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Brain injury after neonatal hypoxia–ischemia in rats: a role of cysteine?
Brain Research 797 Ž1998. 328–332
Research report
Brain injury after neonatal hypoxia–ischemia in rats: a role of cysteine? M. Puka-Sundvall a b
a, )
, M. Sandberg a , H. Hagberg
a,b
Dept. of Anatomy and Cell Biology, UniÕ. of Goteborg, Box 420, 405 30 Goteborg, Sweden ¨ ¨ Dept. of Obstetrics and Gynecology, UniÕ. of Goteborg, Box 420, 405 30 Goteborg, Sweden ¨ ¨ Accepted 7 April 1998
Abstract The aim of this study was to investigate the role of cysteine in development of brain damage after hypoxia–ischemia ŽHI. in neonatal rats. Rat pups were subjected to unilateral carotid ligation and exposure to hypoxia Ž7.7% oxygen. for 60 or 90 min. A subtoxic dose of cysteine were administered before or after HI and the unilateral brain injury was evaluated 14 days after the insult and expressed as ipsilateral weight deficit as % of the contralateral hemisphere. In some experiments the changes of extracellular Že.c.. cysteine in the cerebral cortex were sampled with microdialysis and analyzed with HPLC. Cysteine in a dose of 0.2 mgrg s.c. given before 60 min of HI increased the extent of brain injury by 59%. The effect of posttreatment was limited and dependent on the duration of HI: 0.2 mgrg of cysteine given after 90 min of HI increased the degree of brain injury by 25%, whereas the same dose administered after 60 min of HI was ineffective in spite of that this combination of cysteine and HI resulted in e.c. cysteine concentrations 3–4 times higher than those observed in non-treated HI controls. These data show that subtoxic doses of cysteine administered before or after HI enhances brain injury. However, e.c. cysteine levels exceeding those induced by HI are required which makes a substantial contribution of cysteine in the pathophysiology of HI brain injury in the neonatal rat unlikely. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Neonate; Hypoxia–ischemia; Cysteine; Neurotoxicity; Sulfur-containing amino acid; Excitatory amino acid
1. Introduction L-Cysteine is a sulfur containing amino acid with some properties characteristic for neurotransmitters: it depolarizes neuronal membranes w20x and can be released from adult rat brain slices in a Ca2q - dependent manner w8,30x. It has been implicated that cysteine plays a role in the pathophysiology of stroke w26x and a number of neurological disorders including motor neuron disease, parkinsonism and Alzheimer’s disease w7x. Cysteine is neurotoxic if administered orally or subcutaneously to neonatal rodents w17–19x. Neurotoxicity is strongly age-dependent: 7-day-old rat pups are more vulnerable than the younger animals w3x. There are evidence in support of that cysteine toxicity is mediated by NMDA Ž N-methyl-D-aspartate. glutamatergic
Abbreviations: HI, hypoxia–ischemia; NMDA, N-methyl-D-aspartate; s.c., subcutaneously; e.c., extracellular; PND, postnatal day ) Corresponding author. Fax: q46-31-773-33-30. 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 4 1 5 - 6
receptors: NMDA and cysteine produce the same lesion pattern in retina and NMDA receptor antagonists block cysteine toxicity w20x. The toxicity of cysteine may be related to that it is a weak NMDA agonist w21x, can modulate the NMDA receptor–ion channel complex activity by reduction of the redox site w12x, chelate Zn ions w4x or block glutamate uptake w5x w22x. It seems that a low cysteine concentration activates NMDA receptors while higher concentrations act on both NMDA and non-NMDA receptors w20x. In earlier work we showed that cysteine potentiates glutamate toxicity in vivo w22x. Cysteine involvement in ischemic brain injury in adult animals is implicated by the marked increase of its concentration in the tissue w13,26x and extracellular space w9x during ischemia in the adult brain. In a neonatal rat model of hypoxia–ischemia ŽHI., we have found some elevation of the extracellular cysteine level during HI and reperfusion w24x, which was accompanied with a minor increase of tissue cysteine concentration w23x. The importance of cysteine with respect to HI brain injury has not been investigated so far.
M. Puka-SundÕall et al.r Brain Research 797 (1998) 328–332
2. Materials and methods Hypoxia–ischemia was performed in immature rats according to the method described previously w6,25x. Rat pups ŽPND 7–10, inbred Wistar, both sexes. were anesthetized with halothane in oxygenrnitrous oxide 1:1 Ž3% for initiation, 0.5–1.5% for maintenance.. The right carotid artery was dissected and cut between double ligatures of prolene sutures. After the surgical procedure the wounds were infiltrated with local anesthetics. After 2 h of recovery the animals were exposed to 7.7% O 2 in N2 for 60 min or 90 min in a humidified chamber at 35–368C. In experiments evaluating cysteine toxicity in HI, rat pups of PND 7 were used Ž38 controls and 40 cysteine treated.. Experimental animals were randomized for controlrcysteine treatment and injected with subtoxic dose of cysteine Ž0.2 mgrg in NaCl, s.c.. 10 min prior or 10 min after the HI Žexposition to hypoxia., and littermates Žinjected with 0.9% NaCl s.c. and subjected to HI. served as controls. After the hypoxic exposure and injection the pups were returned to their dams. The brain damage was evaluated after 14 days by comparing the weight of the cerebral hemispheres and expressed as weight deficit of the hemisphere ipsilateral to the ligation as % of the contralateral. In some experiments, extracellular cysteine was sampled with the microdialysis method. In these experiments rat pups of PND 7–10 were used. After ligation of the carotid artery the rat was immobilized and a dialysis probe ŽCMA 11, 1 mm length, CMA Microdialysis, Stockholm, Sweden. was implanted into the parietal cerebral cortex of the hemisphere ipsilateral to the ligation Ž2 mm posterior to bregma, 3 mm lateral to the sagittal suture, 1.5 mm ventral to the cortical surface.. The microdialysis probe was perfused with dialysis fluid containing 150 mM NaCl, 3 mM KCl and 1.2 mM CaCl 2 at a flow rate of 2 m lrmin using perfusion pump ŽCMA 100, CMA Microdialysis..
