EXPERIMENTAL STUDIES

EXPERIMENTAL STUDIES

NITRIC OXIDE SYNTHASE IN ACUTE ALTERATION OF NITRIC OXIDE LEVELS AFTER SUBARACHNOID HEMORRHAGE Fatima A. Sehba, Ph.D. Department of Neurosurgery, Mount Sinai School of Medicine, New York, New York

Igor Chereshnev, M.D. Department of Neurosurgery, Mount Sinai School of Medicine, New York, New York

Saul Maayani, Ph.D. Department of Neurosurgery, Mount Sinai School of Medicine, New York, New York

Victor Friedrich, Jr., Ph.D. Department of Neurobiology, Mount Sinai School of Medicine, New York, New York

Joshua B. Bederson, M.D. Department of Neurosurgery, Mount Sinai School of Medicine, New York, New York

OBJECTIVE: Subarachnoid hemorrhage (SAH) is associated with acute decreases and subsequent recovery of cerebral nitric oxide (NO) levels, but the mechanisms of these alterations are not known. In this study, we measured NO synthase (NOS) protein and kinetics to determine its involvement in the alterations of cerebral NO levels after SAH. METHODS: The endovascular rat model of SAH was used. The number of NOS-1 (neuronal) and NOS-2 (inducible)-positive cells (0–96 h) was determined by counting immunoreactive cells in 8-␮m cryostat sections. The tissue content of active NOS and its kinetic parameters were studied with an enzymatic L-citrulline assay. RESULTS: The number of NOS-1-positive cells increased between 1 and 3 hours after SAH, decreased to and below control values at 6 and 72 hours after SAH, and increased to control values 96 hours after SAH. The number of NOS-2-positive cells increased 1 hour after SAH, decreased to control values at 24 hours, and increased above control values 96 hours after SAH. The Michaelis-Menten kinetic parameters (Vmax, Km, slope) of NOS remained unchanged at 10 and 90 minutes after SAH. CONCLUSION: NOS-1 and -2 proteins undergo a triphasic alteration after SAH, whereas the amount of active NOS and its kinetic parameters remain unchanged during the first 90 minutes after SAH. Depletion of NOS is not involved in the acute alterations of cerebral NO levels after SAH. KEY WORDS: Cerebral ischemia, Nitric oxide, Nitric oxide synthase, Subarachnoid hemorrhage Neurosurgery 55:671-678, 2004

Reprint requests: Joshua B. Bederson, M.D., Department of Neurosurgery, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1136, New York, NY 10029-6574. Email: [email protected] Received, August 20, 2003. Accepted, April 4, 2004.

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DOI: 10.1227/01.NEU.0000134557.82423.B2

itric oxide (NO) plays an important role in maintaining cerebral vascular tone and cerebral blood flow (CBF) (7, 23, 24, 44). Alterations in NO metabolism pathways have been implicated in the pathophysiology of subarachnoid hemorrhage (SAH) (6, 37–39). We have previously shown that cerebral NO levels decrease immediately after SAH and subsequently increase (39). The transient decrease in NO makes SAH unique among all other forms of cerebral ischemia, in which an increase in NO is observed (8, 21, 25, 26, 30, 31, 48). The unique decrease in NO that occurs in SAH may contribute to several pathophysiological events, including unopposed vasoconstriction, decreased CBF, cerebral ischemia, and increased glutamate release (3, 4, 35, 38), which together may augment brain injury after SAH. Administration of an NO donor immediately after experimental

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SAH restores CBF and decreases ischemic glutamate release (38), and administration of NO donors in other models of cerebral ischemia is noted to increase CBF and reduce cerebral infarction size (47, 50). It is therefore likely that interventions that increase cerebral NO levels early in an episode of ischemia may be neuroprotective (10, 17, 28), and a detailed understanding of the mechanisms that regulate NO levels after SAH may contribute to the development of therapeutic interventions. A number of investigators have studied NOS expression in other models of cerebral ischemia (14, 18, 32). Accumulating evidence indicates early increases in NOS-1 (neuronal) and NOS-3 (endothelial) (49) and a delayed increase in NOS-2 (inducible) expression (18, 19), accompanied by increased NO production (21). To determine how NOS is altered after SAH, we studied changes in NOS protein lev-

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els after SAH by examining the amount of active NOS, its kinetic parameters, and the number of NOS-1- and NOS-2positive cells at different time intervals after experimental SAH. We found that the amount of active NOS and its kinetic parameters were unaltered during the first 90 minutes after SAH and that the numbers of NOS-1- and NOS-2-positive cells undergo a complex triphasic change during the first 96 hours after SAH.

