Nitric Oxide Modulates Dopamine Release During Global Temporary Cerebral Ischemia

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1 Nitric Oxide Modulates Dopamine Release During Global Temporary Cerebral Ischemia Ronald A. Kahn, MD*, Jesse Weinberger, Mm, Timothy Brannan, MDt, Alla Prikhojan, MDt, and David L. Reich, MD* Departments of *Anesthesiology and tneurology, The Mount Sinai Medical Center, New York, New York Dopamine (DA) is released in large quantities from the striatum during cerebral ischemia. Along with excitatory neurotransmitters, DA plays a role in cellular neuronal ischemic injury. In this study we examined the role of nitric oxide (NO) in the ischemia-induced release of DA. A microdialysis probe was stereotactically placed into the corpus striatum of 16 Sprague-Dawley rats for DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) level determinations. After probe stabilization, the animals received either flnitro-l-arginine-methyl ester (), a NO synthase inhibitor, or vehicle through the microdialysis probe. Temporary global forebrain ischemia was induced using bilateral carotid artery ligature tightening and controlled hemorrhagic hypotension for 15 min. administration caused a reduction in ischemic estimated extraneuronal DA concentration by 60% (P < 0.005) compared with control. There was an increase in both DOPAC and HVA concentrations during the recovery period compared to baseline values in the control group (P < 0.05). also caused a reduction in HVA concentration compared to vehicle administration during the latter part of recovery (P < 0.05). These data support the concept that ischemic dopamine release may be mediated by NO. This NO-modulated DA release may contribute to the previously reported deleterious neurotoxic effects of NO during ischemia. (Anesth Analg 1995;80: ) T he small- to medium-sized neurons of the dorsolateral corpus striatum are more susceptible to ischemic injury than most other neuronal units (1). Catecholaminergic nerve terminals are more sensitive to ischemic damage than serotoninergic, GABAergic, or glutaminergic neurons (2,3). Dopamine (DA) is released in large quantities by the corpus striatum during anoxia, hypoglycemia, and ischemia (4-7). Excessive neuronal DA release has been implicated as a major factor in the pathogenesis of neuronal damage to the striatum (8-10). In animals in which DA depletion was induced in the corpus striatum, Weinberger et al. (101 and Globus et al. (11) observed a significant reduction in neuronal damage ischemic insult. Breakdown products of DA also may be involved in neuronal injury. DA may be converted to homovanillic acid (HVA) through one of two biochemical pathways. In the first pathway, DA is metabolized Presented in part at the annual meeting of the American Society of Anesthesiology, San Francisco, CA, October Accepted for publication January 23, Address correspondence and reprint requests to Ronald A. Kahn, MD, Department of Anesthesiology, Box 1010, The Mount Sinai Medical Center, One Gustave L. Levy Place, New York, NY to 3,4-dihydroxyphenylacetic acid (DOPAC) by monoamine oxidase and subsequently to HVA by catechol 0-methyltransferase. Alternatively, DA may be converted to 3-methoxytyramine by catechol 0-methyltransferase and subsequently to HVA by monoamine oxidase. During the conversion of DA to DOPAC, hydrogen peroxide, superoxide, and hydroxyl radicals are formed. These byproducts are potentially neurotoxic and may contribute to further neuronal injury during DOPAC synthesis (12). Nitric oxide (NO), produced endogenously by the conversion of L-arginine to L-citrulline by nitric oxide synthase (NOS), is an important modulator of vascular tone, platelet adhesion, and neurotransmission (13). There is increasing evidence that there is significant NO production during cerebral ischemia, and its release, or modulation of its release, may be an important factor in the pathogenesis of neuronal ischemic injury (14). To date, there have been numerous studies of the effects of NO donors and inhibitors of NOS on histopathologic outcome but with conflicting results (15-21). The difference may be due to the differential effects of NO on cerebral blood flow (CBF) and neurotoxicity during ischemia. In this study, we examined a possible mechanism of action of NO during cerebral ischemia. In particular, we examined the effect of NOS inhibition on 1116 Anesth Analg 1995;80: by the International Anesthesia Research Society /95/55.00

