-off that was predefined in the InShape III study) and the overall judgment on the presence of CTEPH were correlated to a final CTEPH diagnosis during 2-year follow-up. Patients with high suspicion of CTEPH in whom diagnosis was not confirmed with RHC were not included in the main analysis but only in the sensitivity analysis. Measures of diagnostic accuracy were calculated with corresponding 95% confidence intervals (95%CI). All statistical tests were performed using SPSS Statistics software .n = 68), or acute PE was diagnosed using a ventilation/perfusion scan (n = 15). Patients’ characteristics at baseline are presented in Table Of the 424 consecutively included PE patients in the InShape II study, index CTPA scans of 341 patients were available and evaluated by six independent radiologists. The remaining 83 patients could not be included because the patients were referred for treatment to one of the study sites after the CTPA had been performed elsewhere (CTEPH was confirmed by RHC in nine of the 341 patients (2.6%), of whom eight had been identified early by the algorithm and one during follow-up (Ten CTPA scans 2.9%) had a suboptimal quality, i.e. inadequate contrast timing in six and motion artefacts in four scans, but all were of sufficient quality to be used in the analysis. Chronic thrombi were present on 74 (22%) of 341 index CTPAs   of 11% (95%CI 5.2–21) and a negative predictive value (NPV) of 98% (95%CI 97–99), accompanied by a positive likelihood ratio (LR) of 4.5 (95%CI 2.0–9.9) and a negative LR of 0.62 (95%CI 0.34–1.1). Overall expert judgment resulted in a PPV of 30% (95%CI 20–41) and a NPV of 99.7% (98–99.9) against a positive LR of 16 (96%CI 9.5–25) and a negative LR of 0.12 (95%CI 0.0–0.8).Associations between CTPA reading in the subgroups are presented in Table In this practice-based cohort of PE patients, a high prevalence of chronic thrombi (22%) and signs of PH (30%) on index CTPAs were observed by expert chest radiologists and were both associated with a future CTEPH diagnosis. Firstly, using a predefined cut-off of at least 3 of 6 radiological predictors present, 4 out of 9 cases (44%) were correctly identified. Secondly, overall expert judgment led to suspicion of CTEPH in 8 of 9 CTEPH cases (89%). Importantly, either way of CTPA reading yielded a considerably high specificity (≥ 90%) for a future CTEPH diagnosis with a positive likelihood ratio of 4.5 and 16, respectively. Subgroup analysis revealed no clear differences between patients with high versus low clinical pre-test probability, between those with or without symptoms, or between sexes.We observed that dedicated assessment of index CTPAs for 6 specific CTPA signs performed by expert chest radiologists was highly specific for a future CTEPH diagnosis, confirming the results of the InShape III study (the derivation study) . In dailIn line with existing literature, our findings show that a careful evaluation of chronic thrombi and signs of PH is a promising approach for earlier detection of CTEPH –19. In CRefined CTPA assessment might not be sufficient as a stand-alone tool for achieving early CTEPH diagnosis. However, it may still play an important role in routine follow-up strategies among acute PE patients. In this setting, in the 2019 European Society of Cardiology Guidelines on acute PE, it was proposed to perform echocardiography in PE patients with persistent dyspnoea, functional limitations, and/or predisposing conditions for CTEPH . PresencFuture studies should incorporate dedicated radiological evaluation in prospective validation of follow-up strategies , 23, 24.In conclusion, dedicated assessment of the presence of signs of chronic clots or PH on CTPAs performed in the setting of suspected acute PE may contribute to earlier detection of CTEPH, validating the results of the InShape III study. Overall expert judgment yielded similar results to focussing on a predefined set of objective radiological predictors only, but performed better in terms of case finding. As stand-alone assessment, expert reading was not sufficient to identify each CTEPH patient, but would have identified the vast majority.Supplementary file1 (DOCX 126 KB)Below is the link to the electronic supplementary material.


