Original Research Paper
Inhibition of neuronal nitric oxide synthase improves microregional O2 balance in cerebral ischemia –reperfusion Oak Z. Chi1, Kang H. Rah1, Sylviana Barsoum1, Xia Liu1, Harvey R. Weiss2 1
Department of Anesthesiology, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ, USA, Department of Neuroscience and Cell Biology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ, USA
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Objectives: Return of regional cerebral blood flow (rCBF) in focal cerebral ischaemia may not ensure proper distribution of blood flow to meet metabolic demand. This study was performed to determine how inhibition of neuronal nitric oxide synthase (NOS) during ischaemia–reperfusion would affect microregional O2 supply/consumption balances and their variation. Methods: Twenty minutes before middle cerebral artery (MCA) occlusion, a NOS inhibitor 7-nitroindazole (7-NI) 50 mg/kg ip (7-NI group) or vehicle (control group) was administered. At 1 hour of ischaemia and 2 hours of reperfusion, rCBF, the size of cortical infarct and arteriolar and venular O2 saturations (20– 60 mm in diameter) using cryomicrospectrophotometry were determined. Results: Ischaemia–reperfusion decreased the average venular O2 saturation and the ratio of O2 supply/ consumption. But, 7-NI treatment improved the average O2 supply/consumption ratio and venular O2 saturation (57.6+ 1.3 vs 52.0+ 3.8%) in ischaemia–reperfusion. The heterogeneity of venular O2 saturations reported as coefficient of variation (CV5100|SD/mean) was much smaller in the 7-NI than the control group (8.8 vs 15.5). The number of veins with low O2 saturation (v50%) was also smaller with the 7-NI (4/70) than the control group (18/70). The size of cortical infarct was smaller with 7-NI treatment. Discussion: Our data suggest that inhibition of neuronal NOS by 7-NI improved microregional O2 balance in the ischaemic–reperfused cortex (IR-C). The improvement in microregional O2 balance could be one of the contributing factors to the reduced size of cortical infarct. Keywords: Regional cerebral blood flow, Ischaemia/reperfusion, Heterogeneity, Venous O2 saturation, Neuroprotection, Neuronal nitric oxide synthase
Introduction Return of regional cerebral blood flow (rCBF) in focal cerebral ischaemia may not promise proper distribution of blood flow to meet metabolic demand or an improved microregional O2 supply/consumption balance or better neurological outcome.1–4 Ischaemia–reperfusion initiates various pathophysiological changes, such as excitation of neurons, activation and induction of various nitric oxide synthases (NOS) and platelet–leucocyte aggregation.1,2,5,6 In cerebral ischaemia, overproduction of nitric oxide from activation of neuronal NOS (nNOS) may lead to neurotoxicity. However, production of nitric oxide from endothelial NOS (eNOS) may protect brain tissue by maintaining rCBF.7–9 Inducible NOS (iNOS) is detected at the later stage of cerebral
Correspondence to: Oak Z. Chi, Department of Anesthesiology, Rutgers Robert Wood Johnson Medical School, 125 Paterson Street, Suite 3100, New Brunswick, NJ 08901-1977, USA. Email: [email protected]
ß W. S. Maney & Son Ltd 2015 DOI 10.1179/1743132815Y.0000000062
ischaemia.10–12 During the early stage of ischaemia– reperfusion, nNOS may play a bigger role than eNOS or iNOS in the production of nitric oxide.6,11,12 The produced nitric oxide works as an intercellular messenger and is involved in various activities, such as control of vascular tone, coupling of cerebral blood flow and metabolism, gene transcription, excitotoxicity, activation of free radical formation and lipid peroxidation.13–16 7-nitroindazole (7-NI) is a relatively specific nNOS inhibitor and is known to produce neuroprotection against excitotoxicity, and focal and global cerebral ischaemia.17–19 nNOS activity and cyclic GMP levels in brain during ischaemia and reperfusion were reported to be coupled with the activation of N-methyl-D-aspartate (NMDA) receptors.13 Inhibition of nNOS attenuated NMDA-induced increase of cerebral O2 consumption and cerebral blood flow.20 We reported that inhibition of NOS by L-NAME, that is a nonspecific NOS inhibitor, mildly improved the
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microregional O2 supply/consumption balance in the ischaemic cortex with permanent middle cerebral artery (MCA) occlusion in spite of lower cerebral blood flow.21 These studies suggest that nitric oxide may affect microregional O2 supply/consumption balance in cerebral ischaemia–reperfusion. In this study, we hypothesised that pretreatment with an nNOS inhibitor would improve microregional O2 supply/consumption balance and could be associated with a decrease in the infarct size in the cerebral ischaemic–reperfused cortex (IR-C) during the early stage of cerebral ischaemia–reperfusion. We tested this hypothesis using cryomicrospectrophotometry in the IR-C of the rats pretreated with 7-NI by determining microvascular arterial (SaO2) and venous O2 saturation (SvO2), and by determining the size of the cortical infarct.
exposed and carefully separated from the adjacent nerve through a midline ventral cervical incision. The tip of a 4.0 monofilament thread was covered with silicon and was inserted into the stump of the external carotid artery. It was advanced approximately 1.7 cm into the internal carotid artery until resistance was felt. The filament was kept in place for 60 minutes to block the MCA. It was removed to allow reperfusion, and then the external carotid artery was closed. After 120 minutes of reperfusion, measurements were performed. Seven animals in each group were selected randomly, then, rCBF and microscopic O2 saturations of small veins and arteries were determined in the IR-C, contralateral cortex (C-C) and pons. To determine the size of cortical infarct, six rats in each group were used.
