Brain Research, 588 (1092) 140-143 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
140
BRES 25244
Short Communications
The oligemic phase of cortical spreading depression is not blocked by tirilazad mesylate (U-74006F) P e t e r J. G o a d s b y Department of Neurology, The Prince Henry Hospital, Little Bay, NSW (Australia) (Accepted 24 March 1992)
Key words: Cerebral blood flow; Laser Doppler: Cat:. Brain injury
Cortical spreading depression is characterised by a wave of depolarization that moves across the cortex leaving in its wake a state of hyperpolarization. Characteristic changes in cerebral blood flow are also seen and these consist of a wave of hyperemia followed by an oligemia, the latter lasting some hours in some experimental animals including the cat. in this study cerebral blood flow was measured using laser Doppler flowmetry in the anesthetised ventilated cat. Spreading depression was initiated with a pin-prick injury prior to or following administration of U74006F (tirilizad mesylat¢; 3 mg/kg, ivi) or an identical volume of vehicle. Laser Doppler probes were placed bilaterally and in each case studied both the hyperemic and oligemic phases of spreading depression were preserved after administration of either U74006F or its vehicle. These data suggest that free radical mechanisms have no significant role in mediating the blood flow changes of spreading depression and are consistent with data in the literature of a quantitative using single-point measurements that again U74006F does not affect spreading depression.
The cortical spreading depression of Leao (SD) was first reported to occur in the exposed rabbit cortex '~'~° and is characterised as a negative shift in cortical DC potential that is accompanied by changes in cerebral blood flow and cerebrovascular reactivity ~. This shift corresponds ionically to a redistribution of K, Na, CI, Ca and H ions that has been carefully assessed by micropipette studies ~'. Several features of SD have been used to characterise it and these include a rate of propagation of 2-6 mm/min, limitation to one hemisphere and a refractory period for further SD of up to 3 min. Recently we have developed a model of SD by monitoring cerebral cortical perfusion with laser Doppler flowmetry (CBFLDF). The changes in blood flow are highly reproducible and offer a relatively simple method to monitor and assess the phenomenon. The observation of both left and right cortices has greatly facilitated the study of the phenomenon with changing conditions or pharmacological intervention. SD has been observed in a number of species including rat 7, cat ~ and in man ~7. Although the latter observation has proved difficult to reproduce 14. The changes can include subcortical structures, such as the
caudate nucleus ~7 and thalamus ~3, which seem to be less quantitatively affected ~a. In association with the ionic changes and shift in DC potential that are the electrophysiological markers of SD distinct changes in cerebral blood flow have been described. Initially flow may increase after SD has been initiated and the increase has been reported to be up to 200% s and postulated to be related to the pre-SD level of blood flow ~'~. This is followed by a prolonged moderate reduction in cerebral blood flow 7't8 that is associated with a marked blunting of cerebrovascular responses to hypercapnia with normal autoregulations. Recently it has been shown that the reduction in cerebral blood flow seen after SD may involve not only the cortex but may also include subcortical structures and even the brainstcm ~2. Moreover, it has been suggested that the anti-oxidant Tirilazad mesylate (U74006F) may block the oligemic phase of SD implying a hitherto unsuggested free-radical role in the phenomenon 4. This latter observation has recently ~ been challenged suggesting some important interaction may be occuring. Laser Doppler flowmetry is ideally suited to address a possible role for the drug by allow-
Correspomlence: P.J. Goadsby, Department of Neurology, The Prince Henry Hospital, Anzac Parade, Little Bay, NSW 2036, Australia.
141 ing sequential and in our hands bilateral measurements contemporaneously. Ten cats weighing 2.8 +_0.4 kg (mean +_ S.D.) were anesthetised with a-choloralose (60 mg/kg, i.p. with supplements of 10 mg/kg i.v.) after induction with 4% halothane. They were intubated and ventilated (Harvard Pump, MA) and paralysed with gallamine triethiodide (6 mg/kg, i.v.). End-expiratory CO 2 and fraction of inspired 0 2 were continuously monitored (DATEX, Finland). The animals were mounted in a sterotaxic device (David Kopf, CA). Polyethylene catheters were also placed into the femoral artery for monitoring blood pressure and into the vein for fluid and drug administration.