This report presents a rare fetal and neonatal complication brain injury  as a consequence of severe fetal anemia resulting from Rhesus (Rh) isoimmunization. A 28-year-old gravida 4 para 3 woman was referred at 21+4 weeks of gestation to the fetal medicine clinic as a case of Rh isoimmunization. Fetal ultrasound showed a normal anatomy scan with normal brain structure, but with severe fetal anemia. The patient was treated with multiple intrauterine transfusions, but still developed complications post-transfusions.This case shows that severe cerebral developmental anomalies can occur because of severe fetal anemia secondary to Rh isoimmunization, such as in this case - ventriculomegaly and encephalomalacia. It has been concluded that proper antenatal counseling and early intervention for severe fetal anemia are beneficial to prevent such complications from occurring. It is crucial to consider appropriate antenatal and postnatal radiological imaging for such cases. ABO type, Rhesus (Rh{D}) status, and antibody screening should be determined in all pregnant women at the initial perinatal visit. The most common antigen causing isoimmunization is Rh(D). Only 17% of Rh(D)-negative women who do not receive prophylaxis immunoglobulin become immunized. Up to 90% cases of Rh isoimmunization can be prevented by routine postpartum immunoglobulin administration to Rh(D)-negative women, and >99% cases can be prevented if the immunoglobulin is given during the third trimester and postpartum.A 28-year-old gravida 4 para 3 woman with two living children presented with a medical history of rheumatic heart disease. The patient underwent aortic valve replacement with a tissue valve in 2012, during which she received many units of blood transfusion and developed Rh isoimmunization . Her first pregnancy was complicated by hydrops due to which she did not receive an intrauterine blood transfusion (IUT) and was delivered via cesarean section (C/S) prematurely. The second pregnancy was complicated by intrauterine fetal death and terminated before 24 weeks of gestation. The patient’s last pregnancy was managed with frequent IUTs, and she delivered a healthy newborn via cesarean section (C/S) at 36 weeks of gestation.During the current pregnancy, the patient presented for the first time at 21 weeks and three days of gestation with an uneventful pregnancy. The anatomy scan revealed normal fetal anatomy without signs of hydrops but with middle cerebral artery-peak systolic velocity (MCA-PSV) at 42.8 cm/s  in uncomplicated procedures. However, the risk increases to about 20% in hydropic fetuses -3% in un. HoweverIn a retrospective study, Pasman et al. have reported 135 IUTs in 56 fetuses, with minimal adverse effects of approximately 10%. They found that severe fetal effects occurred in only 1.5%, and hydrops and IUT in the free lope were a significant predisposing factor. However, IUT in the late gestation was associated with good fetal and neonatal outcomes [The risk of anemia is considered high when MCA-PSV reaches ≥1.5 MoM, whereas levels below this value indicate that the fetus is not anemic or does not call for interventions . MCA-PSVThe LOTUS study found that children (two to 17 years) who received IUT had normal neurodevelopment. This shows the safety of IUTs regardless of the number of procedures, with rare complications mainly related to premature delivery, hydrops fetuses, and low hemoglobin rather than the number of transfusions or procedures itself . Severe IUT is the gold standard for fetal anemia treatment. In cases of Rh isoimmunization, delayed treatment or complicated IUT procedures can cause fetal brain anoxia and brain lesions. Thus, timely and careful intervention by an experienced maternal fetal medicine specialist is crucial for superior fetal and neonatal outcomes.


Pomc) gene and an elevated stress response for multiple generations via the male germline. Male germline-specific effects of FAE on the Pomc gene raises the question if the sex-determining region Y (SRY) may have a role in regulating Pomc gene expression. Using a transgenerational model of FAE in Fischer 344 rats, we determined the role of SRY in the regulation of the Pomc gene. FAEs, like on the Pomc gene, reduced Sry gene expression in sperm and the mediobasal hypothalamus (MBH) in male adult offspring. Fetal alcohol-induced inhibition of Sry gene expression was associated with increased Sry promoter DNA methylation. Additionally, fetal alcohol effects on the Sry gene persisted for three generations in the male germline but not in the female germline. Sry gene knockdown reduced the Pomc gene expression. Sry recruitment onto the Pomc promoter was found to be reduced in the hypothalamus of fetal alcohol-exposed rats compared to control rats. Pomc promoter luciferase activity was increased following Sry overexpression. A site-directed mutagenesis study revealed that SRY binding sites are required for POMC promoter transcription activity. Overall, these findings suggest that SRY plays a stimulatory role in the regulation of Pomc gene expression and may potentially contribute to the fetal alcohol-induced changes in the level of Pomc gene expression for multiple generations.Fetal alcohol exposure (FAE) causes various neurodevelopmental