160 E
1
increased K for instance stroke and TBI could influence
measure of perfusion pressure. A flow meter [11, Gil-
1
the overall response to K . mont Instruments, Barrington, IL, USA, connected to the
The purpose of this investigation was to study the tubing leading to the output reservoir, measured luminal
1
effects of changes in the concentration of extracellular K flow [5]. The luminal perfusate was gassed in the input
on the contractile state of the rat middle cerebral artery. reservoir. In addition, the luminal perfusate traveled
Specifically we tested two hypotheses: first, the constric- through gas permeable silastic tubing in the bath prior to
1
tion produced by increased extracellular K 30–80 mM
perfusing the lumen of each artery to ensure that it was as occurs following TBI in the rat middle cerebral artery
properly gassed and equilibrated to 378C. Samples of PSS MCA is attenuated by the stimulated production of NO in
from the bath and luminal perfusate were analyzed for endothelium and second, that increases in osmolality that
pO , pCO , and pH using a Corning model 280 analyzer
2 2
are observed after TBI [20,45], are responsible for the NO Medfield, MA, USA.
component. The vessels were magnified using an inverted micro-
scope equipped with a video camera and monitor. Outside diameters of the arteries were measured directly from the
video screen.
2. Materials and methods
The arteries were allowed at least 1 h to stabilize before conducting any experiments. During this time period the
2.1. Animals and harvesting arteries diameters decreased to approximately 75 of the initial
diameter after pressurization. This development of sponta- The experimental protocol was approved by the Animal
neous tone was indicative of a viable artery. Protocol Review Committee at Baylor College of Medi-
cine. Male Long Evans rats weighing between 275 and 350 g were anesthetized with isoflurane. After loss of the
2.3. General experimental outline and design righting reflex, each rat was decapitated; the brain was
removed from the cranium and placed in cold physiologi-
1
For concentration–response curves, the K concentra-
cal saline solution PSS. The left and right middle tion of the extraluminal bath was increased by the addition
cerebral arteries MCAs were carefully removed begin- of KCl to the extraluminal bath. Three methods were used
ning at the circle of Willis and continuing distally for
1 1
for increasing K : 1 isotonic K K
where increases
iso
approximately 5–6 mm. Left and right posterior cerebral
1 1
1
in K were offset by decreases in Na , 2 hypertonic K
arteries and segments of second and third order branches
1
K where K
was increased without a concomitant
hyper
of arteries in the mesenteric bed were also harvested.
1
adjustment of Na , and 3 a solution K using K
but
suc iso
with the addition of sucrose to obtain a hypertonic 2.2. Mounting the arteries
solution. The amount of sucrose added was calculated to produce the same osmotic effect as the addition of adding
Arteries were placed in an arteriograph Living Systems KCl to the extraluminal perfusate without any adjustment
Inc., Burlington, VT, USA where a micropipette was of other ions. The diameter was measured 5 min after
inserted into the proximal end of each artery and secured changing the KCl concentrations in the bath.
with a 10-O nylon suture. The lumen was gently perfused One or two concentration–response curves were con-
with PSS to remove blood and other contents and the distal ducted per MCA. Between each concentration–response
end was cannulated with a second glass micropipette and curve, the bath was washed with fresh PSS and the MCA
secured. A segment of each artery lying between branch was allowed 30 min to recover before the next con-
points was positioned between the tips of the two mi- centration–response curve was performed. In studies
cropipettes. Each artery was bathed in PSS which was where two concentration–response curves were conducted,
continually circulated from a reservoir where it was the MCAs were divided into control and experimental
equilibrated with a gas consisting of 20 O and 5 CO groups. In control vessels, two control concentration–
2 2
with a balance of N . After gassing, the pCO and pO response curves were obtained; in the experimental vessel
2 2
2
were approximately 7.37 and 140 mmHg, respectively. one control curve was obtained followed by the ex-
The PSS in the bath was maintained at 378C using a perimental concentration–response curve
L
-NAME, etc..