2 ANESTH ANALG NEUROSURGICAL ANESTHESIA KAHN ET AL ;80: NITRIC OXIDE AND ISCHEMIC DOPAMINE RELEASE Figure 1. Experimental protocol. BASE = baseline; ISC = ischemia: Rl. R2. R3. and R4 = f&r consecutive 15&n intekals after reperfusion; = w-nitro-larginine-methvl ester: CSF = cerebrosui- 1 na i fluid minutes I I I I I I I I I L Time 1 BASE 1 1 DRUG 1,SC 1 RI 1 R2 / R3 1 R4 / - Period: t 15 min i Probe +stabilizationi Artifical CSF vehicle (control) or b DA and DA metabolite release during and after cerebral ischemia using an established model of temporary forebrain ischemia with in vizlo microdialysis in rats. Methods The study protocol was approved by our institution s animal care and use committee. Light-cycled Sprague-Dawley rats ( g) were fasted for 12 h but allowed free access to water prior to the experiment. Anesthesia was induced using intraperitoneal chloral hydrate, 400 mg/kg, and maintained with supplemental chloral hydrate intraperitoneally, 100 mg/kg. All surgical sites were infiltrated with bupivacaine 0.25%. A femoral vein and artery were cannulated for drug infusion, pressure monitoring, and blood withdrawal. Both carotid arteries were dissected from the vagus nerve, isolated, and encircled with a suture ligature. The trachea was orally intubated and ventilation was adjusted using intermittent measurements of Pace,. The pha was maintained within the normal range using sodium bicarbonate as necessary. The electrocardiogram and rectal temperature were monitored and temperature was maintained within the normal range with overhead radiant warming lamps. Cerebral temperature was estimated using a needle temperature probe place epicranially under the temporalis muscle. Epicranial temperature was controlled by radiant lamps to remain between 36.0 and 36.5 C. Each animal was placed into a stereotaxic frame. A 4-mm tip-length microdialysis probe (CMA Microdialysis, Acton, MA) was inserted into the right corpus striatum through a burr hole using predetermined stereotaxic coordinates relative to the bregma Cm. The techniques of measuring extracellular DA, DOPAC, and HVA using cerebral microdialysis during and after global cerebral ischemia have been previously described in reports from our laboratory (23,24). The microdialysis probes were perfused with artificial cerebrospinal fluid (CSF) at a rate of 1.33 pl/min. The artificial CSF consisted of sodium 127 meq/l, potassium 2.5 meq/l, calcium 1.8 meq/l, and chloride 131 meq/l. The artificial CSF was not buffered. The perfusate was fed directly into the injector port of the high-performance liquid chromatography (HPLC) system. Samples were analyzed at 15 min intervals. The HPLC system consisted of a Brownlee C-18 Velosep 3 p reverse phase cartridge (3.2 mm internal diameter X 10 cm), a 20-PL injection loop, and a LC-4B electrochemical detector and cell (Bioanalytical Systems, W. Lafayette, IN). The electrochemical detector was set at +0.7 V and an Ag/AgCl electrode was used. The mobile phase consisted of 93 parts 150 mm monochloroacetic acid (ph 3.0) with 0.7 mm EDTA and 2.0 mm sodium octyl sulfate (paired ion reagent) and 7 parts acetonitrile. The HPLC was calibrated using an external standard. Peak heights and retention times of standard solutions were compared with the reference samples and were used to calculate concentrations of constituents. Prior to each experiment, the uniformity and efficiency of each microdialysis probe were tested by immersion in 0.1 N perchloric acid and 40 mg/l diethylenetriamine pentaacetic acid containing 200 pg/pl of DOPAC, HVA, and DA. An in vitro recovery for each probe was calculated for DA, DOPAC, and HVA prior to each experiment using the following equation: in vitro recovery = recovered concentration standard concentration The brain concentration was estimated by dividing the effluent dialysate concentration by the in vitro recovery. The experimental protocol is graphically summarized in Figure 1. After allowing 60 min for stabilization after probe insertion, baseline effluent was collected (BASE). Animals were randomized to receive either p-nitro-l-arginine-methyl ester () or the vehicle. In order to minimize the systemic effects of administration, the drug or vehicle was given through the microdialysis probe as a continuous infusion beginning 30 min prior to ischemia (ISC) and continuing through the experimental period. The concentration of used was 0.2 mg/ml (dissolved in artificial CSF), and it was given at the rate of 1.33 FLlmin. This dose of was chosen to approximate extracellular concentration

