Cerebroprotective Effect of the Nitric Oxide Synthase Inhibitors,1-(2-Trifluoromethylphenyl) Imidazole and 7-Nitro Indazole,after Transient Focal Cerebral Ischemia in the Rat

Abstract

The novel neuronal nitric oxide synthase inhibitors, 1-(2-trifluoromethylphenyl)imidazole (TRIM) and 7-nitro indazole (7-NI), were used to investigate the role of nitric oxide in a model of transient focal cerebral ischemia in vivo. In halothane-anesthetized rats, the middle cerebral artery (MCA) was occluded for 2 hours using an intravascular thread and then reperfused for 22 hours before histologic evaluation. TRIM (10, 20, or 50 mg/kg), 7-NI (60 mg/kg), TRIM (50 mg/kg) plus L-arginine (300 mg/kg), or L-arginine (300 mg/kg) alone was administered intraperitoneally, either at 5 or 90 minutes after MCA occlusion. Immediate administration (5 minutes after MCA occlusion) of TRIM produced a dose-related reduction in lesion size, which was reversed with L-arginine coadministration. Similarly, delayed administration of TRIM (90 minutes after MCA occlusion, 50 mg/kg) decreased total lesion volume by 48.4% ± 13.0% in comparison to a reduction of 39.3% ± 10.9% when TRIM (50 mg/kg) was administered immediately (5 minutes) after occlusion. 7-NI (60 mg/kg) reduced the total lesion volume by 38.5% ± 13.7% when administered immediately (5 minutes) after MCA occlusion, but had no effect when administration was delayed (90 minutes). Neither TRIM (50 mg/kg) nor 7-NI (60 mg/kg), administered 5 minutes after MCA occlusion, had any significant effect on mean arterial blood pressure throughout the ischemic period or for up to 10 minutes after reperfusion. These results indicate that immediate or delayed administration of the selective neuronal NOS inhibitor TRIM reduces the lesion volume after transient MCA occlusion. In contrast, only immediate administration of 7-NI reduces lesion volume.

Keywords

Nitric oxide synthase 7-Nitro indazole Rat Transient middle cerebral artery occlusion 1-(2-Trifluoromethylphenyl)imidazole

Nitric oxide plays multiple roles in the pathophysiology of focal cerebral ischemia (Dawson, 1994). Focal cerebral ischemia in rats causes an immediate increase in the production of nitric oxide in cerebral tissue (Malinski et al., 1993) and in the appearance of nitric oxide end products in plasma (Kumura et al., 1994). Experiments using conventional nitric oxide synthase (NOS) inhibitors or nitric oxide donors have provided evidence that nitric oxide can act both as a neuroprotective and neurotoxic agent in animal models of focal cerebral ischemia (for reviews see Iadecola et al., 1994; Dawson, 1994; Meldrum, 1996). For example, administration of nitric oxide donors (sodium nitroprusside, 3-morpholinosydnonimine) or l-arginine reduces the size of the ischemic lesion induced by permanent middle cerebral artery (MCA) occlusion in rats (Zhang et al., 1994; Zhang and Iadecola, 1993, 1994; Morikawa et al., 1992a, 1994). These data suggest that an increase in nitric oxide levels during cerebral ischemia may be beneficial in protecting against ischemic cell damage. This may be caused by several biologic actions of nitric oxide. Nitric oxide causes vasodilation, which results in an increase in cerebral blood flow, thereby improving tissue perfusion and reducing ischemic cell damage (Morikawa et al., 1992b, 1994; Zhang et al., 1994). Nitric oxide also inhibits platelet aggregation and leukocyte activation and adhesion, which may prevent additional thrombosis and minimize inflammation in the ischemic brain (Radomski et al., 1987; Bath et al., 1991; Moncada et al., 1991). Provisional data suggest that nitric oxide, administered as sodium nitroprusside, may attenuate platelet activation and improve cerebral blood flow in patients with acute stroke (Butterworth and Bath, 1996). N-Methyl-d-aspartate receptor activation can also be inhibited by nitric oxide, possibly limiting glutamate excitotoxicity by a negative feedback mechanism (Manzoni et al., 1992).

In contrast to these potential beneficial effects, nitric oxide may itself be a mediator of neuronal destruction. Several studies using nonselective NOS inhibitors have shown that NOS inhibition may protect against cerebral ischemic damage in vivo (Nowicki et al., 1991; Nagafuji et al., 1992; Buisson et al., 1993; Ashwal et al., 1993). In vitro, nitric oxide has been shown to exacerbate ischemic cell damage by generating peroxynitrite (Radi et al., 1991), inhibiting iron-containing enzymes (Stadler et al., 1991), and mediating glutamate neurotoxicity (Dawson et al., 1991).