1
circulating water bath and heat exchange column [5]. The concentration–response curves to K
could be re- Luminal or transmural pressure was maintained by
peated at least three times without affecting the response. raising reservoirs, connected to the micropipettes by tygon
For purposes of simplifying each figure, the concentration– tubing, to the appropriate height above each artery. Trans-
response curves for the control MCAs were omitted. mural pressure was adjusted to 85 mmHg. Luminal
In several studies the endothelium was removed by perfusion was adjusted to 100 ml min by setting the two
passing 8 ml of air through the lumen of the vessels [5]. reservoirs at different heights. Pressure transducers be-
Care was taken to ensure that pressure did not exceed 85 tween the micropipettes and the reservoirs provided a
mmHg during this process. Removal of the endothelium
E .M. Golding et al. Brain Research 880 2000 159 –166
161
was confirmed by the absence of a dilation to the addition 3. Results
25
of 10 mol L UK14,304, an a agonist [5].
2
Fig. 1 shows the response of the MCAs to increases in
1
extracellular K . The data are expressed as a percent 2.4. Reagents and drugs
change from the resting diameter; positive numbers signify dilations and negative numbers signify constrictions. The
G 1
Sucrose, ouabain, serotonin 5-HT, and
L
-NAME [N - resting outside diameters K
5.88 mM for K , K
,
hyper iso
nitro-
L
-arginine methyl ester] were obtained from Sigma and K
were 25066 mm n510, 250611 mm n56,
suc 1
St Louis, MO, USA.
L
-NAME was dissolved in PSS and and 23868 mm n57, respectively. At K
concentrations added to both the luminal and extraluminal baths. The PSS
of 21 mM, the MCA diameters increased 15–20 regard-
1
consisted of the following [34]: NaCl 119 mM, NaHCO less of the method of K
addition. At greater concen-
3
24 mM, KCl 4.7 mM, KH PO 1.18 mM, MgSO 1.17 trations the diameters for the K
and K groups
2 4
4 hyper
iso
mM, CaCl 1.6 mM, glucose 5.5 mM, and EDTA 0.026 constricted from the maximum dilation at 21 mM. MCAs
2
mM. in the K
group were significantly more constricted at 51
iso
and 81 mM compared to K and K
Fig. 1A.
hyper suc
The response of the MCAs to K at 51 and 81 mM
hyper
2.5. Statistics prompted us to compare the K
response of the MCA to
hyper
that of another cerebral artery, the posterior cerebral artery Data are expressed as the mean6S.E.M. For comparison
PCA, and a peripheral artery from the mesenteric bed
1
of responses to KCl, the repeated measures analysis of Fig. 1B. K
of 21 mM K in the extraluminal bath
hyper
variance was initially attempted. However, in many of the increased the diameter of the MCAs and PCAs by 2662
comparisons the equal variance or normality tests failed n511 and 2761 n54, respectively. With subsequent
1
indicating that parametric statistical designs were not increases in the K
concentration, the diameter of the appropriate. In these situations, we used the Kruskal–
MCAs and PCAs decreased toward baseline; however, the
1
Wallis non-parametric test with a Bonferroni correction for decrease in diameter with increasing K
concentrations multiple comparisons Minitab, State College, PA, USA.
was sharper with the PCAs than with the MCAs. At 66 and
1
The acceptable level of significance was defined as P, 81 mM K , the PCAs were constricted significantly from
0.05. the resting diameter. The diameter change for the PCAs
Fig. 1. A Concentration–response curves in the rat MCA to hypertonic KCl K , isotonic KCl K
, and isotonic KCl1sucrose K . The resting
hyper iso
suc
diameters for K , K
, and K were 25066 mm n510, 250611 mm n56, and 23868 mm n57, respectively. P,0.05 compared to
hyper iso
suc
corresponding K and K
. B The effects of increasing K concentrations on the diameters of pressurized, perfused rat middle cerebral arteries
hyper suc
hyper
MCA, 21468 mm, n511, posterior cerebral arteries PCA, 196610 mm, n54, and mesenteric arteries Mes A, 306620 mm, n56. The change in diameter is expressed as a percent of the resting diameter. P,0.05 compared to the MCA at the same K
concentration; P,0.05 compared to the
hyper
MCA and PCA at the same K concentration.
hyper
162 E
was significantly different from the MCAs at 51, 66, and
L
-NAME could have been due to the increased tone more
1
81 mM K . In this study the MCAs did not return to constricted MCA and not to the inhibition of NO.
1
baseline; even at 81 mM K the MCAs were significantly
Therefore, to test for this possibility we constricted the
27 26
dilated 662, P,0.05 using Bonferroni correction. MCAs with either serotonin between 10
and 10 M
26
However, in some studies mean diameter was near or or 10
M ouabain. Serotonin and ouabain constricted the slightly below the resting baseline at the highest K
MCAs by 17 n59 and 12 n55, respectively. This
hyper
concentration see Fig. 1A. is compared to a 11 constriction produced by
L
-NAME.
