Anemia Destabilizes the Vascular Glycocalyx and Exacerbates Early Blood–Brain Barrier Injury After Stroke

1. Overview

Scientific Rationale and Importance

Anemia affects up to 30% of patients with acute ischemic stroke and is consistently associated with worse outcomes, yet the underlying vascular mechanism remains unknown. Red blood cells and hemoglobin serve as major circulating antioxidant buffers, contributing to luminal redox homeostasis. The endothelial glycocalyx is a critical luminal structure regulating vascular permeability and barrier stability. We hypothesize that hemoglobin insufficiency disrupts luminal redox balance, destabilizes the endothelial glycocalyx, and exacerbates early blood–brain barrier (BBB) disruption during ischemic stroke.

To test this, we will employ two complementary anemia paradigms — chronic iron-deficiency anemia and acute anti-TER119–mediated red blood cell depletion — in a transient middle cerebral artery occlusion (MCAO) model. At 4.5 hours after stroke onset, we will quantify endothelial glycocalyx ultrastructure by electron microscopy (EM) and BBB permeability by Evans Blue (EB) extravasation.

This preregistered study will establish whether hemoglobin insufficiency directly destabilizes luminal vascular structure during early stroke injury. This study reframes anemia as a primary luminal vascular destabilizer rather than solely a systemic oxygen-delivery deficit.

Facilities and Ethical Approvals

All procedures will be conducted under approved institutional animal care (IACUC) protocols. Stroke surgery, EM preparation, permeability assays, and exploratory biochemical analyses will be performed using existing laboratory infrastructure and core facilities at Stanford University.

Timeline (6 months)

Month 1: Model alignment and QC optimization
Months 2–4: Data collection (both anemia paradigms)
Month 5: Quantitative analysis
Month 6: Preprint preparation and open data release

Data Sharing Commitment

All raw EM measurements, EB quantification data, statistical scripts, inclusion/exclusion logs, and preregistered protocols will be deposited in an open-access repository (e.g., OSF/Zenodo) upon study completion.

2. Introduction

Background

Low hemoglobin (anemia) affects up to 30% of patients presenting with acute ischemic stroke and is consistently associated with worse neurological outcomes^1–3. Despite this strong clinical association, the vascular mechanisms underlying this vulnerability remain undefined.

We propose that hemoglobin insufficiency weakens the vascular lumen — the immediate interface between circulating blood and the blood–brain barrier (BBB). By impairing luminal antioxidant buffering, low hemoglobin may render the endothelium more susceptible to early barrier breakdown following ischemia–reperfusion.

Red blood cells constitute a major circulating antioxidant and redox buffering system^4,5. Reduced hemoglobin levels may therefore disrupt vascular luminal redox balance, increasing oxidative stress at the endothelial surface and promoting activation of glycocalyx-degrading enzymes such as heparanase and matrix metalloproteinases (MMPs)^6–10. The endothelial glycocalyx is a carbohydrate-rich luminal matrix that regulates permeability, leukocyte adhesion, and mechanotransduction across vascular beds, including the cerebral microvasculature^11–13. Disruption of the cerebral endothelial glycocalyx has been linked to BBB dysfunction in experimental stroke models¹⁴.

Glycocalyx alterations have been described in systemic hemodilution and chronic hemoglobinopathies^15,16, suggesting that hemoglobin-dependent redox imbalance may destabilize luminal vascular structure. However, whether hemoglobin insufficiency directly destabilizes the cerebral endothelial glycocalyx during early ischemic stroke, and whether this is associated with increased BBB dysfunction, has not been mechanistically tested.

We focus on the early reperfusion phase (4.5 hours) to capture primary vascular structural effects before extensive secondary injury develops. Prior work from our laboratory demonstrates that this hyperacute timepoint allows reliable detection of quantifiable luminal ultrastructural changes and BBB permeability differences following transient MCAO¹⁷. This window parallels clinically relevant early reperfusion periods in acute stroke management.

Central Hypothesis: Low hemoglobin creates a pro-degradative luminal microenvironment that destabilizes the endothelial glycocalyx and increases early BBB vulnerability following transient ischemic stroke.

Research Questions

Does hemoglobin reduction exacerbate endothelial glycocalyx structural loss and increase BBB permeability during early reperfusion after stroke?
Are the structural and permeability effects consistent across both chronic iron-deficiency anemia and acute red blood cell depletion models?