329
After 1 h of equilibration samples were collected before, during and for 6 h after HI and immediately frozen with b-mercaptoethanol, EDTA and sodium azide Žfinal concentrations in dialysate: 20 mM, 1 mM and 5 mM, respectively.. Animals were sacrificed after 6 h reperfusion. Dialysates were subsequently analyzed for amino acids using liquid chromatography ŽHPLC. as previously described w11x. The effect of 60 min HI Ž n s 11. on changes in extracellular cysteine was evaluated and compared with the effect of HI combined with cysteine administration Ž0.2 mgrg s.c.. 10 min prior Ž n s 5. or 10 min after Ž n s 5. the insult. Extracellular cysteine changes were expressed as percentage of basal level Žbefore HI.. Since mercaptoethanol was used in the procedure any cystine present would be reduced to and measured as cysteine. In separate control experiments, the toxicity of 0.2 mgrg cysteine Žwithout HI. in the brain was evaluated. Rat pups ŽPND 7, inbred Wistar, both sexes. were injected with cysteine Ž0.2 mgrg in NaCl, s.c.. and littermates Žinjected with 0.9% NaCl s.c.. served as controls. The brains were weighted after 14 days Žcontrols: n s 12, cysteine-injected: n s 13.. Some cysteine-treated animals were sacrificed after 24 h Ž n s 5., 48 h Ž n s 3. and 72 h Ž n s 3., brains were dissected, frozen in dry ice-chilled dimethylbutan and stored in y808C until use. Coronal cryostat sections Ž8 m m. were cut at the level of hippocampus and stained with hematoxyline and eosin. Animal experiments were approved by the ethical comŽnos. 29-95 and 116-95.. mittee in Goteborg ¨ The evaluation of cysteine administration on HI-induced brain damage was performed with unpaired Mann– Whitney U test ŽFig. 1.. Wilcoxon signed rank test with Bonferroni correction for multiple comparisons were applied for analysis of the HI effect on e.c. cysteine changes ŽFig. 2.. Kruskal–Wallis test and unpaired Mann–Whitney U test were used to compare e.c. cysteine changes induced
Fig. 1. Brain damage Žexpressed as weight deficit of the ipsilateral hemisphere as a % of the contralateral hemisphere. after HI compared with HI combined with administration of cysteine in a dose of 0.2 mgrg, given s.c. 10 min prior to 60 min HI ŽA., 10 min after 60 min HI ŽB. and 10 min after 90 min HI ŽC.. The values are expressed as mean " S.E.M. Significance of the difference Žcysteineq HI vs. HI only. was tested with unpaired Mann–Whitney U test Ž) p - 0.05, )) p - 0.01..
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M. Puka-SundÕall et al.r Brain Research 797 (1998) 328–332
Fig. 2. Cysteine concentration in microdialysates during HI and reperfusion expressed as a % of basal level. The values are given as mean" S.E.M. Ž ns11.. Significance of cysteine increase Žcompared to basal level. was tested with Wilcoxon signed rank test with Bonferroni correction for multiple comparisons: Ž) p- 0.05..
by HI alone vs. HI combined with cysteine administration ŽFig. 3.. Values are given as mean " standard error ŽS.E.M...
