In cerebral ischemia, there is a transient increase of nitric oxide (NO) production [1,2]. The effects of NO on the outcome of focal cerebral ischemia are complex. NO may exert both beneficial and detrimental effects during cerebral ischemia .
It is reported that NO synthase inhibitors decrease regional cerebral blood flow (rCBF) in both nonischemic and ischemic brain regions [4,5]. Decreased, increased, and unaltered infarction sizes have been reported with the administration of NO synthase inhibitors [6-8]. NO donors appear to increase rCBF in focal cerebral ischemia [9,10]. Administering L-arginine before or after middle cerebral artery (MCA) occlusion increased rCBF and reduced the infarct size . Intracarotid administration of sodium nitroprusside (SNP) or 3-morpholinosydnonimine (SIN-1) increased rCBF and reduced the size of infarction . However, intravenously (IV) administered SNP failed to improve the outcome of stroke or failed to increase the rCBF in the focal ischemic area [11,12]. Multiple factors including the administration route of NO-related drugs are involved in determining the size of the infarction and in the outcome of stroke.
CAS 754 [3-(cis-2,6-dimethyl piperidino)-sydnonimine] is a metabolite of CAS 936 [3-(cis-2,6-dimethyl piperidino)-N-(4-methoxybenzoyl)-sydnonimine]. CAS 936 is a long-lasting vasodilator, and is under investigation as an antianginal drug [13-15]. CAS 754 dilates vessels exclusively via NO and cyclic guanosine monophosphate-dependent mechanisms [16,17]. The slow biotransformation of CAS 936 and the relatively slow rate of NO release from its metabolite CAS 754 are responsible for the long-lasting effects in vivo of CAS 936 on systemic circulation . SNP is a short-acting NO donor .
In this study, we hypothesized that systemically administered CAS 754 would exert more NO-dependent cerebrovasodilating action than systemically administered SNP. This could be related to the relatively slow release of NO which allows a larger proportion of CAS 754 to reach the brain. We compared the effects of CAS 754 and SNP on rCBF and regional cerebral vascular resistance (rCVR) in rats with MCA occlusion at the same mean arterial blood pressure (MAP). Results of our investigation may be useful in the application of CAS 754 as a hypotensive and antianginal drug or in the clinical application of its parent drug CAS 936 for antianginal use.
This study was approved by our institutional animal care and use committee. Data reported here are from 28 adult male Long-Evans rats weighing 330-430 g. They were divided into four groups of seven animals each: control group, CAS 754 (CAS) group, CAS 754 + phenylephrine (CAS + Ph) group, and the SNP group.
All the rats were anesthetized with 2% isoflurane in a mixture of air and oxygen (FIO2 0.25-0.3). A tracheostomy was performed for mechanical ventilation. A femoral artery and two femoral veins were catheterized. The arterial catheter was used to monitor the heart rate and arterial blood pressure, and to anaerobically obtain arterial blood samples to analyze the blood gases and pH. Venous catheters were used to administer14 C-iodoantipyrine for blood flow measurements and to administer CAS 754 (Hoechst-Roussel Pharmaceuticals, Inc., Somerville, NJ), phenylephrine, or nitroprusside. For MCA occlusion, an incision was made at the midpoint between the right eye and the right ear. The temporalis muscle was separated and retracted in order to expose the zygoma and the squamosal bone. The MCA was exposed through a hole 2-3 mm in diameter near the anterior junction of the squamosal and the zygomatic bones. The MCA was occluded near the rhinal fissure [18,19]. After craniotomy, the isoflurane concentration was decreased to 1.4%. Pericranial temperature was monitored with a Thermocouple probe (Omega Engineering Inc., Stamford, CT), and was maintained at 36.5 degrees C with a heat lamp. Body temperature was also monitored and maintained at 37 degrees C with a servocontrolled rectal thermistor probe and a heating lamp. Arterial blood pressure was measured continuously using a Statham P23AA transducer (Gould Instruments, Cleveland, OH) coupled to the arterial catheter, and was recorded on a Beckman R-411recorder (Fullerton, CA). Arterial blood samples were obtained anaerobically and analyzed for PO2, PCO2, and pH on a blood gas analyzer (ABL 330; Radiometer America, Westlake, OH). For the CAS group, CAS 754 4-6 mg/kg (10 mg/mL solution) was administered for 5 min IV, 40 min after the MCA occlusion in order to decrease MAP to 55-60 mm Hg. This low MAP was maintained for 15-20 min before measuring rCBF. For the CAS + Ph group, after the MAP had been decreased to 55-60 mm Hg by CAS 754 administration, phenylephrine (0.5-0.7 mg centered dot kg (-1) centered dot h-1) was infused to keep the MAP at the same level as that of the control group (90-100 mm Hg). For the SNP group, nitroprusside infusion (1.5-2.2 mg centered dot kg-1 centered dot h-1) was started 40 min after MCA occlusion to keep the MAP at 55-60 mm Hg. For the control group, normal saline was administered for 20 min (2.7 mL/h) before measuring rCBF.
