direct electrical stimulation or indirectly by stimulation of the mesencephalic reticular formation (MRF) resulted in a rapid dilatation of pial arterioles and venules, ...
Journal of Cerebral Blood Flow and Metabolism 7:315-326 © 1987 Raven Press, New York
Cortical Vasodilatation Produced by Vasoactive Intestinal Polypeptide (VIP) and by Physiological Stimuli in the Cat Tony L. Yaksh, Jia-Y i Wang, *Y. L. W. Go, with technical assistance by Gail J. Harty Departments of Pharmacology and Neurosllrgical Research, and *Department of Gastroenterology, Mayo Clinic and Foundation, Rochester, Minnesota, U,S,A,
Summary: In chloralose-urethanized cats, vasoactive in testinal peptide (VIP), applied by superfusion in steady state concentration (10-10_10-6 M) onto cortical vessels in situ resulted in a rapid concentration-dependent vaso dilatation in vessels that were mildly constricted by prosta glandin Fzo< (PGFzo
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polarization. As will be discussed later, a promi nent and immediate dilation was evoked by such stimulation, which was invariably greater than the maximum dilation evoked by VIP. During the course of these dose-response studies, blood pressure was not altered by any of the agents added to the superfusate. Measurement of perfusion pH with the addition of the P GF2a and the peptides revealed no changes at these drug con centrations in this buffered solution. T hough no power spectral analysis was per formed, cortical administration of VIP frequently resulted in de synchronization of the EEG patterns. T hese changes occurred 5- [0 min after the onset of vasodilatation and were frequently maintained for periods up to [0- [5 min after the removal of VIP solution.
Time (min) FIG. 2. Characteristics observed in a single cat (1-7-85) of
Vasomotor responses of pial vessels to cortical and
the vasodilatory effects of VIP administered by continuous cortical perfusion at increasing concentrations on pial vessels previously contracted by the coperfusion of prosta glandin F2a (5 X 10-5 M). At the time indicated by the black bar (Cor), direct cortical electrical stimulation was per formed. The dilation is expressed as percent change from nonstimulated diameter and represents the mean and SE of 8 and 6 concurrently measured arterioles and venules, re spectively.
MRF stimuli Activation of cerebral cortex, either by direct electrical stimulation or indirectly with ascending input evoked by the electrical stimulation of the re ticular activating system, results in a reliable in crease in the diameter of pial arterioles and venules and a simultaneous desynchronization of the cor tical electroencephalogram (EEG). Examination of micrographs taken before and during cortical acti vation evoked by direct stimulation or indirectly by stimulating MRF, indicated that the characteristics of the vasodilatation (e .g., hyperemia, increased number of visible collateral and anastomotic channels) were indistinguishable from the phenom enon characterized in Fig. I after focal VIP appli cation. As indicated in Fig. 3, this vasodilation oc-
VIP. The summary of the results of these experi ments carried out in the presence of P GF2a and hy pocarbia-constricted veins is presented in Table 2. As indicated by all measures, VIP activity was comparable when assessed under either condition. At the end of the experiment, as indicated in Fig. 2, direct cortical stimulation was carried out in the ab sence of VIP to examine the effect of electrical de-
TABLE 2. Dose-dependent vasodilation of cortical pial arterioles and veil Illes preconstricted with PGF2«a Venulesd.f
Arteriolesd.!