However, there are conflicting reports showing that nonselective NOS inhibitors have a variety of effects on lesion size after cerebral ischemia. Nitric oxide synthase inhibitors have been shown to reduce, increase, or have no effect on focal ischemic lesions depending on (1) the animal model, (2) the dosing regimens (time, dose, and route), and (3) the vasopressor action of the specific nonselective NOS inhibitor used (Dawson, 1994; Iadecola et al., 1994; Dalkara and Moskowitz, 1994). The development of knockout mice deficient in the different isoforms of NOS (neuronal, endothelial, and inducible) has helped to clarify the role of nitric oxide in cerebral ischemia. Knockout mice with selective endothelial NOS deletion develop a larger infarct after MCA occlusion in comparison to wild-type controls (Huang et al., 1996). In contrast, neuronal NOS knockout mice demonstrate a reduction in the size of the infarct in comparison to wild-type controls after both permanent (Huang et al., 1994) and transient MCA occlusion (Hara et al., 1996). Therefore, neuronal NOS may have a harmful role to play in ischemia but endothelial NOS may be protective.

Novel nitric oxide synthase inhibitors (e.g., 7-nitro indazol [7-NI], 1-(2-trifluoromethylphenyl)imidazole [TRIM], and ARL 17477) that show a greater selectivity for certain NOS isoforms have recently been developed. 7-Nitro indazole is an inhibitor of the neuronal NOS in vitro and exhibits antinociceptive effects in vivo (Moore et al., 1993a,1993b). Although 7-NI also inhibits endothelial NOS in vitro, it is devoid of the vasopressor activity associated with existing nonselective NOS inhibitors such as L-N^G-nitroarginine methyl ester (Moore et al., 1993a, 1993b; Babbedge et al., 1993; Rees et al., 1991). Administration of 7-NI produced a 25% reduction in lesion volume in permanent MCA occlusion in rats, which was reversed by l-arginine coadministration (Yoshida et al., 1994). Similarly, ARL 17477, a potent and selective neuronal NOS inhibitor, decreased infarct volume after transient MCA occlusion in rats (Zhang et al., 1996b). ARL 17477 significantly inhibited cortical neuronal NOS activity by 80% in these rats, and this agent was also devoid of vasopressor activity. 1-(2-Trifluoromethylphenyl)imidazole has recently been described as a potent inhibitor of both the neuronal and inducible NOS isoforms, but with poor activity on endothelial NOS in vitro (Handy et al., 1995). This inhibitor possesses antinociceptive activity but does not alter blood pressure in the mouse (Handy et al., 1995, 1996).

In this study we have used TRIM and 7-NI to further investigate the role of nitric oxide in a reperfusion model of MCA occlusion in the rat. These agents are administered after ischemia, which may be more relevant to the clinical situation. Our data indicate that TRIM and 7-NI are cerebroprotective when administered immediately after MCA occlusion; similarly, TRIM may also be effective when given 90 minutes after MCA occlusion.

MATERIALS AND METHODS

All experiments were carried out under U.K. Home Office regulations. Male Sprague-Dawley rats (300 to 350 g, Charles River, U.K.) were fasted overnight before surgery but allowed free access to water. The MCA was occluded for 2 hours using a method of intravascular occlusion (Koizumi et al., 1986; Zea Longa et al., 1989). For occlusion of the MCA, an occluder thread was prepared. The tip of a 3-0 nylon Ethilon suture (Ethicon Ltd., U.K.) was rounded with sandpaper (P1000) and coated with a silicone compound to a diameter of 0.3 mm and a length of 3 mm. Anesthesia was induced with 3% halothane in oxygen and maintained with 1.75% to 2% halothane in oxygen via a face mask in spontaneously breathing rats. The rectal body temperature was continuously monitored and maintained at 37.5°C throughout the surgery using a rectal probe and automated heat pad (ʻk-tempʼ unit, International Market supplies, U.K.). After a ventral midline incision in the neck, the right common carotid artery (CCA) was isolated and a cannula was inserted to collect blood samples for blood gas analysis immediately before occlusion (Table 1). The right CCA was temporarily clamped, using a microsurgical clip, proximal to the bifurcation of the internal carotid artery and external carotid artery and a small incision was made in the CCA. The occluder thread was inserted, secured and then advanced through the CCA into the internal carotid artery to a distance of 18 to 20 mm (depending on body weight) from the internal carotid artery and external carotid artery bifurcation, until the suture blocked the origin of the MCA. The incision was closed and the rat was allowed to recover. On average the surgery was performed within 25 minutes. After 2 hours of ischemia the intraluminal thread was removed under brief anesthesia (2% halothane in oxygen), to allow reperfusion of the ischemic tissue. A small increase in rectal temperature was observed at this time point but there was no significant difference between treatment groups (Table 1). Because the right CCA was ligated in this model, the ischemic tissue was reperfused via the circle of Willis through the contralateral carotid artery, basilar artery, and collateral circulation of the cerebral arteries.

TABLE 1.