Determination of regional cerebral blood flow Methods This study was approved by our Institutional Animal Care and Use Committee and was conducted in accordance to US Public Health Service Guidelines using the Guide for the Care of Laboratory Animals (DHHS Publication No. 85–23, revised 1996). Twenty-six male Fischer 344 rats weighing 240– 260 g were randomly assigned to two groups: the control (n513) and the 7-NI group (n513). They were initially anaesthetised with 2% isoflurane in an air and oxygen mixture and were mechanically ventilated through a tracheal tube. A femoral artery and vein were cannulated. The venous catheter was used to administer radioactive tracer and normal saline. To monitor heart rate and blood pressure, the arterial catheter was connected to Statham P23Db pressure transducer and an Iworx data acquisition system. Using a Radiometer blood gas analyzer (ABL80), arterial blood samples were analysed for haemoglobin, blood gases and pH. The isoflurane concentration was maintained at 1.4% after MCA occlusion. Body temperature was monitored and maintained at 37uuC+ 0.5 with a servo-controlled rectal thermistor probe and a heating lamp. For pericranial temperature, temporalis muscle temperature was monitored using a thermocouple probe (Omega Engineering, Inc., Stamford, CT, USA), which averaged 37uuC. For the 7-NI group (n513), 7-NI 50 mg/ kg ip was administered 20 minutes before MCA occlusion. For the control group (n513), the same volume of vehicle (DSMOznormal saline) was administered ip. This dose was chosen because previous studies showed that the similar doses of 7-NI decreased NOS activity or increased neuronal survival in various experimental conditions.22,23 For cerebral ischaemia–reperfusion, transient occlusion of the MCA was induced using an intraluminal thread.24,25 The common carotid artery was
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To determine rCBF, 40 mCi of 14C-iodoantipyrine was infused intravenously. Approximately every 3 seconds during next 60 seconds, 20 blood samples were obtained from the arterial catheter that was cut to 20 mm to minimise smearing. Immediately after the last sample was obtained, the animal was decapitated and the head was frozen in liquid nitrogen. While frozen, brain samples were obtained from three regions: IR-C, C-C and pons. These samples were cut into 20 mm thick slices on a microtome-cryostat and then were exposed to X-ray film to obtain an autoradiogram. Using the NIH imageJ program, the cerebral 14C-iodoantipyrine concentrations were determined by reference to eight precalibrated standards (40–1069 nCi/g, Amersham). A minimum of eight optical density measurements were made, each on different sections, for each brain region examined. Blood samples were placed in a tissue solubilizer and 24 hours later put in a counting liquid. These samples were counted on a liquid scintillation counter and were quench corrected. Determinations of rCBF were then calculated using the following previously published equation:20,21 ðT CiðTÞ ¼ lK C A e 2KðT2tÞ dt; o
where Ci(T) represents the tissue concentration of C-iodoantipyrine at the time of decapitation, l is the tissue:blood partition coefficient, CA is the arterial concentration of the tracer, and t is time. K is defined as follows: K5mF/W, where m is the constant related to diffusion, and F/W represents the blood flow per unit mass of tissue. The l-value of 0.8 calculated by Sakurada et al. was used.26 14
Determination of microvascular arterial and venous O2 saturation For determination of microvascular SaO2 and SvO2 alternate sections of brain tissue used for
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measurement of rCBF were used. These cortical sections were from an approximately 5 mm plug taken from the parietal cortex over the MCA. Details of this technique have been published previously.27,28 Briefly, the brain sections were sliced into wafers at {20uuC, and then were mounted with embedding medium in a microtome-cryostat. In nitrogen gas atmosphere, 20 mm thick sections were obtained at {35uuC, and were transferred to precooled glass slides and covered with degassed silicone oil and coverslips. These slides were placed on a microspectrophotometer fitted with a nitrogen gas-flushed cold stage and then readings of optical density were obtained at 568, 560 and 523 nm. This three-wavelength method corrects for the light scattering in the frozen blood. Only vessels in the transverse section were examined so that the path of light would traverse only the blood. The size of the measuring spot was 8 mm in diameter. In each region, five arterioles and 10 venules (20–60 mm in diameter) were used to obtain readings for O2 saturations (SaO2 and SvO2). Arterioles and venules were identified by presence or absence of muscle layers.
Determination of O2 extraction, O2 consumption and O2 supply/consumption ratio The O2 content of blood was determined by multiplying the percent O2 saturation by the haemoglobin concentration times 1.36, the maximal binding capacity of haemoglobin for O2 per gram. Regional O2 extraction was obtained by the difference between the average arterial and venous O2 contents. Using the Fick principle, we calculated the O2 consumption for each region as the product of average blood flow and O2 extraction. This method has been validated in the brain.27 The ratio of O2 supply/consumption in a region was determined by dividing local O2 supply by local O2 consumption:4,20,21 CaO2 £ rCBF rCBFðCaO2 2 CvO2 Þ where CaO2 and CvO2 are the arterial and venous O2 content and rCBF is the rCBF. CaO2 or CvO2 was determined by multiplying SaO2 or SvO2 by haemoglobin (Hb) times 1.36, the maximum binding capacity of Hb for O2 per gram. This equation cancels out Hb, 1.36 and rCBF. This reduces to: SaO2 SaO2 2 SvO2 where SaO2 and SvO2 are the percent oxy-hemoglobin in the arterial and venous blood, respectively.
Determination of size of infarct To determine the size of infarct, the brain was removed from the head and was sliced in coronal
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sections. There were typically three to four slices of approximately 2–3 mm thickness of brain tissue each. For tetrazolium staining, a 0.05% solution of 2,3,5-triphenyltetrazolium chloride (Sigma) solution in PBS was prepared and warmed to 37uuC, where the brain slices were placed in and were incubated for 30 minutes. The tetrazolium chloride solution was then removed and the slices were washed three times in PBS, 1 minute per wash. To keep the slices from drying out, each slice was placed in a small weighing boat with PBS. Then, the boat was placed on a dissecting microscope and a clean slide was placed over the weighing boat. The cortical region of each slice was traced onto the slide. Any infarcted areas were marked by cross-hatching over any areas not marked with tetrazolium staining. The slides were then scanned and the scanned images were measured for total and infarcted areas in the cortex using Image J. Cortical infarcted areas were reported as percent of total cortical area.
Statistical analysis A two-way analysis of variance (ANOVA) was performed for haemodynamic parameters, blood gases, cerebral blood flow and O2 balance to assess the difference between the different regions (used as a repeated measures within treatment) and the difference between the treatments. Analysis was conducted using the general linear model (PROC GLM) from the SAS Institute (Cary, NC, USA). Post hoc testing of multiple comparisons was performed using Tukey’s procedure. An ANOVA was used to analyse the heterogeneity or variability of all measured SvO2 determined. The coefficient of variation (CV) of SvO2 was also reported to describe heterogeneity. The CV was calculated as: SD £ 100 Mean where SD is the standard deviation. A chi-square test was also used to assess differences in the distribution of SvO2 and differences in the number veins with low O2 saturation between groups. Non-paired t-test was used to assess the difference in the size of infarct between the groups. A value of Pv0.05 was considered as statistically significant. All values were expressed as mean + SD.
Results Haemodynamic and blood gas parameters in the control and in the 7-NI group of rats that were used for determination of O2 balance parameters are shown in Table 1. The mean arterial blood pressure and heart rate were similar between the two groups. Arterial blood gases, pH and haemoglobin were not significantly different between the two experimental groups.
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Table 1 Haemodynamic and blood gas values for the isoflurane and the pentobarbital groups just before determination of oxygen balance parameters
Group
Control (n ¼ 7)
Mean blood pressure (mm Hg) Heart rate (beats/minute) Arterial PO2 (mm Hg) Arterial PCO2 (mm Hg) pH Haemoglobin (g/100 ml)
99 ^ 20 293 ^ 30 117 ^ 26 36 ^ 7 7.36 ^ 0.17 9.8 ^ 1.3
7-nitroindazole (n ¼ 7) 110 ^ 13 315 ^ 32 112 ^ 25 38 ^ 5 7.35 ^ 0.06 10.6 ^ 1.8
Values are mean ^ SD.
The haemodynamic, blood gas data and Hb for rats used for determination of size of infarct were not significantly different between the control and 7-NI group and not different from the corresponding groups that were used for O2 balance parameters.
Average rCBF, O2 consumption and extraction Regional cerebral blood flow was not significantly different between the IR-C and the C-C or between the control and the 7-NI group (Fig. 1A). The oxygen consumption was not significantly different between the IR-C and C-C or between the two groups of rats (Fig. 1B). The oxygen extraction of the IR-C was significantly elevated when compared with the C-C in the control group (z24%, Pv0.001) as well as in the 7-NI group (z9%, Pv0.001). There were no significant differences in oxygen extraction between the control and the 7-NI group in either IR-C or C-C (Fig. 1C).