3. Results Of 78 rat pups exposed to HI, two did not survive hypoxia Žcontrols. and three died during the 2 weeks after the insult Žone control and two cysteine-treated.. Induction of HI for 60 min or 90 min in this setting produced brain damage measured as a marked weight deficit of the hemisphere ipsilateral to the ligation after 14 days. A subtoxic dose of cysteine 0.2 mgrg administered
10 min prior to the 60 min insult increased the hemisphere weight deficit from 22 " 3% to 35 " 2% Ž p - 0.01. ŽFig. 1A.. The effect of post-HI administration of cysteine was not as pronounced and depended on the duration of HI. The effect of administration of 0.2 mgrg cysteine after 60 min of HI had no effect on the neuropathological outcome, whereas administration of 0.2 mgrg cysteine after 90 min of HI slightly increased the hemispheric weight deficit from 36 " 3% to 45 " 3% Ž p - 0.05. ŽFig. 1B and C.. There was no difference in the weight of the contralateral hemisphere between control ŽHI. and cysteine-injected animals in any of the experimental groups ŽTable 1.. In control experiments Žno HI. 0.2 mgrg cysteine did not produce any histopathological changes after 24 h, 48 h and 72 h Žnot shown.. After 14 days, however, a slight Ž2.8%. decrease of brain weight as compared to saline-injected animals was observed Ž1038.5 " 11.5 mg and 1068.3 " 6.6 mg, respectively, p - 0.05—Mann–Whitney U test for unpaired data., but there were no differences in the brain to body weight ratio between cysteine- and saline-injected animals Ž3.38 " 0.08% and 3.41 " 0.06%, respectively.. The basal concentration of cysteine in dialysate was 0.070 " 0.009 m M. HI of 60 min duration produced a transient elevation of e.c. cysteine Ž322 " 41% of basal level.. During reperfusion the extracellular level of cysteine remained slightly elevated during 6 h of reflow, Žmean value during 6 h reperfusion: 163 " 9% of basal level. ŽFig. 2.. Administration of 0.2 mgrg cysteine before 60 min of HI caused an increase in the extracellular concentration of cysteine with a maximum level reached after 60 min of HI Ž865 " 243% of basal level. ŽFig. 3A.. Injection of 0.2
Fig. 3. The effect of cysteine administration on HI-induced changes in the e.c. cysteine concentration. The e.c. cysteine levels after 60 min of HI Žblack bars. compared with HI q 0.2 mgrg cysteine Žhatched bars. administered before ŽA. or after ŽB. HI. The changes of cysteine concentrations in dialysates are expressed as a % of the basal level. Arrows indicate the time points of cysteine administration. The values are expressed as mean" S.E.M. and difference between the groups were tested by comparison of the area under the curve with Kruskal–Wallis test and with unpaired Mann–Whitney U test. Significance of differences: HI vs. HI q cysteine pretreatment: p - 0.01, HI vs. HI q cysteine posttreatment: p - 0.01.
M. Puka-SundÕall et al.r Brain Research 797 (1998) 328–332
331
Table 1 The weight of contralateral hemisphere in hypoxic–ischemic rats 14 days after the insult. Data are expressed as mean" S.E.M. The differences between saline- and cysteine-injected animals were non-significant in all the experimental groups, as evaluated by Mann–Whitney U test for unpaired data Contralateral hemisphere weight Žmg.
HI—60 min q pretreatment
HI—60 min q posttreatment
HI—90 min q posttreatment
Saline 0.2 mgrg Cysteine
517.9 " 5.8 502.2 " 8.8 Žn.s..
531.6 " 11.0 527.6 " 14.3 Žn.s..
505.7 " 9.7 504.3 " 6.6 Žn.s..
mgrg cysteine after 60 min of HI caused an increase in extracellular cysteine concentration with maximum after 120 min of reperfusion Ž741 " 202% of basal level, compared to 162 " 12% after HI only. ŽFig. 3B..
4. Discussion The relevance as well as advantages and disadvantages of the used model of perinatal asphyxia have been discussed previously w6,28x. Permanent unilateral ligation of the common carotid artery does not produce any decrease in cerebral blood flow ŽCBF., but ligation combined with systemic hypoxia leads to a pronounced, transient reduction of CBF in the ipsilateral hemisphere w29x. HI produces asymmetrical cerebral growth retardation in ipsilateral hemisphere and does not affect the weight of the contralateral hemisphere w27x or affects it minimally w1x. Therefore we can treat the contralateral hemisphere as a control. In the present experiments extracellular cysteine increased in the cerebral cortex during HI and remained elevated for at least 6 h of reflow ŽFig. 3.. These changes are, however, much smaller than the marked e.c. elevations of cysteine that occurs in adult irreversible cerebral ischemia w9x. HI-induced increase in tissue cysteine concentration observed in neonatal rats w23x is also less pronounced than tissue cysteine elevation in irreversible focal ischemia in adults w13,26x. Nevertheless, cysteine is highly toxic to the immature brain w17–19x and the possibility remained that cysteine toxicity contributed to development of HI brain injury, maybe by modulation of NMDA receptors. This possibility was supported by the fact that a low dose of cysteine Ž0.2 mgrg s.c.., which is not toxic under normal conditions w19x, enhanced HI brain injury considerably if given before the insult and aggravated injury to a limited extent if administered after 90 min of HI. Two different durations of HI Ž60 min and 90 min. were used on purpose to produce different severity of HI brain damage. Cysteine increased injury after 90 min but not 60 min of HI. Increased e.c. cysteine levels during ischemia and reperfusion may contribute to the development of brain damage by enhancing of glutamate toxicity, probably through NMDA receptors. The probable mechanism may be stimulation of the receptor Žas a weak agonist. w21x or receptor sensitization by reducing disulfide bonds w14x. Cysteine may also increase extracellular glutamate concentration by
inhibiting the uptake w5x. Another possibility is that autooxidation of cysteine leads to increased production of oxygen free radicals w15,16x and membrane lipid peroxidation w2x. There are data suggesting that cysteine may also potentiate the effects of nitric oxide w10x. In the study a subtoxic dose of cysteine was used Ž0.2 mgrg.. Previously reported threshold of toxicity in fetal rodents was 0.8 mgrg, and the dose of 0.5 mgrg produced no histopathological changes after 6 and 24 h w19x. In our study 0.2 mgrg cysteine did not affect the weight if the contralateral hemisphere 14 days after HI ŽTable 1. which indicates that the dose used was not toxic. The same dose of cysteine, if applied without HI, produce no histopathological changes in the rat brain up to 72 h, but resulted in slightly decreased brain weight after 14 days compared to saline-injected animals. It may be explained by the whole body growth retardation, since the ratio: brain to body weight did not differ between the saline- and cysteine-injected rats. In order to assess the relevance of these results the e.c. levels of cysteine were measured with microdialysis after cysteine injection qHI and compared with effect of HI alone. These data show that much higher e.c. concentrations of cysteine was found after cysteineq HI than after HI alone ŽFig. 3. which was evident both after pretreatment with cysteine which does aggravate injury and after posttreatment which does not aggravate injury. These results makes it unlikely that the concentrations of cysteine that are present during post-HI reperfusion contribute to the brain lesions. The possibility still remains that lower doses of cysteine administered before the insult exert injurious effect even at levels actually found during HI. However, cysteine and glutamate have the same toxic potency w17x and the latter reaches approximately 5 times higher e.c. concentrations during neonatal HI than cysteine w24x which does not support a critical role for cysteine during HI in neonatal rats. In conclusion, a subtoxic dose of cysteine administered before or after HI aggravates brain injury in immature rats. However, there is no evidence for that the e.c. cysteine levels reached during or after HI contribute to development of brain injury.
