(Stroke. 1996;27:2299-2303.)
© 1996 American Heart Association, Inc.
Articles |
Zinc Protoporphyrin, Zinc Ion, and Protoporphyrin Reduce Focal Cerebral Ischemia
the Departments of Pharmacology (Y.-J.Z., E.F.D.) and Surgery (Neurosurgery) (G.-Y.Y.), University of Michigan (Ann Arbor).
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Abstract |
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Background and Purpose Zinc protoporphyrin pretreatment protects against temporary focal ischemic brain injury in rats. However, it is not known whether the zinc or the protoporphyrin portion of zinc protoporphyrin has effects on focal cerebral ischemia. Hence, all three agents were compared with regard to infarct size and edema in a rat model of middle cerebral artery occlusion.
Methods Four groups of adult male Sprague-Dawley rats were subjected to 2 hours of temporary middle cerebral artery occlusion followed by 22 hours of reperfusion. Each group was pretreated 30 minutes before middle cerebral artery occlusion with 0.9% NaCl, and then three groups were given equimolar doses of zinc protoporphyrin, zinc chloride, or protoporphyrin, respectively. Regional cerebral blood flow in the ischemic cortex was monitored with a laser Doppler flowmeter. Cerebral infarct size, brain water content, and ion content were measured 24 hours after the onset of occlusion.
Results Regional cerebral blood flow during middle cerebral artery occlusion was approximately 9.2% to 13% of baseline in all four groups. Brain water content in the infarcted zone after temporary focal ischemia in control, zinc protoporphyrin, zinc chloride, and protoporphyrin groups was 85.7%, 80.6%, 85.6%, and 81.4%, respectively. Brain sodium content in the same areas in all four groups paralleled the water content. Infarct size in the controls and groups treated with zinc protoporphyrin, zinc, and protoporphyrin was 25.6%, 7.2%, 7.6%, and 7.2%, respectively. Compared with the control group, the infarct volume in all three treated groups was significantly reduced (P<.05).
Conclusions The present results indicate that zinc protoporphyrin, but also zinc and protoporphyrin, contribute to brain-protective effects when administered early in a temporary focal ischemia model. Zinc chloride reduced infarct size but not edema formation when compared with zinc protoporphyrin and protoporphyrin. Zinc ion in vivo has brain-protective effects, confirming in vitro studies previously reported by some but contrary to reports of others. Blood versus brain neuropil and cell body concentrations of zinc ion need to be studied in the future to define the precise role of zinc in the complex mechanisms involved in brain ischemia.
Key Words: cerebral ischemia, focal • middle cerebral artery occlusion • neuroprotection • rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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The brain-protective effects of ZnPP involve multiple mechanisms of action including anti-inflammatory properties as an antagonist of interleukin-1, as well as inhibition of heme oxygenase and other heme-dependent enzymes such as guanylate cyclase and nitric oxide synthase.1 2 3 Kadoya et al4 showed that pretreatment with ZnPP reduces brain infarct volume and edema accumulation after tMCAO. ZnPP had to be given early to enhance recovery from temporary ischemia. The purpose of this study was to compare ZnPP to Zn2+ or PP itself in reducing infarct size and brain edema.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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All of the procedures involving laboratory animals were approved by the University of Michigan Committee on Use and Care of Animals. Forty-eight adult male Sprague-Dawley rats weighing 230 to 300 g (Charles River, Portage, Mich) were randomly divided into four groups to undergo tMCAO for 2 hours and reperfusion for 22 hours. Animals were treated 30 minutes before tMCAO. The experimental animal groups were as follows: group 1 (control) was given 0.9% NaCl 1 mL/kg IP; group 2 received ZnPP in a dose of 50 mg/kg (50 mg/mL) IP; group 3 received ZnCl2 (Zn2+) in an equimolar dose of 10.9 mg/kg (10.9 mg/mL) IP; group 4 received Na2PP (PP) in an equimolar dose of 48.5 mg/kg (48.5 mg/mL) IP. In each group of animals, two sets of experiments were performed: (1) measurement of ischemic brain edema and ion content and (2) measurement of infarct volume using 2% TTC staining. During all experiments, physiological parameters were monitored and controlled. Surface CBF was measured with an LDF (Laserflow BRM,2 Vasamedics Inc). The brain temperature was measured at the temporal muscle using thermocouples (E 450, Omega Engineering Inc), and the rectal temperature was measured with a thermometer (YSI model 73A, Yellow Springs Instrument Co). The temperature was carefully regulated by use of a heating lamp and heating pad to maintain 37±0.5°C. After each experiment, the animal was killed, and brain samples were measured for infarct volume, water content, and ion content.