Cerebral cortical perfusion with laser Doppler flowmetry (CBFLor). The theory and use of LDF as it has been applied in this laboratory has been recently described in detail 3. Briefly, biparietal burr holes were placed using a low speed dental drill with care to avoid damage to the underlying cortex. The dura was left intact for these studies. CBFLo F. was measured with a BPM 403a (TSI Instruments, St Paul, MN) laser Doppler flowmeter and a fine probe mounted in a stereotaxic holder. The probe was carefully placed under microscope control so as to not markedly distort the dura. A small amount of parafin was placed around the probe to prevent drying of the tissue. All probe signals were monitored on-line. On-line monitoring. In order to determine accurately the changes in cerebrovascular dynamics measured with the laser Doppler the physiological variables were monitored on-line by a microcomputer. The blood pressure, heart rate, end-expiratory CO 2, inspiratory O2 and laser Doppler volume, flow and velocity signals were passed to a signal conditioning device and then to an analog-to-digital convertor (LabMaster DMA, Scientific Solutions, OH) in an 80286/80287-based microcomputer (APC IV Powermate II, NEC, Japan). Data could be monitored by averaging over a respiratory pump cycle. The ventilation cycle was monitored by a photoelectric transducer on the pump that was in turn connected to the digital input of the analog-to-digital board. The input of all signals was collected and averaged over the pump cycle and stored as the mean for the pump cycle in the data file. All data were stored on disk for later analysis and plotting. Study design. The purpose of the study was to determine whether a normal cortex responding to hypercapnia by a brisk stimulus-locked hyperemia would have a typical biphasic hyperemic/oligemic spreading depression after tirilazad mesylate administration. The animal was preFared for bilateral flow studies. On the initial side (callc ' ~ide 1) a spreading depression was induced
Spreading depre~i~ after needle stick injury I I I I I
I LDF1
( --.~.) .
.
-
I
-388 300
~ t
I
f",,
I.ILIF2
('-~)
I I Fig. 1. Computer processed data showing a tracing of end-expiratory CO 2 (EECO2) and two laser Doppler flow outputs (LDFI and LDF2) plotted against time (solid bar represents 2 min). Both the CO 2 and laser Doppler data hat been converted to the percentage change from the control period wh,,:h is that period before the vertical dashed line. The changes in CBFLDv are shown following a needle stick injury with the typical hyperemic/oligemic picture of spreading depression.
by a standard 3 mm needle stick injury to the parietal cortex with a 26 gauge needle. The changes of SD were observed and then 30 min later the SD was repeated. Either active compound or vehicle were then randomly administered in doses of 3 mg/kg (i.v.i.) or the vehicle volume equivalent. Hypercapnic vasodilatation was then tested to ensure the integrity of side 2 and a SD was provoked on that side and the response observed. Statistics. The hyperemic and oligemic responses on side 1 before the drug or vehicle administration were compared to the mean maximal responses on side 2 after administration by means of a one-way ANOVA. Included in the comparison was a second SD on side 1 to particularly examine the effect of repeated SD. A significant difference was assessed at the P0.05 level using Dunnett's t-test 2. The baseline physiological data from the ten animals were all well within normal limits for the anesthetised cat. CBFLDr after SD. Needle stick injury resulted in a typical biphasic change in CBFLov at a latency consis-
TABLE I
Effect of vehicle or tirilazad mesylate on the hyperemic and oligemic phases of spreading depression Control (%)
Side I After vehicle
After tirilazad
Side 2 Repeat 1 (%)
297~± 47 20 ± 6
307 -4-49 23 +_ 6
300 + 42 2± 5
(%)
Hyperemia Oligemia
310 + 51 22-4- 7
(%)
! Indicates a second needle stick injury on the same side as the first.