Acknowledgements This work was supported by the Swedish Society for Medical Research, The Royal Society of Arts and Sciences
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M. Puka-SundÕall et al.r Brain Research 797 (1998) 328–332
in Gothenburg, Linnea and Josef Carlsson foundation, Swedish Natural Sciences Research Council, Swedish ˚ Medical Research Council, Sven Jerring foundation, Ake ˚ ´ foundation, Magnus Bergwall Wiberg foundation, Ahlen foundation, Frimurare Barnhus foundation, The Goteborg ¨ Medical Society, Medical Faculty of Goteborg, First May ¨ Flower Annual Campaign.The authors would like to thank Mrs. Anita Palm for her expert help with HPLC analysis.
w13x
w14x
w15x
w16x
References w17x w1x P. Andine, ´ M. Thordstein, I. Kjellmer, C. Nordborg, K. Thiringer, E. Wennberg, H. Hagberg, Evaluation of brain damage in a rat model of neonatal hypoxic–ischemia, J. Neurosci. Methods 35 Ž1990. 253–260. w2x V. Calabrese, N. Ragusa, A. Antico, S. Maangiameli, V. Rizza, Cysteine-induced enhancement of lipid peroxidation in substantia nigra: comparative effect with exogenous administration of reduced glutathione, Drugs Exp. Clin. Res. 23 Ž1997. 25–31. w3x J.G. Csernansky, M.E. Bardgett, J. Labruyere, J.J. Jackson, J.W. Olney, Age-dependent neurotoxic response to L-cysteine, Soc. Neurosci. Abstr. 19 Ž1993. 1659. w4x S. Eimerl, M. Schramm, An endogenous metal appears to regulate NMDA receptor mediated 45 Ca influx and toxicity in cultured cerebellar granule cells, Neurosci. Lett. 137 Ž1992. 198–202. w5x J. Ferkany, J.T. Coyle, Heterogeneity of sodium-dependent excitatory amino acid uptake mechanisms in rat brain, J. Neurosci. Res. 16 Ž1986. 491–503. w6x H. Hagberg, E. Bona, E. Gilland, M. Puka-Sundvall, Hypoxia– ischaemia model in the 7-day-old rat: possibilities and shortcomings, Acta Paediatr. Suppl. 422 Ž1997. 85–88. w7x M.T. Heafield, S. Fearn, G.B. Steventon, R.H. Waring, A.C. Williams, S.G. Sturman, Plasma cysteine and sulphate levels in patients with Motor neurone, Parkinson’s and Alzheimer’s disease, Neurosci. Lett. 110 Ž1990. 216–220. w8x H.J. Keller, K.Q. Do, M. Zollinger, K.H. Winterhalter, M. Cuenod, ´ Cysteine: depolarization-induced release from rat brain in vitro, J. Neurochem. 52 Ž1989. 1801–1806. w9x H. Landolt, T.W. Lutz, H. Langemann, D. Stauble, A. Mendelow¨ itsch, O. Gratzl, C.G. Honegger, Extracellular antioxidants and amino acids in the cortex of the rat: monitoring by microdialysis of early ischemic changes, J. Cereb. Blood Flow Metab. 12 Ž1992. 96–102. w10x K. Laustiola, P. Vuorinen, H. Vapaatalo, T. Metsa-Ketela, ¨ ¨ Effects of cysteine and nitroglycerin on bovine heart guanylate cyclase and tissue cyclic GMP and lactate of rat atria, Eur. J. Pharmacol. 91 Ž1983. 301–304. w11x A. Lehmann, H. Hagberg, O. Orwar, M. Sandberg, Cysteine sulphinate and cysteate: mediators of cysteine toxicity in the neonatal rat brain?, Eur. J. Neurosci. 5 Ž1993. 1398–1412. w12x S.A. Lipton, J.S. Stamler, Actions of redox related congeners of
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nitric oxide at the NMDA receptor, Neuropharmacology 33 Ž1994. 1229–1233. P. Lyrer, H. Landolt, A. Kabiersch, H. Langemann, H. Kaeser, Levels of low molecular weight scavengers in the rat brain during focal ischemia, Brain Res. 567 Ž1991. 317–320. G.A. Mathisen, F. Fonnum, R.E. Paulsen, Contributing mechanisms for cysteine excitotoxicity in cultured cerebellar granule cells, Neurochem. Res. 21 Ž1996. 293–298. A.J. Nappi, E. Vass, Comparative studies of enhanced iron-mediated production of hydroxyl radical by glutathione, cysteine, ascorbic acid, and selected catechols, Biochim. Biophys. Acta 1336 Ž1997. 295–301. K.A. Nath, A.K. Salahudeen, Autooxidation of cysteine generates hydrogen peroxide: cytotoxicity and attenuation by pyruvate, Am. J. Physiol. 264 Ž1993. F306–F314. J.W. Olney, O.L. Ho, Brain damage in infant mice following oral intake of glutamate, aspartate and cysteine, Nature 227 Ž1970. 