Preconstrictor Treatment ne PGF2a"
VIP
\3
Hypocarbiab
VIpe
11
+
Hypocarbiab
TTX VIP
Peptide Peptide Maximum % increase ECmax x 10-6 M ECso X 10-8 M 63 (52-64) 62 (53-71)
2 (8-63) 8 (5-15)
6 (2-17) 5 (2-15)
78 (68-88)
3 (0.8-\3)
3 (\-10)
n 15 16
Peptide Peptide Maximum o/r increase ECmax x 10-6 M ECso X 10-8 M 47 (40-54) 36 (31-41)
I (3-47) 8 (3-20)
5 (2-13) 6.1 (1-32)
41 (29-53)
3 (0.6-16)
I (0.3-4)
5 X 10-6 M PGF2a added to cortical perfusate. Animal maintained at Paco 2 25-28 for duration of dose-response curve. C Number of animals. d Each peptide effect presented represents the mean results of three cats , in which concentration-response curves were carried out at increasing concentrations of VIP. In each cat , 3-4 arterioles (resting diameter in the presence of PGF2a or hypocarbia : 100-200 f.1.m) and 3-5 venules (resting diameter in the absence of drug : 120-225 f.1.m) were studied. Results from each dose-response curve generated with each single arteriole or venule segment in that group were calculated and pooled. Thus , each ECso and ECmax computation repre sents 11-16 dose-response curves. e TTX (10-5 M) added in superfusate. f f.1. ± 95% Cl. a
b
J Cereb Blood Flow Metab, Vol. 7. No.3, 1987
320
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in disproportionate increases in resting diameter. In spite of this clear deviation from the expected values, the slope of the regression lines for the re sponse of the venuoles to either VIP, cortical, or MRF stimulation ranged from -0.05 to -0.07. These values are 0.5-0.25 of those slopes observed for the arteriolar response to these same manipula tions (-0.19 to -0.23).
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FIG. 3. Time course observed in a single cat (2-9-85) of effect
of direct cortical (top) and mesencephalic reticular forma tion (MRF) (bottom) stimulation at the time indicated by the black bar on the diameter of pial arterioles and venules as a function of time. These results were obtained sequentially and represent the mean and SE of four pial arterioles and five pial venules, which were examined under both condi tions.
cured with an extremely short latency. Near-max imal increases in arteriolar diameters were frequently noted in less than 10-15 s after the initi ation of stimulation (see below). P ial vessels re mained dilated throughout the period of stimula tion, and as indicated in Fig. 3, the vasodilatation frequently outlasted the stimulation for up to sev eral minutes. Relationship between resting vessel diameter and the magnitude of the evoked increase
The percent increase in vessel diameter after ei ther VIP (10-6 M), cortical or MRF stimulation (Fig. 4) was inversely proportional to the resting di ameter of the arteriole or venuole. The relationship between percent dilation as a function of the resting diameter during either cortical or MRF stimulation could be described by a linear regression model. Thus, the correlation coefficients for the linear re gression lines, established for either VIP, cortical, or MRF stimulation with arterioles was 0.81-0.89. However, the regression line for the venuoles was less adequately described by a linear regression model, as correlation coefficients were on the order of 0.6. Inspection of the graphs in Fig. 4 indicates that among the smaller venuoles «50 f,Lm), both VIP and the cortical and MRF stimulations resulted
J Cereb Blood Flow Metab, Vol. 7, No.3, 1987
To determine whether or not the dilatory effects of VIP on direct cortical stimulation were modified or mediated by other cortical neurotransmitter systems, phentolamine (10-4 M), propranolol (10-4 M), naloxone (10-4 M), or atropine (10-4 M) were administered into the cortical superfusate 30 min prior to examining the vasodilatory effects of VIP (10-6 M) in the superfusate and direct cortical stim ulation of VIP. Each antagonist was examined in 2 cats, with 3-5 arterioles and venules per animal. The sequence of treatment was alternated in each of the two cats. Thus, in one cat, the paradigm re quired administration of VIP (10-6 M) for 15 min. Thirty minutes after the removal of VIP, direct cor tical stimulation was initiated for 5 min. In the second cat, the sequence was reversed. As indi cated in Table 3, none of the several antagonists at the concentrations employed had any prominent ef fect on the vasodilatory effect of direct cortical stimulation or VIP (one-way analysis of variance F values all < 1.0, df 4/22 and 4/24 for venules and arterioles, respectively). =
Dilatory effects of VIP and electrical stimulation on cortical vessels in the presence of TTX
To determine whether the dilatory effects of VIP or direct cortical stimulation might be secondary to increased neuronal activity, concentration-re sponse curves were carried out (as illustrated in Fig. I) in two cats with a 60-min prior exposure to tetrodotoxin (TTX; 10-5 M in cortical superfusate). P revious studies have shown this treatment to de press MRF-evoked VIP release (Wang et aI., 1986), suggesting an effective blockade of sodium-depen dent conduction. As indicated in Table 2, TTX had no effect on the maximum percent increase, the ECmax, or EC50 for cortically applied VIP as com pared to non-TTX-treated controls. (Two-sample t test: t values for arterioles and venules ranged from 0.301 to 0.615; df 22 and 29 for arterioles and venules, respectively.) Comparable experiments were carried out at a single concentration of VIP (10-6 M) and direct cortical stimulation. As shown in Table 3, TTX again had no effect on VIP -evoked dilation. With =
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VESSEL DIAMETER (uM) FIG. 4. Percentage dilatation in pial arteries (top) and veins (bottom) as a function of pre-VIP diameter, induced by topical VIP
(10-6 M, left), direct cortical stimulation (middle) or MRF stimulation (right). Each dot represents a single vessel. Data represent the summary of results from 16, 8, and 8 cats, respectively.