Microvascular O2 saturation SaO2 was similar in all regions and groups (Table 2). However, the average SvO2 was significantly lower in the IR-C when compared with the C-C in both the
control (Pv0.0001) and the 7-NI group (Pv0.0001). In the IR-C, the average SvO2 of the 7-NI group was significantly higher than that of the control group (Table 2, P v 0.001). The SvO2 was similar between the two groups in the C-C. The ratio of O2 supply/consumption of the IR-C was significantly lower than that of the corresponding C-C in both the control (Pv0.0001) and the 7-NI group (Pv0.0001) (Table 2). The ratio of O2 supply/consumption in the IR-C was significantly higher (Pv0.005) in the 7-NI group than in the control group.
Heterogeneity of SvO2 The distribution of all measured SvO2 was heterogeneous in both IR-C and C-C in both groups (Fig. 2). There was a significant shift to lower values in the IR-C when compared with the C-C in both groups (isoflurane: Pv0.0001, 7-NI: Pv0.0001). The distribution of SvO2 was significantly shifted (Pv0.0001) to higher values with the 7-NI group in the IR-C when compared with the control group. The degree of heterogeneity of SvO2 was analysed by ANOVA and reported as CV (100|SD/mean)
Figure 1 Cerebral blood flow, oxygen consumption and oxygen extraction. C-C: contralateral cortex; IR-C: ischaemic –reperfused cortex; 7-NI: 7-nitroindazole treated group; control: control, vehicle treated group. Values are means 6 SD. *Significantly different from the contralateral cortex
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Table 2 Arterial and venous oxygen saturations (%) and the ratio of oxygen supply and consumption (S/C) in the ischaemic–reperfused and the contralateral cortex of the control and 7-nitroindazole groups Control (n ¼ 7) Group Cortex SaO2 (%) SvO2 (%) S/C ratio
7-nitroindazole (n ¼ 7)
Ischaemic– reperfused
Contralateral
Ischaemic–reperfused
Contralateral
91.2 ^ 2.9 52.0 ^ 3.8* 2.32 ^ 0.23*
93.6 ^ 1.8 62.0 ^ 0.6 2.93 ^ 0.13
92.8 ^ 0.9 57.6 ^ 1.3*† 2.68 ^ 0.92*†
93.2 ^ 0.5 61.7 ^ 0.8 2.96 ^ 0.85
SaO2: arterial O2 saturation; SvO2: venous O2 saturation; S/C ratio: oxygen supply/consumption ratio. Values are mean ^ SD (n ¼ 7). * Significantly different from the contralateral cortex. † Significantly different from the control group.
Figure 2 The distribution of individual microvascular venous oxygen saturation. In the ischemic–reperfused cortex, the heterogeneity of venous oxygen saturation as well as number of veins with oxygen saturation < 50% were much smaller with 7-nitroindazole treatment despite similar regional cerebral blood flow
obtained from the distribution curve (Fig. 2). Ischaemia–reperfusion significantly increased the heterogeneity of SvO2 in both groups (Pv0.0001). 7nitroindazole significantly decreased the heterogeneity of microregional SvO2 (Pv0.0001) in the IR-C when compared with the control group. The values of CV were 15.5 and 6.0 for the IR-C and C-C, respectively, in the control group, and 8.8 and 6.4 for the 7-NI group. The number of veins with oxygen saturation below 50% was significantly less in the 7-NI than in the control group (4 out of 70 vs 18 out of 70, chi-square: Pv0.005) in the IR-C.
Size of cortical infarct The size of cortical infarct measured as percentage out of total cortical area determined at the end of a 2-hour reperfusion was significantly smaller with the 7-NI than the control group (10.5 + 1.3% vs 13.5+ 1.7%, Pv0.01).
Pons We also compared the effects of the 7-NI in O2 balance in the remote cerebral region, the pons. There were no significant differences in the cerebral blood flow (control: 136+ 55 vs 7-NI: 116+ 35 ml/minute/ 100 g) in the pons between the two experimental groups. The oxygen extraction (control: 4.3+ 0.6 vs
7-NI: 4.5+ 0.7 ml O2/100 ml) was also similar between the two groups in the pons. The oxygen consumption of the pons between the groups was not significantly different either (control 5.8+ 2.7 vs 7NI 5.2+ 1.8 ml O2/minute/100 g).
Discussion Our data demonstrated that ischaemia–reperfusion decreased the average microvascular SvO2 with increased heterogeneity. In the IR-C, with 7-NI treatment, the average SvO2 became higher with a decrease in the number of veins with low O2 saturation and with smaller heterogeneity without a significant difference in rCBF or the average O2 consumption. It suggests improved microregional O2 balance. The size of cortical infarct determined at 2 hours after reperfusion was also smaller with 7-NI treatment. Our data showed restoration of rCBF with reperfusion in both groups as shown in other studies.4,24 7nitroindazole treatment almost completely restored rCBF in the IR-C. Our data agree with previous reports that in contrast to non-specific NOS inhibition, nNOS inhibitor did not significantly affect cortical cerebral blood flow in transient cerebral ischaemic models.12 Overall, the rCBF, O2 consumption and O2 extraction were similar between the groups. Our data suggest
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that without alterations of average rCBF and O2 consumption, the microvascular SvO2 or microregional O2 balance could be heterogeneous. In the equation for O2 supply/consumption ratio described in the Methods section, since rCBF and Hb are cancelled out and SaO2 is essentially constant and independent of ischaemic status or regions, individual microvascular SvO2 would reflect the final product of microregional O2 supply/consumption balance.4,21,27 SvO2 variability in this study represents the heterogeneity of O2 supply/consumption ratio on microregional basis. Ischaemia–reperfusion produces various pathophysiological reactions, such as platelet–leucocyte aggregation, release of excitatory amino acid neurotransmitters and inflammatory agents, release of free radicals, generation of interstitial and cellular oedema and activation of various nitric oxide synthases.1,2,5,6 It is possible that reperfusion caused microvascular blockage or hyperaemia and caused irregular distribution of rCBF. Ischaemia–reperfusion may have rendered neurons excited or left them normal or made them non-functioning or dead, which caused increase or decrease in the O2 consumption of various microscopic areas. This anatomic and functional heterogeneity could have caused mismatch between O2 supply and consumption, thus producing heterogeneity of microvascular SvO2. Higher microvascular SvO2 may represent higher O2 supply/consumption ratio in that microregion. However, higher SvO2 would not mean better neuronal survival since neurons could be inactive or dead. Even though the average microvascular SvO2 was similar, however, the increase of the number of individual microvascular veins with low SvO2 may be harmful for neuronal survival. Studies on transgenic nNOS knock-out mouse and on the rats treated with 7-NI indicate that activation of nNOS contributed to ischaemic cell damage after cerebral ischaemia.7,29 Various mechanisms of neuroprotection by 7-NI were suggested, such as decrease in lipid peroxidation and, free radicals and decreased NMDA activity. Attenuation of increased cerebral metabolism by NMDA may be one of the factors of neuroprotection. Various biological targets of nitric oxide were reported such as O2, guanylate cyclase, Ras, NADH ubiquinone or succinate oxide reductases, creatinine kinase, cis-aconitase, activation of PARS and cytochrome oxidase.11,30 Our data suggest that decreasing nitric oxide by inhibition of neuronal NOS could optimise the energy supply/consumption balance at the cellular level and contribute to producing beneficial effects on neuronal survival. Our previous study showed that the NMDAinduced increase in rCBF and O2 consumption was attenuated by 7-NI.20 However, 7-NI did not affect