609–611. J.W. Olney, O.L. Ho, V. Rhee, Cytotoxic effects of acidic and sulphur containing amino acids on the infant mouse central nervous system, Exp. Brain Res. 14 Ž1971. 61–76. J.W. Olney, O.L. Ho, V. Rhee, B. Schainker, Cysteine-induced brain damage in infant and fetal rodents, Brain Res. 45 Ž1972. 309–313. J.W. Olney, C. Zorumski, M.T. Price, J. Labruyere, L-cysteine, a bicarbonate-sensitive endogenous excitotoxin, Science 248 Ž1990. 596–599. J.R. Pace, B.M. Martin, S.M. Paul, M. Rogawski, High concentrations of neutral amino acids activate NMDA receptor currents in rat hippocampal neurons, Neurosci. Lett. 141 Ž1992. 97–100. M. Puka-Sundvall, P. Eriksson, M. Nilsson, M. Sandberg, A. Lehmann, Neurotoxicity of cysteine: interaction with glutamate, Brain Res. 705 Ž1995. 65–70. M. Puka-Sundvall, E. Gilland, E. Bona, A. Lehmann, M. Sandberg, H. Hagberg, Development of brain damage after neonatal hypoxia– ischemia: excitatory amino acids and cysteine, Metab. Brain Dis. 11 Ž1996. 109–123. M. Puka-Sundvall, M. Sandberg, H. Hagberg, Brain injury after hypoxia–ischemia: relationship to extracellular levels of excitatory amino acids and cysteine, Brain Res. 750 Ž1997. 325–328. J.E. Rice, R.C. Vannucci, J.B. Brierley, The influence of immaturity on hypoxic–ischemic brain damage in the rat, Ann. Neurol. 9 Ž1981. 131–141. A. Slivka, G. Cohen, Brain ischemia markedly elevates levels of the neurotoxic amino acid, cysteine, Brain Res. 608 Ž1993. 33–37. J. Towfighi, C. Housman, R.C. Vannucci, D.F. Heitjan, Effects of unilateral perinatal hypoxic–ischemic brain damage on the gross development of opposite cerebral hemisphere, Biol. Neonate 65 Ž1994. 108–118. R.C. Vannucci, Experimental models of perinatal hypoxic–ischemic brain damage, APMIS 101 Ž1993. 89–95, Suppl. 40. R.C. Vannucci, D.T. Lyons, F. Vasta, Regional cerebral blood flow during hypoxia–ischemia in immature rats, Stroke 19 Ž1988. 245– 250. L. Zangerle, M. Cueenod, K.H. Winterhalter, K.Q. Do, Screening of ¨ thiol compounds: depolarization-induced release of glutathione and cysteine from rat brain slices, J. Neurochem. 59 Ž1992. 181–189.
Research report
Brain injury after neonatal hypoxia–ischemia in rats: a role of cysteine? M. Puka-Sundvall a b
a, )
, M. Sandberg a , H. Hagberg
a,b
Dept. of Anatomy and Cell Biology, UniÕ. of Goteborg, Box 420, 405 30 Goteborg, Sweden ¨ ¨ Dept. of Obstetrics and Gynecology, UniÕ. of Goteborg, Box 420, 405 30 Goteborg, Sweden ¨ ¨ Accepted 7 April 1998
Abstract The aim of this study was to investigate the role of cysteine in development of brain damage after hypoxia–ischemia ŽHI. in neonatal rats. Rat pups were subjected to unilateral carotid ligation and exposure to hypoxia Ž7.7% oxygen. for 60 or 90 min. A subtoxic dose of cysteine were administered before or after HI and the unilateral brain injury was evaluated 14 days after the insult and expressed as ipsilateral weight deficit as % of the contralateral hemisphere. In some experiments the changes of extracellular Že.c.. cysteine in the cerebral cortex were sampled with microdialysis and analyzed with HPLC. Cysteine in a dose of 0.2 mgrg s.c. given before 60 min of HI increased the extent of brain injury by 59%. The effect of posttreatment was limited and dependent on the duration of HI: 0.2 mgrg of cysteine given after 90 min of HI increased the degree of brain injury by 25%, whereas the same dose administered after 60 min of HI was ineffective in spite of that this combination of cysteine and HI resulted in e.c. cysteine concentrations 3–4 times higher than those observed in non-treated HI controls. These data show that subtoxic doses of cysteine administered before or after HI enhances brain injury. However, e.c. cysteine levels exceeding those induced by HI are required which makes a substantial contribution of cysteine in the pathophysiology of HI brain injury in the neonatal rat unlikely. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Neonate; Hypoxia–ischemia; Cysteine; Neurotoxicity; Sulfur-containing amino acid; Excitatory amino acid