3 1118 NEUROSURGICAL ANESTHESIA KAHN ET AL. NITRIC OXIDE AND ISCHEMIC DOPAMINE RELEASE ANESTH ANALG 1995;80: Table 1. In vitro probe recovery for DA, DOPAC, and HVA Control DA k 3 DOPAC 23 -e HVA Values expressed as percent recovery t SD. DA = dopamine; DOPAC = 3,4-dihydroxyphenylacetic acid; HVA = homovanillic acid; = fl-nitro+arginine-methyl ester. when 30 mg/kg is administered systemically, as used by other investigators (15). Effluent microdialysate was collected during the latter 15-min period (DRUG). Heparin 50 U was administered, and temporary global cerebral ischemia was induced by bilateral carotid ligature tightening and controlled hemorrhagic hypotension to a mean arterial pressure (MAP) of 40 mm Hg for 15 min (ISC). After the ischemit period, the carotid arteries were released and blood was reinfused. DA and DA metabolites were measured for 1 h in 15-min epochs (Rl-R4). Concentrations of DA, DOPAC, and HVA are expressed as estimated extracellular fluid concentration in pg/pl. All data are presented as the mean + SD. Change in estimated extracellular concentration of DOPAC and HVA compared to baseline values were calculated for each animal. Differences in physiologic variables, DA, DOPAC, and HVA concentrations were determined by analysis of variance. Post-hoc analysis was performed using Student s t-tests with a Bonferroni s correction to correct for multiple comparisons. Intragroup differences were compared with paired t-tests, while intergroup testing was unpaired. A two-tailed P value less than 0.05 was considered significant. Results A summary of the in vitro probe recovery for DA, DOPAC, and HVA is presented in Table 1. There were eight animals in each group. Physiologic data are summarized in Table 2. Although the baseline MAP of the control group was lower than the group (P < 0.05), there were no other significant differences between the two groups during any study period. Since We assumed that was distributed throughout total body water, which was estimated as 60% of the rat s weight. If a rat of kg is used, (30 mg/kg). (0.250 kg) mg of is administered systemically (if a dose of 30 mg/kg is used). If this quantity is distributed evenly throughout the total body water, the extracellular concentration of would be (30 mg/kg). (0.250 kg) (0.60 L/kg). (0.250 kg) or 0.05 mg/ml. If the probe recovery is approximately 25%, a concentration of 0.2 mg/ml would be required to be perfused through the probe to deliver 0.05 mg/ml extraneuronally. both pressures were within normal limits, it is unlikely that this difference is clinically significant. There were no significant increases in MAP after administration and a significant reduction of MAP during ischemia. Estimated extracellular concentrations of DA are presented in Figure 2. During baseline measurements, DA levels were low in both groups. Although no differences in DA were detected during drug administration, DA was close to the lower limits of detectability. Induction of ischemia resulted in a marked increase in DA in both groups, but there was significant attenuation of the DA increase with administration (P < 0.005). The DA concentration remained significantly higher during Rl, R2, and R3 (P < 0.05). Estimated extracellular concentrations of DOPAC are presented in Figure 3. No differences in DOPAC were noted between the two groups. There was no significant change in DOPAC with drug or vehicle administration from baseline values. During ischemia, there was a significant decrease in DOPAC compared to baseline in both groups (P < 0.05). During recovery, there was increasing DOPAC in the control group during R3 and R4 (P < 0.05). There were no discernable differences between the pattern of DOPAC extracellular levels between the groups, despite the absence of statistically significant increases in DOPAC levels in the group during R3 and R4 (compared to baseline). The pattern of estimated extracellular concentrations of I-WA mirrored DOPAC (Figure 4). There was no significant change in HVA concentration during drug administration. During ischemia and Rl, there was a significant decrease in HVA concentration compared to baseline values during vehicle administration and administration (P < 0.05). During R3 and R4, there was a significant increase in I-WA compared to baseline values in the control group (P < 0.05). There were significant intergroup differences during Rl and R4, which revealed a greater change from baseline values with vehicle administration compared with administration (P < 0.05). Discussion In this study, we examined the effects of NOS inhibition on extracellular DA and DA metabolite concentrations during temporary forebrain ischemia in the rat. During the baseline period, there was no detectable difference in DA concentration between the two groups. Because the baseline levels were at the lower levels of sensitivity, a true difference between these groups cannot be excluded. administration significantly attenuated ischemic DA recovery during ischemia and subsequent recovery periods.