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rCBF or O2 consumption in the normal contralateral cortex. It was reported that at 2 hours of reperfusion, the NMDA was still activating NOS activity.13 These studies suggest the possibility that nNOS could affect neuronal O2 consumption as well as rCBF in the early stage of ischaemia–reperfusion. 7-nitroindazole decreased nitric oxide production and did not significantly affect cerebral blood flow or oxy-haemoglobin concentration during kainate-induced seizure, but produced less damage to the hippocampus.23 However, the abnormal discharges of EEG during kainate-induced seizure were attenuated. They used laser Doppler flow metre for rCBF determination and near infrared spectrophotometry for measuring cerebral oxygenation by determining the concentration of oxy-haemoglobin (HbO2). These techniques can only determine global or regional blood flow or oxygenation.23 These studies suggest that inhibition of neuronal nitric oxide could modify microregional neuronal function, distribution of rCBF or O2 supply/consumption balance without significant changes in global or regional CBF, or HbO2 to produce better neuronal survival during cerebral ischaemia–reperfusion. It has been reported that the NMDA receptor antagonist MK-801 remarkably reduced the ischaemia-enhanced NOS activity and cyclic GMP level in the brain hemisphere during reperfusion.13 7-nitroindazole significantly decreased membrane lipid peroxidation during the early stage of reperfusion. Ischaemia-evoked activation of glutamatergic system stimulates nitric oxide-dependent signal transmission.13 Our previous study suggests not only 7-NI that could affect nNOS, which is distal to NMDA in the NMDA-nitric oxide pathway and affect cerebral O2 balance but also suggests the possibility of retrograde effect of nitric oxide on NMDA.20 It has been reported that elevation of nitric oxide production led to mitochondrial dysfunction, glutamate release and excitotoxicity.31 Since NMDA receptors are activated during ischaemia– reperfusion, it is possible that the increase of O2 consumption by NMDA activation in the ischaemia– reperfusion could be decreased by 7-NI and affect microregional O2 supply/consumption balance. That could contribute to higher average microvascular SvO2 and the higher O2 supply/consumption ratio in the condition of similar rCBF to the control. Similar average O2 consumption between the control and the 7-NI group suggest that more neurons survived with 7-NI treatment in the IR-C. Inhibition of nNOS by ARL 17477 at the dose that did not affect rCBF or MABP but significantly decreased NOS activity decreased the size of infarct that was determined 7 days after transient MCA occlusion in rats.32 The results of our study are similar to this study that showed no significant changes in rCBF.
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Improved microregional O2 balance could have contributed to decrease in the size of infarct in both studies. Our data suggest that 7-NI could decrease neuronal damage during reperfusion through improved microscopic O2 supply/consumption balance despite no significant change in rCBF or O2 consumption. Decreasing the number of veins with low O2 saturation may decrease neuronal damage. 7-nitroindazole significantly decreased the percentage of the area of cortical infarct out of the total cortical area. This effect at 2 hours of reperfusion does not necessarily mean a long-term effect, but other studies showed neuroprotective effects of nNOS inhibitors at the later stage of cerebral ischaemia.13,17,32 Our study suggests that this improved microregional O2 supply/consumption balance reflected by a decrease in the number of veins with low SvO2 could be one of the contributory factors decreasing the size of infarct in the early stage of reperfusion, at least in the cortical area. Differences in mean arterial blood pressure, rCBF and Hb between groups even though statistically not significant may have affected our data. The reported rCBF values are from regions, not from microregions. However, the equation used for O2 supply/ consumption balance cancels out Hb as well as rCBF. SaO2 was also essentially constant and independent of ischaemic status. Therefore, the individual SvO2 should represent the final product of the O2 supply/consumption balance in these microregions. The limitation in our measurements of rCBF and O2 extraction/consumption was reported previously.27,28 This technique is one of the few methods that give rapid, accurate, quantitative assessment of cerebral O2 balance in small microregions of normal and ischaemic cortex that has drainage fields of cerebral veins of 20–60 mm in diameter. In conclusion, with 7-NI pretreatment in the IR-C, the average microvascular SvO2 was higher and the number of veins with low O2 saturation and heterogeneity of SvO2 were smaller without a significant difference in average rCBF and O2 consumption. Our major finding is that 7-NI improved microregional O2 balance in cerebral ischaemia–reperfusion. The size of cortical infarct was smaller with 7-NI that was determined at 2 hours of reperfusion. Our data suggest that this improved microregional O2 supply/consumption balance with 7-NI may have contributed to the decreased size of cortical infarct during the early stage of reperfusion.
Disclaimer statements Contributors OZC: took part in design, experiments, data analysis and writing. KHR: took part in design, data analysis and writing. SB: took part in design,
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data analysis and writing. XL: took part in experiment and data analysis. HRW: took part in design, experiment, data analysis and writing. Funding Funding was provided by the intramural departmental sources. Conflict of interest Authors do not have any conflicts of interests to be disclosed. Ethics approval This study was approved by our Institutional Animal Care and Use Committee and was conducted in accordance to US Public Health Service Guidelines using the Guide for the Care of Laboratory Animals (DHHS Publication No. 85-23, revised 1996).
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19 Yoshida T, Limmroth V, Irikura K, Moskowitz MA. The NOS inhibitor, 7-nitroindazole, decreases focal infarct volume but not the response to topical acetylcholine in pial vessels. J Cereb Blood Flow Metab. 1994;14:924–9. 20 Chi OZ, Liu X, Weiss HR. Effects of inhibition of neuronal nitric oxide synthase on NMDA-induced changes in cerebral blood flow and oxygen consumption. Exp Brain Res. 2003;148:256–60. 21 Wei HM, Chi OZ, Liu X, Sinha AK, Weiss HR. Nitric oxide synthase inhibition alters cerebral blood flow and oxygen balance in focal cerebral ischemia in rats. Stroke. 1994;25:445–50. 22 Ishida A, Trescher WH, Lange MS, Johnston MV. Prolonged suppression of brain nitric oxide synthase activity by 7-nitroindazole protects against cerebral hypoxic-ischemic injury in neonatal rat. Brain Dev. 2001;23:349–54. 23 Monte´cot C, Rondi-Reig L, Springhetti V, Seylaz J, Pinard E. Inhibition of neuronal (type 1) nitric oxide synthase prevents hyperaemia and hippocampal lesions resulting from kainateinduced seizures. Neuroscience. 1998;84:791–800. 24 Lipsanen A, Jolkkonen J. Experimental approaches to study functional recovery following cerebral ischemia. Cell Mol Life Sci. 2011;68:3007–17. 25 Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989;20:84–91.