Physiologic variables

	Immediate (5 min)				Delayed (90 min)		Immediate (5 min)		Delayed (90 min)
	Saline 10 mL/kg	TRIM 50 mg/kg	L-Arginine 300 mg/kg	L-Arginine 300 mg/kg + TRIM 50 mg/kg	Saline 10 mL/kg	TRIM 50 mg/kg	Vehicle 10 mL/kg	7-NI 60 mg/kg	7-NI 60 mg/kg
Blood gases at occlusion
pH	7.34 ± 0.04	7.33 ± 0.02	7.33 ± 0.03	7.32 ± 0.03	7.35 ± 0.03	7.35 ± 0.02	7.30 ± 0.04	7.31 ± 0.03	7.30 ± 0.06
Paco₂ (mm Hg)	41.5 ± 4.2	40.6 ± 5.4	45.1 ± 4.6	43.9 ± 3.2	44.7 ± 4.4	44.3 ± 5.3	45.7 ± 6.5	45.3 ± 4.28	45.0 ± 3.1
Pao₂ (mm Hg)	375 ± 55	337 ± 48	348 ± 48	375 ± 51	402 ± 54	406 ± 59	387 ± 52	450 ± 48	434 ± 54
Temperature at reperfusion (2 hours after occlusion)
°C	38.4 ± 0.4	38.0 ± 0.6	38.6 ± 0.2	38.3 ± 0.3	38.5 ± 0.3	38.6 ± 0.4	38.4 ± 0.4	38.1 ± 0.8	37.7 ± 0.6
Neurological score after occlusion*
1 hour	3.0 ± 0.0	2.4 ± 0.5†	3.0 ± 0.0	3.0 ± 0.0	3.0 ± 0.0	3.0 ± 0.0	3.0 ± 0.0	3.0 ± 0.0	3.0 ± 0.0
24 hours	1.9 ± 0.3	1.0 ± 0.7†	1.8 ± 0.4	1.8 ± 0.4	1.7 ± 0.5	1.4 ± 0.5	1.3 ± 0.8	0.9 ± 0.8	1.4 ± 0.9
n	16	9	9	9	7	7	7	8	8

TRIM, 1-(2-trifluoromethylphenyl)imidazole; 7-NI, 7-nitro inidazole

Values are mean ± SD. “Immediate” indicates drug administration 5 minutes after MCA occlusion and “delayed” indicates administration 90 minutes post-MCA occlusion.

From Bederson et al., 1986b.

†

P < 0.05 in comparison to the corresponding time-matched saline treated group (analysis of variance and Kruskal-Wallis Test).

After MCA occlusion, neurologic examination was performed in the conscious rat at 1, 4, and 24 hours after MCA occlusion, using a 4-point scoring system (Bederson et al., 1986b). The neurologic deficit was assessed as follows: grade 1, forelimb flexion; grade 2, decreased resistance to lateral push; or grade 3, gait disturbances leading to circling or walking to the left; grade 4, no neurologic deficit (Bederson et al., 1986b). Successful occlusion was demonstrated by an animal with a grade 3 deficit. All vehicle- or saline-treated rats exhibited left circling 1 hour (grade 3) after MCA occlusion (Table 1).

Blood pressure measurements

In a separate group of animals the effect of saline, TRIM, or 7-NI on arterial blood pressure was assessed. Surgery was performed as described above for MCA occlusion, and blood pressure was measured via the right carotid artery using a MacLab/8s (ADInstruments Ltd., U.K.) and recorded by an Apple Macintosh computer (Powerbook 520c, Apple Ltd., U.K.). Blood pressure was monitored during ischemia and reperfusion in the anesthetized rat (maintenance, 1% to 1.5% halothane). This group of animals were not allowed to recover from the anesthesia at the end of this experiment.

Determination of lesion size

Neuropathologic evaluation was performed at 24 hours after the induction of ischemia. The animals were anesthetized using 3% halothane and decapitated. The brains were removed and examined to confirm the position of the intravascular thread and the absence of subarachnoid hemorrhage or arterial penetration with the thread. Five of 97 animals were excluded from the study because they demonstrated signs of subarachnoid hemorrhage after brain removal. The brains were sectioned coronally with a Jacobowitz rat brain slicer (Zivic-Miller Lab, Inc., Allison Park, PA U.S.A.) at 2-mm intervals and incubated for 30 minutes in 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for vital staining (Bederson et al., 1986a). Then the sections were fixed by immersion in 10% formalin solution. The brain sections were scanned using a conventional, high-resolution flatbed scanner system (Agfa Ltd., U.K.) and lesion size was determined using an in-house semiautomated image analysis system (Dispim software). Areas not stained red with TTC were considered lesioned. The total and lesion size (mm²) was obtained for each coronal section. Lesion volume (mm³) for each section was obtained by multiplying the lesion size by the slice thickness (2 mm). Total brain lesion volume was calculated as the sum of each slice volume.

Drug treatment

Animals were randomly assigned to drug or vehicle-treated groups. In group 1, TRIM (10, 20, or 50 mg/kg), l-arginine (300 mg/kg), TRIM (50 mg/kg) plus l-arginine (300 mg/kg), or saline (10 mL/kg) was injected intraperitoneally 5 minutes after MCA occlusion. In group 2, TRIM (50 mg/kg) or saline (10 mL/kg) was injected 90 minutes after MCA occlusion. In group 3, 7-NI (60 mg/kg) or the vehicle (0.25% methylcellulose, 10 mL/kg) was injected intraperitoneally 5 or 90 minutes after MCA occlusion. In group 4 (blood pressure experiments), saline (10 mL/kg), TRIM (50 mg/kg), or 7-NI (60 mg/kg) was injected 5 minutes after MCA occlusion.