4 ANESTH ANALG NEUROSURGICAL ANESTHESIA KAHN ET AL ;80: NITRIC OXIDE AND ISCHEMIC DOPAMINE RELEASE Table 2. Summary of Physiologic Data Baseline Drug Ischemia Recovery MAP (mm Hg) % k Rectal temperature ( t t Brain temperature ( C) I! PHa 7.39 c ? c 0.04 Pace, (mm Hg) Pao, (mm Hg) k t MAP = mean arterial pressure; = NG-nitro-L-arginine-methyl ester. * P i 0.05 compared to group. Dopamine Concentration DOPAC Concentraton (% change from baseline) % dmq.3 from bamlin* 40 T Figure 2. Estimated extraneuronal dopamine concentration. + +P < compared to control. +P < 0.05 compared to control. ISC = ischemia; Rl, R2, R3, and R4 = four consecutive 15-min intervals after reperfusion; = No-nitro-L-arginine-methyl ester. These results support the hypothesis that DA release by dopaminergic neurons during cerebral ischemia is an active phenomenon. Since NOS inhibition reduced DA release, NO may modulate ischemic release of DA from dopaminergic neurons. Most likely, NO is an intermediary in the mechanism of ischemic DA release. The reduction of NO production by during ischemia may cause inhibition of DA release through another mechanism. This modulation of ischemic DA release may explain some of the described neurotoxic effects of NO. DA and other catecholamines may play a direct role in the pathogenesis of neuronal injury. In vitro and in uivo preparations demonstrate that catecholamines are neurotoxic (25,26). Reduction of extraneuronal DA release may attenuate cerebral ischemic injury. Weinberger et al. (10) induced DA depletion in gerbils. Figure 3. Estimated extraneuronal DOPAC concentration. Baseline concentrations of DOPAC in the control group were 1152 k 434 and in the group. *P < 0.05 compared to baseline. ISC = ischemia; Rl, R2, R3, and R4 = four consecutive 15-min intervals after reperfusion; = fl-nitro-l-arginine-methyl ester; DOPAC = 3,4-dihydroxyphenylacetic acid. After bilateral forebrain ischemia, damage to the dopaminergic and other adjacent nerve terminals were substantially attenuated in the DA depleted group compared with the control group. They concluded that DA is intimately related to the evolution of ischemit cerebral injury. These results were supported by Globus et al. (8,111. After creation of a unilateral substantia nigra lesion, virtually eliminating dopaminergic input to the striatum, reversible cerebral ischemia was induced. An early postischemic resumption of glucose metabolism without a corresponding increase in CBF was observed in the nonlesioned group. They speculated that DA release resulted in this metabolic uncoupling (Le., mismatch between CBF and

5 1120 NEUROSURGICAL ANESTHESIA KAHN ET AL. ANESTH ANALG NITRIC OXIDE AND ISCHEMIC DOPAMINE RELEASE 19!35;80: HVA Concentration ( % change from baseline) % change WJm baedme M * Tlms Pemd Figure 4. Estimated extraneuronal HVA concentration. Baseline concentrations of HVA in the control group were 956? 328 and in the erouu. P < 0.05 comuared to baseline. tp < 0.05 between-group &ffe;ence. ISC = ischkmia; Rl, R2, R3, and R4 = four consecutive 15-min intervals after reperfusion; = p-nitro-l-arginine-methyl ester; HVA = homovanillic acid. cerebral metabolic rate), thus exacerbating neuronal injury. Several investigators have demonstrated a possible protective effect of NO during ischemia by potentiation of CBF. Sancesario et al. (15) described worsening hippocampal neuronal damage after high-dose chronic NOS inhibition. Yamamoto et al. (16) and Kuluz et al. (17) also report worsening of ischemic injury with after focal cerebral ischemia. Morikawa et al. (18) published further evidence for the protective effect of NO. After administration of L- arginine (a precursor of NO) they observed an increase in regional CBF after middle cerebral artery occlusion