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J Neurosurg 87:307–314, 1997
Microcirculation after cerebral venous
occlusions as
assessed by laser Doppler scanning
HIROYUKI NAKASE, M.D., OLIVER S.
KEMPSKI, M.D., PH.D., AXEL HEIMANN, D.V.M.,TOSHIKAZU TAKESHIMA,
M.D., AND JAROSLAV TINTERA, PH.D.
Institutes for Neurosurgical
Pathophysiology and Neuroradiology, Johannes
Gutenberg–University of Mainz, Mainz, Germany
Reombotic technique provides a minimally
invasive, clinically relevant, and reproducible model suited to
study the pathophysiology of CVCDs. In this study, the effects
of venous occlusion on regional cortical blood flow and the
brain (rat
skull microcirculation,,,) damage that ensues were evaluated. Cortical vein occlusion
was induced by photoactivation of rose bengal via 100-mm
fiberoptic illumination. The cerebral venous flow pattern was
examined using fluorescence angiography until 90 minutes after
venous occlusion, and regional cerebral blood flow (rCBF) was
determined at 48 locations by using laser Doppler scanning.
Histological damage was assessed 48 hours after vein occlusion.
Occlusion of two cortical veins (Group T; seven animals) was
compared with single-vein occlusion and its ensuing brain damage
(Group S; five animals) and with sham-operated control (five
animals). An rCBF reduction occurred 30 minutes after occlusion
in Group T and was more extensive than the decrease in Group S
after 60 minutes. Observation frequency histograms based on
local CBF data obtained in Group T demonstrated that local CBF
at some sites decreased to a level below the ischemic threshold
within 90 minutes. Six of the seven rats (rat
skull microcirculation,,,)in Group T had a
growing venous thrombus with extravasation of fluorescein. The
resulting infarction was significantly larger in Group T (9.8 6
4.5% of the hemispheric area) than in Group S (only 3 6 1.5% of
the hemispheric area). In conclusion, microcirculation
(rat
skull microcirculation,,,)
perturbations occur early after venous occlusion and result in
the formation of a venous thrombus accompanied by local ischemia
and severe venous infarction. The extent of vein occlusion
determines the resulting brain damage. Based on the results of
this study, the authors conclude that CVCDs may be attenuated by
prevention of venous thrombus progression together with the use
of protective measures against the consequences of
ischemia.search on cerebral venous circulation disturbances (CVCDs)
has been limited partly by the paucity of animal
models that produce consistent venous infarction.
KEY WORDS • cerebral blood flow • cortical
vein occlusion • laser Doppler scanning • rat
(rat
skull microcirculation,,,)
To date, the consequences of venous occlusion in
the brain have been underestimated in neurosurgical practice,
and experimental studies of ischemic injury have focused
primarily on the effects of arterial occlusion. Recently,
however, more attention has been paid to brain
(rat
skull microcirculation,,,) injury following
perturbations of the cerebral venous circulation because
increasing numbers of neurosurgical operations are performed in
older-aged patients and because of the development of skull base
neurosurgery. 7,8,12,17–19,24,30,32 Therefore, neurosurgical phlebology is gradually maturing as a specialty. Cerebral blood
volume (CBV) increases after sinus vein thrombosis (SVT) or
cortical vein occlusion. Intracranial hypertension from edema,
reductions of regional cerebral blood flow (rCBF), and brain
damage can develop secondarily.4,7–9,27,30,33 More recently,
local (l)CBF monitoring has been shown to be useful for
predicting brain damage subsequent to SVT or cortical vein
occlusion.19,33 To continue this line of research, further study
must be made of the contribution of ischemia mechanisms to the
pathophysiological consequences of cerebral venous circulation
disturbances (CVCDs). Recently we introduced a potentially
useful rat (rat
skull microcirculation,,,) model of photochemically induced venous occlusion
that allows selective occlusion of bridging or cortical
veins.18,19 We demonstrated that this nonmechanical method of
cortical vein occlusion resulted in a decrease in lCBF, followed
by typical pathological changes in the rat brain(rat
skull microcirculation,,,). Following
occlusion of a single vein, this experimental approach is
characterized by a high variability in symptoms and histological
brain injury in 30% of the animals, whereas, after occlusion of
two adjacent veins, brain damage is evident in more than 90% of
the animals.19 Therefore, the occlusion of a single vein is an
appropriate model for studies of the variable pathophysiology of
venous occlusion observed experimentally as well as in patients,
whereas the occlusion of two adjacent veins is a reproducible
approach for studies on the pathophysiology and therapy of CVCDs.
In the current investigation, patho-physiological aspects of the
cortical microcirculation (rat
skull microcirculation,,,) after CVCDs were studied by means of a
laser Doppler (LD) scanning technique10,14,31,33 in rats with
occlusion of two cortical veins. The results were compared with
data from single-vein occlusion experiments. The quantitative
assessment of brain injury provides the basis for a comparison
of experiments with occlusion of one and two cortical veins.