regard to direct cortical stimulation, TTX not only failed to block the effects of direct depolarization, but significantly enhanced the magnitude of arteri olar dilation (Table 3). The dilation of the venules
TABLE 3. Effects of pharmacolo!;ical anta!;onists
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the percent change q{pial arterioles and venllle,l' durin!; the application of VIP on direct cortical stimulation a Treatment VIP (10-6 M) Arteriole
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also appeared to be augmented, but this was not statistically significant. Comparison of the dilatory effects of VIP and cortical electrical stimulation
Systematic comparison of the maximum vasodi latory effect produced by VIP (10-6 M), a dose pro ducing maximal dilation (see Table 2), versus direct cortical activation was permitted by the control (CSF) experiments outlined above. In these experi ments, with populations of vessels of comparable size, the mean arteriolar dilation evoked by VIP was 56% of control, which is less than that evoked by cortical stimulation (91%); (paired t test; t 3.21, df 9, p < 0.01). A similar statistical differ ence was observed with the effects of these manip ulations on the venules (paired t test; t 3.11, df 8, p < 0.01). Further support for this difference can be assessed by the joint consideration of all ap plicable data from all groups. Of 66 arteries and 72 veins examined (n 18 cats), in 61 and 69, respec tively, the maximum effects of direct cortical stimu lation were greater than those produced by 10-6 M VIP. =
Control (CSF)
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+ Phentolamine
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and venules measured in each animal. as described in the text. Mean arte riole and venule diameter for vessels, from all groups, prior to stimulation was
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Effects of VIP antisera of vasodilatation elicited by cortical activation
Because of the lack of specific antagonist for the VIP receptor, a high-titer VIP antiserum was used
J Cereb Blood Flow Metah. Vol. 7, No.3, 1987
T. L. YAKSH ET AL.
322
tion evoked by cortical stimulation. Thus, within I min after stimulation, in excess of 50% of the max imum effect was observed. VIP antisera (l :25) alone had no effect on resting venule or arteriole diameter after 40 min. As shown, however, there was a clear delay in the initial dilation, which pla teaued shortly at a level that was about 30-40% below that produced in the presence of NRS. With continued stimulation, there was invariably a sub sequent rapid elevation for the duration of the stim ulation. Figure 5 also presents a similar experiment (cat 11-6-84) showing the effects of NRS and VIP anti sera on the dilation evoked by MRF stimulation. A comparable delay in the onset of stimulation and the appearance of a shoulder in the dilation time course was also seen in these MRF-evoked in-
in an attempt to block the vasodilatation elicited by electrical stimulation and by exogenous VIP. In such experiments, normal rabbit serum from a non immunized rabbit was used as a control serum to ascertain whether or not any effects observed were due to the nonspecific effect of sera proteins. The VIP antiserum or the normal rabbit serum, in ap propriate concentrations, was given and maintained (preincubated) in cortical cups 40 min before exog enous VIP or electrical stimulation was applied to allow for diffusion of serum proteins. Figure 5 presents one of four cats (cat 11-1-84) showing the effects of normal rabbit serum (NRS), VIP antisera, and subsequently NRS on the dilation of arterioles and venules evoked by cortical stimu lation. NRS had no effect on resting vessel diam eter and did not noticeably alter the degree of dila-
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