MATERIALS AND METHODS All experimental procedures and protocols used in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the Mount Sinai Medical Center.

Animal Model of SAH Male Sprague-Dawley rats (300–400 g) underwent experimental SAH using the endovascular suture model developed in this laboratory (3, 4). Briefly, rats were anesthetized with chloral hydrate (35 mg/kg intraperitoneally), intubated transorally, ventilated, and maintained on inspired halothane (1 to 2% in oxygen-supplemented room air) and placed on a homeothermic blanket (Harvard Apparatus, Natick, MA) attached to a rectal temperature probe set to maintain body temperature at 37°C. Rats were positioned in a stereotactic frame, and the femoral artery was exposed and cannulated for blood gas and blood pressure monitoring. For measurement of intracranial pressure (ICP), the atlanto-occipital membrane was exposed and cannulated, and the cannula was affixed with methylmethacrylate cement to a stainless steel screw implanted in the occipital bone (2). CBF was measured by a laser-Doppler flowmeter (0.8-mm-diameter probes; Model P-433; Vasamedics, Inc., St. Paul, MN) advanced under stereotactic control to the epidural surface exposed by small burr holes over the middle cerebral artery territory 5 mm lateral to the midline at the coronal suture. SAH was induced by advancing a suture retrogradely through the ligated right external carotid artery and distally through the internal carotid artery until the suture perforated the intracranial bifurcation of the internal carotid artery. This event was confirmed by a sudden rise in ICP and bilateral decrease in CBF (3). Animals were monitored for 20 minutes before SAH and 60 minutes after SAH. As animals regained consciousness and were able to breath spontaneously, they were returned to their cages. Animals were killed at random at 1 to 96 hours for immunohistochemical measurements (n ⫽ 3–7 per time point) or at 10 to 90 minutes (n ⫽ 3–6 per time point) for measurements of NOS activity after SAH. To increase survival rate, surgery was limited by not measuring ICP in animals killed 24 hours after SAH. In this study, nonoperated animals were used as controls. Preliminary experiments showed no increase in NOS-1 and -2 immunoreactivity in sham-operated animals. Additional experiments used nonoperated controls to limit the number of surgeries.

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NOS Immunohistochemistry Tissue Preparation Animals were anesthetized and perfused transcardially with chilled 4% buffered paraformaldehyde, and brains were removed and fixed at 4°C in paraformaldehyde for at least 24 hours. Coronal slices 3.0 mm thick were embedded in paraffin. Adjacent sections (5 ␮m) were used for NOS-1 and NOS-2 immunohistochemistry. Nonoperated animals were used as controls; in preliminary experiments, no increase in NOS-1 or -2 immunostaining between sham (6, 24, and 96 h) and nonoperated animals was found.

Immunohistochemistry Rabbit anti-human (NOS-1 [K-20]: sc-1025) and rabbit antihuman (NOS-2 [N-20]: sc-651; both Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were used to detect NOS-1 and NOS-2, respectively. Preliminary studies using Western blot analysis showed complete absorption of NOS-1 and -2 bands on addition of their respective blocking peptides (NOS-1, sc1025 P, and NOS-2, sc-651 P; Santa Cruz Biotechnology). Antibody binding was visualized by use of biotinylated secondary antibody, avidin-horseradish peroxidase, and diaminobenzidine (Santa Cruz Biotechnology). Sections from nonoperated animals and sections from SAH brains with no primary antibody were used as positive and negative controls, respectively, in each stain set. Cell Counting. Marked changes in NOS-1 and NOS-2 immunostaining (Fig. 1) after SAH were readily visible at low magnification. For cell counting, images of frontal and basal

FIGURE 1. Photomicrographs of immunostained sections. NOS-1 and -2 immunoreactivity increases after SAH. Representative sections of the rat cerebral cortex immunostained for NOS-1 or NOS-2 protein 1 or 3 hours after SAH are shown. NOS-1- and -2-positive cells are sparse in controls as compared with SAH animals. Some microvessels also showed NOS-1 immunoreactivity after SAH.