142 Spreedin9 depressim~ after needle stick ( r e p o t )
£~02 (~...,)
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Fig. 2. Computer processed plot similar to Fig. I. A repeat needle stick injury fails to elicit a further oligemia.
tent with a rate of propagation of 3-5 mm/min. A typical response is shown in Fig. 1, while the mean data for the cohort examined are in Table I. Repetition of the needle prick 30 min after the first insertion always provoked an additional SD but this second SD never had an oligemic component (Fig. 2). Tirilazad mesylate. Administration of either tirilazad mesylate or the appropriate volume of its vehicle did not alter resting CBFLD F. Needle stick injury contralateral to the initial side used (side 2) produced in every case tested a normal SD response with both hyperemic and oligemic phases that were no different from the control responses (d2, s = l.l, P < 0.01; Figs. 3 and 4, Table l). These data clearly demonstrate that tirilazad mesylate or vehicle do not affect any part of the CBFLD ~ changes associated with needle stick injury-induced SD. Indeed it is clear that once the cortex has had an SD induced that it neither responds to hypercapnia nor does it express an oligaemia if subsequent SD is elicited. The suggestion that tirilazad mesylate could block KCI-induced SD oligaemia in rat leads to the possibil-
Fig. 4. Computer processed tracing similar to Fig. 1 showing no change in either the hyperemic or oligemic phases of SD after pre-treatment with vehicle.
ity that lipid peroxidation and free radical generation may be involved in least the cerebral blood flow changes seen with S D 4. The data presented here using CBFLD F and the complementary data from Duckrow ~ in the rat indicate that this may not be the case. Indeed, indomethacin despite enhancing the vasodilator phase of SD does not appear to affect the constictor phase mS. Furthermore release of local metabolites also does not seem to play a significant role in SD as has been shown very elegantly by Busija et ai. m6. What could account for these opposite results? The original study employed the method of hydrogen clearance to determine cerebral blood flow. It is possible that the changes in flow that were measured are deeper in the cortex that the 2.5 mm 3 examined with the Doppler technique. If this were the case then the autoradiographic approach of Duckrow ~ should have had the same results. On the other hand if temporal resolution was important for looking at the changes and it is certainly true that the changes in cerebral blood flow with SD have important temporal patterns than perhaps these were better ap-
I
158
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l
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(~)
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~.
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150
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l'-'\ /
,
, ) -IooL
'-[
LDP). . . . . . . (,-4 -lOU L
Fig. 3. Computer processed plot as for Fig. ! showing the response to hypercapnia after a unilateral spreading depression. The physiological vasodilator response is blocked on the side ipsilateral to the SD.
~_ I
LDFI (~,)
I I
-~58
'-"~
A.
[_ A
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. . . .
liI
Fig. 5. Computer processed tracing similar to Fig. 1 showing typical cortical blood flow changes of SD despite the presence of tirilazad mesylate (3 mg/kg, i.v.i.).