which was associated with a reduction in infarct volume 24 h after ischemia. Seemingly contradictory evidence for a neuroprotective role of NOS inhibitors has been suggested by other investigators (19-21). In a cat model of focal ischemia, administration before and after ischemia resulted in a marked decrease in neuronal injury in the caudate, but not the cortex, compared to control animals (21). We suggest that the differential protection seen in the caudate may be due to the inhibition of DA release observed in the current study. DA neurons are abundant in the caudate and less concentrated in the cortex. If NOS inhibition caused decreased DA release, the greatest area of neuronal protection would be seen in the area with the greatest dopaminergic innervation. Differences between the outcome of these studies may be explained by the differential effects of NO; NO increases CBF and also modulates neurotoxic neurotransmitter release. In models of ischemia where there is a greater ischemic penumbra, the cerebral vasodilation mediated by NO may supplement the CBF and decrease the degree of cerebral injury. In contrast, the increase in the CBF afforded by NO may not be significant in ischemic models with a smaller ischemic penumbra. The neurotoxic effects of NO may be mediated by neurotransmitter release, excitotoxicity, or free radical production. N-Methyl-d-aspartate increases the in vitro basal DA release from striatial slices (27). This release can be blocked by NOS inhibition and attenuated by addition of hemoglobin (a NO scavenger). Exposure of exogenous cell cultures to NO results in increased DA release. These observations give support that DA release is in response to excitatory neuronal input, and the release of DA is mediated by NO. Using an in bivo model, Strasser et al. (28) examined the effect of NO inhibition on DA release during ischemia. The investigators infused a NOS inhibitor or vehicle into the gerbil striatum before inducing global ischemia. They observed an attenuation of DA release with NOS inhibition. During ischemia, NOS inhibition did not effect DA release. The differing results from this study compared to the present study may be related to the dose of NOS inhibitor used. The higher dose of NOS inhibitor used by Strasser et al. (28) may have potentiated the decrease in CBF during ischemia, thus increasing extraneuronal DA release. In this model, a NOS inhibitor was injected directly into the neuronal region of study. Although this technique allowed separation of the systemic effects of NO with the local effects of NO, associated beneficial cerebral vasoactive effects of NO during ischemia may not be seen. The increase in CBF afforded by NO may decrease the ischemic penumbra and improve neurologic outcome. In addition, since was locally administered, a lower NOS dose was used; and the systemic effects of NOS will be attenuated. The hypertensive effects normally seen with NOS inhibition may increase cerebral perfusion pressure and possibly affect eventual outcome. In conclusion, an ischemia-induced increase in extracellular DA concentration was attenuated by NOS inhibition at pharmacologic doses. These data support the concept that ischemic DA release may be mediated by NO. These results are consistent with other in vitro and in vim data indicating a modulator effect of NO on DA release. This NO-modulated DA release may contribute to the deleterious neurotoxic effects of NO during ischemia, particularly the selective damage seen in the striatum. References 1. Ginsberg MD, Graham DI, Busto R. Regional glucose utilization and blood flow following graded forebrain ischemia in the rat: correlation with neuropathology. Ann Neurol 1985;18:

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