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cortex, striatum, and hippocampus (Morrell Labophot-2; ⫻4 objective; Morrell, Melville, NY) were captured under constant conditions and analyzed with IPLAB (IPLab, Version 3.0; Scanalytics, Inc., Fairfax, VA), which segments images on the basis of pixel values. For segmentation, images were thresholded by intensity to eliminate background, and the identified objects were gated by size. Our experience indicates that this approach is effective for counting cell profiles in this material, and the results are comparable to those obtained by manual counting. Cell counts (NOS-1, 18–150 per 3⫻106-␮m2 field; NOS-2, 10–200 per 3⫻106-␮m2 field) were recorded as number per unit area and are presented as mean ⫾ standard error of the mean (SEM) % nonoperated control values. These values document the number of profiles in sections and are influenced by cell size. For that reason, changes in the number of cells per volume of tissue may differ somewhat in magnitude from the counts reported here. To determine whether cell counts were normally distributed, a Kolmogorov-Smirnov normality test was performed. This demonstrated a normal distribution in each brain area. In addition, standard normal probability plots were prepared. These plots also confirmed a normal distribution. For this reason, statistical evaluation was performed using a one-way analysis of variance followed by post hoc Fisher’s protected least significant difference test.

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150 ␮l of 3.4% sodium dodecyl sulfate in 0.05 mol/L sodium citrate, pH 2.5 (final assay, pH 4). [14C]citrulline (0.25 mmol/L final concentration, containing approximately 3000 dpm) was added as internal standard to each tube, samples were purified onto Dowex AG50 columns equilibrated with 0.1 mol/L sodium citrate, pH 4, and the radioactivity of the eluate was measured by scintillation counting (Beckman ZLS500 or Beckman LS 6500; Beckman Instruments, Fullerton, CA). NOS activity was defined as the 100 ␮mol/L nitroargininesensitive production of 3H materials from [3H]arginine, which has been shown to be identical to [3H]citrulline (43). Standard kinetic parameters, Km and Vmax, were calculated by nonlinear least-squares curve fitting of all measured points to the Michaelis-Menten equation plus a straight line derived from the linear regression equation for the nitroarginine blank values.

Physiological Data Acquisition CBF, ICP, and mean arterial blood pressure data were recorded continuously starting 10 minutes before and 10 to 60 minutes after SAH using customized analog-to-digital conversion hardware and software (Labview, Version 4.0; National Instruments, Austin, TX) and stored at a rate of 0.25 Hz.

RESULTS

Measurement of NOS Activity Sample Preparation

NOS Immunohistochemistry

Animals were killed 10 or 90 minutes (n ⫽ 3–6 min) after SAH surgery. Nonoperated animals (n ⫽ 3) were used as controls. Cerebral cortex, cerebellum, striatum, or hippocampus was dissected from each side of the brain. Ipsilateral (side of SAH induction; right hemisphere) and contralateral (side opposite to SAH induction; left hemisphere) tissues were pooled for assay. Each assay contained tissue from one animal only. Samples were homogenized in ice-cold buffer containing (in mmol/L) sucrose 300, ethylenediamine tetra-acetic acid 0.5, dithiothreitol 1.0, and N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid 20; pH 7.6. The homogenate was centrifuged at 100,000 ⫻ g for 1 hour at 4°C.

NOS-1- and NOS-2-immunoreactive cells were counted in sections of basal and frontal cerebral cortex, striatum, and the CA1 region of both ipsilateral (right) and contralateral (left) hemispheres relative to the site of SAH induction. Changes in NOS-1- and -2-positive immunoreactivity were similar in the two hemispheres; consequently, we illustrate data from the ipsilateral hemisphere only.