143
preciated by hydrogen clearance. Laser Doppler has better temporal resolution than hydrogen clearance however, being able to update the flow signal every 100 ms compared to some seconds being required for a hydrogen washout curve at best. The laser Doppler gives the advantage of being able to test the hypercapnic cerebral vasodilator response, if it is not present it is highly likely that a SD has been provoked and from the data in this study this alone will eliminate the oligemic phase. Since when using hydrogen clearance a needle stick injury must be executed to implant the probe for its measurements, the possibility of a SD occuring must be high. Indeed without explicit evidence that hypercapnic vasodilatation is present acute hydrogen clearance data must be viewed with a good measure of suspicion and caution. It is possible that such a situation explains these conflicting studies. A further technical consideration might be the different cortical regions measured by each method. Laser Doppler measures very superficial cortex while being an implanted method hydrogen clearance by necessity measures at a deeper level in the cortex. Perhaps these different regions have subtle differences in the role of lipid peroxidation in the manifestations of SD. Another possible explanation for the different results might be the different species, cat being used here while rat was used in the initial study. Again given the rat autoradiographic study ~ this must be a remote possibility. The other major consideration might be the dose of the drug used. There is some evidence that tirilazad has U-shaped dose-response curve (Hall, personnal communication). ' The dose of 3 mg/kg would seem reasonable and a more detailed dosing study might be in order but could only be justified by a clear demonstration that in rat with intact hypercapnic vasodilatation there was the reported effect on SD. In summary, in the anesthetised cat needle stick injury produced a very reproducible hyperemic/ oligemic response in the cerebral cortical blood flow that could not be attenuated in normal cortex by tirilazad mesylate. The oligemic phase of the response is not present on subequent needle sticks indicating that accidental induction of a SD would be sufficient to produce a blockade of the oligaemia that might then be attributed to a pharmacological manipulation. These data support the view that the changes in brain blood flow seen with SD are not due in any crucial part to lipid peroxidation or mediated by free radicals. Tirilazad mesylate (U74006F) was a generous gift from the Upjohn Co and valuable discussion on these experiments with Dr E. Hall was appreciated. The author thanks Karen Hoskin for her excellent technical assistance and Dr Brad Duckrow for his very useful discus-
sions concerning the data. The author also thanks the Department of Medical Illustration, University of New South Wales, for photographing all figures. This programme has been supported by the National Health and Medical Research Council of Australia and by grants from Warren and Cheryl Anderson, The J.A.W. Perini Family Trust, the Basset Trust, the Ramacciotti Foundation and the Australian Brain Foundation. P.J.G. is a Wellcome Senior Research Fellow.
1 Duckrow, R.B. and Beard, D.C., Cortical hypoperfusion associated with spreading depression persists in awake rats after treatment with tirizalad mesylate (U-74006F), Proc. Soc. Neurosci, 17 (1991) 865. 2 Dunnett, C.W., A multiple comparison procedure for comparing several treatments with a control, J. Am. Stat. Assoc., 50 (1955) 1096-1121. 3 Goadsby, P.J., Characteristics of facial nerve elicited cerebral vasodilatation determined with laser Doppler flowmetry, Am. J. Physiol., 260 ( 1991 ) R255- R262. 4 Hall, E.D. and Smith, S.L., The 21-aminosteroid antioxidant tirilazad mesylate, U-74006F, blocks cortical hypoperfusion following spreading depression, Bra#l Res., 553 (1991) 243-248. 5 Hansen, A.J., Quistorff, B., and Gjedde, A., Relationship between local changes in cortical blood flow and extracellular K + during spreading depression, Acta Physiol. Scand., 109 (1980) I-6. 