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26 Sakurada O, Kennedy C, Jehle J, Brown JD, Carbin GL, Sokoloff L. Measurement of local cerebral blood flow with iodo-C14-antipyringe. Am J Physiol. 1978;234:H59–H66. 27 Buchweitz-Milton E, Weiss HR. Effect of MCA occlusion on brain O2 supply and consumption determined microspectrophotometrically. Am J Physiol. 1987;253:H454–60. 28 Zhu NH, Weiss HR. Oxy- and carboxyhemoglobin saturation determination in frozen small vessels. Am J Physiol. 1991;260:H626–31. 29 Panahian N, Yoshida T, Huang PL, Hedley-Whyte ET, Dalkara T, Fishman MC, et al. Attenuated hippocampal damage after global cerebral ischemia in mice mutant in neuronal nitric oxide synthase. Neuroscience. 1996;72:343–54. 30 Yun HY, Dawson VL, Dawson TM. Neurobiology of nitric oxide. Crit Rev Neurobiol. 1996;10:291–316. 31 McNaught KS, Brown GC. Nitric oxide causes release of glutamate from brain synaptosomes. J Neurochem. 1998;70:1541–6. 32 Zhang ZG, Reif D, Macdonald J, Tang WX, Kamp DK, Gentile RJ, et al. ARL 17477, a potent and selective neuronal NOS inhibitor decreases infarct volume after transient middle cerebral artery occlusion in rats. J Cereb Blood Flow Metab. 1996;16:599–604.
Inhibition of neuronal nitric oxide synthase improves microregional O2 balance in cerebral ischemia –reperfusion Oak Z. Chi1, Kang H. Rah1, Sylviana Barsoum1, Xia Liu1, Harvey R. Weiss2 1
Department of Anesthesiology, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ, USA, Department of Neuroscience and Cell Biology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ, USA
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Objectives: Return of regional cerebral blood flow (rCBF) in focal cerebral ischaemia may not ensure proper distribution of blood flow to meet metabolic demand. This study was performed to determine how inhibition of neuronal nitric oxide synthase (NOS) during ischaemia–reperfusion would affect microregional O2 supply/consumption balances and their variation. Methods: Twenty minutes before middle cerebral artery (MCA) occlusion, a NOS inhibitor 7-nitroindazole (7-NI) 50 mg/kg ip (7-NI group) or vehicle (control group) was administered. At 1 hour of ischaemia and 2 hours of reperfusion, rCBF, the size of cortical infarct and arteriolar and venular O2 saturations (20– 60 mm in diameter) using cryomicrospectrophotometry were determined. Results: Ischaemia–reperfusion decreased the average venular O2 saturation and the ratio of O2 supply/ consumption. But, 7-NI treatment improved the average O2 supply/consumption ratio and venular O2 saturation (57.6+ 1.3 vs 52.0+ 3.8%) in ischaemia–reperfusion. The heterogeneity of venular O2 saturations reported as coefficient of variation (CV5100|SD/mean) was much smaller in the 7-NI than the control group (8.8 vs 15.5). The number of veins with low O2 saturation (v50%) was also smaller with the 7-NI (4/70) than the control group (18/70). The size of cortical infarct was smaller with 7-NI treatment. Discussion: Our data suggest that inhibition of neuronal NOS by 7-NI improved microregional O2 balance in the ischaemic–reperfused cortex (IR-C). The improvement in microregional O2 balance could be one of the contributing factors to the reduced size of cortical infarct. Keywords: Regional cerebral blood flow, Ischaemia/reperfusion, Heterogeneity, Venous O2 saturation, Neuroprotection, Neuronal nitric oxide synthase
Introduction Return of regional cerebral blood flow (rCBF) in focal cerebral ischaemia may not promise proper distribution of blood flow to meet metabolic demand or an improved microregional O2 supply/consumption balance or better neurological outcome.1–4 Ischaemia–reperfusion initiates various pathophysiological changes, such as excitation of neurons, activation and induction of various nitric oxide synthases (NOS) and platelet–leucocyte aggregation.1,2,5,6 In cerebral ischaemia, overproduction of nitric oxide from activation of neuronal NOS (nNOS) may lead to neurotoxicity. However, production of nitric oxide from endothelial NOS (eNOS) may protect brain tissue by maintaining rCBF.7–9 Inducible NOS (iNOS) is detected at the later stage of cerebral
Correspondence to: Oak Z. Chi, Department of Anesthesiology, Rutgers Robert Wood Johnson Medical School, 125 Paterson Street, Suite 3100, New Brunswick, NJ 08901-1977, USA. Email: [email protected]
ß W. S. Maney & Son Ltd 2015 DOI 10.1179/1743132815Y.0000000062
ischaemia.10–12 During the early stage of ischaemia– reperfusion, nNOS may play a bigger role than eNOS or iNOS in the production of nitric oxide.6,11,12 The produced nitric oxide works as an intercellular messenger and is involved in various activities, such as control of vascular tone, coupling of cerebral blood flow and metabolism, gene transcription, excitotoxicity, activation of free radical formation and lipid peroxidation.13–16 7-nitroindazole (7-NI) is a relatively specific nNOS inhibitor and is known to produce neuroprotection against excitotoxicity, and focal and global cerebral ischaemia.17–19 nNOS activity and cyclic GMP levels in brain during ischaemia and reperfusion were reported to be coupled with the activation of N-methyl-D-aspartate (NMDA) receptors.13 Inhibition of nNOS attenuated NMDA-induced increase of cerebral O2 consumption and cerebral blood flow.20 We reported that inhibition of NOS by L-NAME, that is a nonspecific NOS inhibitor, mildly improved the
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microregional O2 supply/consumption balance in the ischaemic cortex with permanent middle cerebral artery (MCA) occlusion in spite of lower cerebral blood flow.21 These studies suggest that nitric oxide may affect microregional O2 supply/consumption balance in cerebral ischaemia–reperfusion. In this study, we hypothesised that pretreatment with an nNOS inhibitor would improve microregional O2 supply/consumption balance and could be associated with a decrease in the infarct size in the cerebral ischaemic–reperfused cortex (IR-C) during the early stage of cerebral ischaemia–reperfusion. We tested this hypothesis using cryomicrospectrophotometry in the IR-C of the rats pretreated with 7-NI by determining microvascular arterial (SaO2) and venous O2 saturation (SvO2), and by determining the size of the cortical infarct.
exposed and carefully separated from the adjacent nerve through a midline ventral cervical incision. The tip of a 4.0 monofilament thread was covered with silicon and was inserted into the stump of the external carotid artery. It was advanced approximately 1.7 cm into the internal carotid artery until resistance was felt. The filament was kept in place for 60 minutes to block the MCA. It was removed to allow reperfusion, and then the external carotid artery was closed. After 120 minutes of reperfusion, measurements were performed. Seven animals in each group were selected randomly, then, rCBF and microscopic O2 saturations of small veins and arteries were determined in the IR-C, contralateral cortex (C-C) and pons. To determine the size of cortical infarct, six rats in each group were used.