Materials and solutions

1-(2-Trifluoromethylphenyl)imidazole was obtained from Lancaster Synthesis Ltd., U.K. and dissolved in warm (37°C) physiologic saline (0.9%) at a concentration of 5 mg/mL. 7-Nitro indazole (Lancaster Synthesis Ltd.) was suspended in 0.25% wt/vol methylcellulose (400 cp, Sigma Ltd., U.K.) in HPLC-grade distilled water at 37°C, using sonication, at a concentration of 5 mg/mL. 2,3,5-Triphenyltetrazolium chloride was obtained from Sigma.

Statistical analysis

Data are presented as mean ± SD and were evaluated by analysis of variance and Tukey's test. Neurologic data were analyzed using Kruskal-Wallis followed by Dunn's procedure. Statistical significance was accepted at the P < 0.05 level.

RESULTS

Immediate or delayed NOS inhibition on lesion size

Immediate administration of TRIM (5 minutes after MCA occlusion) produced a dose-related inhibition of ischemic lesion size when measured at 24 hours after MCA occlusion (Fig. 1). The highest dose of TRIM (50 mg/kg) significantly reduced total lesion volume by 39.3% ± 10.9% in comparison to saline-treated animals (Table 2). Similarly, delayed administration of TRIM (50 mg/kg, 90 minutes) significantly reduced the total lesion size by 48.4% ± 13.0%. There was no significant difference in the reduction in lesion size between immediate or delayed TRIM-treated (50 mg/kg) groups (Table 2). Also, immediate (5 minutes) administration of TRIM (50 mg/kg) produced a significant (P < 0.05) improvement in the neurologic score at 1 and 24 hours after MCA occlusion, in comparison to saline-treated animals (Table 1). The neurologic score (1 or 24 hours after MCA occlusion) after delayed administration of TRIM was not significantly (P > 0.05) different from the appropriate saline-treated group.

FIG. 1.

Dose-response curve for 1-(2-trifluoromethylphenyl)imidazole- (TRIM-) versus saline-treated rats. Graph shows the percent lesion size for each coronal section (2 mm) from anterior to posterior. Values are means and SD. Saline (•, 10 mL/kg, n = 14) or TRIM at 10 mg/kg (□, n = 5), 20 mg/kg (△, n = 7), or 50 mg/kg (▪, n = 9) was administered 5 minutes after MCA occlusion. Administration of TRIM (50 mg/kg) significantly reduced the lesion size (*P < 0.05) as compared with the saline-treated group (analysis of variance and Tukey's test).

TABLE 2.

Effect of NOS inhibitors on lesion volume (mm³) resulting from transient MCA occlusion

	Lesion volume (mm³)
	Striatum	Cortex	Total	n
Saline (10 mL/kg)	76.9 ± 13.9	166.0 ± 60.3	243.0 ± 70.0	16
TRIM + 5 min (50 mg/kg)	51.1 ± 24.1*	94.3 ± 61.5*	145.5 ± 70.1*	9
TRIM + 90 min (50 mg/kg)	63.2 ± 14.1	51.6 ± 39.8*	114.8 ± 42.8*	7
Vehicle (10 mL/kg)	66.8 ± 14.4	119.1 ± 75.5	200.3 ± 66.5	7
7-NI + 5 min (60 mg/kg)	53.4 ± 12.4*	69.7 ± 55.6*	123.2 ± 77.5*	8
7-NI + 90 min (60 mg/kg)	69.6 ± 12.3	140.6 ± 65.6	210.2 ± 75.6	8

Values are mean ± SD.

P < 0.05 in comparison to appropriate vehicle or saline treated group (analysis of variance and Tukey's test).

Administration of 7-NI (60 mg/kg, intraperitoneally) produced a significant 38.5% ± 13.7% reduction in total lesion volume when given immediately (5 minutes) after MCA occlusion, but no significant (P > 0.05) cerebroprotection was observed on delayed administration (90 minutes) (Table 2 and Fig. 2). Neither immediate nor delayed administration of 7-NI had any significant (P > 0.05) effect on neurologic score in comparison to vehicle-treated group (Table 1).

FIG. 2.

Effect of 7-nitro indazole (7-NI) on lesion size. Graph shows percent lesion size for each coronal section (2 mm) from anterior to posterior. Values are means and SD. Vehicle (•, 10 mL/kg, n = 7) or 7-NI (60 mg/kg) were administered at 5 (▪, n = 8) or 90 (△, n = 8) minutes after MCA occlusion. Immediate (5 minutes) administration of 7-NI significantly (*P < 0.05) reduced lesion size as compared with the vehicle-treated group (analysis of variance and Tukey's test).

