The Role Of The Glycocalyx And Magnesium In Delayed Cerebral Ischemia After Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) caused by a ruptured aneurysm accounts for only
5% of strokes, but occurs at a fairly young age and carries a worse prognosis
than other types of stroke. Because of this, the loss of productive life years
from SAH is as large as that from ischemic stroke, the most frequent subtype of
stroke.
Delayed cerebral ischemia (DCI) is consistently the leading cause of death and
disability, adversely affecting more than one in five of all patients who have
suffered SAH and survived. The characteristics of DCI show resemblance with
eclampsia, a common, potentially life-threatening, complication of pregnancy.
At present, the pathophysiology of DCI is incompletely understood. In a
meta-analysis of all randomized clinical trials, magnesium shows a tendency to
reduce the occurrence of DCI and poor outcome after SAH. A large phase III
trial, the MASH-II (Magnesium in Aneurysmal Subarachnoid Hemorrhage) is
currently being conducted investigating the potential beneficial effects of
magnesium in SAH.
The endothelial glycocalyx is a layer of complex sugars lining the endothelium
thereby providing a first line defence mechanism between flowing blood and the
vessel wall. The glycocalyx regulates vascular permeability for macromolecules
and is involved in vessel tone regulation by mediating shear-dependent NO
release. In addition, the glycocalyx has potent anti-thrombotic effects.
Because of recent experiments and previous observations we believe that damage
to the endothelial glycocalyx may play a pivotal role in pre-eclampsia.
Perturbation of the glycocalyx in experimental models leads to increased
vascular leakage, proteinuria and a prothrombotic state. Moreover plasma
hyaluronan levels appear to be significantly increased in pre-eclamptic women
compared to healthy pregnant controls. Finally, it has been demonstrated that
magnesium is required for inhibition of hyaluronidase, an important glycocalyx
degrading enzyme, thereby preserving glycocalyx volume. The as yet unexplained
beneficial effects of magnesium sulfate in pre-eclampsia could therefore rely
on magnesium-related preservation of glycocalyx volume, but this may also play
a role in DCI after SAH. There are, however, no reports on the role of the
glycocalyx in the development of DCI in SAH patients.
As sulfate may add to the biosynthesis (*sulfation*) of heparan sulfate, their
may be a dual role of magnesium sulfate (MgSO4) in preserving and stimulating
glycosaminoglycan synthesis.
Based on findings of recently performed experiments we hypothesize that
perturbation of the endothelial glycocalyx contributes to impaired flow
mediated dilatation and increased permeability of the endothelial surface layer
in SAH patients. Additionally magnesium administration results in preservation
of glycocalyx volume and diminishes glycocalyx shedding.

Primary objective: The general aim of this study is to elucidate the role of
the glycocalyx in the pathogenesis of DCI in SAH and to study the effects of
magnesium on glycocalyx volume.
Main objectives of this proposal are:
- To assess whether the glycocalyx is affected in SAH patients and is related
to its severity and the development of DCI.
- To evaluate whether glycocalyx perturbation is associated with vasomotion
changes as non-invasively assessed by finger arterial waveform registration, in
SAH patients.
- To determine whether the proposed beneficial effects of magnesium may be
explained by its ability to preserve glycocalyx volume and prevent further
glycocalyx shedding.

A total of 40 SAH patients will be enrolled of which at least twenty patients
are included in the MASH II study (MEC 07.241) to allow comparison of MgSO4
treatment and placebo on glycocalyx shedding and -volume. In addition 20
control subjects matched for age and gender will be included to assess
differences in glycocalyx perturbation in SAH patients. The subgroup from the
MASH-II trial is randomized for 20 day treatment with magnesium sulphate or
placebo. MASH-II randomization is performed in block-sizes of 4 patients.
Patients or their legally-appropriate substitute decision maker will be
approached to participate in the study. They will be informed about the
rationale of this study, possible risks and study burden. Eligible candidates
or their legally-appropriate substitute decision maker who are willing to
participate will be asked to provide informed consent. The following
non-invasive measurements will be performed on admission, i.e. prior to
initiation of treatment with MgSO4 and at 3, 7 and 20 days following admission:
1. glycocalyx dimensions will be visualized using SDF imaging, 2. noninvasive
finger arterial pressure waveform registration by Nexfin (BMeye, Amsterdam, The
Netherlands) will be used for continuous monitoring of heart rate, blood
pressure, cardiac stroke volume, cardiac output, and peripheral vascular
resistance. 24-hour urine samples will be collected. Finally, blood samples
will be drawn with routine laboratory assessments on admission. Additionally,
laboratory assessments for measurement of coagulation, glycocalyx constituents
and enzymes will be performed on admission, i.e. prior to initiation of
treatment with MgSO4, and at 3, 7 and 20 days following admission. When
patients are discharged before the final measurements on day 20, but have
completed all prior measurements, their participation will be concluded as
such, i.e. the day 20 measurements will not be performed.
Approximately three months after the initial SAH included subjects will be
contacted for a short telephonic questionnaire to asses for clinical outcome
defined by the RANKIN scale.
Hypertension is a major risk factor for subarachnoid hemorrhage.(16) Treated
and untreated, age and gender matched control subjects will be recruited from
the hypertension out-patient clinic of the AMC, Amsterdam and from the patients
next of kin. By choosing this control group we should be able to correct for
possible hypertension related, shear-stress induced, changes in glycocalyx
perturbation. Additionally, relatives to SAB patients will be asked to
participate as control subjecs. Control subjects will undergo one session of
non-invasive measurements, additionally a single set of venous blood samples
will be taken, totaling 40 ml of blood.

Virtually all measurements, with the exemption of bloodsampling, performed in
this study are non-invasive. We will attempt to combine blood drawing for
research purposes with blood drawings for regular patient care, thus minimizing
additional discomfort. If possible, blood will we aspirated from intra-venous
lines inserted for regular patient care. Control subjects will undergo a single
venapuncture, with risk of a hematoma at the puncture site. The techniques used
in this study consist of normal light (SDF-imaging) or localized pressure
(finger arterial waveform registration) and are considered safe. The discomfort
and risks associated with these measurements are comparable to an abdominal
ultrasound examination.