deficits in offspring, including reduced expression of the stress regulatory proopiomelanocortin ( Pomc DNA methylation via recruitment of MeCP2 on Pomc promoter  is transmitted from father to sons  and sperm. We also conducted Sry gene knockdown experiments in vitro and in vivo and determined its effect on Pomc expression. Additionally, SRY recruitment onto the Pomc promoter was assessed in MBH of FAE rats. Furthermore, we determined SRY binding motifs on Pomc promoter and evaluated SRY regulation through its binding sites using promoter luciferase activity. The results of these studies are presented here.Fetal alcohol exposure (FAE) has been shown to elevate neuroendocrine response of the hypothalamic-pituitary-adrenal (HPA) axis to stress , partly promoter . Interesgermline , suggest to sons  and has  to sons . The sexises SRY . SRY is ises SRY . SRY alsises SRY . SRY binises SRY . We hypoad libitum (AD), a liquid diet containing ethanol , or pair-fed  an isocaloric liquid control diet . Previous studies have shown that the peak blood ethanol concentration is achieved in the range of 120–150 mg/dl in pregnant dams fed with this ethanol-containing liquid diet  and housed in a controlled condition at a constant temperature of 22°C and 12-h light/dark cycles throughout the study. These rats were bred in our animal facility and used for this study. On gestational day (GD) 7 through 21, rats were fed rat chow uid diet . AF and 2.Neural stem cells were prepared and differentiated to POMC/β-endorphin neurons using cAMP and pituitary adenylate cyclase-activating polypeptide (PACAP) and maintained in cultures as we described previously . The HEKPomc and Sry in rat MBH and sperm were measured by quantitative real-time polymerase chain reaction (RT-PCR) (SYBR Green assay). Total RNA from MBH and sperm were extracted using an RNeasy kit . Total RNA (1 μg) was converted to first-strand complementary DNA (cDNA) using a high-capacity cDNA reverse transcription kit (Life Technologies). The primer sequences used for Pomc, Sry, Gapdh, 18S, and Rpl19 are given in Pomc, Sry) and the three reference genes  were measured using the standard curve method. Target-gene expression was normalized with reference gene expression levels.Gene expression levels of Sry knockdown as it was described  in a 12-well plate, and oligo 0.4 μg was added in 0.4 ml of medium in each well  and incubated in a 37°C incubator with 7.5% CO2. The cells were harvested 48 h after transfection, and we pooled the cells from two wells in triplicate. Gene expression for Sry and Pomc was measured as it is described in section “Materials and Methods.” For in vivo knockdown experiments, oligos were injected into the third ventricle of the rats via intracerebroventricular infusion as we described earlier (n = 6) for each group were used for the experiment. Oligo mix prepared in artificial CSF (1 μg/μl) was injected 2 μl into each rat at the rate of 1 μl/min. Rats were allowed to recover, were sacrificed 72 h after oligo injection, and then brain tissue was collected. MBH was isolated from the brain and analyzed for Sry and Pomc gene expression as it was described in section “Materials and Methods.”Sense and antisense oligos  were used for escribed . The seq earlier . Male raPomc promotor-specific ChIP PCR primers  following instructions from the manufacturer. MBH collected from the brain tissue was placed on a small petri dish on ice and was minced into tiny pieces. Proteins were crosslinked with DNA from a tissue sample in 1% formaldehyde in PBS with protease inhibitors at room temperature for 30 min. The crosslinking was quenched with 0.125M glycine, and the sample was collected by centrifugation and washed twice with ice-cold PBS. The sample was suspended in SDS lysis buffer (with protease inhibitor) and homogenized to get uniform cell suspension. The crosslinked DNA was sheared into fragments the size of about 400–500 bp with a setting of 30% amplitude, with 15 s pulse on, 30 s pulse off for 10–12 cycles using the Qsonica ultrasonic cell disruptor . The sample was centrifuged at 13,000 × g for 10 min, and we collected sonicated cell supernatant. The sample was diluted 10-fold in ChIP dilution buffer, and we used an aliquot for input. The remaining diluted sample was precleared with salmon sperm DNA/protein-A agarose. A ChIP assay was performed with a precleared sonicated sample with rabbit polyclonal SRY antibody  (10 μg/sample) using a ChIP assay kit  as specified in the manufacturer’s instructions. Equal concentration of the normal rabbit IgG was employed as negative control. The eluted ChIP samples and input sonicated samples were reverse crosslinked with 5M NaCl and incubated at 65°C overnight. DNA was extracted from these samples using a DNeasy micro kit (Qiagen). Pomc promoter region was PCR amplified from rat MBH genomic DNA using primers as listed in SRY binding sites of the Pomc promoter. All three mutants were designed by altering SRY binding site 1 (mutant 1), binding site 2 (mutant 2), and 1 and 2 together . We used PGL3 vector-containing wild-type Pomc promoter as a template to make these mutants using primers whose sequences are given in Pomc promoter wild-type or mutant plasmid DNA along with either Sry overexpression plasmid or