Striatal and cortical lesion volumes were analyzed to investigate further the area of the ischemic lesion that was affected by NOS inhibition. Immediate (5 minutes) administration of either TRIM or 7-NI reduced the lesion volume in both the striatum and the cortex (Table 2). The reduction in the size of the cortical lesion was, however, more pronounced in comparison to the striatal lesion. Delayed administration of TRIM, but not 7-NI, also decreased the size of the striatal and cortical lesion (Table 2).

l-Arginine reversal of TRIM cerebroprotection

Immediate administration (5 minutes) of l-arginine (300 mg/kg) alone or coadministration of l-arginine with TRIM (50 mg/kg) had no significant (P > 0.05) effect on the total lesion volume or the neurologic score (1 or 24 hours after MCA occlusion), in comparison to saline-treated animals (Fig. 3; Table 1). Therefore, l-arginine significantly reversed the cerebroprotective effect of TRIM (Fig. 3).

FIG. 3.

l-Arginine reversal of TRIM-induced cerebroprotection. Bar graph shows the total lesion volume (mm³), and values are mean ± SD. Saline (10 mL/kg, n = 11), TRIM (50 mg/kg, n = 9), l-arginine (300 mg/kg, n = 9), or TRIM (50 mg/kg) plus l-arginine (300 mg/kg, n = 9) was administered 5 minutes after MCA occlusion. Administration of TRIM significantly reduced the total lesion volume in comparison with the saline-treated group (*P < 0.05). l-Arginine alone and l-arginine plus TRIM were not significantly different from saline-treated groups (analysis of variance and Tukey's test).

Effect of NOS inhibition on blood pressure

Middle cerebral artery occlusion caused a transient reduction in mean arterial blood pressure, which returned to baseline levels before intraperitoneal injections at 5 minutes after MCA occlusion. Neither saline, TRIM, nor 7-NI had any significant effect on blood pressure during either the ischemic or reperfusion periods after MCA occlusion (Table 3). These results are in keeping with previous studies in normal animals, which have shown no significant (P > 0.05) change in blood pressure on administration of TRIM or 7-NI (Handy et al., 1995; Moore et al., 1993a).

TABLE 3.

Effect of saline, TRIM, or 7-NI on mean arterial blood pressure after transient MCA occlusion in the anesthetized rat

Time (min)	Saline 10 mL/kg	TRIM 50 mg/kg	7-NI 60 mg/kg
0	87 ± 4	89 ± 9	87 ± 10
5	94 ± 6	93 ± 4	93 ± 10
10	97 ± 8	91 ± 6	96 ± 12
15	98 ± 10	92 ± 5	95 ± 11
20	97 ± 6	94 ± 4	92 ± 11
25	94 ± 4	94 ± 3	95 ± 8
30	94 ± 4	95 ± 4	97 ± 8
45	91 ± 3	94 ± 4	96 ± 7
60	93 ± 5	95 ± 6	98 ± 10
75	93 ± 6	92 ± 6	99 ± 13
90	92 ± 7	94 ± 9	100 ± 13
105	89 ± 3	93 ± 8	98 ± 10
120	85 ± 6	89 ± 4	98 ± 13
125	86 ± 5	88 ± 8	94 ± 8
130	87 ± 5	85 ± 7	93 ± 8
n	4	4	4

Values are mean ± SD; P > 0.05 (analysis of variance and Tukey's test). Arterial pressure values (mm Hg) are given as follows: MCA occlusion was performed at t = 0 minutes, test agents were injection at t = 5 minutes, and reperfusion was performed at t = 120 minutes.

DISCUSSION

This study demonstrates that inhibition of NOS with either TRIM or 7-NI reduces the size of the ischemic lesion measured 24 hours after transient focal cerebral ischemia. Immediate administration of TRIM (5 minutes after occlusion) produced a dose-related inhibition of the lesion size after reversible MCA occlusion. Delayed administration (90 minutes after occlusion, i.e., 30 minutes before reperfusion) of the highest dose of TRIM (50 mg/kg) also reduced the size of the ischemic lesion. The observation that TRIM is protective even after delayed treatment is similar to a previous study using the novel selective neuronal NOS inhibitor, ARL 17477, which decreased infarct volume after transient MCA occlusion in rats when administered at the time of reperfusion (120 minutes after occlusion; Zhang et al., 1996b). These results show that there is a modest therapeutic time window for NOS inhibition in reversible focal cerebral ischemia and confirm previous findings that suggest a detrimental role for neuronally synthesized nitric oxide in the acute stages of focal cerebral ischemia. Immediate administration of TRIM also partially reduces the deficits in motor function observed 1 and 24 hours after transient MCA occlusion, which is described as a decrease in the neurologic score in comparison to saline-treated groups. These results provide evidence that TRIM not only protects against the physical ischemic damage, indicated by TTC staining, but also reduces the deficit in neurologic function observed after transient focal cerebral ischemia. More recently, our group has confirmed the cerebroprotective effect of immediate administration of TRIM using magnetic resonance imaging (Haga et al., 1997).