Anesthesia was induced by inhalation of 2.5% halothane in a 30/70-O2/N2O gas mixture. After intubation, ventilation was maintained with 1% halothane. The femoral artery was cannulated with PE-50 tubing to allow continuous monitoring of arterial blood pressure and sampling of arterial blood gases, pH, and glucose. Systemic arterial blood pressure was maintained above 90 mm Hg by adjusting the halothane concentration. tMCAO was produced using a modification of the method of Zea-Longa et al.5 Briefly, using an operating microscope with enhanced visualization, the left CCA was exposed through a midline incision. The branches of the ECA were isolated and coagulated along with the terminal lingual and maxillary branches. The ICA was then isolated, and its extracranial branch, the pterygopalatine artery, was ligated close to its origin. Thus, the ICA, which is the only extracranial branch of the CCA, remained patent. A 3-cm length of 3-0 nylon suture with a slightly enlarged and rounded tip was introduced into the transected lumen of the ECA and gently advanced from the ECA into the ICA. The distance from the tip of the suture to the bifurcation of the CCA is about 19 to 20 mm in adult rats. For reperfusion, the suture was withdrawn back into the ECA to restore ICA-MCA blood flow.
For the CBF measurements, a BPM2 LDF monitor (Vasamedics Inc) equipped with a small caliber probe of 0.7-mm diameter (P-433, Vasamedics Inc) was used. LDF was performed on both cerebral cortices. Point A was placed 5 mm lateral in the contralateral hemisphere, and point B was placed 5 mm lateral in the ipsilateral ischemic hemisphere. Both points were 1.5 posterior to bregma. After the rat was mounted in a stereotaxic frame, the skull was exposed through a midline skin incision. Then two holes were drilled, and a thin bone layer was carefully removed to prevent injury to the cortex. The dura remained intact. Each probe was held in a micromanipulator and stereotactically advanced to gently touch the intact dura mater. To obtain a clearer optic medium between the LDF probe and the cortex and to maintain the brain temperature at 37°C to 37.5°C, warmed 0.9% NaCl was slowly rinsed around the probe during the experiment.6 Ten-minute stable baseline LDF readings were obtained before occlusion of all the sites described above; then tMCAO was performed. LDF values from continuous digital display were averaged over 5-second intervals and recorded every 30 minutes during the tMCAO until 15 minutes after reperfusion. The CBF values were calculated and expressed as percentage of baseline values (milliliters per 100 g per minute). If CBF increased to >35 mL/100 g per minute during the tMCAO, the animal was excluded from the study.
Samples for brain water content were removed from flattened cortical mantels using 7-mm and 10-mm cork borers to obtain tissue from the center, intermediate, and outer zones of the ischemic cerebral cortex and from corresponding areas of the contralateral nonischemic cortex.7 8 The tissue samples were weighed to an accuracy of ±0.0001 mg to obtain wet weight (W). The samples were then dried in a gravity oven (Blue M Electric Co) at 100°C for 24 hours and reweighed to obtain dry weight (D). Water content was expressed as percent wet weight and was calculated as (W-D)/Wx100. The dehydrated section was digested in 1 mL of 1N nitric acid for 1 week. A 0.2-mL aliquot was then removed and diluted to 2 mL with deionized water and 3 mmol/L CsCl2 solution. The Na+ and K+ contents of this solution were measured with atomic absorption spectroscopy (IL943 Automatic Flame Photometer, Instrumentation Laboratory Inc). Flame conditions and detection wavelengths were optimized for sensitivity and linearity.
The area of cerebral infarction was quantified by an image analysis system.9 After 22 hours of reperfusion, the brains were removed without external perfusion. Six coronal slices of 2, 4, 6, 8, 10, and 12 mm distal from the frontal pole were dissected using a brain slicer (Activational System Co). All brain slices were incubated with an immersion technique using TTC solution (in Dulbecco's phosphate buffer, pH 7.4) for 20 minutes at 37°C as described previously.10 After incubation, all of the samples were fixed in 10% buffered formaldehyde solution. The slices were then photographed, and the total cross-sectional area of the infarcted tissue was measured in square millimeters using a computer-assisted planimeter (National Institutes of Health 1.55). The infarct areas were expressed as a percentage of the total ischemic hemisphere area. Total size of the cerebral stroke was calculated as the sum of the infarcted areas from the frontal pole through all six slices.