1. Introduction L-Cysteine is a sulfur containing amino acid with some properties characteristic for neurotransmitters: it depolarizes neuronal membranes w20x and can be released from adult rat brain slices in a Ca2q - dependent manner w8,30x. It has been implicated that cysteine plays a role in the pathophysiology of stroke w26x and a number of neurological disorders including motor neuron disease, parkinsonism and Alzheimer’s disease w7x. Cysteine is neurotoxic if administered orally or subcutaneously to neonatal rodents w17–19x. Neurotoxicity is strongly age-dependent: 7-day-old rat pups are more vulnerable than the younger animals w3x. There are evidence in support of that cysteine toxicity is mediated by NMDA Ž N-methyl-D-aspartate. glutamatergic
Abbreviations: HI, hypoxia–ischemia; NMDA, N-methyl-D-aspartate; s.c., subcutaneously; e.c., extracellular; PND, postnatal day ) Corresponding author. Fax: q46-31-773-33-30. 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 4 1 5 - 6
receptors: NMDA and cysteine produce the same lesion pattern in retina and NMDA receptor antagonists block cysteine toxicity w20x. The toxicity of cysteine may be related to that it is a weak NMDA agonist w21x, can modulate the NMDA receptor–ion channel complex activity by reduction of the redox site w12x, chelate Zn ions w4x or block glutamate uptake w5x w22x. It seems that a low cysteine concentration activates NMDA receptors while higher concentrations act on both NMDA and non-NMDA receptors w20x. In earlier work we showed that cysteine potentiates glutamate toxicity in vivo w22x. Cysteine involvement in ischemic brain injury in adult animals is implicated by the marked increase of its concentration in the tissue w13,26x and extracellular space w9x during ischemia in the adult brain. In a neonatal rat model of hypoxia–ischemia ŽHI., we have found some elevation of the extracellular cysteine level during HI and reperfusion w24x, which was accompanied with a minor increase of tissue cysteine concentration w23x. The importance of cysteine with respect to HI brain injury has not been investigated so far.
M. Puka-SundÕall et al.r Brain Research 797 (1998) 328–332
2. Materials and methods Hypoxia–ischemia was performed in immature rats according to the method described previously w6,25x. Rat pups ŽPND 7–10, inbred Wistar, both sexes. were anesthetized with halothane in oxygenrnitrous oxide 1:1 Ž3% for initiation, 0.5–1.5% for maintenance.. The right carotid artery was dissected and cut between double ligatures of prolene sutures. After the surgical procedure the wounds were infiltrated with local anesthetics. After 2 h of recovery the animals were exposed to 7.7% O 2 in N2 for 60 min or 90 min in a humidified chamber at 35–368C. In experiments evaluating cysteine toxicity in HI, rat pups of PND 7 were used Ž38 controls and 40 cysteine treated.. Experimental animals were randomized for controlrcysteine treatment and injected with subtoxic dose of cysteine Ž0.2 mgrg in NaCl, s.c.. 10 min prior or 10 min after the HI Žexposition to hypoxia., and littermates Žinjected with 0.9% NaCl s.c. and subjected to HI. served as controls. After the hypoxic exposure and injection the pups were returned to their dams. The brain damage was evaluated after 14 days by comparing the weight of the cerebral hemispheres and expressed as weight deficit of the hemisphere ipsilateral to the ligation as % of the contralateral. In some experiments, extracellular cysteine was sampled with the microdialysis method. In these experiments rat pups of PND 7–10 were used. After ligation of the carotid artery the rat was immobilized and a dialysis probe ŽCMA 11, 1 mm length, CMA Microdialysis, Stockholm, Sweden. was implanted into the parietal cerebral cortex of the hemisphere ipsilateral to the ligation Ž2 mm posterior to bregma, 3 mm lateral to the sagittal suture, 1.5 mm ventral to the cortical surface.. The microdialysis probe was perfused with dialysis fluid containing 150 mM NaCl, 3 mM KCl and 1.2 mM CaCl 2 at a flow rate of 2 m lrmin using perfusion pump ŽCMA 100, CMA Microdialysis..