NOS Assay A modified citrulline production assay (43) using [3H]arginine (New England Nuclear, Boston, MA) purified with a Dowex AG50 ion exchange resin (Bio-Rad Laboratories, Hercules, CA) was used. The initial velocity of NOS catalytic reaction was determined in mixture a containing (in mmol/L) CaCl2 0.45, reduced nicotinamide adenine dinucleotide phosphate 0.1, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid 20, sucrose 60, and dithiothreitol 1.0, with 150–900 ␮g/ml protein homogenate and [3H]arginine as follows. Triplicate determinations were performed at seven values of [arginine], from 0.25 to 20 ␮mol/L, a range spanning 1 log unit above and below the Km of NOS (ⵑ2 ␮mol/L). Blanks contained 100 ␮mol/L of nitroarginine. Tubes were incubated for 10 to 30 minutes at 24°C, and the reaction was stopped by addition of

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NOS-1 Control Animals. In control animals, NOS-1-positive cells displayed neuronal shapes (fusiform, triangular, multipolar, spiny; Fig. 1). These cells were present in cortex, in striatum, and in hippocampus. Intense NOS-1 staining was found primarily in the perikarya; however, occasional cell processes ⬎10 ␮m in length were also stained. In the frontal and basal cortex, NOS-1 immunoreactivity was observed in isolated cortical neurons distributed throughout the different cortical layers. Striatal NOS-1-positive neurons showed the typical pattern for spiny nitrinergic neurons. In hippocampal CA1, a small number of interneurons intermingled among unstained pyramidal neurons showed NOS-1 immunoreactivity. SAH Animals. After SAH, the intensity of NOS-1 staining in large neurons increased, and a marked increase in the number of NOS-1-positive cells with neuronal structure but smaller size was noted bilaterally in all brain regions compared with nonoperated controls (Fig. 1). In cortex and hippocampus, NOS-1 immunoreactivity remained largely in the cell bodies. However, in striatum, many cell processes (⬍200


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␮m in length) also became positive for NOS-1. Thus, counting of NOS-1-positive cells required not only thresholding of images by intensity to eliminate background but also identifying counted objects by size gating to eliminate small profiles such as dendrites. NOS-1 immunoreactivity in microvessels was also observed after SAH but not in controls. Cell Numbers. In cerebral cortex, the number of NOS-1positive cells per unit area increased at 1 and 3 hours after SAH in frontal (Fig. 2A) and basal (Fig. 2B) cortex compared with controls (P ⬍ 0.05). The duration of increase in the NOS-1-positive cells was 6 to 24 hours. NOS-1-positive cells decreased below control values in all cortical areas at 72 hours after SAH and increased to control values at 96 hours (Fig. 2, A and B). In striatum, the number of NOS-1-positive cells increased significantly at 1 hour and peaked at 3 hours after SAH (P ⬍ 0.05). NOS-1-positive cells remained elevated 6 hours after SAH and then decreased to and below control values at 24 and 72 hours after SAH, respectively. A second increase in NOS-1-positive cells was observed at 96 hours after SAH (Fig. 2C). In hippocampus, NOS-1-positive cells remained unchanged during the first 6 hours after SAH. In the ipsilateral CA1 region, a trend toward decrease followed by a significant decrease in NOS-1-positive cells was observed at 48 (P ⫽ 0.06), 72, and 96 hours (P ⬍ 0.05) after SAH (Fig. 2D). In the contralateral CA1 region, NOS-1-positive cells decreased at 24 hours (P ⬍ 0.05) and increased to control values 48 hours after SAH (data not shown).

NOS-2 Control Animals. Little NOS-2 immunoreactivity was seen in brains from control animals, as reported elsewhere (40).

