Main FindingsConclusion

1
Vascular Remodeling in Cerebral Aneurysms
Measured by Polarizing Microscopy
Brittany N. Balint1, Supervised by Peter B.
Canham, PhD2,3 Helen M. FINLAY2 1Department of
Physiology and Pharmacology, University of
Western Ontario, London, Canada

2Department of Medical Biophysics, University of
Western Ontario, London, Canada

3Robarts Research
Institute, London, Canada
Measurements/Results
Summary of Results
Introduction / Rationale
Saccular aneurysms (Fig.1), which are the most
frequent form of brain aneurysms, commonly arise
near the flow divider of a branching artery. They
.
Example Polarizing Micrographs Picrosirius red
staining
are possibly caused by hypertension and weakening
of blood vessel walls, resulting in a localized,
balloon-like protrusion in the wall of the weak
vessel. There are common features among saccular
aneurysms, as they are all made up of multiple
layers of collagen, and each layer is different
in terms of its thickness and its strength 1,2.
This is quite different from artery walls, which
contain elastin while aneurysms have barely any
elastin, and are very stiff, elastically. A
fusiform aneurysm (Fig. 2) is a localized
widening of an artery wall.
SA Area 1
SA Area 2
B (nm)
B (nm)
Fig.1 Saccular aneurysm 6.
As the aneurysm enlarges over months or years,
the orientation of the collagen in each layer
changes in order to adapt to new collagen that is
added to the expanding layers. This results in
the original layers, the ones furthest from the
lumen, to be mechanically stronger as the
collagen in these layers undergoes cross-linking,
which significantly adds to their strength. If
the aneurysm enlarges to a size of about 5-10 mm,
which can be detected by medical imaging, it is
considered at risk for rupture 1. If a saccular
aneurysm does rupture, the patient will likely
suffer a severe headache, seizures, and/or
movement and speech disorders, and may die within
minutes. Fortunately, most lesions do not grow
large enough to be at risk for rupture, and
therefore, only a small percentage of aneurysms
actually rupture and cause harm 3. .

Layer
Layer
Saccular aneurysm Area 1
Saccular aneurysm Area 3
SA Area 3
SA Area 4
B (nm)
B (nm)
Through the services of pathology, we were able
to acquire saccular aneurysm tissue, and also
tissue from a fusiform aneurysm that was used as
a comparison. Both of the lesions that we
obtained were the cause of patient death, and
they were used to study the properties of
aneurysms, mainly in regards to the birefringent
properties and the strength of the various layers
within the vessel.
Layer
Layer
Fusiform aneurysm Area 2
Fusiform aneurysm (thin region) Area 1
SA Area 5
SA Area 6
Fig. 2 Fusiform aneurysm 5.
B (nm)
B (nm)
Data Pathway Saccular Aneurysm Area 1
Objectives
1. To highlight the layered collagen fine
structure within brain aneurysm tissue from
lesions that have been the cause of death, as
normal procedures in pathology in regards to
reporting tissue structure do not show detail
about the extracellular matrix (ECM). 2.
 To quantitatively evaluate the diversity of ECM
collagen at various areas along the perimeter of
the lesions, as well as the differences in
collagen that exist from the most outer layers
to the lumenal layer.
Layer
Layer
Methods
FA Area 1
FA Area 2
Tissue source Two aneurysms were investigated
a 1.5 cm saccular aneurysm (SA) (which ruptured,
causing death) and a 3 cm fusiform aneurysm (FA).
The SA was from a 67 year old f. and was found on
the anterior communicating artery (ACoA). The FA
was found on the vertebral artery (VA) of a 79
year old f., and the size of the lesion (causing
brainstem pressure) ultimately lead to her death.
Tissue preparation Both lesions were wax
embedded, sectioned at 5µm, and stained with
Picrosirius red, which is a birefringent
enhancer. Experimental procedure 6 areas (from
outer layer to lumenal layer) around the
perimeter of the SA wall were marked off for
microscopy as representative samples. A similar
approach was done for the FA (2 areas were used).
Each area (corridor) was divided into sections
(layers) based on the varying orientations of
collagen within the lesion (Fig. 3).
Consequently, as a result of subjective
interpretation of the histological sections, the
different corridors had different numbers of
regional layers depending on how many
orientations the collagen at that specific
location took on. Starting with area 1, the
birefringence (B) of each layer was measured 10
times, and the mean B of each layer within an
area was calculated. The same was done with areas
2-6. The B between layers was compared, as well
as B within layers at different locations around
the aneurysm. The birefringent properties of the
SA were compared with that of the FA.
B (nm)
B (nm)
Layer
Layer
Fig. 3 Mean Birefringence of each layer studied
within the outlined corridors (see Fig. 2) of the
saccular aneurysm and the fusiform aneurysm
investigated. Generally, B is greatest in the
outermost layers and lowest in the lumenal layer.
Main Findings/Conclusion