Immediate administration of 7-NI (5 minutes after MCA occlusion) decreased the total lesion volume at a dose that does not alter blood pressure and that has been shown previously to reduce neuronal NOS activity by 50% in the cerebral cortex (Yoshida et al., 1994). The cerebroprotective effect of 7-NI is similar to a previous report in which 7-NI (50 mg/kg) was shown to inhibit lesion volume by 25% after permanent MCA occlusion, an effect that was reversed by l-arginine (Yoshida et al., 1994). In our study, 7-NI reduces both striatal and cortical lesion sizes, which confirms findings using the permanent MCA occlusion model (Yoshida et al., 1994). The apparently greater effect of 7-NI reported here in the reversible model (38% reduction) compared with 25% in the permanent model of MCA occlusion, if significant, could reflect the difference in the severity of the ischemic time periods or the slightly higher dose of 7-NI used in our study. In contrast to the cerebroprotective effect of TRIM (administered 90 minutes after occlusion), delayed administration of 7-NI showed no significant effect on the lesion size. It is unclear why TRIM, but not 7-NI, was protective when administered 90 minutes after occlusion, especially because 7-NI is a more potent nNOS inhibitor than TRIM in vitro (Handy et al., 1996). One possibility is that TRIM is more soluble than 7-NI and thus may have a higher bioavailability than 7-NI.

Interestingly, l-arginine alone had no effect on lesion size. This finding matches other reports that l-arginine does not improve histologic outcome in rats subjected to either a distal occlusion of the MCA using photothrombotic techniques (Prado et al., 1996) or permanent MCA occlusion (Buisson et al., 1993; Iadecola et al., 1995a). In contrast, delayed administration of l-arginine at 24, 48, and 72 hours after MCA occlusion enlarged the infarct by 29% after transient focal cerebral ischemia (Zhang et al., 1996a). There are however several reports that l-arginine reduces ischemic lesion volume after permanent MCA occlusion in spontaneously hypertensive rats (Morikawa et al., 1992a; Hamberg et al., 1993) and in normotensive Sprague-Dawley rats (Morikawa et al., 1994). l-Arginine also increases cerebral blood flow in ischemic (Hamberg et al., 1993; Morikawa et al., 1994) and nonischemic rats (Dalkara et al., 1994; Prado et al., 1996). The protective effect of l-arginine is thought to be mediated by nitric oxide-dependent cerebrovascular dilation increasing blood flow to the penumbral region of the ischemic lesion (Morikawa et al., 1994; Dalkara et al., 1994). These studies, which demonstrated neuroprotection with l-arginine, used multiple dosing regimes or intravenous infusions (Morikawa et al., 1992a, 1994). It is likely that l-arginine did not have any protective effect in our study because it was given as a single intraperitoneal injection after MCA occlusion.

It has been suggested that the mechanism of action of both TRIM and 7-NI is through the inhibition of NOS, preventing the conversion of l-arginine to nitric oxide (Handy et al., 1995; Moore et al., 1993a; Yoshida et al., 1994). Neither TRIM or 7-NI had any effect on arterial blood pressure at the highest doses used, which is consistent with other studies in the mouse and rat (Handy et al., 1995; Moore et al., 1993a, 1993b; Rees et al., 1991; Smith et al., 1996). These results suggest that endothelial NOS is not inhibited to any significant extent during this transient model of focal cerebral ischemia. Also, coadministration of l-arginine with TRIM reversed the protective effect observed with TRIM alone, thereby suggesting that the mechanism of action of TRIM is through the inhibition of NOS. Direct measurement of NOS activity in vivo would help to confirm the mechanism of neuroprotection by TRIM.

Slight hyperthermia, which could contribute to the development of the ischemic lesion, was observed at the time of reperfusion, and previously a reduction in body temperature has been shown to be neuroprotective (Chen et al., 1992). However, TRIM and 7-NI are unlikely to be cerebroprotective as a result of a reduction in body temperature because at the time of reperfusion there was no temperature difference between control and drug-treated groups. Similarly, 24 hours after MCA occlusion no difference in body temperature was observed (unpublished communication). Also, neither agent had any effect on systemic blood pressure on intraperitoneal administration. Therefore, we can conclude that there should not be any major change in body temperature because of any potential hemodynamic effects of TRIM or 7-NI.

Although both TRIM and 7-NI have been shown to be relatively selective inhibitors of neuronal NOS, a possible effect on inducible NOS should be considered (Moore et al., 1993a, 1993b; Babbedge et al., 1993; Handy et al., 1995). Expression of brain inducible NOS mRNA, however, begins at 12 hours and peaks 48 hours after MCA occlusion, and inducible NOS catalytic activity is observed only 48 hours after ischemia (Iadecola et al., 1995b). Therefore, it is unlikely that TRIM or 7-NI alters inducible NOS activity, because the protocol used in our study measured lesion size only 24 hours after occlusion. Also, both agents were administered in the acute stages (<2 hours) of ischemic cell damage, before expression of the inducible NOS protein.

In conclusion, inhibition of NOS with TRIM or 7-NI elicited protection against ischemic cell damage after transient focal cerebral ischemia in rats. These results confirm previous observations that neuronally derived nitric oxide is detrimental in the early stages of transient focal cerebral ischemia in vivo.

Footnotes

Abbreviations used

Acknowledgment

The authors thank Dave Plummer (UCH, London) for access to the suite of image display (Dispim) software.