Animal Preparation
Seventeen male Wistar rats(rat
skull microcirculation,,,), each weighing between
260 and 340g, were used for this study: five rats
(rat
skull microcirculation,,,) acted as
sham-operated controls; seven rats were used for the two-vein
occlusion experiments; and data from five rats(rat
skull microcirculation,,,), which had
suffered brain damage after onevein occlusion in a previous
study,18 were used for comparison. The animals were given free
access to food and water in their standard environment prior to
surgery. The methods used here have been described in detail
previously.18 Each animal was lightly anesthetized by ether
inhalation; anesthesia was maintained by intraperitoneal
injection of chloral hydrate (36 mg/100 g wt) after
premedication with 0.5 mg atropine. Rectal temperature was kept
close to 37°C in all animals. Spontaneous ventilation was
maintained throughout the experiment. Polyethylene catheters
were placed into the tail artery and the right femoral vein. The
arterial catheter was used for continuous monitoring of arterial
blood pressure and for blood gas analysis, whereas the venous
line was used for administration of fluids and drugs. The PaO2,
PaCO2, and arterial pH were monitored. The head () of each animal
was placed in a stereotactic frame. Aided by an operating
microscope, a 1.5-cm midline skin incision was made and a
cranial window (4.5 mm 3 6 mm) was created over the right
frontoparietal region using a high-speed drill. The drill tip
was cooled during the craniotomy by continuous irrigation with
physiological saline. The dura mater was left intact and the
right frontoparietal cortex was exposed.
Fluorescence Angiography
Fluorescence angiography was performed in all
rats(rat
skull microcirculation,,,). Epicortical vessel structures were studied using a 2% Na+–fluorescein
solution
and an excitation source with a wave length of 450 to 490 nm and
a No. 12 filter block. A photomacroscope furnished with a 50-W
mercury lamp and fluorescence filter was used for fluorescence
angiographic studies before and 30 and 90 minutes after
induction of
venous occlusion. The images were recorded on super-video home
system (S-VHS) tape. To minimize damage by fluorescence
excitation, illumination was restricted to angiography.
Histological Preparation
Two days after the operation the rats(rat
skull microcirculation,,,) were each
given an injection of 2% Evans blue dye solution (1 ml/kg) after
general anesthesia
had been induced with chloral hydrate. After 1 hour, the rats
(rat
skull microcirculation,,,)were subjected to perfusion fixation with 4% paraformaldehyde.
The
brains were removed from the skull and embedded in paraffin to
obtain coronal sections of the frontoparietal region. Sections
were
stained with hematoxylin and eosin. For quantitative assessment
of brain injury, the histological sections were projected onto
the screen of a computer using a color charge-coupled device
camera and a genlock interface. The optical system was
calibrated by using a microscopic ruler. Self-programmed
computer software allowed us to measure distances and areas of
infarction. The size of the infarction was estimated by
evaluating three sections from each brain: the section
demonstrating the largest infarction area and sections obtained
0.4 mm anterior and posterior to it. These areas were averaged
and expressed as ratios of the ipsilateral and contralateral
hemispheric size determined from the respective section.
Discussion
Rat (rat
skull microcirculation,,,)Cortical Vein Occlusion Using the Photochemical Thrombotic Technique
Until recently, very little
was known about the pathophysiology of CVCDs. The paucity of
suitable animal models has, in part, impaired the development of
research on CVCDs, and technical difficulties have precluded
studies of the selective occlusion of bridging and cortical
veins thus far. We introduced a rat (rat
skull microcirculation,,,)model of cortical vein
occlusion involving the use of the photochemical thrombotic
technique, which has little local or systemic side effects, is
easy to perform, and is clinically relevant. Moreover, the rat
(rat
skull microcirculation,,,)model clearly has some advantages over large-animal models.19 It
is also a model for the intraoperative sacrifice of cortical
veins during neurosurgical operations. The photothrombotic
occlusion technique was originally established to perform
arterial occlusions.
The underlying mechanism is
aggregation of platelets at the endothelial luminal surface
after photochemical induction
of local singlet oxygen production. Platelet–endothelial cell
and platelet–platelet interactions are integral to the model.
Nakayama, et al.,20 who occluded the rat (rat
skull microcirculation,,,) middle cerebral artery
by using this technique and recanalized it by topical
application of the calcium entry blocker nimodipine demonstrated
that the resulting endothelial damage consisted only of a mild
luminal discontinuity and surface projections. In addition, we
could not detect any cell damage after exposure of cultured
endothelial cells to rose bengal and light (data not shown).
Furthermore, we have already verified the absence of any
influence on cerebral parameters (for instance, CBF, CBV
fraction, and histological structures) and global physiological
parameters (such as systemic blood pressure, heart rate,
arterial pH,PaO2, PaCO2, and hematocrit) by means of either
irradiated controls without dye injection or controls with dye
injection and no illumination.18,19 However, after rose bengal
application, the brain is temporarily sensitive to light
wavelengths of 500 to 600 nm, and much care should be taken to
avoid illumination of tissues and other vessels next to the
target vein.
Conclusions
One of the pathophysiological consequences of
CVCDs is certainly hypoperfusion of circumscribed brain areas
drained by the occluded veins. In some of these territories the
supply of blood may fall below a critical threshold,resulting in
an ischemic or hypoxemic lesion. The comparison of a model of
solitary vein occlusion with one of dual vein occlusion has
shown that the extent of vein occlusion determines the size of
parenchymal injury. Moreover, inhibition of the growth of the
thrombus together with antiischemic measures could impede the
development of CVCDs. The use of the photochemical dye technique
to produce cerebral venous occlusion is a worthwhile addition to
study circulation perturbations of the brain(rat
skull microcirculation,,,).
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