SAH Animals. After SAH, the number of NOS-2-positive cells increased dramatically at some postoperative time points (P ⬍ 0.0001; Fig. 1). NOS-2-positive cells were present in all layers of cortex and throughout the striatum and hippocampus. Two types of cells showed NOS-2 immunoreactivity after SAH: 1) cells that exhibit neuronal structure and 2) smaller cells that did not exhibit neuronal structure and are consistent with glia. Cell Numbers. In cerebral cortex, NOS-2-positive cells peaked in number per area of section 1 hour after SAH in both frontal and basal cortex, remained elevated for 6 hours, and decreased to control values 48 hours after SAH (P ⬍ 0.05). A second increase in NOS-2-positive cells was observed 96 hours after SAH (Fig. 2, A and B). In striatum, NOS-2-positive cells increased 1 hour after SAH and remained elevated for 6 hours (P ⬍ 0.05). A decline in NOS-2-positive cells below control values was observed 24 hours after SAH. NOS-2-positive cells returned to control values 48 hours after SAH and stayed at control values at 96 hours after SAH (Fig. 2C). In hippocampus, NOS-2-positive cells increased 1 hour after SAH remained elevated on the ipsilateral side for 3 hours (Fig. 2D; P ⬍ 0.05). In the contralateral CA1 region, NOS-2-positive cells decreased transiently below control values at 24 hours and recovered by 48 hours (data not shown). In the ipsilateral CA1 region, NOS-2-positive cells increased to and above control values at 24 and 96 hours after SAH, respectively (Fig. 2D).

Measurement of NOS Enzymatic Activity Approximately 30% of basal NOS activity was located in membranes and the rest in cytosol. In control animals, the Vmax of NOS in both soluble cytosolic and membrane-bound particulate fractions varied across regions (data not shown). Km (2.4 ⫾ 0.60 ␮mol/L) and slope index (1.20 ⫾ 0.03) of NOS remained constant across regions and in the cytosolic and particulate fractions. No significant change in Vmax or Km of NOS was found at 10 or 90 minutes after SAH compared with control in cytosolic or particulate fractions of any of the brain areas studied (P ⬎ 0.05; Fig. 3).

DISCUSSION

FIGURE 2. Graphs showing regional differences in NOS-1- and -2-positive cells after SAH. Data are mean ⫾ SEM, from ipsilateral basal (A) and frontal (B) cortex, striatum (C), or CA1 regions of hippocampus (D). *†, significantly different from respective controls (P ⬍ 0.05).


The present study examined NOS kinetics and immunostaining after SAH. We found that NOS MichaelisMenten Vmax and Km were unchanged for at least 90 minutes after SAH, indicating that NOS synthetic potential remains stable during the initial phase of NO depletion. The number of NOS-1- and NOS-2-positive

FIGURE 3. Bar graph showing that NOS activity is not altered after SAH. Represented is Vmax of NOS in striatum of controls or SAH animals. SAH did not have any effect on Vmax of NOS. In addition, neither Km nor slope index of NOS was affected by SAH (data not shown). Data are mean ⫾ SEM.



cells (immunoreactive profiles in sections of brain) underwent a triphasic pattern of change during the first 96 hours after SAH. This pattern involved an initial increase (Phase I, 1–6 h), a decrease back to control values (Phase II, 6–72 h), and a transient decrease in NOS-1- (Phase III, 72 h) and increase in NOS-2positive cells (96 h) after SAH (Fig. 4). We have previously shown that cerebral NO levels decrease acutely after SAH and recover 180 minutes later (39). In addition, using a nonselective NOS inhibitor (NG-nitro-l-arginine methyl ester), we demonstrated that preinhibition of NO synthesis did not potentiate the fall in CBF seen after SAH (36). However, the same treatment 60 minutes after SAH limited CBF recovery. This observation underlines the observation that NO and its vasodilatory effect are absent during the first hour after SAH. A number of mechanisms could account for the initial decrease in cerebral NO levels specific to SAH. These include scavenging by hemoglobin, changes in the amount or activity of NOS, and depletion of substrate or cofactors. Previous studies have reported that NOS enzymatic activity is (13) or is not (22, 42) changed after SAH. In the present study, we found no change in Michaelis-Menten Vmax and Km, indicating that NOS was not irreversibly inactivated or altered in its kinetics during the first phase of acute NO depletion. It should be noted that our in vitro data do not preclude an in vivo reduction in NO production because of substrate or cofactor depletion or changes in pH after SAH. In this regard, direct data on the in vivo production of NO would be desirable. Nevertheless, our data are consistent with the hypothesis that, after SAH, NO is depleted by hemoglobin scavenging and that NO levels recover as this hemoglobin is metabolized into methemoglobin, which has a slower rate of binding NO or slowly dissociating nitrosylhemoglobin (39). At rest, 1 to 2% of cortical neurons in the rat are NOS-1 immunoreactive (5), and 95% of total NOS catalytic activity is from NOS-1 (15). NOS-1 immunoreactive neurons are large fusiform, triangular, or large multipolar and are scattered throughout the different layers of cortex (20). NOS-1-positive small neu-