Hypotheses

H1: Anemic mice will exhibit reduced glycocalyx thickness and coverage at 4.5h post-MCAO stroke compared to non-anemic controls.
H2: Anemic mice will demonstrate increased Evans Blue extravasation at 4.5h post-MCAO stroke.
H3: The direction of effect will be consistent across chronic iron-deficiency and acute RBC depletion models.
Impact Statement

This preregistered study will define whether hemoglobin-dependent luminal biology directly modulates early vascular barrier stability after ischemic stroke and will generate effect size estimates to guide subsequent mechanistic and translational studies.

3. Methods

Adult male C57BL/6J mice (10–12 weeks old) will be used. To minimize hormonal variability and maximize statistical power in this 6-month proof-of-concept study, initial experiments will focus on males; sex as a biological variable will be incorporated in subsequent investigations. All animal procedures will be conducted in accordance with institutional and federal guidelines for the care and use of laboratory animals. The study will be performed under approved Institutional Animal Care and Use Committee (IACUC) protocols at Stanford University. All efforts will be made to minimize animal suffering, including appropriate anesthesia, analgesia, and predefined humane endpoints.

Anemia Induction

Two complementary anemia paradigms will be implemented following prior studies¹⁸.

Chronic iron-deficiency model: Three-week-old mice will be maintained for 8 weeks on an iron-deficient diet (Envigo Teklad TD.110592); controls will receive iron-replete diet (TD.190878). Hemoglobin will be quantified using Drabkin reagent prior to stroke induction using the same assay across all groups. Anemia will be defined as ≥25% reduction in hemoglobin relative to age-matched controls. Animals not meeting predefined anemia criteria will be excluded prior to randomization.

Acute RBC depletion model: Adult mice will receive 45 µg anti-TER119 antibody (i.p.) 2 days prior to MCAO; controls receive IgG2b isotype. Hemoglobin will be quantified prior to surgery to confirm comparable reduction (≥25% decrease). Anti-TER119 induces rapid clearance of circulating erythrocytes independent of iron metabolism.

Using both paradigms allows determination of whether observed vascular effects are attributable to hemoglobin reduction per se rather than iron-specific systemic adaptations.

Stroke Model

Transient middle cerebral artery occlusion (tMCAO) will be performed for 30 minutes followed by reperfusion using a standardized intraluminal filament technique. This model is well established in our laboratory¹⁷.

Mice will be anesthetized with isoflurane (5% induction, 2% maintenance) and body temperature maintained at 36.5–37°C using a feedback-controlled heating system. After 30 minutes, the filament will be withdrawn to initiate reperfusion. Postoperative analgesia will be provided with buprenorphine (0.1 mg/kg, s.c.), and hydration support will be administered as needed. Animals will recover in a temperature-controlled environment before returning to home cages. Cerebral blood flow (CBF) will be monitored using laser Doppler flowmetry. A ≥70% reduction in CBF during occlusion will be required for inclusion. Animals exhibiting procedural complications such as vessel rupture or subarachnoid hemorrhage will be excluded from analysis. All mice must survive to the predefined 4.5-hour endpoint to be included in outcome assessment.

Experiments will be randomized, and investigators will remain blinded to group allocation during surgery, outcome assessment (electron microscopy and Evans Blue quantification), and statistical analysis.

Experimental Groups

Each anemia paradigm will include:

Sham + Control

Sham + Anemia

MCAO + Control

MCAO + Anemia

Sample size is described in the Sample Size Justification section below.

Electron Microscopy for Endothelial Glycocalyx Assessment

TEM will be performed following protocols established in our laboratory ¹⁷. At 4.5 hours after stroke onset, mice will be transcardially perfused with sodium cacodylate buffer followed by fixation in a glutaraldehyde–paraformaldehyde solution optimized for glycocalyx preservation, including lanthanum nitrate to stabilize and enhance visualization of luminal glycocalyx structures. Transcardial perfusion will be performed using a standardized, pressure-controlled setup to maintain constant perfusion pressure across animals, minimizing artifactual glycocalyx loss, with identical parameters applied across groups. Brain tissue samples from the ischemic hemisphere will be post-fixed in osmium tetroxide, dehydrated through graded ethanol, embedded in resin, and sectioned at approximately 80 nm thickness. Ultrathin sections will be stained with uranyl acetate and lead citrate and examined using a transmission electron microscope operated at 120 kV. Digital images will be acquired at predefined magnifications suitable for luminal surface analysis. For each animal, three predefined non-overlapping microvascular fields within the ischemic cortex will be analyzed in a blinded manner. To ensure structural comparability, analyses will be restricted to capillary-sized microvessels (luminal diameter <10 µm), which represent the principal site of blood–brain barrier exchange. Vessel diameter will be documented during image acquisition to minimize variability attributable to vascular caliber. All image acquisition and quantification will be performed by investigators blinded to experimental group allocation.