ZnPP as used by Kadoya et al4 was given intraperitoneally in a dose of 50 mg/kg. The drug was obtained from the Aldrich Chemical Co, Inc, converted to the disodium salt with 0.1N NaOH, and dissolved in 25 mmol/L potassium phosphate buffered 0.9% NaCl. Zn2+ and disodium PP were also obtained from the Aldrich Chemical Co, Inc. Dosage calculations were as follows: molecular weight of ZnPP=626.03, of Zn2+=136.28, and of Na2PP=606.64; the dose of ZnPP used by Kadoya et al was 50 mg/kg. Hence, 626.03 mg/1 mmol=50 mg/x, and x=0.0798 mmol of ZnPP. To deliver 0.0798 mmol of Zn2+, one should administer 50 mgx136.28/626.03=10.88 mg/kg. To deliver 0.0798 mmol of Na2PP, one should administer 50 mgx606.64/626.03=48.45 mg/kg. Zn2+ and Na2PP were dissolved in 25 mmol/L potassium phosphate–buffered saline to duplicate the first study as closely as possible.
The data are expressed as the mean±SE of the mean or the mean±SD. Statistical differences among groups were identified using ANOVA and Dunnett's correction for multiple comparisons. A value of P<.05 was considered to represent a significant difference.
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Results |
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The cardiovascular and respiratory data from all four treatment groups are given in Table 1
. The values were measured during the period of occlusion and were all in the normal range. Temporalis muscle temperature was regulated at 37°C to 37.5°C. Rectal temperature was maintained at a mean of 37.5°C during the experiments in all four groups.
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Introduction of a suture to block the blood supply to the territory of the MCA produced a similar decrease in relative surface CBF in all four groups (Table 2). The mean percentage of baseline CBF in the contralateral hemisphere was comparable, remaining around 100% in all four groups. During tMCAO, CBF in the ipsilateral hemisphere was reduced to 9.2% to 13% of baseline in each temporary ischemic group of rats. After reperfusion, CBF returned to 91% to 97% of baseline in each group. There were no significant differences among the groups. It should be noted that immediately after reperfusion there was no consistent pattern of hyperperfusion.
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The mean±SE water content in the core, intermediate, and outer zones of ischemic cortex from the control, ZnPP-, Zn2+-, and PP-treated groups are illustrated in Fig 1. The H2O content in the contralateral hemisphere was comparable. For example, the water content in the core in the control group was 78.8±0.4%, and in the other groups it was 79±0.2% (P>.05, data not shown). Compared with the control group, the increase of water in the ischemic cores and intermediate zones in both ZnPP- and PP-treated groups was significantly less (core: ZnPP=80.6±1.1%, PP=81.4±1.1% versus control=85.7±1.2%; intermediate zone: ZnPP=80.5±0.9%, PP=80±0.4% versus control=83.2±0.9%; P<.05). The increase in water content in the ischemic tissue was compared with shifts in Na+ and K+ (Fig 2). The increases in Na+ content in the ZnPP- and PP-treated groups were less than those in the control group in the core zone (ZnPP=303±77, PP=408±85 versus control=827±128 µEq/g dry wt, P<.05). There was a significant difference in K+ content between the ZnPP-treated and control groups (core zone: ZnPP=418.8±36.1 versus control=218±45.8 µEq/g dry wt, P<.05). The changes of water, Na+, and K+ contents in the Zn2+-treated group were similar to those in the control group.
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P<.01 compared with control 0.9% NaCl group.
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The method used for inducing tMCAO yielded reproducible infarction areas and volumes in the distribution territory of the rat MCA as measured by mitochondrial dehydrogenase staining with TTC.9 As shown in Figs 3 and 4, the infarct areas and volumes were significantly smaller in all three treated groups compared with control. In the control group, the infarct volume was 25.6±3.5%, whereas in the ZnPP, Zn2+, and PP groups, the infarct volumes were 7.3±2.5%, 7.6±2%, and 7.15±2.6%, respectively (P<.05).