FIGURE 4. Graph showing temporal changes in the number of NOS-1- and -2-positive cells in sections of rat brain after SAH. NOS-1- and -2-positive cells showed similar pattern of change for the first 48 hours after SAH. At 72 and 96 hours after SAH, NOS-1-positive cells decreased below and to the control values, and NOS-2-positive cells increased above control values 96 hours after SAH. Data are accumulation (mean ⫾ SEM) of NOS-1- or -2-positive profiles in all three regions in both hemispheres. †*, significantly different from respective controls (P ⬍ 0.05).

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rons are present in the cortex of humans and other primates, but their presence in intact rat and mouse is controversial (46). We did not observe NOS-1-positive small neurons in our control animals. NOS-1 is also expressed in cerebral blood vessels (41). NOS-2 is found at low levels in rat brain under normal conditions (40) and is expressed in response to injury by astrocytes, microglia cells, neurons, and to a lesser extent, endothelial cells (11, 29). Cerebral injuries lead to altered expression of all three NOS isoforms (1, 9, 14), but the temporal and isoform patterns vary with the type of brain injury (9). For example, in permanent focal ischemia, NOS-1 messenger ribonucleic acid (mRNA) is increased by 15 minutes and NOS-1 protein by 1 hour after the insult (49). In transient 2-hour focal ischemia, expression of NOS-1 and -2 proteins is increased by 15 minutes after reperfusion (14). During oxygen-and-glucose-deprived global ischemic injury, the expression of NOS-1 and NOS-2 proteins is increased by 4 hours after reperfusion (1). Similarly, expression of all three NOS isoforms is increased by 6 hours and peaks at 12 hours after traumatic brain injury (9). In the present study, NOS-1-positive cell profiles were markedly increased in staining intensity and in number 1 to 3 hours after SAH. This increase may indicate a response to reduced CBF or an acute stress-induced response. The increase in number of NOS-1 cells after SAH occurred primarily in small neurons, whereas the intensity of staining increased in larger neurons. Similar findings were reported by Alonso et al. (1) in a study of oxygen- and glucose-deprived global ischemia. They observed an increase in intensity of NOS-1-stained cortical neurons during a 30-minute period of ischemia and an increase in the number of smaller neurons positive for NOS-1 during the first 6 hours of reperfusion (1). The decrease toward control values in NOS-1-positive cells observed 72 hours after SAH could be a result of recovery of CBF during this period or of resolution of the initial stress-induced increase, or it may represent a loss of cortical neurons. Cortical cells are known to be affected primarily by acute SAH (16). Although NOS-1-positive neurons maybe resistant to a hypoxicischemic insult, the mechanism of this protection does not prevent their necrotic death (12, 49). Degeneration of NOS-1positive neurons 7 days after transient focal ischemia has been demonstrated in adult rats (49) and in neonatal rats (12) 24 hours after hypoxic-ischemic injury. NOS-2-positive cell profiles increased within 1 hour after SAH, returned to control values at 72 hours after SAH, and increased again at 96 hours. An increase in NOS-2 mRNA and protein was reported by Sayama et al. (33) in rat cerebral vascular tissues 1 day after SAH. They found newly expressed NOS-2 to be enzymatically active, and because its inhibition ameliorated SAH-induced vasoconstriction, they suggested that NOS-2 contributes directly to delayed vasoconstriction after SAH (34). In other ischemic injury models, increased expression of NOS-2 (12 h to 2 or 4 d) is accompanied by large increases in NO production and is deleterious (18, 19). In the present study, NOS enzymatic data indicated that the increase in total NOS-2-positive cells 1 hour after SAH was not accompanied by an increase in total tissue content of active NOS.