Evans Blue Assay

Blood–brain barrier (BBB) permeability will be quantified using Evans Blue (EB)¹⁷, an albumin-binding tracer that does not cross the intact BBB. At 2.5 hours post-stroke, mice will receive 2% Evans Blue (4 mL/kg i.v.). After 2 hours circulation, animals will be perfused with PBS. Ipsilateral hemispheres will be homogenized, dye extracted in dimethylformamide, and absorbance measured at 620 nm. EB concentration will be calculated from standard curves and normalized to tissue weight. Liver EB will confirm equivalent systemic delivery.

Primary Outcomes

The primary endpoints of this study are designed to quantify both structural and functional indices of early blood–brain barrier vulnerability at 4.5 hours following transient MCAO.

First, endothelial glycocalyx ultrastructure will be assessed by transmission electron microscopy in microvessels from the ischemic hemisphere. Quantitative measurements will include (1) glycocalyx thickness (nm), measured at standardized perpendicular intervals along the endothelial luminal surface, and (2) glycocalyx coverage (%), defined as the proportion of luminal endothelial surface exhibiting an identifiable glycocalyx layer. Per-animal values will be averaged across fields prior to statistical comparison.

Second, blood–brain barrier permeability will be quantified using Evans Blue extravasation. Evans Blue concentration will be calculated from a standard curve and normalized to ipsilateral brain tissue weight (µg/g) to allow quantitative comparison across groups.

Plasma and residual brain tissue will be preserved for exploratory analyses of vascular injury markers, including oxidative stress indicators and glycocalyx-shedding mediators (e.g., MMPs, heparanase), contingent upon available resources. As an initial mechanistic bridge between hemoglobin insufficiency and glycocalyx degradation, we may quantify selected redox markers (e.g., 4-hydroxynonenal [4-HNE], malondialdehyde [MDA], or plasma total antioxidant capacity) to assess whether anemia shifts the microvascular environment toward a pro-oxidative state. These exploratory measurements are intended to inform future mechanistic studies and will not modify predefined primary endpoints or statistical plans. Additional contributors to glycocalyx instability in anemia, such as altered hemorheology or shear stress changes, may be addressed in subsequent investigations.

Statistical Analysis

Data will be analyzed using GraphPad Prism or R. Normality will be assessed using the Shapiro–Wilk test. For normally distributed data, group comparisons will be performed using two-way ANOVA (Stroke × Anemia), followed by planned comparisons between MCAO + Control and MCAO + Anemia groups. For non-normally distributed data, non-parametric tests (Mann–Whitney U) will be used.

Primary interpretation will focus on differences between anemic and control animals within the MCAO condition. Analyses will be performed separately for each anemia paradigm. Sham groups are included to provide baseline context. Statistical significance will be set at p < 0.05 for each predefined primary endpoint.

IF data violate homogeneity of variance assumptions, Welch’s correction will be applied. IF batch effects are detected, batch will be included as a covariate. IF one EM field is technically unusable, remaining fields will be averaged.

Sample Size Justification

This 6-month proof-of-concept study is designed to estimate effect sizes and detect moderate-to-large differences. We will use n = 6–8 mice per group, consistent with prior variability observed in our laboratory and established experimental stroke standards. Based on prior coefficients of variation (~20–25%) in our measurements, this sample size is expected to provide reasonable sensitivity to detect moderate-to-large (≥30%) differences at α = 0.05. Formal power considerations were guided by these empirical variability estimates.

4. Pilot Data (Optional)

Feasibility of the proposed work is strongly supported by our established vascular injury platform and prior technical experience.

Our laboratory has extensive experience with the transient MCAO model, hyperacute timepoint tissue collection, Evans Blue quantification, and endothelial glycocalyx ultrastructural analysis using transmission electron microscopy. These methodologies are described in detail in our recent preprint¹⁷, where we successfully implemented:

(1) reproducible tMCAO with defined reperfusion timepoints,

(2) quantitative Evans Blue permeability assays (see Fig. 2H in the preprint), and

(3) high-resolution endothelial glycocalyx ultrastructural imaging (see Fig. 2E in the preprint).

These data demonstrate our ability to reliably detect early luminal structural vulnerability and BBB permeability changes at hyperacute timepoints. The imaging infrastructure, EM processing pipeline, and quantitative analysis workflows are fully operational in our laboratory.

