Dominik J. Schaer

BACKGROUND: After aneurysmal subarachnoid hemorrhage (aSAH), erythrocyte lysis within cerebrospinal fluid (CSF) releases cell-free hemoglobin, a potential driver of secondary brain injury (SBI). Whether hemolysis precedes SBI and how its temporal associations compare with those of inflammation and neuroglial injury remain unclear. METHODS: In this biomarker profiling study of the prospective multicenter HeMoVal cohort, 259 patients with aSAH requiring external ventricular drainage contributed 2,411 serial CSF samples over days 1–14 after hemorrhage. We quantified 22 analytes using spectrophotometry, immunoassay, and mass spectrometry; 19 were assigned to five prespecified disease pathway composites. Day- and site-adjusted generalized estimating equations estimated associations with same-day SBI and future first SBI (angiographic vasospasm, delayed cerebral ischemia, or delayed ischemic neurological deficit); a day-wise landmark analysis assessed robustness. RESULTS: On the day of sampling, SBI was associated with four pathways, with the largest estimates for inflammation and neuroglial injury. Within the prespecified +72-hour horizon, only hemolysis (OR 1.40 per SD; 95% CI, 1.17–1.68) and the erythrophagocyte response (OR 1.28; 1.07–1.54) had FDR-significant forward associations with first SBI. Hemolysis had the largest forward estimate at every horizon; landmark analysis was concordant (pooled OR 1.86; 95% bootstrap CI, 1.37–2.65). The protective scavenger proteins haptoglobin and hemopexin declined to fitted plateaus of 2.4% and 12.5% of day-1 concentrations, versus ∼36% for plasma-protein references. CONCLUSION: In a scavenger-depleted CSF compartment, hemolysis and the erythrophagocyte response were associated with subsequent SBI. These findings identify hemolysis as a candidate upstream process and support testing sustained CSF hemoglobin scavenging to prevent SBI. TRIAL REGISTRATION: ClinicalTrials.gov NCT04998370 . FUNDING: CSL Behring; Swiss National Science Foundation; University of Zurich

Objectives To validate whether cerebrospinal fluid oxyhaemoglobin (CSF-Hb), measured from external ventricular or lumbar drains, is associated with secondary brain injury (SAH-SBI) after aneurysmal subarachnoid haemorrhage (aSAH) and to assess its value as a real-time monitoring biomarker. Design Pre-registered multicentre prospective observational cohort study. Setting Eight neurosurgical tertiary centres in Switzerland, Germany and Austria between August 2021 and June 2024. Participants 366 patients with aSAH (mean age 58 years; 65% women). Of these, 260 provided cerebrospinal fluid (CSF) samples via external ventricular drain (EVD; 2467 samples, median 10 days per patient) and 66 via lumbar drain (LD; 379 samples, median 6 days). Interventions Daily CSF samples were collected via EVD or LD from day 1 to day 14 after haemorrhage; no therapeutic interventions were tested. Main outcome measures CSF-Hb and its metabolites were analysed post hoc in a blinded manner. The primary outcome was SAH-SBI, defined as a composite of angiographic vasospasm (aVSP), delayed cerebral ischaemia (DCI) and delayed ischaemic neurological deficits (DIND), assessed daily over 14 days. Secondary outcomes included temporal CSF-Hb profiles and associations with aneurysm location, haematoma volume, intraventricular haemorrhage, chronic hydrocephalus and 3 month functional outcome. Results CSF-Hb showed a delayed peak pattern: concentrations were low after aSAH, rose to a maximum on day 10 (EVD-derived CSF-Hb median 11.3 µM, IQR 2.64 to 25.90) and then declined. Larger haematoma volume (pConclusions In this preregistered multicentre validation study, EVD-derived CSF-Hb did not perform as a robust real-time monitoring biomarker for SAH-SBI, showing limited same-day discrimination and substantial between-centre heterogeneity. These findings argue against clinical implementation of CSF-Hb point-measurement as a single-parameter biomarker. Higher CSF methaemoglobin was associated with SAH-SBI; this hypothesis-generating observation requires prospective confirmation and motivates continued investigation of haemolysis-related pathways. Future work using the HeMoVal biobank will apply multi-marker, pathway-level analyses to define haemolysis-related biomarker signatures and provide a platform for robust external validation of future candidates. Trial registration number NCT04998370.