Determination of regional cerebral blood flow Methods This study was approved by our Institutional Animal Care and Use Committee and was conducted in accordance to US Public Health Service Guidelines using the Guide for the Care of Laboratory Animals (DHHS Publication No. 85–23, revised 1996). Twenty-six male Fischer 344 rats weighing 240– 260 g were randomly assigned to two groups: the control (n513) and the 7-NI group (n513). They were initially anaesthetised with 2% isoflurane in an air and oxygen mixture and were mechanically ventilated through a tracheal tube. A femoral artery and vein were cannulated. The venous catheter was used to administer radioactive tracer and normal saline. To monitor heart rate and blood pressure, the arterial catheter was connected to Statham P23Db pressure transducer and an Iworx data acquisition system. Using a Radiometer blood gas analyzer (ABL80), arterial blood samples were analysed for haemoglobin, blood gases and pH. The isoflurane concentration was maintained at 1.4% after MCA occlusion. Body temperature was monitored and maintained at 37uuC+ 0.5 with a servo-controlled rectal thermistor probe and a heating lamp. For pericranial temperature, temporalis muscle temperature was monitored using a thermocouple probe (Omega Engineering, Inc., Stamford, CT, USA), which averaged 37uuC. For the 7-NI group (n513), 7-NI 50 mg/ kg ip was administered 20 minutes before MCA occlusion. For the control group (n513), the same volume of vehicle (DSMOznormal saline) was administered ip. This dose was chosen because previous studies showed that the similar doses of 7-NI decreased NOS activity or increased neuronal survival in various experimental conditions.22,23 For cerebral ischaemia–reperfusion, transient occlusion of the MCA was induced using an intraluminal thread.24,25 The common carotid artery was
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To determine rCBF, 40 mCi of 14C-iodoantipyrine was infused intravenously. Approximately every 3 seconds during next 60 seconds, 20 blood samples were obtained from the arterial catheter that was cut to 20 mm to minimise smearing. Immediately after the last sample was obtained, the animal was decapitated and the head was frozen in liquid nitrogen. While frozen, brain samples were obtained from three regions: IR-C, C-C and pons. These samples were cut into 20 mm thick slices on a microtome-cryostat and then were exposed to X-ray film to obtain an autoradiogram. Using the NIH imageJ program, the cerebral 14C-iodoantipyrine concentrations were determined by reference to eight precalibrated standards (40–1069 nCi/g, Amersham). A minimum of eight optical density measurements were made, each on different sections, for each brain region examined. Blood samples were placed in a tissue solubilizer and 24 hours later put in a counting liquid. These samples were counted on a liquid scintillation counter and were quench corrected. Determinations of rCBF were then calculated using the following previously published equation:20,21 ðT CiðTÞ ¼ lK C A e 2KðT2tÞ dt; o
where Ci(T) represents the tissue concentration of C-iodoantipyrine at the time of decapitation, l is the tissue:blood partition coefficient, CA is the arterial concentration of the tracer, and t is time. K is defined as follows: K5mF/W, where m is the constant related to diffusion, and F/W represents the blood flow per unit mass of tissue. The l-value of 0.8 calculated by Sakurada et al. was used.26 14
Determination of microvascular arterial and venous O2 saturation For determination of microvascular SaO2 and SvO2 alternate sections of brain tissue used for
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measurement of rCBF were used. These cortical sections were from an approximately 5 mm plug taken from the parietal cortex over the MCA. Details of this technique have been published previously.27,28 Briefly, the brain sections were sliced into wafers at {20uuC, and then were mounted with embedding medium in a microtome-cryostat. In nitrogen gas atmosphere, 20 mm thick sections were obtained at {35uuC, and were transferred to precooled glass slides and covered with degassed silicone oil and coverslips. These slides were placed on a microspectrophotometer fitted with a nitrogen gas-flushed cold stage and then readings of optical density were obtained at 568, 560 and 523 nm. This three-wavelength method corrects for the light scattering in the frozen blood. Only vessels in the transverse section were examined so that the path of light would traverse only the blood. The size of the measuring spot was 8 mm in diameter. In each region, five arterioles and 10 venules (20–60 mm in diameter) were used to obtain readings for O2 saturations (SaO2 and SvO2). Arterioles and venules were identified by presence or absence of muscle layers.
Determination of O2 extraction, O2 consumption and O2 supply/consumption ratio The O2 content of blood was determined by multiplying the percent O2 saturation by the haemoglobin concentration times 1.36, the maximal binding capacity of haemoglobin for O2 per gram. Regional O2 extraction was obtained by the difference between the average arterial and venous O2 contents. Using the Fick principle, we calculated the O2 consumption for each region as the product of average blood flow and O2 extraction. This method has been validated in the brain.27 The ratio of O2 supply/consumption in a region was determined by dividing local O2 supply by local O2 consumption:4,20,21 CaO2 £ rCBF rCBFðCaO2 2 CvO2 Þ where CaO2 and CvO2 are the arterial and venous O2 content and rCBF is the rCBF. CaO2 or CvO2 was determined by multiplying SaO2 or SvO2 by haemoglobin (Hb) times 1.36, the maximum binding capacity of Hb for O2 per gram. This equation cancels out Hb, 1.36 and rCBF. This reduces to: SaO2 SaO2 2 SvO2 where SaO2 and SvO2 are the percent oxy-hemoglobin in the arterial and venous blood, respectively.
Determination of size of infarct To determine the size of infarct, the brain was removed from the head and was sliced in coronal
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sections. There were typically three to four slices of approximately 2–3 mm thickness of brain tissue each. For tetrazolium staining, a 0.05% solution of 2,3,5-triphenyltetrazolium chloride (Sigma) solution in PBS was prepared and warmed to 37uuC, where the brain slices were placed in and were incubated for 30 minutes. The tetrazolium chloride solution was then removed and the slices were washed three times in PBS, 1 minute per wash. To keep the slices from drying out, each slice was placed in a small weighing boat with PBS. Then, the boat was placed on a dissecting microscope and a clean slide was placed over the weighing boat. The cortical region of each slice was traced onto the slide. Any infarcted areas were marked by cross-hatching over any areas not marked with tetrazolium staining. The slides were then scanned and the scanned images were measured for total and infarcted areas in the cortex using Image J. Cortical infarcted areas were reported as percent of total cortical area.
Statistical analysis A two-way analysis of variance (ANOVA) was performed for haemodynamic parameters, blood gases, cerebral blood flow and O2 balance to assess the difference between the different regions (used as a repeated measures within treatment) and the difference between the treatments. Analysis was conducted using the general linear model (PROC GLM) from the SAS Institute (Cary, NC, USA). Post hoc testing of multiple comparisons was performed using Tukey’s procedure. An ANOVA was used to analyse the heterogeneity or variability of all measured SvO2 determined. The coefficient of variation (CV) of SvO2 was also reported to describe heterogeneity. The CV was calculated as: SD £ 100 Mean where SD is the standard deviation. A chi-square test was also used to assess differences in the distribution of SvO2 and differences in the number veins with low O2 saturation between groups. Non-paired t-test was used to assess the difference in the size of infarct between the groups. A value of Pv0.05 was considered as statistically significant. All values were expressed as mean + SD.
