Glycocalyx assessment in temporal lobe epilepsy

Epilepsy is the most common neurological problem after stroke. Most patients
can be treated with antiepileptic drugs, but 30% is pharmacoresistant. Since so
many patients are affected, this is a major problem, both medically and
socioeconomically.

The most frequent type of epilepsy is temporal lobe epilepsy (TLE). Despite
more than 50 years of extensive research, the pathophysiology of epilepsy has
not been elucidated yet. Over the years, we have learned several things, e.g.
that febrile seizures in childhood and traumatic brain injury (TBI) are the
most important risk factors for developing temporal lobe epilepsy, that the
sclerotic hippocampus (present in 60% of TLE patients) is site for mossy fiber
sprouting and granular cell dispersion, resulting in disturbed neuronal
networks in which deep nuclei such as the thalamus play a role, and that after
a seizure, the blood brain barrier opens and inflammation occurs. However, we
still do not know if and how inflammation leads to epilepsy, nor why some
develop TLE after having suffered from FS or TBI earlier in life, while others
do not. We hypothesize that the cerebral microcirculation, and in particular
the glycocalyx, may play a role.

The glycocalyx is a thin gellayer lining the endothelium on its luminal side.
It consists of a skeleton that is bound to the endothelium and is made up of
proteoglycans and glycoproteins, and of several soluble molecules from the
endothelium and plasma, such as free proteoglycans, antithrombin III and
cytokines. This endothelial glycocalyx appears to play a central role in
vascular homeostasis, and protects the endothelium from circulating blood. In
the brain, it is the first barrier between the blood and the brain, and can be
considered part of the BBB. The BBB has been shown to be affected in some way
in epilepsy, but in what way exactly is unclear. No data on glycocalyx in
epilepsy are available. Experimental studies have shown that loss of glycocalyx
increases vascular permeability, and that inflammation can disrupt the
glycocalyx. Furthermore, experimental TBI affects the glycocalyx.

No studies on glycocalyx in (temporal lobe) epilepsy patients have been
published so far. Maastricht offers the unique combination of an enormous
experience with glycocalyx measurements and the opportunity to perform these
measurements in brain tissue in an in vivo situation.

Primary objective of this study: To establish glycocalyx properties of temporal
lobe epilepsy (TLE) patients.

Secondary objectives of this study:
- To establish differences in glycocalyx properties of glycocalyx between TLE
patients and controls.
- To establish the correlation between sublingual glycocalyx measurements and
cortical glycocalyx measurements in TLE patients and controls.
- To establish differences in glycocalyx properties between TLE patients with
hippocampal sclerosis and those without hippocampal sclerosis.
- To establish differences in glycocalyx between TLE patients with a history of
febrile seizures and/or traumatic brain injury and those without a history of
febrile seizures and/or traumatic brain injury.
- To establish the correlation of glycocalyx properties in TLE patients to
epilepsy-specific factors such as type of seizures and seizure frequency.
- To establish the examination of viability of small cortical arteries in an
in-vitro setting using an arteriography.
- To establish the examination of the neurovascular unit of small cortical
arteries in an in-vitro setting using an arteriograph and electrical field
stimulation.
- To establish the examination of the glycocalyx of small cortical arteries in
an in-vitro setting using an arteriograph and 2-photon microscopy and to
correlate this to the in-vivo results.

In our department, we treat 30-40 patients per year for medically refractory
localisation-related epilepsy by resective brain surgery. These patients
constitute a group of epilepsy patients that suffer from localisation-related
epilepsy, and in whom it is expected that they will be seizure free or have a
major seizure reduction after resection of the seizure focus in the brain. The
prior extensive diagnostic examinations and possible surgery indication is
appointed by the Academisch Centrum voor Epilepsie.
In most cases, the seizure focus is located in the mesiotemporal region, in
casu amygdala and hippocampus. The hippocampus is located on the mesial side of
the temporal lobe. After performing a temporal lobe resection plus resection of
the (sclerotic) hippocampus and amygdala, around 70% of the patients will be
rendered seizure-free. During surgery, the (anterior part) temporal lobe is
fully exposed. This gives us the unique opportunity to use the SDF camera
directly on the cortical and hippocampal surface, in order to directly measure
glycocalyx in cortical and hippocampal vessels, respectively. This may increase
surgery time (normally three to four hours) with maximally five minutes.
The control population consists of patients that require a (small) craniotomy
or burr hole surgery for tumor resection or biopsy, and patiens between 18 and
60 years that require a craniotomy because of neurovascular surgery, like
arteriovenous malformations of aneurysms. In these cases it is also possible to
use the same probe on the cortical surface prior to performing the corticotomy.

The camera has been used successfully on human tissue in vivo before, mainly on
kidney and sublingual.

Method
The study is an observational case-control study. In order to include
sufficient patients, the study will take four years.

Measurements
The glycocalyx measurement will take place by means of a small video-microscoop
(SDF camera, MicroVision Medical, Amsterdam, CE-certified, Maastricht
University equipment registration number H.08IHVI06265). Using GlycoCheck ICU
analysing software, red blood cell (RBC) column width will be measured
automatically in approximately 3000 vessel segments with a diameter of 5-30
micrometer. Subsequently, the perfused boundary region (PBR) will be calculated
for all 3000 vessel segments.

In all patients (TLE and control) a standard sublingual glycocalyx measurement
will be performed as measurement 1:
Measurement 1: Directly following anesthesia induction the measurement will
take place in the operation room. The patient is not aware of this measurement.

