Neuroprotective effects of flunarizine and lamotrigine on hypoxic-ischemic brain damage in fetal rats
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《中华医药杂志》英文版
Neuroprotective effects of flunarizine and lamotrigine on hypoxic-ischemic brain damage in fetal rats(pdf)
Department of Pediatric, Affiliated Fourth People's Hospital,Guangdong Medical College, Shenzhen,Guangdong Province 518033，China
Correspondence to HE Li,Department of Pediatric, Affiliated Fourth People's Hospital,Guangdong Medical College, Shenzhen,Guangdong Province 518033，China
E-mail:fhlily 2002@ yahoo.com.cn
[Abstract] Objective To investigate the neuroprotective effects of flunarizine(FNZ),lamotrigine(LTG) and the combination of two drugs on hypoxic-ischemic brain damage in fetal rats.Methods 40 cases of pregnant Wistar rats(pregnancy for 20 days) were randomly divided into FNZ group, LTG group, FNZ+ LTG group and control group. Drugs were administered through feeding tube 3 days before the hypoxic-ischemic operation once a day. The dosages to the first three groups respectively were: FNZ 0.5 mg/kg·d，LTG 10 mg/kg·d，FNZ 0.5 mg/kg·d + LTG 10 mg/kg·d. Three hours after the third dose was administered,8 cases of pregnant rats of each group were operated for fetal rats hypoxic-ischemic brain damage model,2 cases for control.5 fetal rats were taken out of each pregnant rat.0.3 ml serum were taken from fetal rats 6,12,24,48 h respectively after the birth. The concentration of neuro specific enolase(NSE),and S-100 protein(S-100) in serum was detected by enzyme immunoassay.The concentration of brain-specific creatine kinase (CK-BB) in serum was detected by electrochemistry radiation method.Results The concentrations of serum NSE,S-100 protein and CK-BB of hypoxic-ischemic fetal rats were markedly higher than that of no ischemic group. The concentrations of serum NSE,S-100 and CK-BB protein in FNZ group, LTG group, FNZ+ LTG group were distinctively lower than that of control group, there were significant difference between drug groups and control group,and between combined drugs group with single drug group(P<0.01).Conclusions Preventive antenatal oral FNZ and LTG has positive neuroprotective effects on intrauterine hypoxic-ischemic brain damage. The effects of the combination of two drugs are much better.
[Key words] flunarizine;lamotrigine;hypoxic-ischemic brain damage;neuro specific enolase; S-100 protein;brain-specific creatine kinase
INTRODUCTION
The diphenylpiperazine flunarizine(FNZ) is classified as a type IV voltage-dependent calcium channel antagonist. It has been widely used in adult ischemic cerebrovascular diseases.Small doses of FNZ was effective on hypoxic-ischemic brain damage in newborn animals[1].Lamotrigine(LTG) is a powerful antiepileptic drug. Study shows that LTG and its ramifications possesses the neuroprotective effects[2].
At present,seldom report are about the application of FNZ and LTG in intrauterine hypoxic-ischemic brain damage.In this study, we treated late pregnant rats with FNZ,LTG, observed the variation of serum neuro specific enolase(NSE), S-100 protein(S-100),brain-specific creatine kinase(CK-BB) after the hypoxic-ischemic brain damage operation.The aim of this study is to investigate the neuroprotective effects of preventive antenatal taking medicine and new methods in preventing neonatal hypoxic-ischemic encephalopathy(HIE).
MATERIALS AND METHODS
Materials
40 cases of pregnant Wistar rats(pregnancy for 20 days,offered by Experimental Animal Center in Guangzhou Medical College) were randomly divided into FNZ group, LTG group, FNZ+ LTG group and control group. Drugs were administered through feeding tube 3 days before the hypoxic-ischemic operation once a day. The dosages to the first three groups respectively were: FNZ 0.5 mg/kg·d(capsula,5 mg/cap, XIAN-Janssen pharmaceutical Ltd.)，LTG 10 mg/kg·d(tablet,25 mg/tablet,GlaxoWellcome Inc.)，FNZ 0.5 mg/kg·d+LTG 10 mg/kg·d.