Results Haemodynamic and blood gas parameters in the control and in the 7-NI group of rats that were used for determination of O2 balance parameters are shown in Table 1. The mean arterial blood pressure and heart rate were similar between the two groups. Arterial blood gases, pH and haemoglobin were not significantly different between the two experimental groups.
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Table 1 Haemodynamic and blood gas values for the isoflurane and the pentobarbital groups just before determination of oxygen balance parameters
Group
Control (n ¼ 7)
Mean blood pressure (mm Hg) Heart rate (beats/minute) Arterial PO2 (mm Hg) Arterial PCO2 (mm Hg) pH Haemoglobin (g/100 ml)
99 ^ 20 293 ^ 30 117 ^ 26 36 ^ 7 7.36 ^ 0.17 9.8 ^ 1.3
7-nitroindazole (n ¼ 7) 110 ^ 13 315 ^ 32 112 ^ 25 38 ^ 5 7.35 ^ 0.06 10.6 ^ 1.8
Values are mean ^ SD.
The haemodynamic, blood gas data and Hb for rats used for determination of size of infarct were not significantly different between the control and 7-NI group and not different from the corresponding groups that were used for O2 balance parameters.
Average rCBF, O2 consumption and extraction Regional cerebral blood flow was not significantly different between the IR-C and the C-C or between the control and the 7-NI group (Fig. 1A). The oxygen consumption was not significantly different between the IR-C and C-C or between the two groups of rats (Fig. 1B). The oxygen extraction of the IR-C was significantly elevated when compared with the C-C in the control group (z24%, Pv0.001) as well as in the 7-NI group (z9%, Pv0.001). There were no significant differences in oxygen extraction between the control and the 7-NI group in either IR-C or C-C (Fig. 1C).
Microvascular O2 saturation SaO2 was similar in all regions and groups (Table 2). However, the average SvO2 was significantly lower in the IR-C when compared with the C-C in both the
control (Pv0.0001) and the 7-NI group (Pv0.0001). In the IR-C, the average SvO2 of the 7-NI group was significantly higher than that of the control group (Table 2, P v 0.001). The SvO2 was similar between the two groups in the C-C. The ratio of O2 supply/consumption of the IR-C was significantly lower than that of the corresponding C-C in both the control (Pv0.0001) and the 7-NI group (Pv0.0001) (Table 2). The ratio of O2 supply/consumption in the IR-C was significantly higher (Pv0.005) in the 7-NI group than in the control group.
Heterogeneity of SvO2 The distribution of all measured SvO2 was heterogeneous in both IR-C and C-C in both groups (Fig. 2). There was a significant shift to lower values in the IR-C when compared with the C-C in both groups (isoflurane: Pv0.0001, 7-NI: Pv0.0001). The distribution of SvO2 was significantly shifted (Pv0.0001) to higher values with the 7-NI group in the IR-C when compared with the control group. The degree of heterogeneity of SvO2 was analysed by ANOVA and reported as CV (100|SD/mean)
Figure 1 Cerebral blood flow, oxygen consumption and oxygen extraction. C-C: contralateral cortex; IR-C: ischaemic –reperfused cortex; 7-NI: 7-nitroindazole treated group; control: control, vehicle treated group. Values are means 6 SD. *Significantly different from the contralateral cortex
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Table 2 Arterial and venous oxygen saturations (%) and the ratio of oxygen supply and consumption (S/C) in the ischaemic–reperfused and the contralateral cortex of the control and 7-nitroindazole groups Control (n ¼ 7) Group Cortex SaO2 (%) SvO2 (%) S/C ratio
7-nitroindazole (n ¼ 7)
Ischaemic– reperfused
Contralateral
Ischaemic–reperfused
Contralateral
91.2 ^ 2.9 52.0 ^ 3.8* 2.32 ^ 0.23*
93.6 ^ 1.8 62.0 ^ 0.6 2.93 ^ 0.13
92.8 ^ 0.9 57.6 ^ 1.3*† 2.68 ^ 0.92*†
93.2 ^ 0.5 61.7 ^ 0.8 2.96 ^ 0.85
SaO2: arterial O2 saturation; SvO2: venous O2 saturation; S/C ratio: oxygen supply/consumption ratio. Values are mean ^ SD (n ¼ 7). * Significantly different from the contralateral cortex. † Significantly different from the control group.
Figure 2 The distribution of individual microvascular venous oxygen saturation. In the ischemic–reperfused cortex, the heterogeneity of venous oxygen saturation as well as number of veins with oxygen saturation < 50% were much smaller with 7-nitroindazole treatment despite similar regional cerebral blood flow
obtained from the distribution curve (Fig. 2). Ischaemia–reperfusion significantly increased the heterogeneity of SvO2 in both groups (Pv0.0001). 7nitroindazole significantly decreased the heterogeneity of microregional SvO2 (Pv0.0001) in the IR-C when compared with the control group. The values of CV were 15.5 and 6.0 for the IR-C and C-C, respectively, in the control group, and 8.8 and 6.4 for the 7-NI group. The number of veins with oxygen saturation below 50% was significantly less in the 7-NI than in the control group (4 out of 70 vs 18 out of 70, chi-square: Pv0.005) in the IR-C.
Size of cortical infarct The size of cortical infarct measured as percentage out of total cortical area determined at the end of a 2-hour reperfusion was significantly smaller with the 7-NI than the control group (10.5 + 1.3% vs 13.5+ 1.7%, Pv0.01).
Pons We also compared the effects of the 7-NI in O2 balance in the remote cerebral region, the pons. There were no significant differences in the cerebral blood flow (control: 136+ 55 vs 7-NI: 116+ 35 ml/minute/ 100 g) in the pons between the two experimental groups. The oxygen extraction (control: 4.3+ 0.6 vs
7-NI: 4.5+ 0.7 ml O2/100 ml) was also similar between the two groups in the pons. The oxygen consumption of the pons between the groups was not significantly different either (control 5.8+ 2.7 vs 7NI 5.2+ 1.8 ml O2/minute/100 g).