References

Ashwal

Cole

Osborne

Pearce

(1993) Low dose l-NAME reduces infarct volume in the rat MCAO/reperfusion model. J Neurosurg Anesthesiol 5:241–259.

Babbedge

Bland-Ward

Hart

Moore

(1993) Inhibition of rat cerebellar nitric oxide synthase by 7-nitro indazole and related substituted indazoles. Br J Pharmacol 110:225–228.

Bath

PMW

Hassall

Gladwin

Palmer

RMJ

Martin

(1991) Nitric oxide and prostacyclin. Divergence of inhibitory effects on monocyte chemotaxis adhesion to endothelium in vitro. Arterioscler Thromb 11:254–260.

Bederson

Pitts

Germano

Nishimura

Davis

Bartkowski

(1986a) Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17:1304–1308.

Bederson

Pitts

Tsuji

Nishimura

Davis

Bartkowski

(1986b) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17:472–476.

Buisson

Margaill

Callebert

Plotkine

Boulu

(1993) Mechanisms involved in the neuroprotective activity of a nitric oxide synthase inhibitor during focal cerebral ischemia. J Neurochem 61:690–696.

Butterworth

Bath

PMW

(1996) The effect of nitric oxide on platelet function in acute ischaemic stroke (Abstr). Cerebrovasc Dis 6(Suppl 2):6.

Chen

Chopp

Zhang

Garcia

(1992) The effect of hypothermia on the transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 12:621–628.

Dalkara

Moskowitz

(1994) The complex role of nitric oxide in the pathophysiology of focal cerebral ischemia. Brain Pathol 4:49–57.

10.

Dalkara

Yoshida

Irikura

Moskowitz

(1994) Dual role of nitric oxide in focal cerebral ischemia. Neuropharmacology 33:1447–1452.

11.

Dawson

(1994) Nitric oxide and focal cerebral ischemia: multiplicity of actions and diverse outcome. Cerebrovasc Brain Metab Rev 6:299–324.

12.

Dawson

TMD

London

Bredt

Snyder

(1991) Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc Natl Acad Sci USA 88:6368–6371.

13.

Escott

Beech

Meldrum

Williams

SCR

Bath

PMW

(1997) Delayed treatment of reversible focal cerebral ischaemia with 7-nitroindazole in rats. J Hum Hypertens (in press).

14.

Haga

Escott

Beech

Smart

Kinchesh

Bath

PMW

Meldrum

Williams

(1997) The combined application of MRI and 19F MRS to the evaluation of a trifluorinated, nitric oxide synthase inhibitor in a rodent model of focal cerebral ischaemia. Submitted to Proceedings of the International Society for Magnetic Resonance in Medicine, 5th Scientific meeting, Vancouver, 1997, pp 1267.

15.

Hamberg

Huang

Hunter

Caramia

Moskowitz

Rosen

(1993) Time effects of l-arginine on cerebral hemodynamics and ischemic volume during acute focal ischemia demonstrated by dynamic MRI. In: Proceedings of the Society of Magnetic Resonance in Medicine, 12th Annual Scientific Meeting, August 1993, New York, Vol. 1, pp 397.

16.

Handy

RLC

Harb

Wallace

Gaffen

Whitehead

Moore

(1996) Inhibition of nitric oxide synthase by 1-(2-trifluoromethylphenyl) imidazole (TRIM) in vitro: antinociceptive and cardiovascular effects: Br J Pharmacol 119:423–431.

17.

Handy

RLC

Wallace

Gaffen

Whitehead

Moore

(1995) The antinociceptive effect of 1-(2-trifluoromethylphenyl)imidazole (TRIM), a potent inhibitor of neuronal nitric oxide synthase in vitro, in the mouse. Br J Pharmacol 116:2349–2350.

18.

Hara

Huang

Panahian

Fishman

Moskowitz

(1996) Reduced brain edema and infarction volume in mice lacking the neuronal isoform of nitric oxide synthase after transient MCA occlusion. J Cereb Blood Flow Metab 16:605–611.

19.

Huang

Panahian

Dalkara

Fishman

Moskowitz

(1994) Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 265:1883–1885.

20.

Huang

Fishman

Moskowitz

(1996) Focal cerebral ischemia in mice deficient in either endothelial (eNOS) or neuronal nitric oxide (nNOS) synthase (Abstr). Stroke 27:173.

21.

Iadecola

Pelligrino

Moskowitz

Lassen

(1994) Nitric oxide synthase inhibition and cerebrovascular regulation. J Cereb Blood Flow Metab 14:175–192.

22.

Iadecola

Zhang

(1995a) Inhibition of inducible nitric oxide synthase ameliorates cerebral ischemic damage Am J Physiol 268:R286–R292.

23.

Iadecola

Zhang

Casey

Ross

(1995b) Inducible nitric oxide synthase expression in brain following cerebral ischemia. J Cereb Blood Flow Metab 15:378–384.

24.

Koizumi

Yoshida

Nakazawa

Ooneda

(1986) Experimental studies of ischemic brain edema: 1. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Jpn J Stroke 8:1–8.

25.