•  
• The birefringence, and hence, tensile strength,
  of the aneurysm is generally greatest in the
  outermost layers, and least in the lumenal
  layers. This is likely due to the cross-linking
  of collagen fibers in the older (outermost)
  layers of the aneurysm as new collagen is added
  when the lesion expands.
• Area 6 of the SA was largely inconsistent in
  terms of collagen arrangement, and hence did not
  follow the same general trends as the other
  corridors (consistent with the idea that the
  aneurysm structure is not uniform).
• A slight increase in B indicates a great increase
  in tensile strength (Fig. 5), as the two are
  related exponentially.
• The tensile strength of a tendon is 60-100 MPa,
  and in a previous study 1, artery wall strength
  was found to be as great as 35 MPa. The highest
  tensile strength measured in this study was the
  strength of (fusiform) Corridor 2, section a (20
  MPa). So, the aneurysms generally showed
  significantly lower tensile strength than tendons
  and artery walls.
• The highest B was found in the outermost layer of
  the FA (Area 2). This is possibly due to a
  compensatory mechanism (for strength) since the
  lesion was composed of generally less layers (3)
  than the SA.
• In comparing the SA and the FA, it can be noted
  that the FA generally has less layers than the
  SA. Since the artery wall is composed of 3 main
  layers, it is possible that the FA more closely
  resembles the artery wall than the SA (consistent
  with the idea that the FA is a swelling of the
  artery wall SA made entirely from new, hence
  more layers).

Area 1
Area 4
Table 1. Example of measurements taken in this
study. Ten measurements of B were taken at each
section, the mean and SD were calculated, as well
as tensile strength and p-values between adjacent
layers. Thickness of each layer was measured.
Area 3
Area 6
Birefringence (nm)
Area 5
T. Strength (MPa)
Area 2
Fig. 3 Circle of Willis 3. Arterial network at
base of brain, commonly home to cerebral
aneurysms. occurrence of aneurysms, blue
indicates location of aneurysms used in this
study.
Fig. 4 Tracing of SA of study. 6 areas were
chosen, each divided into sections (indicated by
a lowercase letter) based on orientation of
collagen and changes in birefringence.
Acknowledgements
This study was funded by the Canadian Institutes
of Health Research. Many thanks to supervisors
Dr. PB Canham and HM Finlay, as their services
made this study possible.
,
Collagen is birefringent (does not have equal
refractive indices in all directions) and
therefore, when plane polarized light (through
the use of a polarized light microscope) travels
through it, it divides into 2 rays (each
traveling at different velocities), which
vibrate at 90
References
Layer
Layer
1 Canham PB, Finlay HM, Kiernan JA, Ferguson
GG. Layered structure of saccular aneurysms
assessed by collagen birefringence. Neurol. Res.
1999 21618-626. 2 Canham PB, Ferguson GG. A
mathematical model for the mechanics of saccular
aneurysms. Neurosurg. 1985 17 291-295 3
Ferguson GG. Physical Factors in the Inhibition,
Growth and Rupture of Human Intracranial Saccular
Aneurysms (PhD thesis). UWO press 1970 9. 4
Macdonald JD, Finlay HM, Canham PB. Directional
wall strength in saccular brain aneurysms from
polarized light microscopy. Ann. of Biomed. Eng.
2000 28 533-542 5 www.mayoclinic.org 6
www.wrha.mb.ca
.
Fig. 5. Example of the primary data for Area 1
(SA) and the derived strength data. Each corridor
was divided into layers according to B and
orientation of collagen within the layers. At
each layer, 10 measurements of B were taken and
the mean and SD were calculated. The tensile
strength of each layer was calculated according
to the equation 1 Tensile Strength (MPa)
0.0031B2.33 The thickness of each layer was
measured (to investigate correlation between
thickness and B). Students t-tests were
performed between adjacent layers to determine
whether or not a significant difference in B
existed between the layers.
from each other. This causes phase retardation
between the rays, to which extent depends
directly on the thickness of the birefringent
material and the strength of the B within the
material. A Senarmont comp-ensator is used to
measure the phase retardation, and thus, the
birefringence of the collagen.
polarizer
analyzer
Birefringent fabric
light source