6 Kraig, R.P. and Nicholson, C., Extracellular ionic variations during spreading depression, Neuroscience, 3 ( ! 978) 1045-1059. 7 Lauritzen, M., Jorgensen, M.B., Diemer, N.H., Gjedde, A. and Hansen A.,I., Persistent oligaemia of rat cerebral cortex in the wake of spreading depression, Ann. NeuroL, 12 (1982) 469-474. 8 Lauritzen, M., Long-lasting reduction of cortical blood flow of the rat brain after spreading depression with preserved autoregulation and impaired CO 2 response, J. Cereb. Blood Flow Metab., 4 (1984) 546-554. 9 Leap, A.A.P., Spreading depression of activity in cerebral cortex, J. Neurophysiol., 7 (1944) 359-390. 10 Leap, A.A.P., Pial circulation and spreading activity in the cerebral cortex, J. NeurophysioL, 7 (1944) 391-396. I! Marshall, W.H., Spreading cortical depression of Leap, Physiol. Rec., 39 (1959) 239-288. 12 Mraovitch, S., Calando, Y. and Seylaz, J., Long-lasting cerebral blood flow and metabolic changes within the limbic and brainstem regions following cortical spreading depression in rat, J. Cereb. Blood Flow Metab., 9 (1989) $508. 13 Mraovitch, S., Calando, Y., Goadsby, P.J. and Seylaz, J., Subcortical cerebral blood flow and metabolic changes elicited by cortical spreading depression in rat, Cephalalgia, 12 (1992) in press. 14 Piper, R.D., Matheson, J.M., Hellier, M., Vonau, M., Lambert, G.A., Olausson, B. and Lance, J.W., Cortical spreading depression is not seen intraoperatively during temporal Iobectomy in humans, Cephalalgia, 11 (Suppl 11)(1991) I. 15 Shibata, M., Leffler, C.W. and Busija, D.W., Cerebral hemodynamics during cortical spreading depression in rabbits, Bra#t Res., 530 (1990) 267-274. 16 Shibata, M., Leffler, C.W. and Busija, D.W., Evidence against parenchymal metabolites directly promoting pial arteriolar dilation during cortical spreading depression in rabbits, Brabz R,,s. Bull., 26 (1991) 753-758. 17 Sramka, M., Brozek, G., Bures, J. and Nadvornik, P., Functional ablation by spreading depression: possible use in human stereotactic neurosurgery, Appl. NeurophysioL, 40 (1977) 48-61. 18 Tomida, S., Wagner, H.G., Klatzo, I. and Nowak, T.S., Effect of acute electrode placement on regional CBF in the gerbil: a comparison of blood flow measured by hydrogen clearance, [3H]-nicotine, and [14C]-iodo-antipyrine techniques, J. Cereb. Blood Flow Metab., 9 (1989) 79-86. 19 Wahl M., Lauritzen, M. and Schilling, L., Changes of cerebrovascular reactivity after cortical spreading depression in cats and rats, Brain Res.,411 (1987) 72-80.
140
BRES 25244
Short Communications
The oligemic phase of cortical spreading depression is not blocked by tirilazad mesylate (U-74006F) P e t e r J. G o a d s b y Department of Neurology, The Prince Henry Hospital, Little Bay, NSW (Australia) (Accepted 24 March 1992)
Key words: Cerebral blood flow; Laser Doppler: Cat:. Brain injury
Cortical spreading depression is characterised by a wave of depolarization that moves across the cortex leaving in its wake a state of hyperpolarization. Characteristic changes in cerebral blood flow are also seen and these consist of a wave of hyperemia followed by an oligemia, the latter lasting some hours in some experimental animals including the cat. in this study cerebral blood flow was measured using laser Doppler flowmetry in the anesthetised ventilated cat. Spreading depression was initiated with a pin-prick injury prior to or following administration of U74006F (tirilizad mesylat¢; 3 mg/kg, ivi) or an identical volume of vehicle. Laser Doppler probes were placed bilaterally and in each case studied both the hyperemic and oligemic phases of spreading depression were preserved after administration of either U74006F or its vehicle. These data suggest that free radical mechanisms have no significant role in mediating the blood flow changes of spreading depression and are consistent with data in the literature of a quantitative using single-point measurements that again U74006F does not affect spreading depression.