One hour after MCA occlusion,14 C-iodoantipyrine (Amersham, Arlington Heights, IL) was infused into the venous catheter by means of an infusion pump (Sage Instruments, Cambridge, MA). At the time of entry of the isotope into the venous circulation, the arterial catheter was cut to a length of 15-20 mm to minimize smearing in the sampling catheter. Timed blood samples were withdrawn from the arterial catheter and collected approximately every 3 s in capillary tubes. These samples were collected over a period of 60 s. The rat was decapitated at the moment the last sample was obtained. The head was quickly frozen in liquid nitrogen for later analysis. While frozen, the head was cut in the midsagittal plane, and the following regions were dissected and weighed: ischemic cortex, contralateral cortex, olfactory bulb, hippocampus, basal ganglia, hypothalamus, cerebellum, pons, and medulla. Blood and tissue samples were then placed in a tissue solubilizer (Soluene; Packard, Downers Grove, IL) and 24 h later, they were put in a counting fluid (Dimiscint; National Diagnostic, Manville, NJ) and agitated. These samples were counted using a Beckman LS-230 liquid scintillation counter. Quench curves were prepared using carbon tetrachloride while the isotope counts were adjusted for color and quench correction.
Regional blood flow was calculated using a computer program based on the following equation: Equation 1 where Ci(T) equals the tissue concentration of14 C-iodoantipyrine at the time of decapitation; lambda equals the tissue:blood partition coefficient; CA is the arterial concentration of the tracer; and t equals time. K is defined as K = mF/W, where m is the constant related to diffusion and F/W equals the blood flow per unit mass of tissue. The lambda value of 0.8 calculated by Sakurada et al.  was used. rCBF was expressed in mL centered dot min-1 centered dot 100 g-1. rCVR was calculated as the ratio of MAP to rCBF and expressed in mm Hg centered dot min centered dot 100 g centered dot mL-1. We assumed that the effects of the vasoactive substances that we used on regional cerebral venous pressure were negligible in comparison to changes in MAP.
A factorial analysis of variance was applied for the measurements performed in order to determine the difference between brain regions and between groups. Multiple comparisons were made using Duncan's post-hoc procedure. All values are expressed as mean +/- SD. A P value < 0.05 was considered statistically significant.
Blood gases and hemodynamic variables for the four groups are presented in Table 1. Blood pressure significantly decreased after administering CAS 754 or nitroprusside. Phenylephrine significantly increased blood pressure in the CAS 754-treated animals. MAP was similar between the control and the CAS + Ph groups and between the CAS and the SNP groups. PaO2 and PaCO2 were similar among the four groups of rats. The pH of the SNP group was higher than that of the rest of the groups. Heart rate was similar among the four groups.
The average rCBF of the eight nonischemic brain regions under isoflurane anesthesia was 121 +/- 15 mL centered dot min-1 centered dot 100 g-1. Administering CAS 754 significantly increased the rCBF by 34% (162 +/- 39 mL centered dot min-1 centered dot 100 g-1). However, nitroprusside failed to increase rCBF (114 +/- 15 mL centered dot min-1 centered dot 100 g-1). The rCBF of the CAS group was significantly higher than that of the SNP group (+ 42%). Infusion of phenylephrine in the CAS group did not change the average rCBF (165 +/- 21 mm Hg).
In most of the nonischemic brain regions (contralateral cortex, hippocampus, hypothalamus, cerebellum, and pons), the rCBF was higher in the CAS or CAS + Ph group than in the control or SNP group Figure 1. In the control or CAS group, the rCBF in the cerebellum or medulla was higher than in most of the remaining nonischemic regions. In the CAS + Ph or SNP group, the rCBF in the cerebellum was higher than in most of the remaining nonischemic regions.
In the control group, occlusion of the MCA decreased the rCBF of the ischemic cortex (IC) by 56% when compared with the contralateral cortex (CC) (IC 51 +/- 7, CC 116 +/- 28 mL centered dot min-1 centered dot 100 g-1, respectively) Figure 1. Administering CAS 754 did not increase the rCBF of the IC (61 +/- 13 mL centered dot min-1 centered dot 100 g-1), which was 40% of that of the corresponding CC. The rCBF of the SNP group (53 +/- 18 mL centered dot min-1 centered dot 100 g-1) was similar to that of the control animals and was 44% of that of the corresponding CC. Infusion of phenylephrine in the CAS group did not significantly affect the rCBF of the IC (52 +/- 24 mL centered dot min-1 centered dot 100 g-1) nor did it affect the rCBF of the CC.