Kumura

Kosaka

Shiga

Yoshimine

Hayakawa

(1994) Elevation of plasma nitric oxide end products during focal cerebral ischemia and reperfusion in the rat. J Cereb Blood Flow Metab 14:487–491.

26.

Malinski

Bailey

Zhang

Chopp

(1993) Nitric oxide measured by a porphyrinic microsensor in rat brain after transient middle cerebral artery occlusion. J Cereb Blood Flow Metab 13:355–358.

27.

Manzoni

Prezeau

Marin

Deshager

Bockaert

Fagni

(1992) Nitric oxide-induced blockade of NMDA receptors. Neuron 8:653–662.

28.

Meldrum

(1996) The role of nitric oxide in ischemic damage. In: Advances in Neurology. Vol. 17: Cellular and Molecular Mechanisms of Ischemic Brain Damage ( Siesjö

Wieloch

, eds), Philadelphia, Lippincott-Raven, pp 355–363.

29.

Moncada

Palmer

Higgs

(1991) Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109–142.

30.

Moore

Babbedge

Wallace

Gaffen

Hart

(1993a) 7-Nitro indazole, an inhibitor of nitric oxide synthase, exhibits anti-nociceptive activity in the mouse without increasing blood pressure. Br J Pharmacol 108:296–297.

31.

Moore

Wallace

Gaffen

Hart

Babbedge

(1993b) Characterization of the novel nitric oxide synthase inhibitor 7-nitro indazole and related indazoles: antinociceptive and cardiovascular effects. Br J Pharmacol 110:219–224.

32.

Morikawa

Huang

Moskowitz

(1992a) l-Arginine decreases infarct size caused by middle cerebral artery occlusion in SHR. Am J Physiol 263:H1632–H1635.

33.

Morikawa

Rosenblatt

Moskowitz

(1992b) l-Arginine dilates rat pial arterioles by nitric oxide-dependent mechanisms and increases blood flow during focal cerebral ischemia. Br J Pharmacol 107:905–907.

34.

Morikawa

Moskowitz

Huang

Yoshida

Irikura

Dalkara

(1994) l-Arginine promotes nitric oxide-dependent vasodilation, increases regional cerebral blood flow, and reduces infarction volume in the rat. Stroke 25:429–435.

35.

Nagafuji

Matsui

Koide

Asano

(1992) Blockade of nitric oxide formation by Nω-nitro-l-arginine mitigates ischemic brain edema and subsequent cerebral infarction in rats. Neurosci Lett 147:159–162.

36.

Nowicki

Duval

Poignet

Scatton

(1991) Nitric oxide mediates neuronal death after focal cerebral ischemia in the mouse. Eur J Pharmacol 204:339–340.

37.

Prado

Watson

Zhao

Yao

Busto

Dietrich

Ginsberg

(1996) l-Arginine does not improve cortical perfusion or histological outcome in spontaneously hypertensive rats subjected to distal middle cerebral artery photothrombotic occlusion. J Cereb Blood Flow Metab 16:612–622.

38.

Radi

Beckman

Bush

Freeman

(1991) Peroxynitrite oxidation of sulfhydryls. J Biol Chem 266:4244–4250.

39.

Radomski

Palmer

RMJ

Moncada

(1987) Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 2:1057–1058.

40.

Rees

Palmer

RMJ

Moncada

(1991) Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc Natl Acad Sci USA 86:3375–3378.

41.

Smith

Man

Yip

Tang

Chapman

Meldrum

(1996) Anticonvulsant effects of 7-nitro indazole in rodents with reflex epilepsy may result from l-arginine accumulation or a reduction in nitric oxide or l-citrulline formation. Br J Pharmacol 119:165–173.

42.

Stadler

Billiar

Curran

Stuehr

Ochoa

Simmons

(1991) Effect of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes. Am J Physiol 260:C910–C916.

43.

Yoshida

Limmroth

Irikura

Moskowitz

(1994) The NOS inhibitor, 7-nitroindazole, decreases focal infarct volume but not the response to topical acetylcholine in pial vessels. J Cereb Blood Flow Metab 14:924–929.

44.

Zea Longa

Weinstein

Carlson

Cummins

(1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84–91.

45.

Zhang

Iadecola

(1993) Nitroprusside improves blood flow and reduces brain damage after focal ischemia. Neuroreport 4:559–562.

46.

Zhang

Iadecola

(1994) Reduction of focal cerebral ischemic damage by delayed treatment with nitric oxide donors. J Cereb Blood Flow Metab 14:574–580.

47.

Zhang

White

Iadecola

(1994) 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 14:217–226.

48.

Zhang

Casey

Ross

Iadecola

(1996a) Aminoguanidine ameliorates and l-arginine worsens brain damage from intraluminal middle cerebral artery occlusion. Stroke 27:317–323.

49.

Zhang

Reif

Macdonald

Tang

Kamp

Gentile

Shakespeare

Murray

Chopp

(1996b) ARL 17477, a potent and selective neuronal NOS inhibitor decreases infarct volume after transient cerebral artery occlusion in rats. J Cereb Blood Flow Metab 16:599–604.