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After 1 h of equilibration samples were collected before, during and for 6 h after HI and immediately frozen with b-mercaptoethanol, EDTA and sodium azide Žfinal concentrations in dialysate: 20 mM, 1 mM and 5 mM, respectively.. Animals were sacrificed after 6 h reperfusion. Dialysates were subsequently analyzed for amino acids using liquid chromatography ŽHPLC. as previously described w11x. The effect of 60 min HI Ž n s 11. on changes in extracellular cysteine was evaluated and compared with the effect of HI combined with cysteine administration Ž0.2 mgrg s.c.. 10 min prior Ž n s 5. or 10 min after Ž n s 5. the insult. Extracellular cysteine changes were expressed as percentage of basal level Žbefore HI.. Since mercaptoethanol was used in the procedure any cystine present would be reduced to and measured as cysteine. In separate control experiments, the toxicity of 0.2 mgrg cysteine Žwithout HI. in the brain was evaluated. Rat pups ŽPND 7, inbred Wistar, both sexes. were injected with cysteine Ž0.2 mgrg in NaCl, s.c.. and littermates Žinjected with 0.9% NaCl s.c.. served as controls. The brains were weighted after 14 days Žcontrols: n s 12, cysteine-injected: n s 13.. Some cysteine-treated animals were sacrificed after 24 h Ž n s 5., 48 h Ž n s 3. and 72 h Ž n s 3., brains were dissected, frozen in dry ice-chilled dimethylbutan and stored in y808C until use. Coronal cryostat sections Ž8 m m. were cut at the level of hippocampus and stained with hematoxyline and eosin. Animal experiments were approved by the ethical comŽnos. 29-95 and 116-95.. mittee in Goteborg ¨ The evaluation of cysteine administration on HI-induced brain damage was performed with unpaired Mann– Whitney U test ŽFig. 1.. Wilcoxon signed rank test with Bonferroni correction for multiple comparisons were applied for analysis of the HI effect on e.c. cysteine changes ŽFig. 2.. Kruskal–Wallis test and unpaired Mann–Whitney U test were used to compare e.c. cysteine changes induced
Fig. 1. Brain damage Žexpressed as weight deficit of the ipsilateral hemisphere as a % of the contralateral hemisphere. after HI compared with HI combined with administration of cysteine in a dose of 0.2 mgrg, given s.c. 10 min prior to 60 min HI ŽA., 10 min after 60 min HI ŽB. and 10 min after 90 min HI ŽC.. The values are expressed as mean " S.E.M. Significance of the difference Žcysteineq HI vs. HI only. was tested with unpaired Mann–Whitney U test Ž) p - 0.05, )) p - 0.01..
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Fig. 2. Cysteine concentration in microdialysates during HI and reperfusion expressed as a % of basal level. The values are given as mean" S.E.M. Ž ns11.. Significance of cysteine increase Žcompared to basal level. was tested with Wilcoxon signed rank test with Bonferroni correction for multiple comparisons: Ž) p- 0.05..
by HI alone vs. HI combined with cysteine administration ŽFig. 3.. Values are given as mean " standard error ŽS.E.M...
3. Results Of 78 rat pups exposed to HI, two did not survive hypoxia Žcontrols. and three died during the 2 weeks after the insult Žone control and two cysteine-treated.. Induction of HI for 60 min or 90 min in this setting produced brain damage measured as a marked weight deficit of the hemisphere ipsilateral to the ligation after 14 days. A subtoxic dose of cysteine 0.2 mgrg administered
10 min prior to the 60 min insult increased the hemisphere weight deficit from 22 " 3% to 35 " 2% Ž p - 0.01. ŽFig. 1A.. The effect of post-HI administration of cysteine was not as pronounced and depended on the duration of HI. The effect of administration of 0.2 mgrg cysteine after 60 min of HI had no effect on the neuropathological outcome, whereas administration of 0.2 mgrg cysteine after 90 min of HI slightly increased the hemispheric weight deficit from 36 " 3% to 45 " 3% Ž p - 0.05. ŽFig. 1B and C.. There was no difference in the weight of the contralateral hemisphere between control ŽHI. and cysteine-injected animals in any of the experimental groups ŽTable 1.. In control experiments Žno HI. 0.2 mgrg cysteine did not produce any histopathological changes after 24 h, 48 h and 72 h Žnot shown.. After 14 days, however, a slight Ž2.8%. decrease of brain weight as compared to saline-injected animals was observed Ž1038.5 " 11.5 mg and 1068.3 " 6.6 mg, respectively, p - 0.05—Mann–Whitney U test for unpaired data., but there were no differences in the brain to body weight ratio between cysteine- and saline-injected animals Ž3.38 " 0.08% and 3.41 " 0.06%, respectively.. The basal concentration of cysteine in dialysate was 0.070 " 0.009 m M. HI of 60 min duration produced a transient elevation of e.c. cysteine Ž322 " 41% of basal level.. During reperfusion the extracellular level of cysteine remained slightly elevated during 6 h of reflow, Žmean value during 6 h reperfusion: 163 " 9% of basal level. ŽFig. 2.. Administration of 0.2 mgrg cysteine before 60 min of HI caused an increase in the extracellular concentration of cysteine with a maximum level reached after 60 min of HI Ž865 " 243% of basal level. ŽFig. 3A.. Injection of 0.2
Fig. 3. The effect of cysteine administration on HI-induced changes in the e.c. cysteine concentration. The e.c. cysteine levels after 60 min of HI Žblack bars. compared with HI q 0.2 mgrg cysteine Žhatched bars. administered before ŽA. or after ŽB. HI. The changes of cysteine concentrations in dialysates are expressed as a % of the basal level. Arrows indicate the time points of cysteine administration. The values are expressed as mean" S.E.M. and difference between the groups were tested by comparison of the area under the curve with Kruskal–Wallis test and with unpaired Mann–Whitney U test. Significance of differences: HI vs. HI q cysteine pretreatment: p - 0.01, HI vs. HI q cysteine posttreatment: p - 0.01.