The cortical spreading depression of Leao (SD) was first reported to occur in the exposed rabbit cortex '~'~° and is characterised as a negative shift in cortical DC potential that is accompanied by changes in cerebral blood flow and cerebrovascular reactivity ~. This shift corresponds ionically to a redistribution of K, Na, CI, Ca and H ions that has been carefully assessed by micropipette studies ~'. Several features of SD have been used to characterise it and these include a rate of propagation of 2-6 mm/min, limitation to one hemisphere and a refractory period for further SD of up to 3 min. Recently we have developed a model of SD by monitoring cerebral cortical perfusion with laser Doppler flowmetry (CBFLDF). The changes in blood flow are highly reproducible and offer a relatively simple method to monitor and assess the phenomenon. The observation of both left and right cortices has greatly facilitated the study of the phenomenon with changing conditions or pharmacological intervention. SD has been observed in a number of species including rat 7, cat ~ and in man ~7. Although the latter observation has proved difficult to reproduce 14. The changes can include subcortical structures, such as the
caudate nucleus ~7 and thalamus ~3, which seem to be less quantitatively affected ~a. In association with the ionic changes and shift in DC potential that are the electrophysiological markers of SD distinct changes in cerebral blood flow have been described. Initially flow may increase after SD has been initiated and the increase has been reported to be up to 200% s and postulated to be related to the pre-SD level of blood flow ~'~. This is followed by a prolonged moderate reduction in cerebral blood flow 7't8 that is associated with a marked blunting of cerebrovascular responses to hypercapnia with normal autoregulations. Recently it has been shown that the reduction in cerebral blood flow seen after SD may involve not only the cortex but may also include subcortical structures and even the brainstcm ~2. Moreover, it has been suggested that the anti-oxidant Tirilazad mesylate (U74006F) may block the oligemic phase of SD implying a hitherto unsuggested free-radical role in the phenomenon 4. This latter observation has recently ~ been challenged suggesting some important interaction may be occuring. Laser Doppler flowmetry is ideally suited to address a possible role for the drug by allow-
Correspomlence: P.J. Goadsby, Department of Neurology, The Prince Henry Hospital, Anzac Parade, Little Bay, NSW 2036, Australia.
141 ing sequential and in our hands bilateral measurements contemporaneously. Ten cats weighing 2.8 +_0.4 kg (mean +_ S.D.) were anesthetised with a-choloralose (60 mg/kg, i.p. with supplements of 10 mg/kg i.v.) after induction with 4% halothane. They were intubated and ventilated (Harvard Pump, MA) and paralysed with gallamine triethiodide (6 mg/kg, i.v.). End-expiratory CO 2 and fraction of inspired 0 2 were continuously monitored (DATEX, Finland). The animals were mounted in a sterotaxic device (David Kopf, CA). Polyethylene catheters were also placed into the femoral artery for monitoring blood pressure and into the vein for fluid and drug administration.
Cerebral cortical perfusion with laser Doppler flowmetry (CBFLor). The theory and use of LDF as it has been applied in this laboratory has been recently described in detail 3. Briefly, biparietal burr holes were placed using a low speed dental drill with care to avoid damage to the underlying cortex. The dura was left intact for these studies. CBFLo F. was measured with a BPM 403a (TSI Instruments, St Paul, MN) laser Doppler flowmeter and a fine probe mounted in a stereotaxic holder. The probe was carefully placed under microscope control so as to not markedly distort the dura. A small amount of parafin was placed around the probe to prevent drying of the tissue. All probe signals were monitored on-line. On-line monitoring. In order to determine accurately the changes in cerebrovascular dynamics measured with the laser Doppler the physiological variables were monitored on-line by a microcomputer. The blood pressure, heart rate, end-expiratory CO 2, inspiratory O2 and laser Doppler volume, flow and velocity signals were passed to a signal conditioning device and then to an analog-to-digital convertor (LabMaster DMA, Scientific Solutions, OH) in an 80286/80287-based microcomputer (APC IV Powermate II, NEC, Japan). Data could be monitored by averaging over a respiratory pump cycle. The ventilation cycle was monitored by a photoelectric transducer on the pump that was in turn connected to the digital input of the analog-to-digital board. The input of all signals was collected and averaged over the pump cycle and stored as the mean for the pump cycle in the data file. All data were stored on disk for later analysis and plotting. Study design. The purpose of the study was to determine whether a normal cortex responding to hypercapnia by a brisk stimulus-locked hyperemia would have a typical biphasic hyperemic/oligemic spreading depression after tirilazad mesylate administration. The animal was preFared for bilateral flow studies. On the initial side (callc ' ~ide 1) a spreading depression was induced
Spreading depre~i~ after needle stick injury I I I I I
I LDF1
( --.~.) .
