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Haptoglobin administration into the subarachnoid space prevents hemoglobin-induced cerebral vasospasm
Michael Hugelshofer, … , Emanuela Keller, Dominik J. Schaer
Michael Hugelshofer, … , Emanuela Keller, Dominik J. Schaer
Published August 27, 2019
Citation Information: J Clin Invest. 2019;129(12):5219-5235. https://doi.org/10.1172/JCI130630.
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Research Article Neuroscience Vascular biology

Haptoglobin administration into the subarachnoid space prevents hemoglobin-induced cerebral vasospasm

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Abstract

Delayed ischemic neurological deficit (DIND) is a major driver of adverse outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH), defining an unmet need for therapeutic development. Cell-free hemoglobin that is released from erythrocytes into the cerebrospinal fluid (CSF) is suggested to cause vasoconstriction and neuronal toxicity, and correlates with the occurrence of DIND. Cell-free hemoglobin in the CSF of patients with aSAH disrupted dilatory NO signaling ex vivo in cerebral arteries, which shifted vascular tone balance from dilation to constriction. We found that selective removal of hemoglobin from patient CSF with a haptoglobin-affinity column or its sequestration in a soluble hemoglobin-haptoglobin complex was sufficient to restore physiological vascular responses. In a sheep model, administration of haptoglobin into the CSF inhibited hemoglobin-induced cerebral vasospasm and preserved vascular NO signaling. We identified 2 pathways of hemoglobin delocalization from CSF into the brain parenchyma and into the NO-sensitive compartment of small cerebral arteries. Both pathways were critical for hemoglobin toxicity and were interrupted by the large hemoglobin-haptoglobin complex that inhibited spatial requirements for hemoglobin reactions with NO in tissues. Collectively, our data show that compartmentalization of hemoglobin by haptoglobin provides a novel framework for innovation aimed at reducing hemoglobin-driven neurological damage after subarachnoid bleeding.

Authors

Michael Hugelshofer, Raphael M. Buzzi, Christian A. Schaer, Henning Richter, Kevin Akeret, Vania Anagnostakou, Leila Mahmoudi, Raphael Vaccani, Florence Vallelian, Jeremy W. Deuel, Peter W. Kronen, Zsolt Kulcsar, Luca Regli, Jin Hyen Baek, Ivan S. Pires, Andre F. Palmer, Matthias Dennler, Rok Humar, Paul W. Buehler, Patrick R. Kircher, Emanuela Keller, Dominik J. Schaer

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Figure 6

Haptoglobin blocks translocalization of Hb from CSF into the brain parenchyma.

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Haptoglobin blocks translocalization of Hb from CSF into the brain paren...
(A) Representative fluorescent images of sheep brain sections (120 μm) after injection of TCO-labeled Hb (left panels) and Hb-haptoglobin complexes (right panels), stained for nuclei (blue) and the labeled compound (tetrazine-Cy5, yellow). Hb translocates from the ventricular system through the ependymal barrier and from the subarachnoid space through the glia limitans into the brain interstitial space (images are representative for n = 4 animals). This delocalization cannot be observed when Hb is bound to haptoglobin (images are representative for n = 5 animals). Fluorescent Hb-haptoglobin complexes can be recognized only along penetrating cortical vessels. In Section A, a small amount of Hb-haptoglobin complexes was injected directly into the brain parenchyma at the tip of the external ventricular drain catheter, serving as a positive control for the staining and imaging procedure (arrow head). Whole-slide scans were produced by stitching single images obtained at ×10 magnification. Scale bars are indicated in the figure. (B) Before animals were sacrificed, CSF samples from Hb- and Hb-haptoglobin–treated sheep were collected from the subarachnoid space and analyzed by SEC-HPLC, indicating typical elution profiles of cell-free Hb and large Hb-haptoglobin complexes. CSF sample of a sheep before infusion (blue trace), CSF sample of a sheep infused with Hb (red trace), CSF sample of a sheep infused with Hb-haptoglobin complexes (green trace). (C) The same CSF samples were reacted ex vivo with Cy5 tetrazine and analyzed by SDS page. The bands represent a/b globin chains in cell-free Hb (Hb) and Hb-haptoglobin complex (Hb-Hp). Under nonreducing conditions, the specific ladder pattern of the different type 2-2 haptoglobin polymers is clearly visible when analyzed on a gel optimized for high resolution of large proteins (7.5%).
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