1
Unlike the MCAs and PCAs, the pressurized perfused The response to 21 mM K
after constriction with either
1
mesenteric arteries did not dilate to increased K , due serotonin or ouabain was similar to the response in control
most likely to the absence of K s on the vascular smooth MCAs Fig. 3 or after treatment with
L
-NAME Fig. 2A.
ir 1
1
muscle. Additions of K produced significant constrictions
At K concentrations greater than 21 mM, the response in
beginning at 36 mM K and the arteries continued to
ouabain-treated MCAs was similar to the control response.
hyper
contract with increasing concentrations of K Fig. 1B.
However, the 5-HT-treated group showed more persistent
hyper 25
1
Fig. 2 shows the effects of
L
-NAME 10 M, an
dilations at increased K concentrations, not less, than the
1
inhibitor of nitric oxide synthase, on the response to K ,
control group. Clearly, the more constricted MCAs at K
hyper
K , and K
.
L
-NAME did not affect the percent change concentrations above 21 mM in the
L
-NAME treated
iso suc
in diameter to K 21 and 36 mM; however, it did
vessels was not due to the more constricted state of the
hyper 1
produce a significant reduction in the diameter compared vessel prior to the addition of K .
1
to the control response at K concentrations of 51, 66, and
Fig. 4 describes studies designed to determine if the
25
81 mM Fig. 2A. Addition of 10 M
L
-NAME, sig- endothelium was the source of the NO involved with the
nificantly reduced the diameter by 11 n59, P50.01, see K
response described in Fig. 2A. The endothelium was
hyper
legend Fig. 2A.
L
-NAME had no effect on the K removed by passing air through the lumen [5] and was
iso 1
concentration–response curve at any K concentration
confirmed in each vessel with UK14,304, an a agonist
2
Fig. 2B. However,
L
-NAME significantly constricted the which dilates MCAs through a mechanism requiring intact
MCAs compared to the control response at K con-
endothelium [5,6]. Before removal of the endothelium
suc 25
centrations of 51 and 81 mM Fig. 2C in a manner similar 10
M UK14,304 elicited a 12 dilation; after removal to that of
L
-NAME on the K concentration response.
with air the dilation was reduced to 1 P,0.001. After
hyper
The altered response to K after the addition of
removal of the endothelium the concentration–response
hyper
25
Fig. 2. A The effects of 10 M
L
-NAME, a nitric oxide synthase inhibitor, on the hypertonic KCl K concentration–response curve in rat middle
hyper
cerebral arteries. The resting diameters before the addition of K in the control and
L
-NAME groups were 21863 mm n59 and 19367 mm n59,
hyper 25
respectively. P,0.05 compared to Control response. B The effect of 10 M
L
-NAME on the isotonic KCl K concentration–response curve. Resting
iso
diameters before the addition of K in the Control and
L
-NAME groups were 250611 mm n56 and 213611 mm n56, respectively. C The effect of
iso 25
10 M
L
-NAME on the isotonic KCl1sucrose K concentration–response curve. Resting diameters before the addition of K
in the Control and
suc suc
L
-NAME groups were 23868 mm n57 and 18167 mm n56, respectively. P,0.05 compared to corresponding Control response.
E .M. Golding et al. Brain Research 880 2000 159 –166
163
of the endothelium appeared similar
L
-NAME group in
2
Fig. 2A and Endo in Fig. 4A, we conducted further
experiments to determine if the endothelium accounted for the entire NO involved in the response. Concentration–
1
response curves for K were conducted in MCAs after
2
removal of the endothelium in the absence Endo , Fig.
2
4B and presence of
L
-NAME Endo 1
L
-NAME, Fig. 4B. Although the addition of
L
-NAME appeared to constrict the vessels slightly more at 51, 66, and 81 mM
1
K , the differences were not statistically significant from
2
the Endo Control group. NO participating in the response
to K was derived predominantly, if not exclusively,
hyper
from the endothelium. Fig. 5 shows that MCAs dilated when the osmolality
was increased by adding sucrose to the extraluminal bath. The addition of
L
-NAME significantly attenuated the dilation indicating that NO is involved with the dilation
due to increased osmolality.
Fig. 3. The effects of constricting MCAs with either serotonin 5-HT,
27 26
26
between 10 and 10
M or ouabain 10 M on the hypertonic KCl
4. Discussion