Importantly, the current proposal tests a distinct biological variable (hemoglobin reduction) and does not reuse or reinterpret prior data. Rather, the preprint establishes technical feasibility and provides proof that our platform can detect the structural and permeability endpoints proposed here. Together, these data support the feasibility of completing the proposed proof-of-concept study within the 6-month funding period.

5. Budget

The majority of requested funds are allocated to electron microscopy reagents and core facility usage, which represent the primary structural endpoint of this study. Established laboratory infrastructure and personnel support are already available and will enable project completion within the proposed timeframe.

Category	Estimated Cost (USD)
Mice and housing (anemia cohorts)	$3,000
Iron-deficient and control diets	$1,000
Anti-TER119 and isotype control antibodies	$1,200
Evans Blue dye and related consumables	$500
Electron microscopy reagents and core facility usage	$4,300
Total	$10,000

Reference

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2. Li, Z., Zhou, T., Li, Y., Chen, P. & Chen, L. Anemia increases the mortality risk in patients with stroke: A meta-analysis of cohort studies. Sci. Rep. 6, 26636 (2016).

3. Desai, A. et al. Impact of anemia on acute ischemic stroke outcomes: A systematic review of the literature. PloS One 18, e0280025 (2023).

4. Möller, M. N. et al. Oxidants and Antioxidants in the Redox Biochemistry of Human Red Blood Cells. ACS Omega 8, 147–168 (2023).

5. Daraghmeh, D. N. & Karaman, R. The Redox Process in Red Blood Cells: Balancing Oxidants and Antioxidants. Antioxidants 14, 36 (2024).

6. Masola, V., Greco, N., Gambaro, G., Franchi, M. & Onisto, M. Heparanase as active player in endothelial glycocalyx remodeling. Matrix Biol. Plus 13, 100097 (2022).

7. Noda, K. et al. Heparanase inhibition preserves the endothelial glycocalyx in lung grafts and improves lung preservation and transplant outcomes. Sci. Rep. 11, 12265 (2021).

8. Jacquet, C. et al. Matrix metalloproteinase-9 mediates endothelial glycocalyx degradation and correlates with severity of hemorrhagic fever with renal syndrome. iScience 28, 113262 (2025).

9. Ali, M. M., Mahmoud, A. M., Le Master, E., Levitan, I. & Phillips, S. A. Role of matrix metalloproteinases and histone deacetylase in oxidative stress-induced degradation of the endothelial glycocalyx. Am. J. Physiol. - Heart Circ. Physiol. 316, H647–H663 (2019).

10. Ko, K. et al. Ischemic stroke disrupts the endothelial glycocalyx through activation of proHPSE via acrolein exposure. J. Biol. Chem. 295, 18614–18624 (2020).

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12. Kutuzov, N., Flyvbjerg, H. & Lauritzen, M. Contributions of the glycocalyx, endothelium, and extravascular compartment to the blood-brain barrier. Proc. Natl. Acad. Sci. U. S. A. 115, E9429–E9438 (2018).

13. Dancy, C., Heintzelman, K. E. & Katt, M. E. The Glycocalyx: The Importance of Sugar Coating the Blood-Brain Barrier. Int. J. Mol. Sci. 25, 8404 (2024).

14. Zhu, J. et al. Glycocalyx is critical for blood-brain barrier integrity by suppressing caveolin1-dependent endothelial transcytosis following ischemic stroke. Brain Pathol. 32, e13006 (2022).

15. van Beers, E. J. et al. Sickle cell patients are characterized by a reduced glycocalyx volume. Haematologica 93, 307–308 (2008).

16. Ergin, B. et al. Hemodilution causes glycocalyx shedding without affecting vascular endothelial barrier permeability in rats. J. Clin. Transl. Res. 5, 243–252 (2020).

17. Chen, H. et al. Luminal Vascular Dysfunction Drives Rapid Blood Brain Barrier Injury in Hyperglycemic Stroke: Key Roles for Luminal Glycocalyx and Complement. bioRxiv 2025.06.26.661858 (2025) doi:10.1101/2025.06.26.661858.

18. Cottarelli, A. et al. Low Hemoglobin Causes Hematoma Expansion and Poor Intracerebral Hemorrhage Outcomes. Stroke 56, 1234–1242 (2025).

Anemia Destabilizes the Vascular Glycocalyx and Exacerbates Early Blood–Brain Barrier Injury After Stroke

Anemia Destabilizes the Vascular Glycocalyx and Exacerbates Early Blood–Brain Barrier Injury After Stroke

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