The average rCVR of the eight nonischemic brain regions under isoflurane anesthesia was 0.81 +/- 0.10 mm Hg centered dot min centered dot 100 g centered dot mL-1. CAS 754 decreased the average rCVR of these regions by 51% (0.40 +/- 0.07). Nitroprusside decreased the average rCVR of these regions by 28% (0.58 +/- 0.11). The average rCVR of the CAS group was significantly lower than that of the SNP group (- 31%). In the CAS group, infusion of phenylephrine increased the average rCVR of these regions by 68% (0.67 +/- 0.10). In the control, CAS, and SNP groups, the rCVR of olfactory bulb was highest when compared with the remaining nonischemic brain regions. In the CAS + Ph group, there was no significant difference among the brain regions. In most of the nonischemic brain regions including the CC, the rCVR of the control group was higher than that of the remaining groups. In most of the nonischemic brain regions, the rCVR of the CAS group tended to be lower than that of the SNP group (statistically significant in the CC, hippocampus, cerebellum, and pons; Figure 2).
Calculated rCVR of the IC was highest in the CAS + Ph group followed by the control, and then the CAS and the SNP groups in decreasing order. There was no significant difference in the rCVR between the CAS and the SNP groups Figure 2.
The results of our study showed that, in the CAS group, the average rCBF of the nonischemic brain regions was higher by 42%, and the average rCVR was lower by 31%, than that of the SNP group at an infusion rate producing the same MAP. However, in the IC, neither CAS 754 nor SNP affected rCBF. In the CAS group, infusion of phenylephrine to increase the MAP to the level of the control group did not change rCBF either in the nonischemic brain regions or in the IC.
Our previous study on rats demonstrated that changes in the rCVR after administering NG-nitro-L-arginine methyl ester (L-NAME) under anesthesia with 1.4% isoflurane were similar to those in a conscious state . Todd et al.  also reported that L-NAME produced a dose-related reduction of the rCBF during isoflurane anesthesia and during anesthesia with a low and high dose of pentobarbital. However, L-NAME did not alter the relative differences among different anesthetics. Although these studies suggest that NO may not be the primary mediator of anesthetic effects on the rCBF, other authors reported that NO contributes to cerebral hyperemia induced by isoflurane . In our study, all the animals were anesthetized with the same concentration of isoflurane. The effects of isoflurane on NO activities should be the same in each of the animal groups.
CAS 936 (pirsidomine) is a new long-lasting vasodilator which is under investigation as an antianginal drug. It causes less tolerance than nitrates . It is reported that CAS 936 has both NO dependent and independent vasodilating actions in vitro . However, CAS 754, which is an active metabolite formed by enzymatic hydrolysis of CAS 936, dilates vessels exclusively via NO  and affects both arteries and veins . In vitro, the rate of release of NO from CAS 754 is slower than the rate of NO release from another sydnonimine, SIN-1 (0.03 vs 1.8 micro Meter NO/min calculated for 0.1 mM NO donor concentration) . It is conceivable that a slow biotransformation of CAS 936 and a slow rate of NO release from its metabolite, CAS 754, as compared with SIN-1, are responsible for the long-lasting in vivo systemic effects of CAS 936 .
The reason that systemically administered CAS 754 affected cerebral vasculature more than SNP did is not clear. We speculate that the following mechanisms may play a role: 1) This result may be due to a relatively slower rate of NO release from CAS 754. It appears that much of CAS 754 has time to reach the brain before it releases NO. 2) CAS 754 may work through a binding site in the cerebrovasculature and then release NO. 3) Some of CAS 754 may cross the blood-brain barrier and release NO. 4) The cerebral vasculature could also be more sensitive to CAS 754 than to SNP. Regardless of the reason, the mechanism appears to have a central, rather than a peripheral, origin. In our study, SNP decreased MAP to the same degree as CAS 754. However, the average rCBF was not significantly changed by SNP infusion when compared with the control group. Other studies have also shown either no significant change [24,25] or an increase in rCBF  during hypotension with SNP infusion. Since Zhang et al.  showed that intracarotid administration of SNP increased rCBF in the focal ischemic area, we speculate that systemically administered SNP takes more time to reach the brain than an intracarotid injection of SNP. Therefore, the ability of SNP to yield NO in the brain may be significantly decreased. Intracarotid infusions may deliver a higher concentration of NO.