.
-
I
-388 300
~ t
I
f",,
I.ILIF2
('-~)
I I Fig. 1. Computer processed data showing a tracing of end-expiratory CO 2 (EECO2) and two laser Doppler flow outputs (LDFI and LDF2) plotted against time (solid bar represents 2 min). Both the CO 2 and laser Doppler data hat been converted to the percentage change from the control period wh,,:h is that period before the vertical dashed line. The changes in CBFLDv are shown following a needle stick injury with the typical hyperemic/oligemic picture of spreading depression.
by a standard 3 mm needle stick injury to the parietal cortex with a 26 gauge needle. The changes of SD were observed and then 30 min later the SD was repeated. Either active compound or vehicle were then randomly administered in doses of 3 mg/kg (i.v.i.) or the vehicle volume equivalent. Hypercapnic vasodilatation was then tested to ensure the integrity of side 2 and a SD was provoked on that side and the response observed. Statistics. The hyperemic and oligemic responses on side 1 before the drug or vehicle administration were compared to the mean maximal responses on side 2 after administration by means of a one-way ANOVA. Included in the comparison was a second SD on side 1 to particularly examine the effect of repeated SD. A significant difference was assessed at the P0.05 level using Dunnett's t-test 2. The baseline physiological data from the ten animals were all well within normal limits for the anesthetised cat. CBFLDr after SD. Needle stick injury resulted in a typical biphasic change in CBFLov at a latency consis-
TABLE I
Effect of vehicle or tirilazad mesylate on the hyperemic and oligemic phases of spreading depression Control (%)
Side I After vehicle
After tirilazad
Side 2 Repeat 1 (%)
297~± 47 20 ± 6
307 -4-49 23 +_ 6
300 + 42 2± 5
(%)
Hyperemia Oligemia
310 + 51 22-4- 7
(%)
! Indicates a second needle stick injury on the same side as the first.
142 Spreedin9 depressim~ after needle stick ( r e p o t )
£~02 (~...,)
1~_1|
1 -
--
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-L
-
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~
Fig. 2. Computer processed plot similar to Fig. I. A repeat needle stick injury fails to elicit a further oligemia.
tent with a rate of propagation of 3-5 mm/min. A typical response is shown in Fig. 1, while the mean data for the cohort examined are in Table I. Repetition of the needle prick 30 min after the first insertion always provoked an additional SD but this second SD never had an oligemic component (Fig. 2). Tirilazad mesylate. Administration of either tirilazad mesylate or the appropriate volume of its vehicle did not alter resting CBFLD F. Needle stick injury contralateral to the initial side used (side 2) produced in every case tested a normal SD response with both hyperemic and oligemic phases that were no different from the control responses (d2, s = l.l, P < 0.01; Figs. 3 and 4, Table l). These data clearly demonstrate that tirilazad mesylate or vehicle do not affect any part of the CBFLD ~ changes associated with needle stick injury-induced SD. Indeed it is clear that once the cortex has had an SD induced that it neither responds to hypercapnia nor does it express an oligaemia if subsequent SD is elicited. The suggestion that tirilazad mesylate could block KCI-induced SD oligaemia in rat leads to the possibil-
Fig. 4. Computer processed tracing similar to Fig. 1 showing no change in either the hyperemic or oligemic phases of SD after pre-treatment with vehicle.