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), Zn2+ (
), and PP (
). n=6 in each group;
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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The data obtained in this study are both expected and unexpected. The brain-protective effects of ZnPP pretreatment on temporary focal cerebral ischemia in rats confirm our previous findings.4 Although ZnPP appeared slightly more effective than PP, the latter was statistically comparable in reducing both water and Na+ content in the ischemia area, as well as reducing infarct volume. There are several PP derivatives that are coordinated to different metals. These have different affinities for heme oxygenase, nitric oxide synthase, and interleukin-1 receptors. The fact that PP alone is brain protective suggests that a metal coordinated within the PP ring is not essential. Zinc does not exist as a free cation in ZnPP because it is an integral part of that molecule. In view of the possibility that ZnPP could be catabolized to free Zn2+, the latter was used as a control treatment. Although Zn2+ had no effect on the water and Na+ content in the ischemic area, it did reduce infarct area and volume. The fact that elevated Zn2+ reduces infarct size in vivo, as shown in this study, is of great theoretical significance because the NMDA receptor for glutamic acid contains a Zn2+ binding site. Zn2+ is a selective noncompetitive antagonist of NMDA in cultured neurons for the phencyclidine-related N-(1-[2-thienyl]cyclohexyl)piperidine binding site11 12 13 14 and in neurotoxicity studies.15 Zn2+ differs from Mg2+ in that the latter reduces NMDA currents in a voltage-dependent manner, whereas the former reduces NMDA channel open time.15 Zn2+ acts at a separate site from other NMDA antagonists. Furthermore, Zn2+ blocks
-aminobutyric acid receptors,13 as well as voltage-gated Ca2+ channels.16 The former effect could enhance while the latter reduces ischemic injury. The toxic effects of too much extracellular Zn2+ are very important to note. Koh et al17 have shown that after a 10-minute period of forebrain ischemia produced by clamping both CCAs plus systemic hypotension (50±5 mm Hg) in pentobarbital-anesthetized Long-Evans rats, extracellular Zn2+ accumulated into postsynaptic hippocampal hilal and CA1 pyramidal neurons before their degeneration. When the influx of chelatable extracellular Zn2+ into postsynaptic neurons was blocked by intraventricular injection of a Zn2+ chelating agent, neurodegeneration was prevented. Therefore, the toxic influx of too much Zn2+ might be another mechanism of neuronal death after transient global ischemia. More research is needed on the relationship of plasma Zn2+ levels with those in the neuropil and neuronal cell bodies during acute ischemic brain injury. The present research indicates that moderately elevated plasma Zn2+ levels do not make matters worse and in fact appear to be protective. The fact that some concentrations of blood Zn2+ in vivo have a protective effect on infarct volume is very important regarding the possible mechanisms of action of this ion. There is also a potential therapeutic lead from the finding that some concentrations of Zn2+ are protective. There is evidence that certain tricyclic antidepressants such as desmethylimipramine have properties similar to those of Zn2+ and may bind to the same site.14 This latter observation should be pursued for newer therapeutic agents in the treatment of brain ischemia. The similar protective effectiveness of ZnPP and PP was surprising. ZnPP was predicted to be far superior in view of its multiple actions as an interleukin-1 antagonist,3 as an inhibitor of heme oxygenase1 (which cleaves heme into biliverdin and CO), or as an inhibitor of nitric oxide synthase to prevent nitric oxide formation.2 The actions of nitric oxide in cerebral ischemia are well known.18 The present research was initiated with the hypothesis that ZnPP itself was the most efficacious brain-protective agent. Obviously, that hypothesis must now be rejected, and further research must be undertaken on the mechanisms of action of PP. In addition, the stability of ZnPP compared with PP must be studied, especially under the solubilization conditions that were used. A major shortcoming of this research is the failure to study each agent in a dose-effect manner. The dose of 50 mg/kg of ZnPP was chosen because it was shown to be effective.4 Equimolar doses of both Zn2+ and PP were used on the basis that the molecule of ZnPP should be compared with the Zn2+ and PP molecules. Future studies must pursue the dose-effect relationships and related issues to explain the results of the present study.
One should note that there appears to be no correlation between infarct volume, brain water, and Na+ content with the Zn2+ treatment. However, with ZnPP and PP alone there is a reasonable correlation. It raises the issue of the relative importance of Na+ under these circumstances. All three agents were given as pretreatments. A posttreatment approach has been conducted with ZnPP in which the agent was found to be ineffective.4 A similar posttreatment study needs to be performed with PP and Zn2+ to determine whether these agents would still be effective in reducing infarct volume.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Psychopharmacology Research Fund (361024). The authors would like to thank Dr A. Lorris Betz, Director of the Neurosurgical Research Laboratory at the University of Michigan, for permission to use those facilities.
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Footnotes |
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Reprint requests to Edward F. Domino, MD, Department of Pharmacology, A220E MSRBIII, University of Michigan, Ann Arbor, MI 48109-0632.