Continuation of method in TLE patients:
Measurement 2: When the cortex is fully exposed, measurement two can take
place. Location: operation room.
Measurement 3: Usually the hippocampus will be exposed thirty minutes following
cortical resection. When fully exposed, the hippcampal measurement is
performed. Location: operation room.

Continuation of method in control patients:
Measurement 2: When the cortex is fully exposed, measurement two can take place
. Location: operation room.

All glycocalyx measurement procedures that are performed will lead to a delay
of surgery time of maximally 10 minutes. Following the final measurement
(measurement 3 in TLE patients and measurement 2 in control patients) the
patient has reached the end point of this study.

A significant volume of brain tissue is resected during TLE surgery. Part of
the cortex and hippocampus that were used for glycocalyx measurements will be
transferred to the microcirculation lab where other (in vitro) experiments on
vascular permeability and glycocalyx quality will be performed. Other parts of
the cortex, and part of the hippocampus, are stored in our biobank, while the
rest of the hippocampus and cortex are sent to the department of pathology, for
standard histopathological analysis (e.g. to determine the degree of
hippocampal sclerosis). Using this minimally invasive device with minimal time
investment on epilepsy patients and others undergoing brain surgery, gives us
the unique opportunity to gather direct information on brain microcirculatory
function.

Additional information on the in vitro experiments
Correlation to in vitro experiments: epilepsy patients
During a standard temporal lobectomy including amygdalohippocampectomie
resected cortical, amygdala and hippocampus are partly resected en bloc. Once
resected this tissue is divided in several pieces for clinical and research
matters. The tissue is considered as remnant tissue.

For regular clinical purposes one part of the cortical tissue, one part of the
hippocampus and the whole of the amygdala (too small to be further divided) is
forwarded to the department of pathology. Important pathological parameters
like focal cortical dysplasia and (degree of) hippocampal sclerosis are
determined by the pathologist. Since these parameters are important for
especially research considerations, they will be included in the compiled
clinical data and are included as secondary study parameters/end points.

Another part of the cortical and hippocampal tissue is freshly frozen on dry
ice. This part will be stored in our bio-bank (-80C refrigerator storage). The
tissue in our bio-bank might be used for future study ideas, by example for
genetic analysis. When studies on this tissue will be elaborated, additional
METC approval will be requested.

A third part of the cortical and hippocampal tissue is fixated using
Formaldehyde. After two days of fixation the tissue is embedded in Paraffine.
This tissue is stored in the histological biobank.
The histological biobank for this tissue is located at the MHeNS lab. All
tissue is stored in a locked closet located on the locked room of GH. Only OS,
GH and RH have a key for opening this closet. When the tissue for
neuropathological review was not conclusive or too little to review, the
histological biobank tissue can be used for re-review. Tissue stored in the
histological biobank can be used for future (histological) experiments. When
studies on this tissue will be elaborated, additional METC approval will be
requested.

A fourth part of the cortical tissue (hippocampal vascular structures are
currently too small for handling) of the tissue is stored in a HEPES-buffer
which is important for conservation of vascular features. Direct vascular
dynamics related experiments can then be performed on this tissue. This tissue
is only stored for a maximum of 48 hours. After this time period vascular
dynamical experiments can not be considered viable.
Interesting microvascular features that we want to include is neurovascular
unit viability of vascular segments in the resected cortical tissue. Using
microdissection arterial segments are isolated. Next, an arterial segment is
mounted in an organ chamber between two glass cannulas and exposed to a
continuous distending pressure of 70mmHg. Strong vasoconstrictive agents will
then be applied, followed by strong vasodilative agents. In this way, viability
of vascular wall dynamics is being observed. Once viability is confirmed,
several receptor agonists and antagonists will be applied to further determine
the necessary vasoconstrictive and vasodilatative agents/cascade. Also,
vascular permeability can be determined more thoroughly by comparing responses
to intraluminal and extraluminal application of vasoconstrictive agents.

If viable, supplementary extraluminal electrical field stimulation can be
applied. Vasoconstriction as a function of the neurovascular unit can be
mimicked in this way. Again several receptor agonists and antagonists will be
applied additionally to further determine the necessary vasoconstrictive and
vasodilatative agents/cascade. Since glycocalyx properties are very easily
disrupted, viability of the included vessels is of importance for further
glycocalyx analysis.

Glycocalyx an

Sublingual glycocalyx measurement is a non-invasive, short measurement using a
SDF camera. All measurements are performed when the patient is under general
anesthesia, which makes the burden low while the risk is minimal. The second
and third glycocalyx measurement is performed on cerebral vessel segments
during surgery. This is a non-invasive technique, comparable with
intraoperative cerebral ultrasound, carrying minimal risk and zero burden for
the patient.

Possible risk includes local contusion of the cortex or hippocampus. This has
no consequences for the patient since the tissue possibly damaged will be
resected anyway, with the exception of the neurovascular indications in which
no tissue is resected regularly.

Surgery time will be extended by a maximum of 10 minutes as a consequence of
the measurements, a time-investment we deem justified when taking into account
the huge amount of information we are provided with.

Given the burden of disease for epilepsy patients, that is the consequence of
our lack of pathophysiological knowledge, and given the fact that our proposed
study, which is non-invasive and extremely low risk/low burden for the patient,
will give us unique information on the pathophysiology of epilepsy, we think it
is justified to conduct the study