M. Puka-SundÕall et al.r Brain Research 797 (1998) 328–332
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Table 1 The weight of contralateral hemisphere in hypoxic–ischemic rats 14 days after the insult. Data are expressed as mean" S.E.M. The differences between saline- and cysteine-injected animals were non-significant in all the experimental groups, as evaluated by Mann–Whitney U test for unpaired data Contralateral hemisphere weight Žmg.
HI—60 min q pretreatment
HI—60 min q posttreatment
HI—90 min q posttreatment
Saline 0.2 mgrg Cysteine
517.9 " 5.8 502.2 " 8.8 Žn.s..
531.6 " 11.0 527.6 " 14.3 Žn.s..
505.7 " 9.7 504.3 " 6.6 Žn.s..
mgrg cysteine after 60 min of HI caused an increase in extracellular cysteine concentration with maximum after 120 min of reperfusion Ž741 " 202% of basal level, compared to 162 " 12% after HI only. ŽFig. 3B..
4. Discussion The relevance as well as advantages and disadvantages of the used model of perinatal asphyxia have been discussed previously w6,28x. Permanent unilateral ligation of the common carotid artery does not produce any decrease in cerebral blood flow ŽCBF., but ligation combined with systemic hypoxia leads to a pronounced, transient reduction of CBF in the ipsilateral hemisphere w29x. HI produces asymmetrical cerebral growth retardation in ipsilateral hemisphere and does not affect the weight of the contralateral hemisphere w27x or affects it minimally w1x. Therefore we can treat the contralateral hemisphere as a control. In the present experiments extracellular cysteine increased in the cerebral cortex during HI and remained elevated for at least 6 h of reflow ŽFig. 3.. These changes are, however, much smaller than the marked e.c. elevations of cysteine that occurs in adult irreversible cerebral ischemia w9x. HI-induced increase in tissue cysteine concentration observed in neonatal rats w23x is also less pronounced than tissue cysteine elevation in irreversible focal ischemia in adults w13,26x. Nevertheless, cysteine is highly toxic to the immature brain w17–19x and the possibility remained that cysteine toxicity contributed to development of HI brain injury, maybe by modulation of NMDA receptors. This possibility was supported by the fact that a low dose of cysteine Ž0.2 mgrg s.c.., which is not toxic under normal conditions w19x, enhanced HI brain injury considerably if given before the insult and aggravated injury to a limited extent if administered after 90 min of HI. Two different durations of HI Ž60 min and 90 min. were used on purpose to produce different severity of HI brain damage. Cysteine increased injury after 90 min but not 60 min of HI. Increased e.c. cysteine levels during ischemia and reperfusion may contribute to the development of brain damage by enhancing of glutamate toxicity, probably through NMDA receptors. The probable mechanism may be stimulation of the receptor Žas a weak agonist. w21x or receptor sensitization by reducing disulfide bonds w14x. Cysteine may also increase extracellular glutamate concentration by
inhibiting the uptake w5x. Another possibility is that autooxidation of cysteine leads to increased production of oxygen free radicals w15,16x and membrane lipid peroxidation w2x. There are data suggesting that cysteine may also potentiate the effects of nitric oxide w10x. In the study a subtoxic dose of cysteine was used Ž0.2 mgrg.. Previously reported threshold of toxicity in fetal rodents was 0.8 mgrg, and the dose of 0.5 mgrg produced no histopathological changes after 6 and 24 h w19x. In our study 0.2 mgrg cysteine did not affect the weight if the contralateral hemisphere 14 days after HI ŽTable 1. which indicates that the dose used was not toxic. The same dose of cysteine, if applied without HI, produce no histopathological changes in the rat brain up to 72 h, but resulted in slightly decreased brain weight after 14 days compared to saline-injected animals. It may be explained by the whole body growth retardation, since the ratio: brain to body weight did not differ between the saline- and cysteine-injected rats. In order to assess the relevance of these results the e.c. levels of cysteine were measured with microdialysis after cysteine injection qHI and compared with effect of HI alone. These data show that much higher e.c. concentrations of cysteine was found after cysteineq HI than after HI alone ŽFig. 3. which was evident both after pretreatment with cysteine which does aggravate injury and after posttreatment which does not aggravate injury. These results makes it unlikely that the concentrations of cysteine that are present during post-HI reperfusion contribute to the brain lesions. The possibility still remains that lower doses of cysteine administered before the insult exert injurious effect even at levels actually found during HI. However, cysteine and glutamate have the same toxic potency w17x and the latter reaches approximately 5 times higher e.c. concentrations during neonatal HI than cysteine w24x which does not support a critical role for cysteine during HI in neonatal rats. In conclusion, a subtoxic dose of cysteine administered before or after HI aggravates brain injury in immature rats. However, there is no evidence for that the e.c. cysteine levels reached during or after HI contribute to development of brain injury.
Acknowledgements This work was supported by the Swedish Society for Medical Research, The Royal Society of Arts and Sciences
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in Gothenburg, Linnea and Josef Carlsson foundation, Swedish Natural Sciences Research Council, Swedish ˚ Medical Research Council, Sven Jerring foundation, Ake ˚ ´ foundation, Magnus Bergwall Wiberg foundation, Ahlen foundation, Frimurare Barnhus foundation, The Goteborg ¨ Medical Society, Medical Faculty of Goteborg, First May ¨ Flower Annual Campaign.The authors would like to thank Mrs. Anita Palm for her expert help with HPLC analysis.
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