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However, it is possible that the subsequent increase in NOS2-positive cells at 96 hours is accompanied by increased NO levels, which could contribute to cytotoxicity. In the present study, total in vitro tissue NOS activity (Vmax) did not increase at a time when the numbers of NOS-1- and NOS-2-positive cells increased. A similar pattern has been noted in glucose-and oxygen-deprived ischemic rats (1). The lack of change in Vmax with increased NOS-1- and -2immunoreactive cells may indicate inactivation of NOS by various peptidases that may have been activated during ischemia (21). It could also be that the newly formed NOS protein lacks catalytic activity. A time lapse of more then 24 hours between increase in NOS-2 mRNA verses increase in enzyme activity is found after middle cerebral artery occlusion (19). Hence, it seems that a complete understanding of the significance of our findings requires understanding of regulation of NOS enzyme activity, substrate and cofactor availability, and the extent of dimerization, which is not currently available. In this regard, it is of interest that the intensity and localization of NO production could be detected visually in this model by anti-citrulline immunostaining (27). The increase we observed in NOS-2-positive cells 96 hours after SAH is of special interest. Studies have linked NOS-2-related increases in NO production after SAH to delayed vasospasm (34, 45) and after cerebral ischemia to neuronal toxicity (17). Our data suggest that induction of NOS-2 may contribute to late-phase cytotoxicity after SAH.

CONCLUSION In summary, we have demonstrated that NOS-1 and -2 undergo a triphasic pattern of expression after SAH and that NOS Km is unaltered during the initial increase in NOS-1- and NOS-2-positive cells. We conclude that the unique depletion and subsequent restoration of cerebral NO levels after SAH does not involve depletion of NOS.

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Acknowledgments This study was supported by Grants R29-NS-35904-01 and RO1-NS-42264-01 from the National Institutes of Health, National Institute of Neurological Disorders and Stroke (to JBB).

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haracterization of nitric oxide synthase (NOS)-1 and NOS-2 activity and expression after subarachnoid hemorrhage (SAH) is necessary to help understand the role of nitric oxide (NO)-mediated phenomena in the pathophysiology of acute vasoconstriction and ischemic injury after SAH. This knowledge is a prerequisite before therapeutic interventions that manipulate the pathophysiology of NO can be undertaken. Previous work from Dr. Bederson’s laboratory helped define the temporal course of NO availability immediately after SAH and its effects on cerebral blood flow. Using an endovascular suture model of SAH in a rat, the present study investigated the activity and expression of NOS-1 and NOS-2 isoforms during the first 96 hours of SAH. Immunostaining for NOS-1- and NOS-2-positive cells and kinetic determination of NOS by a citrulline assay were performed. The investigators present evidence for a triphasic pattern of change in the number of NOS-1- and NOS-2-positive cells during the first 96 hours after SAH in a rodent model. In addition, they determined that the kinetic parameters of NOS-1 and NOS-2 remained unchanged during the first 90 minutes after SAH. However, the lack of change in NOS kinetics requires further investigation. The authors analyzed NOS activity in vitro. Investigation into in vivo production of NO is necessary to determine whether decreased NO levels immediately after SAH can be completely explained by hemoglobin scavenging, as the authors have hypothesized. As the authors acknowledge, decreased NOS activity could be because of lack of substrate or cofactor depletion. Other studies have shown an increase in inducible NOS activity in SAH. Suzuki et al. (1) demonstrated that hemin, a breakdown product of hemoglobin, was capable of stimulating the expression of inducible NOS. Thus, future studies will need to confirm the findings of the present study of no change in NOS kinetics during the acute phase of SAH. Susan C. Williams E. Sander Connolly, Jr. New York, New York

1. Suzuki S, Kassell NF, Lee KS: Hemin activity of an inducible isoform of nitric oxide synthase in vascular smooth-muscle cells. J Neurosurg 83:862–866, 1995.