Our data suggest that CAS 754 is a more effective cerebral vasodilator than SNP at the same MAP in the nonischemic brain regions. In order to use CAS 754 as a hypotensive drug under anesthesia or as an antianginal drug, more research should be conducted to clarify the relationship between the intracranial pressure and CAS 754.
In this study, we used an IV route to administer the drugs because this route is used in clinical situations. Our study failed to show any improvement of rCBF in the IC with either CAS 754 or an SNP treatment. A possible explanation for this finding is that blood is shunted away from the ischemic to the nonischemic tissue by the vasodilating effects of SNP or CAS 754 (steal syndrome). Due to a slower blood flow rate of collateral circulation to the ischemic area than to the nonischemic tissue, NO could be released before these drugs reach the ischemic area. The amount of drugs supplied also could be decreased in the ischemic area when compared with the amount supplied to the nonischemic area. It is also possible that vessels are already maximally dilated in the ischemic area, and that NO donors may not exert any further effects. The effects of NO on vasodilation could be altered by the interaction with free radicals, or by a disturbed cyclic guanosine monophosphate system in the ischemic area of the brain.
The mechanisms of release of NO from CAS 754 and from SIN-1 are similar, that is, oxygen is required in this process [15,27,28]. In contrast, SNP releases NO by simple dissociation . In the ischemic area of the brain, the amount of oxygen available is decreased. Previous studies with molsidomine showed that the in vivo oxidative capacity is large enough to guarantee a sufficient NO release, even under ischemic conditions in the heart . Bohn and Schonafinger  showed that nitrogen with a purity of 99.99% containing less than 0.01% oxygen released NO from a buffered SIN-1 solution (which is a metabolite of molsidomine). Therefore, we speculated that the release of NO from CAS 754 would not be affected by focal ischemia.
Our study showed that in the CAS-treated animals, phenylephrine infusion did not affect the average rCBF of the nonischemic brain regions, nor did it affect the rCBF of the IC. However, our previous study demonstrated that phenylephrine increased rCBF under isoflurane anesthesia at higher blood pressure . It has been reported that CAS 754 inhibited norepinephrine-induced vascular contraction in vitro . Our study showed that peripheral vessels responded to phenylephrine by increasing the MAP. In contrast, the rCBF was not affected by phenylephrine. The reason for the lack of response to phenylephrine in the rCBF of the CAS 754-treated animals is not clear. Nevertheless, the failure of CAS 754 to improve the rCBF of the IC does not appear to be caused by hypotension induced by CAS 754.
In conclusion, our study demonstrates that CAS 754 is a more effective cerebral vasodilator than nitroprusside when administered systemically. In the IC, neither CAS 754 nor nitroprusside improved rCBF.
The authors wish to thank Patricia A. Sheffield, MA, for her expert editorial assistance.
1. Malinski T, Bailey F, Zhang ZG, Chopp M. Nitric oxide measured by a porphyrinic microsensor in rat brain after transient middle cerebral artery occlusion. J Cereb Blood Flow Metab 1993;13:355-8.
2. Kader A, Franzzini VI, Solomon RA, Trifiletti RR. Nitric oxide production during focal cerebral ischemia in rats. Stroke 1993;24:1709-16.
3. Choi DW. Nitric oxide: foe or friend to the injured brain? Proc Natl Acad Sci USA 1993;90:9741-3.
4. Wei HM, Chi OZ, Liu X, et al. Nitric oxide synthase inhibition alters cerebral blood flow and oxygen balance in focal cerebral ischemia in rats. Stroke 1994;25:445-50.
5. Nishikawa T, Kirsch JR, Koehler RC, et al. Effect of nitric oxide synthase inhibition on cerebral blood flow and injury volume during focal ischemia in cats. Stroke 1993;24:1717-24.
6. Buisson A, Plotkine M, Boulu RG. The neuroprotective effect of a nitric oxide inhibitor in a rat model of focal cerebral ischaemia. Br J Pharmacol 1992;106:766-7.
7. Kuluz JW, Prado RJ, Dietrich WD, et al. The effect of nitric oxide synthase inhibition on infarct volume after reversible focal cerebral ischemia in conscious rats. Stroke 1993;24:2023-9.
8. Dawson DA, Kusumoto K, Graham DI, et al. Inhibition of nitric oxide synthesis does not reduce infarct volume in a rat model of focal cerebral ischaemia. Neurosci Lett 1992;142:151-4.
9. Morikawa E, Moskowitz MA, Huang Z, et al. L-Arginine infusion promotes nitric oxide-dependent vasodilation, increases regional cerebral blood flow, and reduces infarction volume in the rat. Stroke 1994;25:429-35.
