52 L
lated that elevated ET-1 levels that occur during the fluid replacement of blood taken through arterial catheter.
various phases e.g. hypoxic vs. reperfusion periods of Core
body temperatures
were servo-controlled
at ischemic stroke may compromise tissue perfusion of the
3760.28C via a heating pad connected to the temperature ischemic area in different ways. Under normoxic con-
controller Acadia Clinical. ditions, topical or intraparenchymal application of ET-1 or
ET-3 induces potent and long-lasting vasoconstriction of 2.1.2. Induction of focal cerebral ischemia with ET-1
cerebral vessels to the point of inducing severe ischemia Procedures for the induction of focal cerebral ischemia
and consequently
irreversible ischemic
injury using ET-1 Sigma, St. Louis, MO, USA were modified
[9,10,12,32,41,46,52], but the vasoconstrictive property of from those published previously [9,10,52]. After catheteri-
ET-1 under hypoxic and reoxygenation conditions is not zation of right femoral artery and vein, experimental
known. In addition, pharmacological studies have attempt- animals were placed in a stereotaxic frame David Kopf
ed to correlate the hypoperfusion effects of ET-1 during Instruments. An incision was made along the midline of
hypoxic-reperfusion periods
of the
ischemic stroke
the scalp. Two burr holes were made, one for a Laser [2,8,35,36,49] with its resultant pathophysiological effects
Doppler Pencil Probe P-434 using coordinates of: an- on cerebral tissues during these different phases of is-
terior 0.70 mm from bregma, lateral 20.18 mm from chemia, but the correlation remains inconsistent [33,37]
midline, and ventral 6.0 mm from dura, according to the and largely unknown.
Paxinos and Watson Rat Atlas [38] and the other for the The purpose of the present study was to determine: a
23-gauge guide cannula coordinates noted below in the the vasoactive effects of exogenous ET-1 given intrastriat-
right frontal hemisphere. A Laser Doppler probe was ally during normoxic, hypoxic 12 O , or reoxygenation
attached to the guide cannula and was 2.0 mm lateral from
2
periods on cerebral blood flow; and b if the timing the guide cannula. Relative changes of striatal cerebral
between ET-1 administration and hypoxia altered the blood flow CBF
were continuously monitored via the
LDF
resultant neuronal damage. Laser Doppler perfusion monitor Laserflo Model BPM
403A, TSI, MN, USA throughout the experiment in all groups. After steady state readings were obtained for
2. Materials and methods approximately 2–5 min, baseline control measurements of
CBF were recorded for 30 min before intrastriatal
LDF
2.1. Animal preparation administration of sterile saline SS or ET-1. Since relative
changes, but not absolute values, of CBF are reliably
LDF
2.1.1. General measured with Laser Doppler flowmetry [6,17,41], the
Experiments were done using Long–Evans rats aged relative change in cerebral blood flow over time following
from 9 to 11 weeks obtained from Charles River Lab- SS, ET-1, hypoxia, or reoxygenation were determined
oratories, Montreal, Quebec. Experimental animals were from respective baseline control values for each animal.
maintained under constant environmental conditions with ambient temperatures of 21618C, relative humidity of
2.2. Experimental protocol 30, and a 12 h light and dark cycle. Animals had free
access to standard food pellets and tap water. The Guide- Rats were randomly assigned to one of six groups:
lines of the Canadian Council of Animal Care in conjunc- Group 1, sterile saline SS 1 ml was stereotaxically
tion with the University Animal Care Committee were injected into the striatum and acted as a control group
followed for all experimental procedures. without hypoxia n510; Group 2, 1 ml of ET-1 40
Animals were anesthetized with halothane 4 for pmol, as based on former studies for focal lesion induc-
induction and 1.2 for maintenance via nose cone in a tion [9,10,52], was stereotaxically injected into the
mixture of 30 oxygen, balance nitrogen. Halothane levels striatum but no hypoxia was induced n58; Group 3, SS
were decreased to 0.7 in those groups receiving 35-min 1 ml was given into the striatum during normoxic period
of hypoxia 12 O balance N . Halothane concentration followed by the 35-min period of hypoxia 12 O
2 2
2
was strictly controlled to minimize its vasodilatory effects balance N n55; Group 4, 1 ml of ET-1 40 pmol was
2
since pilot studies showed a concentration-dependent given during normoxic period followed by the 35-min
increase in striatal CBF over time data not shown. The period of hypoxia n58; Group 5, 1 ml of ET-1 40 pmol
right femoral artery and vein were cannulated using PE-50 was given intrastriatally during the 35-min hypoxic period
tubing. The arterial catheter was connected to a Statham n58; and Group 6, 1 ml of ET-1 40 pmol was injected
pressure transducer PD23ID to continuously record mean into the striatum during reoxygenation period following
arterial blood pressure MABP and to collect serial hypoxia n510. All stereotaxic injections of saline or
arterial blood samples at 15, 65, and 90 min for blood gas ET-1 were delivered over 30 s into the striatum using the
analysis of pH, pCO , and pO Nova Biomedical, Stat
coordinates of: anterior 0.70 mm from bregma, lateral
2 2
Profile 5 at normoxic 30 and hypoxic 12 con- 20.38 mm from midline, and ventral 26.0 mm from dura,
centrations of oxygen. The venous catheter was used for according to the Paxinos and Watson Rat Atlas [38]. The
L . Park, J. Thornhill Brain Research 883 2000 51 –59
53
needle was left in place for a further 5 min to prevent physiological variables, CBF
and infarct volume were
LDF
reflex up the needle tract. Hypoxia was induced via a nose analyzed using the Student’s unpaired t-test, one-way or
cone with 12 oxygen balanced in nitrogen for 35 min. the two-way analysis of variance ANOVA with repeated
Schematic presentation of experimental protocol for each measures, or Mann–Whitney U-tests as appropriate.
of the six groups is shown in Fig. 1. Statistical significance was accepted at the level of 0.01 or
0.05 of probability. All values in the text and figures are 2.3. Histopathological assessment
presented as mean6S.E.M. In all instances, n refers to the number of individual animals in which observations were
Three days after the experiment, the striatal lesions of all made.
groups were assessed. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital 100 mg
kg and the brains were perfusion-fixed transcardially with
3. Results