Objectives To validate whether cerebrospinal fluid oxyhaemoglobin (CSF-Hb), measured from external ventricular or lumbar drains, is associated with secondary brain injury (SAH-SBI) after aneurysmal subarachnoid haemorrhage (aSAH), and to assess its value as a real-time monitoring biomarker. Design Preregistered multicentre prospective observational cohort study. Setting Eight neurosurgical tertiary centres in Switzerland, Germany, and Austria, between August 2021 and June 2024. Participants 366 patients with aSAH (mean age 58 years; 65% women). Of these, 260 provided cerebrospinal fluid (CSF) samples via external ventricular drain (EVD; 2,467 samples, median 10 days per patient) and 66 via lumbar drain (LD; 379 samples, median 6 days). Interventions Daily CSF samples were collected via EVD or LD from day 1 to day 14 after haemorrhage; no therapeutic interventions were tested. Main outcome measures CSF-Hb and its metabolites were analysed post hoc in a blinded manner. The primary outcome was SAH-SBI, defined as a composite of angiographic vasospasm (aVSP), delayed cerebral ischaemia (DCI), and delayed ischaemic neurological deficits (DIND), assessed daily over 14 days. Secondary outcomes included temporal CSF-Hb profiles and associations with aneurysm location, haematoma volume, intraventricular haemorrhage, chronic hydrocephalus, and 3-month functional outcome. Results CSF-Hb showed a delayed peak pattern: concentrations were low after aSAH, rose to a maximum on day 10 (EVD-derived CSF-Hb median 11.3 μM, IQR 2.64 to 25.90), and then declined. Larger haematoma volume (pConclusions In this preregistered multicentre validation study, EVD-derived CSF-Hb did not perform as a robust real-time monitoring biomarker for SAH-SBI, showing limited same-day discrimination and substantial between-centre heterogeneity. These findings argue against clinical implementation of CSF-Hb point-measurement as a single-parameter biomarker. In contrast, CSF methaemoglobin remained consistently associated with SAH-SBI, supporting the mechanistic relevance of haemolysis-related pathways. Future work using the HeMoVal biobank will apply multi-marker, pathway-level analyses to define haemolysis-related biomarker signatures and provide a platform for robust external validation of future candidates. Study registration ClinicalTrials.gov NCT04998370 ; date of registration 10 August 2021.

Hemorrhagic stroke triggers secondary brain injury through the red blood cell toxins hemoglobin (Hb) and heme, which fuel iron-driven lipid peroxidation and neuronal injury. We sought to use organotypic brain-slice cultures to dissect how the high-affinity scavenger proteins haptoglobin (Hp) and hemopexin (Hpx) modulate this cascade. By day 7 of culture, slices remained structurally intact, metabolically active, and responsive to oxidative stress, enabling precise toxin exposure studies. Isotopic ⁵⁸Fe tracing revealed that upon cell-free Hb and heme exposures, heme-iron accumulated in brain slices and heightened lipid peroxidation. In contrast, Hpx neutralized heme, nearly abolishing iron deposition, while Hp partially reduced Hb-driven iron accumulation. Both scavengers attenuated lipid peroxidation and reduced neuronal cell death. Transcriptomic profiling revealed that free toxins increased oxidative stress and neuroinflammatory activation markers, whereas Hpx suppressed the expression of heme-induced genes. Remarkably, HbHp complexes triggered a strong Nrf2-centered adaptive program that enhanced iron metabolism and glutathione synthesis. Integrating five readouts─iron accumulation, lipid peroxidation, neuronal cell death, heme-stress transcripts, and Nrf2/metabolic transcripts─via bootstrap-based principal component analysis yielded two orthogonal axes. An oxidative toxicity axis (PC1) captured iron-driven reactive oxygen species and cell death, while a metabolic adaptation axis (PC2) reflected Nrf2-mediated reprogramming. Free toxins clustered at the toxic extreme on PC1, and heme-Hpx aligned near baseline. HbHp shifted slices upward on PC2, reducing neuronal loss through safe adaptation. These findings establish that Hpx neutralizes free heme, whereas Hp stabilizes Hb and elicits cytoprotective gene expression, offering a rational, dual-scavenger strategy to mitigate secondary brain injury in hemorrhagic stroke.

Tumor-associated macrophages (TAMs) can switch between immune-activating and cancer-promoting states; yet, the stress pathways that lock them into pro-cancerous states remain obscure. In MC38 colon tumors, repeated anti-CD40 or radiotherapy created necrosis that split TAMs into peripheral Cxcl9+ and peri-necrotic Spp1+ subsets. Spatial transcriptomics, single-cell RNA-seq, and Keap1-deficient mice showed that the latter are NRF2 ^high “stress-TAMs”, with immunosuppressive and tumor-promoting activity. The same NRF2 activation gradient separates pro-inflammatory CXCL9+ and anti-inflammatory SPP1+ TAMs across diverse human cancers. NRF2 ^high TAMs silence IFN-STAT1 programmes, lose MHC-II and chemokine expression, fail to expand T cells, drive tumor cell invasion in 3D co-cultures, and foster metastasis. Constitutive hematopoietic NRF2 activation accelerated the growth of therapy-naïve MMTV-PyMT breast tumors and markedly impaired anti-CD40 efficacy in MC38 subcutaneous and lung-metastasis models. Conversely, macrophage-specific Nrf2 deletion restored immunogenic TAMs and potentiated anti-CD40 and anti-PD-1 treatments. Thus, NRF2 constitutes a stress-response axis that fixes TAMs in a pro-cancer, therapy-resistant state; inhibiting it could revive the macrophage–T–cell amplification loop and broaden immunotherapy responses. GRAPHICAL