Received June 17, 1996; revision received August 19, 1996; accepted September 6, 1996.
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References |
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3. Nagai H, Kitagaki K, Kuwabara K, Koda A. Anti-inflammatory properties of zinc protoporphyrin disodium (ZN-PP-2Na). Agents Actions. 1992;37:273-283.[Medline] [Order article via Infotrieve]
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Kadoya C, Domino EF, Yang GY, Stern JD, Betz AL. Preischemic but not postischemic zinc protoporphyrin treatment reduces infarct size and edema accumulation after temporary cerebral ischemia in rats. Stroke. 1995;26:1035-1038.
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Zea-Longa E, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989;20:84-91.
6. Dirnagl U, Kaplan B, Jacewicz M, Pulsinelli W. Continuous measurement of cerebral cortical blood flow by laser-Doppler flowmetry in a rat stroke model. J Cereb Blood Flow Metab. 1989;9:589-596.[Medline] [Order article via Infotrieve]
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Betz AL, Coester HC. Effect of steroids on edema and sodium uptake of the brain during focal ischemia in rats. Stroke. 1990;21:1199-1204.
8. Martz D, Beer M, Betz AL. Dimethylthiourea reduces ischemic brain edema without affecting cerebral blood flow. J Cereb Blood Flow Metab. 1990;10:352-357.[Medline] [Order article via Infotrieve]
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Imaizumi S, Woolworth V, Fishman RA, Chan PH. Liposome-entrapped superoxide dismutase reduces cerebral infarction in cerebral ischemia in rats. Stroke. 1990;21:1312-1317.
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Peters S, Koh J, Choi DW. Zinc selectively blocks the action of N-methyl-D-aspartate on cortical neurons. Science. 1987;236:589-593.
12. Christine CW, Choi DW. Effect of zinc on NMDA receptor-mediated channel currents in cortical neurons. J Neurosci. 1990;10:108-116.[Abstract]
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Reynolds IJ, Miller RJ. Guanine nucleotides are competitive inhibitors of N-methyl-D-aspartate at its receptor site both in vitro and in vivo. J Pharmacol Exp Ther. 1989;250:162-169.
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Winegar BD, Lansman JB. Voltage-dependent block by zinc of single calcium channels in mouse myotubes. J Physiol (Lond). 1990;425:563-578.
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Dawson TM, Dawson VL. Nitric oxide: actions and pathological roles. Neuroscientist. 1995;1:7-18.
Editorial Comment
Anesthesiology/Critical Care MedicineThe Johns Hopkins UniversitySchool of MedicineBaltimore, Md
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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This is an interesting and potentially important manuscript (mechanistically) that determines whether the major role of ZnPP in reducing cerebral infarct size and brain edema is due to Zn2+ or PP itself. It is fairly well known that ZnPP pretreatment protects against temporary focal ischemic brain injury, but the authors indicate that it is unknown whether Zn2+ or the PP portion of ZnPP have effects on local cerebral ischemia itself. Thus, the authors compared the effects of all three agents on infarct size and cerebral edema in a rat model of MCA occlusion. The authors conclude appropriately that ZnPP has the greatest protective effect when administered early in temporary focal ischemia. However, the other portions of the molecule, Zn and PP, also contribute significantly to the brain-protective effects. The fact that the brain-protective effects of ZnPP pretreatment are positive in the temporary focal cerebral ischemia model is not new and confirms the author's previous findings. Interestingly enough, PP itself also reduced both water and Na+ content in the ischemic area, as well as infarct volume. The idea that PP alone is protective suggests that a metal coordinated with the PP ring is not essential. The unexpected finding of this study was that Zn2+ had no effect on the water and Na+ content in the ischemic area; however, it did reduce infarct area and volume. The idea that Zn2+ reduced infarct size is of some significance because the NMDA receptor for glutamic acid does contain a Zn2+-binding site, and thus this mechanism may be involved. The other important aspect is that there was no correlation between infarct volume with brain water and Na+ and Na+ with Zn2+. So why in the case of Zn2+ is these no correlation, but in the case of ZnPP and PP alone there does seem to be a reasonable correlation under the circumstances of these experiments? This may say something quite important about the importance of the Na+ ion under these circumstances. Finally, while it is important that the authors demonstrated that these agents were effective when administered before treatment, it would be of importance to demonstrate that these agents could be effective when given after ischemia, particularly if any therapeutic options are going to be involved.
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Data shows % baseline of regional CBF measurements during ischemia and reperfusion. Data are mean±SD, 6 rats in each group.
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