10. Zhang F, White JG, Iadecola C. Nitric oxide donors increase blood flow and reduce brain damage in focal ischemia: evidence that nitric oxide is beneficial in the early stages of cerebral ischemia. J Cereb Blood Flow Metab 1994;14:217-26.
11. Gelb AW, Boisvert DP, Tang C, et al. Primate brain tolerance to temporary focal cerebral ischemia during isoflurane- or sodium nitroprusside-induced hypotension. Anesthesiology 1989;70:678-83.
12. Fitch W, Pickard JD, Tamura A, Graham DI. Effects of hypotension induced with sodium nitroprusside on the profound reduction in cerebral blood flow during focal cerebral ischaemia in the rat. Br J Anaesth 1990;64:498-502.
13. Bohn H, Martorana PA, Schonafinger K. Cardiovascular effects of the new nitric oxide donor, pirsidomine. Hemodynamic profile and tolerance studies in anesthetized and conscious dogs. Eur J Pharmacol 1992;220:71-8.
14. Mulsch A, Hecker M, Mordvintcev PI, et al. Enzymic and nonenzymic release of NO accounts for the vasodilator activity of the metabolites of CAS 936, a novel long-acting sydnonimine derivative. Naunyn-Schmiedebergs Arch Pharmacol 1993;347:92-100.
15. Siegfried MR, Erhardt J, Rider T, et al. Cardioprotection and attenuation of endothelial dysfunction by organic nitric oxide donors in myocardial ischemia-reperfusion. J Pharmacol Exp Ther 1992;260:668-75.
16. Bohn H, Beyerle R, Martorana PA, Schonafinger K. CAS 936, a novel sydnonimine with direct vasodilating and nitric oxidedonating properties: effects on isolated blood vessels. J Cardiovasc Pharmacol 1991;18:522-7.
17. Schini VB, Bond R, Gao Y, et al. The sydnonimine C87-3754 evokes endothelium-dependent relaxations and prevents endothelium-dependent contractions in blood vessels of the dog. J Cardiovasc Pharmacol 1993;22:S10-6.
18. Brint S, Jacewicz M, Kiessling M, et al. Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries. J Cereb Blood Flow Metab 1988;8:474-85.
19. Coyle P, Jokelainen PT. Differential outcome to middle cerebral artery occlusion in spontaneously hypertensive stroke-prone rats (SHRSP) and Wistar Kyoto (WKY) rats. Stroke 1983;14:605-11.
20. Sakurada O, Kennedy C, Brown JD, et al. Measurement of local cerebral blood flow with indo-C14-antipyrine
. Am J Physiol 1978;234:H59-66.
21. Wei HM, Weiss HR, Sinha AK, Chi OZ. Effects of nitric oxide synthase inhibition on regional cerebral blood flow and vascular resistance in conscious and isoflurane-anesthetized rats. Anesth Analg 1993;77:880-5.
22. Todd MM, Wu B, Warner DS, Maktabi M. The dose-related effects of nitric oxide synthase inhibition on cerebral blood flow during isoflurane and pentobarbital anesthesia. Anesthesiology 1994;80:1128-36.
23. Moore LE, Kirsch JR, Helfaer MA, et al. Nitric oxide and prostanoids contribute to isoflurane-induced cerebral hyperemia in pigs. Anesthesiology 1994;80:1328-37.
24. Pinaud M, Souron R, Lelausque JN, et al. Cerebral blood flow and cerebral oxygen consumption during nitroprusside-induced hypotension to less than 50 mm Hg. Anesthesiology 1989;70:255-60.
25. Bunemann L, Jensen KA, Riisager S, Thomsen LJ. Cerebral blood flow and metabolism during hypotension induced with sodium nitroprusside and metoprolol. Eur J Anaesthesiol 1991;8:197-201.
26. Lu GP, Kaul DK, Feldman SM, et al. Sodium nitroprusside (SNP) hypotension: intracranial pressure (ICP) and hemodynamics in pial arteriole in the rat. Microcirc Endothelium Lymphatics 1990;6:315-41.
27. Bohn H, Schonafinger K. Oxygen and oxidation promote the release of nitric oxide from sydnonimines. J Cardiovasc Pharmacol 1989;14:S6-12.
28. Southam E, Garthwaite J. Comparative effects of some nitric oxide donors on cyclic GMP levels in rat cerebellar slices. Neurosci Lett 1991;130:107-11.
29. Rudolph W, Dirschinger J. Effectiveness of molsidomine in the long term treatment of exertional angina pectoris and congestive heart failure. Am Heart J 1985;109:670-4.