ity that lipid peroxidation and free radical generation may be involved in least the cerebral blood flow changes seen with S D 4. The data presented here using CBFLD F and the complementary data from Duckrow ~ in the rat indicate that this may not be the case. Indeed, indomethacin despite enhancing the vasodilator phase of SD does not appear to affect the constictor phase mS. Furthermore release of local metabolites also does not seem to play a significant role in SD as has been shown very elegantly by Busija et ai. m6. What could account for these opposite results? The original study employed the method of hydrogen clearance to determine cerebral blood flow. It is possible that the changes in flow that were measured are deeper in the cortex that the 2.5 mm 3 examined with the Doppler technique. If this were the case then the autoradiographic approach of Duckrow ~ should have had the same results. On the other hand if temporal resolution was important for looking at the changes and it is certainly true that the changes in cerebral blood flow with SD have important temporal patterns than perhaps these were better ap-
I
158
Spreadin9 d e p r e s s i o n a f t e r U740e6F ( i v i )
l
£EC02 (~._)
(~)
-tin L
\
LDFt
~.
-158
J i
150
I
l'-'\ /
,
, ) -IooL
'-[
LDP). . . . . . . (,-4 -lOU L
Fig. 3. Computer processed plot as for Fig. ! showing the response to hypercapnia after a unilateral spreading depression. The physiological vasodilator response is blocked on the side ipsilateral to the SD.
~_ I
LDFI (~,)
I I
-~58
'-"~
A.
[_ A
(~"-") [ -15o
--~~.-_~_~-_.
. . . .
liI
Fig. 5. Computer processed tracing similar to Fig. 1 showing typical cortical blood flow changes of SD despite the presence of tirilazad mesylate (3 mg/kg, i.v.i.).
143
preciated by hydrogen clearance. Laser Doppler has better temporal resolution than hydrogen clearance however, being able to update the flow signal every 100 ms compared to some seconds being required for a hydrogen washout curve at best. The laser Doppler gives the advantage of being able to test the hypercapnic cerebral vasodilator response, if it is not present it is highly likely that a SD has been provoked and from the data in this study this alone will eliminate the oligemic phase. Since when using hydrogen clearance a needle stick injury must be executed to implant the probe for its measurements, the possibility of a SD occuring must be high. Indeed without explicit evidence that hypercapnic vasodilatation is present acute hydrogen clearance data must be viewed with a good measure of suspicion and caution. It is possible that such a situation explains these conflicting studies. A further technical consideration might be the different cortical regions measured by each method. Laser Doppler measures very superficial cortex while being an implanted method hydrogen clearance by necessity measures at a deeper level in the cortex. Perhaps these different regions have subtle differences in the role of lipid peroxidation in the manifestations of SD. Another possible explanation for the different results might be the different species, cat being used here while rat was used in the initial study. Again given the rat autoradiographic study ~ this must be a remote possibility. The other major consideration might be the dose of the drug used. There is some evidence that tirilazad has U-shaped dose-response curve (Hall, personnal communication). ' The dose of 3 mg/kg would seem reasonable and a more detailed dosing study might be in order but could only be justified by a clear demonstration that in rat with intact hypercapnic vasodilatation there was the reported effect on SD. In summary, in the anesthetised cat needle stick injury produced a very reproducible hyperemic/ oligemic response in the cerebral cortical blood flow that could not be attenuated in normal cortex by tirilazad mesylate. The oligemic phase of the response is not present on subequent needle sticks indicating that accidental induction of a SD would be sufficient to produce a blockade of the oligaemia that might then be attributed to a pharmacological manipulation. These data support the view that the changes in brain blood flow seen with SD are not due in any crucial part to lipid peroxidation or mediated by free radicals. Tirilazad mesylate (U74006F) was a generous gift from the Upjohn Co and valuable discussion on these experiments with Dr E. Hall was appreciated. The author thanks Karen Hoskin for her excellent technical assistance and Dr Brad Duckrow for his very useful discus-
sions concerning the data. The author also thanks the Department of Medical Illustration, University of New South Wales, for photographing all figures. This programme has been supported by the National Health and Medical Research Council of Australia and by grants from Warren and Cheryl Anderson, The J.A.W. Perini Family Trust, the Basset Trust, the Ramacciotti Foundation and the Australian Brain Foundation. P.J.G. is a Wellcome Senior Research Fellow.
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