empty vector (PCDNA3.0) using Lipofectamine 3000 reagent (Thermo Fisher Scientific). All cells were also transfected with a plasmid-carrying renilla luciferase for normalization purposes. After 24 h of transfection, we measured firefly luciferase and renilla luciferase activity using the Dual Glo luciferase assay system (Promega) in a BioTech model synergy HT plate reader . Eight samples were used in each group in transfection studies. Luciferase activity was measured in duplicates.HEK293 cells were seeded 50,000 cells/well in a 96-well plate. The cells were transfected with 250 ng of reporter vector or vector-containing post hoc analysis using the Newman-Keuls post hoc test. P < 0.05 was considered significant. The significant differences between two groups were analyzed using the unpaired t-test, and two-tailed p-value < 0.05 was considered significant.Data were analyzed using Prism 5.0 (GraphPad Software). The data shown in the figures are mean ± standard error mean (SEM). The significant differences between different treatment groups were assessed with one-way analysis of variance (ANOVA) with Pomc promoter DNA methylation and suppresses Pomc gene expression in the MBH and in sperm . We measured the expression of Sry in F2 and F3 male offspring of male and female germlines to determine whether the fetal alcohol effect is transmitted through successive generations via germline. Like in the F1 generation, the F2 male progeny of male germlines (AFM) showed a significant reduction in Sry mRNA levels in the MBH  in the Pomc promoter. We cloned the Pomc promoter upstream of firefly luciferase in a basic reporter luciferase vector  promoter sequence compared to vector. We also found that in mutant 1 (mut1: binding site1 mutated) and in mutant 2 (mut2: binding site2 mutated), SRY increased Pomc promoter luciferase activity similarly to that of the wild-type plasmid  that comprises SRY on β-endorphin, a functional peptide of POMC  in vivo. Additionally, ChIP assay data showed that SRY is enriched on the Pomc promoter in the MBH, and its enrichment is significantly reduced in fetal alcohol-exposed male offspring. Furthermore, we confirmed the specificity of SRY binding on Pomc promoter by mutating SRY binding sites using site-directed mutagenesis and showing that SRY binding on Pomc promoter is required for its action. These data suggest for the first time that SRY regulates Pomc gene expression by binding on the Pomc promoter.We demonstrated in this study that Pomc promoter to enhance its activity. A bioinformatics web tool used with a stringent threshold of more than 95% sequence similarity predicted three SRY binding sites  located  upstream of exon1. We found that SRY-increased Pomc promoter activity was unchanged when binding sites 1 or 2 were mutated individually, but it was lost when both of these two sites were mutated simultaneously, indicating a requirement of SRY binding to sites 1 and 2 to promote Pomc gene expression. A similar mechanism has been reported in a study where SRY binding to two sites on the Tcf21 promoter are shown to be required for Tcf21 gene transcription during male gonadal sex determination (The present data also identify the sites where SRY binds to mination .Sry expression and promoter methylation correlate with Pomc in the hypothalamus and sperm. Other evidence from our study, such as Sry knockdown altering Pomc expression, SRY binding on Pomc promoter, and SRY-induced Pomc promoter activity, clearly suggests that SRY plays a role in the regulation of Pomc. However, our study did not conclude whether SRY mediates the FAE-induced male germline transmission of POMC expression changes in the hypothalamus for multiple generations. Further studies determining the role of SRY on FAE-modified Pomc transcription in F2 and F3 offspring are needed to establish the role of this transcription factor in the transgenerational effects of FAE.Our data highlights the novel finding that FAE-induced alteration of The germline transmission of phenotypic and epigenetic traits was reported mostly in animal studies. Some human studies provided evidence for intergenerational transmission of risk variants with the paternal lineage . StudiesPomc gene expression through the male germline.In conclusion, the current study provide some insights to our understanding of the mechanism of fetal alcohol-induced male germline transmission. More studies are required to understand the exact mechanism by which SRY plays a critical role in transmitting fetal alcohol-induced changes of All relevant data are included in this manuscript. Any requests for materials will be fulfilled upon request through appropriate means.All rat studies were performed with approved protocols in compliance with the Association for the Assessment and Accreditation of Laboratory Animal Care and Institutional Animal Care and Use Program, Rutgers, The State University of New Jersey Rutgers University.DS designed the project and wrote the manuscript. OG helped in research design, participated in breeding and feeding the animals, contributed to sample collections, conducted biochemical assays, and helped in writing the manuscript. EM participated in conducting biochemical assays. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.