Discussion Our data demonstrated that ischaemia–reperfusion decreased the average microvascular SvO2 with increased heterogeneity. In the IR-C, with 7-NI treatment, the average SvO2 became higher with a decrease in the number of veins with low O2 saturation and with smaller heterogeneity without a significant difference in rCBF or the average O2 consumption. It suggests improved microregional O2 balance. The size of cortical infarct determined at 2 hours after reperfusion was also smaller with 7-NI treatment. Our data showed restoration of rCBF with reperfusion in both groups as shown in other studies.4,24 7nitroindazole treatment almost completely restored rCBF in the IR-C. Our data agree with previous reports that in contrast to non-specific NOS inhibition, nNOS inhibitor did not significantly affect cortical cerebral blood flow in transient cerebral ischaemic models.12 Overall, the rCBF, O2 consumption and O2 extraction were similar between the groups. Our data suggest
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that without alterations of average rCBF and O2 consumption, the microvascular SvO2 or microregional O2 balance could be heterogeneous. In the equation for O2 supply/consumption ratio described in the Methods section, since rCBF and Hb are cancelled out and SaO2 is essentially constant and independent of ischaemic status or regions, individual microvascular SvO2 would reflect the final product of microregional O2 supply/consumption balance.4,21,27 SvO2 variability in this study represents the heterogeneity of O2 supply/consumption ratio on microregional basis. Ischaemia–reperfusion produces various pathophysiological reactions, such as platelet–leucocyte aggregation, release of excitatory amino acid neurotransmitters and inflammatory agents, release of free radicals, generation of interstitial and cellular oedema and activation of various nitric oxide synthases.1,2,5,6 It is possible that reperfusion caused microvascular blockage or hyperaemia and caused irregular distribution of rCBF. Ischaemia–reperfusion may have rendered neurons excited or left them normal or made them non-functioning or dead, which caused increase or decrease in the O2 consumption of various microscopic areas. This anatomic and functional heterogeneity could have caused mismatch between O2 supply and consumption, thus producing heterogeneity of microvascular SvO2. Higher microvascular SvO2 may represent higher O2 supply/consumption ratio in that microregion. However, higher SvO2 would not mean better neuronal survival since neurons could be inactive or dead. Even though the average microvascular SvO2 was similar, however, the increase of the number of individual microvascular veins with low SvO2 may be harmful for neuronal survival. Studies on transgenic nNOS knock-out mouse and on the rats treated with 7-NI indicate that activation of nNOS contributed to ischaemic cell damage after cerebral ischaemia.7,29 Various mechanisms of neuroprotection by 7-NI were suggested, such as decrease in lipid peroxidation and, free radicals and decreased NMDA activity. Attenuation of increased cerebral metabolism by NMDA may be one of the factors of neuroprotection. Various biological targets of nitric oxide were reported such as O2, guanylate cyclase, Ras, NADH ubiquinone or succinate oxide reductases, creatinine kinase, cis-aconitase, activation of PARS and cytochrome oxidase.11,30 Our data suggest that decreasing nitric oxide by inhibition of neuronal NOS could optimise the energy supply/consumption balance at the cellular level and contribute to producing beneficial effects on neuronal survival. Our previous study showed that the NMDAinduced increase in rCBF and O2 consumption was attenuated by 7-NI.20 However, 7-NI did not affect
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rCBF or O2 consumption in the normal contralateral cortex. It was reported that at 2 hours of reperfusion, the NMDA was still activating NOS activity.13 These studies suggest the possibility that nNOS could affect neuronal O2 consumption as well as rCBF in the early stage of ischaemia–reperfusion. 7-nitroindazole decreased nitric oxide production and did not significantly affect cerebral blood flow or oxy-haemoglobin concentration during kainate-induced seizure, but produced less damage to the hippocampus.23 However, the abnormal discharges of EEG during kainate-induced seizure were attenuated. They used laser Doppler flow metre for rCBF determination and near infrared spectrophotometry for measuring cerebral oxygenation by determining the concentration of oxy-haemoglobin (HbO2). These techniques can only determine global or regional blood flow or oxygenation.23 These studies suggest that inhibition of neuronal nitric oxide could modify microregional neuronal function, distribution of rCBF or O2 supply/consumption balance without significant changes in global or regional CBF, or HbO2 to produce better neuronal survival during cerebral ischaemia–reperfusion. It has been reported that the NMDA receptor antagonist MK-801 remarkably reduced the ischaemia-enhanced NOS activity and cyclic GMP level in the brain hemisphere during reperfusion.13 7-nitroindazole significantly decreased membrane lipid peroxidation during the early stage of reperfusion. Ischaemia-evoked activation of glutamatergic system stimulates nitric oxide-dependent signal transmission.13 Our previous study suggests not only 7-NI that could affect nNOS, which is distal to NMDA in the NMDA-nitric oxide pathway and affect cerebral O2 balance but also suggests the possibility of retrograde effect of nitric oxide on NMDA.20 It has been reported that elevation of nitric oxide production led to mitochondrial dysfunction, glutamate release and excitotoxicity.31 Since NMDA receptors are activated during ischaemia– reperfusion, it is possible that the increase of O2 consumption by NMDA activation in the ischaemia– reperfusion could be decreased by 7-NI and affect microregional O2 supply/consumption balance. That could contribute to higher average microvascular SvO2 and the higher O2 supply/consumption ratio in the condition of similar rCBF to the control. Similar average O2 consumption between the control and the 7-NI group suggest that more neurons survived with 7-NI treatment in the IR-C. Inhibition of nNOS by ARL 17477 at the dose that did not affect rCBF or MABP but significantly decreased NOS activity decreased the size of infarct that was determined 7 days after transient MCA occlusion in rats.32 The results of our study are similar to this study that showed no significant changes in rCBF.
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Improved microregional O2 balance could have contributed to decrease in the size of infarct in both studies. Our data suggest that 7-NI could decrease neuronal damage during reperfusion through improved microscopic O2 supply/consumption balance despite no significant change in rCBF or O2 consumption. Decreasing the number of veins with low O2 saturation may decrease neuronal damage. 7-nitroindazole significantly decreased the percentage of the area of cortical infarct out of the total cortical area. This effect at 2 hours of reperfusion does not necessarily mean a long-term effect, but other studies showed neuroprotective effects of nNOS inhibitors at the later stage of cerebral ischaemia.13,17,32 Our study suggests that this improved microregional O2 supply/consumption balance reflected by a decrease in the number of veins with low SvO2 could be one of the contributory factors decreasing the size of infarct in the early stage of reperfusion, at least in the cortical area. Differences in mean arterial blood pressure, rCBF and Hb between groups even though statistically not significant may have affected our data. The reported rCBF values are from regions, not from microregions. However, the equation used for O2 supply/ consumption balance cancels out Hb as well as rCBF. SaO2 was also essentially constant and independent of ischaemic status. Therefore, the individual SvO2 should represent the final product of the O2 supply/consumption balance in these microregions. The limitation in our measurements of rCBF and O2 extraction/consumption was reported previously.27,28 This technique is one of the few methods that give rapid, accurate, quantitative assessment of cerebral O2 balance in small microregions of normal and ischaemic cortex that has drainage fields of cerebral veins of 20–60 mm in diameter. In conclusion, with 7-NI pretreatment in the IR-C, the average microvascular SvO2 was higher and the number of veins with low O2 saturation and heterogeneity of SvO2 were smaller without a significant difference in average rCBF and O2 consumption. Our major finding is that 7-NI improved microregional O2 balance in cerebral ischaemia–reperfusion. The size of cortical infarct was smaller with 7-NI that was determined at 2 hours of reperfusion. Our data suggest that this improved microregional O2 supply/consumption balance with 7-NI may have contributed to the decreased size of cortical infarct during the early stage of reperfusion.
Disclaimer statements Contributors OZC: took part in design, experiments, data analysis and writing. KHR: took part in design, data analysis and writing. SB: took part in design,
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data analysis and writing. XL: took part in experiment and data analysis. HRW: took part in design, experiment, data analysis and writing. Funding Funding was provided by the intramural departmental sources. Conflict of interest Authors do not have any conflicts of interests to be disclosed. Ethics approval This study was approved by our Institutional Animal Care and Use Committee and was conducted in accordance to US Public Health Service Guidelines using the Guide for the Care of Laboratory Animals (DHHS Publication No. 85-23, revised 1996).
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