I

n an established murine model, the authors investigated the causes of NO alterations after SAH. They have previously demonstrated an immediate and sharp decline in NO after SAH in mice, and the loss of this important endogenous vasodilator might be an important part of the reason behind the acute reduction in cerebral blood flow and cerebral ischemia observed during this same interval. In this study, they measured changes in NOS protein levels by examining the amount of active NOS and its kinetic parameters (by enzymatic assay) and the number of NOS-1 (neuronal)- and NOS-2 (inducible)-positive cells (by immunohistochemistry). They


SEHBA

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found that the different NOS proteins fluctuate in a complex triphasic pattern but that overall cumulative NOS activity itself does not change significantly. Reductions in NO levels after acute SAH are therefore a result of other causes, including possibly scavenging by hemoglobin. This study offers another piece in the puzzle of pathophysiological events surrounding acute aneurysm rupture that this investigative team is unraveling. J. Max Findlay Edmonton, Alberta, Canada

T

his is a continuation of the work on NO in the rat endovascular perforation SAH model performed in the laboratory of Dr. Bederson. The authors had previously shown that NO levels decrease acutely after SAH in this model and recover approximately 3 hours after SAH. The present experiments sought to gain insight into the mechanism of the decrease. The authors found that overall NOS activity does not change in the brain, suggesting that NO production may be unaltered. The limitation that was acknowledged by the authors was that activity in vitro may not accurately reflect activity in vivo because of alterations in substrate supply and cofactors. There was a complex change in immunohistochemical staining for two of the three NOS isoforms that could result in no overall change in activity. The authors examined, by immunohistochemistry, neuronal and inducible but not endothelial NOS, which also would need to be examined in their model. In addition, the site of NO production may be critical to biological processes mediated by it; therefore, overall activity assays may not give the complete picture. The investigators suggest measuring NO production itself, some-

thing that we have tried to accomplish in vivo in the primate model of SAH but that is very difficult to do (2). The specificity of the antibodies for the NOS isoforms was not clarified, so it cannot be known with certainty what isoforms were examined. In addition, as the authors note, no data are provided on the levels of endothelial NOS. Immunohistochemistry is relatively qualitative in any case, so more weight has to be placed on the functional assays. The data are an important addition to the vasospasm literature. They need to be reconciled with the theory of Pluta (3) that early in vasospasm, there is a loss of neuronal NOS activity that contributes to vasospasm and that the vasospasm increases endothelial shear stress, leading to up-regulation of endothelial NOS. Further important factors are believed to be bilirubin oxidation products (1) and asymmetrical dimethyl arginine (3). The time course of changes, however, may be very different in the rat compared with higher species, highlighting the bias of this reviewer that the dog is the lowest species from which useful information can be obtained about true, delayed, or chronic cerebral vasospasm. R. Loch Macdonald Chicago, Illinois 1. Clark JF, Reilly M, Sharp FR: Oxidation of bilirubin produces compounds that cause prolonged vasospasm of rat cerebral vessels: A contributor to subarachnoid hemorrhage-induced vasospasm. J Cereb Blood Flow Metab 22:472– 478, 2002. 2. Macdonald RL, Zhang ZD, Curry D, Elas M, Aihara Y, Halpern H, Jahromi BS, Johns L: Intracisternal sodium nitroprusside fails to prevent vasospasm in nonhuman primates. Neurosurgery 51:761–768, 2002. 3. Pluta RM: Pathophysiology of delayed vasospasm after SAH: New hypothesis and implications for treatment, in Macdonald RL (ed): Cerebral Vasospasm: Proceedings of the 8th International Conference. New York, Thieme, 2004.

In-training Liaison The Congress of Neurological Surgeons exists for the purpose of promoting public welfare through the advancement of neurosurgery by a commitment to excellence in education and by a dedication to research and scientific knowledge. —Mission Statement, Congress of Neurological Surgeons Inherent in this commitment is a critical charge to serve the needs of the in-training individual. Considering the importance of this vital group within the neurosurgical community, the Journal has established a position within its board structure termed In-training Liaison. The individual holding this position will act as a spokesperson especially addressing the needs and concerns of individuals in in-training positions globally, as they relate to journal content and perspective. The current individual holding this position is: John S. Kuo, M.D., Ph.D. Issues attendant to in-training matters should be conveyed to Dr. John S. Kuo at the Department of Neurological Surgery, LAC/USC Medical Center, 1200 N. State Street, Suite 5046, Los Angeles, CA 90033. Tel: 323/226-7421; Fax: 323/226-7833; email: kuo5577 @hotmail.com.