Metabolic reprogramming and mitochondrial dynamics are pivotal in prostate cancer (PCa) progression and treatment resistance, making them essential targets for therapeutic intervention. In this study, we investigated the effects of the androgen receptor antagonist apalutamide (ARN) and the mitochondrial electron transport chain complex I inhibitor IACS-010759 (IACS) on the mitochondrial network architecture and dynamics in PCa cells. Treatment with ARN and/or IACS induced significant changes in mitochondrial morphology, particularly elongation, in androgen-sensitive PCa cells. Additionally, ARN and IACS modulated the mitochondrial fission and fusion processes, indicating a convergence of metabolic and androgen-signaling pathways in shaping mitochondrial function. Notably, the combination treatment with ARN and IACS resulted in increased apoptotic cell death and mitochondrial oxidative stress selectively in the androgen-sensitive PCa cells. Our findings highlight the therapeutic potential of targeting mitochondrial metabolism in prostate cancer and emphasize the need for further mechanistic understanding to optimize treatment strategies and improve patient outcomes.

Secondary brain injury (SBI) occurs with a lag of several days post-bleeding in patients with aneurysmal subarachnoid hemorrhage (aSAH) and is a strong contributor to mortality and long-term morbidity. aSAH-SBI coincides with cell-free hemoglobin (Hb) release into the cerebrospinal fluid. This temporal association and convincing pathophysiological concepts suggest that CSF-Hb could be a targetable trigger of SBI. However, sparse experimental evidence for Hb’s neurotoxicity in vivo defines a significant research gap for clinical translation. We modeled the CSF-Hb exposure observed in aSAH patients in conscious sheep, which allowed us to assess neurological functions in a gyrencephalic species. Twelve animals were randomly assigned for 3-day bi-daily intracerebroventricular (ICV) injections of either Hb or Hb combined with the high-affinity Hb scavenger protein haptoglobin (Hb-Hp, CSL888). Repeated CSF sampling confirmed clinically relevant CSF-Hb concentrations. This prolonged CSF-Hb exposure over 3 days resulted in disturbed movement activity, reduced food intake, and impaired observational neuroscores. The Hb-induced neurotoxic effects were significantly attenuated when Hb was administered with equimolar haptoglobin. Preterminal magnetic resonance imaging (MRI) showed no CSF-Hb-specific structural brain alterations. In both groups, histology demonstrated an inflammatory response and revealed enhanced perivascular histiocytic infiltrates in the Hb-Hp group, indicative of adaptive mechanisms. Heme exposure in CSF and iron deposition in the brain were comparable, suggesting comparable clearance efficiency of Hb and Hb-haptoglobin complexes from the intracranial compartment. We identified a neurological phenotype of CSF-Hb toxicity in conscious sheep, which is rather due to neurovascular dysfunction than structural brain injury. Haptoglobin was effective at attenuating CSF-Hb-induced neurological deterioration, supporting its therapeutic potential.

Background Secondary brain injury (SBI) occurs with a lag of several days post-bleeding in patients with aneurysmal subarachnoid hemorrhage (aSAH) and is a strong contributor to mortality and long-term morbidity. aSAH-SBI coincides with cell-free hemoglobin (Hb) release into the cerebrospinal fluid. This temporal association and convincing pathophysiological concepts suggest that CSF-Hb could be a targetable trigger of SBI. However, sparse experimental evidence for Hb’s neurotoxicity in vivo defines a significant research gap for clinical translation. Methods We modeled the CSF-Hb exposure observed in aSAH patients in conscious sheep, which allowed us to assess neurological functions. Twelve animals were randomly assigned for three-day bi-daily intracerebroventricular (ICV) injections of either Hb or Hb combined with the high-affinity Hb scavenger protein haptoglobin (Hb-Hp, CSL888). Results Repeated CSF sampling confirmed clinically relevant CSF-Hb concentrations. This prolonged CSF-Hb exposure over three days resulted in disturbed movement activity, reduced food intake, and impaired observational neuroscores. The Hb-induced neurotoxic effects were significantly attenuated when Hb was administered with equimolar haptoglobin. Preterminal magnetic resonance imaging (MRI) showed no CSF-Hb-specific structural brain alterations. In both groups, histology demonstrated an inflammatory response and revealed enhanced perivascular histiocytic infiltrates in the Hb-Hp group, indicative of adaptive mechanisms. Heme exposure in CSF and iron deposition in the brain were comparable, suggesting comparable clearance efficiency of Hb and Hb-haptoglobin complexes from the intracranial compartment. Conclusion We identified a neurological phenotype of CSF-Hb toxicity in conscious sheep, which is rather due to neurovascular dysfunction than structural brain injury. Haptoglobin was effective at attenuating CSF-Hb-induced neurological deterioration, supporting its therapeutic potential.