Preparation for Animal Model[3,4]
Three hours after the third dose,8 cases of pregnant rats from each group were operated for fetal rats hypoxic-ischemic brain damage model.2 cases for control.Under inhaled aerosol anaesthesia, abdominal midline incision were made, unilateral uterine artery was ligated.10 minutes later,uterus incision was made.5 fetal rats were taken out from each pregnant rat and were kept warm.
Measurement of Serum NSE,S-100,CK-BB
10 newborn rats were taken 6,12,24,48 h after birth respectively.At each time,plasma were collected by cutting rats' heads and centrifuged at 3000 rpm for 3 min. 0.3 ml serum samples were collected and stored at -30 ℃ for analysis. The concentration of neuro specific enolase(NSE ),S-100 protein(S-100) in serum was detected by enzyme immunoassay.The concentration of brain-specific creatine kinase (CK-BB) in serum was detected by electrochemistry radiation method. All procedures were performed according to the manufacturer's instructions.
Statistical Analysis
The data were expressed as x±s. The SPSS 11.0 for windows was used. One-way ANOVA was used to evaluate the differences between the groups. Correlation analysis was also made. The P value < 0.01 was considered statistically significant.
RESULTS
The concentrations of serum NSE,S-100 and CK-BB of hypoxic-ischemic fetal rats were markedly higher than that of no ischemic group. The concentrations of serum NSE,S-100 and CK-BB in FNZ group, LTG group, FNZ+ LTG group were distinctively lower than that of control group, there were significant difference between drug groups and control group and between combined drugs group with single drug group(P<0.01)(Table 1,2,3).
Table 1 Fetal Rat Serum NSE at Different Times after Ischemia (μg/ml，x±s，n＝10)
Compared with control group *P<0.01,compared with FNZ+LTG △P<0.01
Table 2 Fetal Rat Serum S-100 at Different Times after Ischemia (μg/ml，x±s，n＝10)
Compared with control group *P<0.01,compared with FNZ+LTG △P<0.01
Table 3 Fetal Rat Serum CK-BB at Different Times after Ischemia (μg/ml，x±s，n＝10)
Compared with control group *P<0.01,compared with FNZ+LTG △P<0.01
DISCUSSION
Several biochemical markers in cerebrospinal fluid have been associated with cerebral complications in infants after perinatal asphyxia.Recent investigations suggest that serum NSE,S-100 and CK-BB are capable of identifying patients with a risk of developing neonatal hypoxic-ischemic encephalopathy(HIE)[5].
NSE is the neuronal form of the intracytoplasmic glycolytic enolase. It is found in neuronal cell bodies, axons, and neuroendocrine cells.S-100 is a dimeric acidic calcium-binding protein constituting a major component of the cytosol of various cell types which predominantly present in astrocytes and Schwann cells[6]. CK-BB is found in both neurons and astrocytes.
NSE,S-100,CK-BB in serum or cerebrospinal fluid varies constantly after brain damage.They may be released during hypoxia or asphyxia.Measurement of their concentration may aid the early clinical diagnosis of hypoxic ischemic stress and susceptibility to brain damage during the perinatal period. Also,it is beneficial to the evaluation of curative effect and prognosis.
Birth asphyxia remains a considerable problem in perinatal medicine with an incidence around 0.6%~0.8% of births[7].In addition to pulmonary, renal, and cardiac dysfunction, HIE develops in one third of asphyxiated newborns.The early prediction of outcome is necessary to identify infants with a higher risk for brain damage for neuroprotective interventions aiming to limit the extent of brain injury[8].
The complicated pathogenesis of hypoxic-ischemic brain injury in the fetus and neonate has been understood considerably over the last two decades related to both clinical and laboratory observations[9,10].Though it is contributed some factors,a fundamental process believed to be responsible for hypoxic-ischemic damage to neurons is called excitotoxicity which refers to cell death mediated by excessive stimulation of extracellular excitatory amino acid receptors[11].
Normally these receptors mediate physiological excitatory effects of the dicarboxylic acid glutamate,one of the most ubiquitous and versatile neurotransmitters in the brain. When excessively stimulated by combinations of elevated synaptic levels of glutamate and membrane depolarization associated with ischemia,channels associated with these receptors allow a lethal flood of Ca2+ and sodium to enter neurons.Direct effects of Ca2+ flooding and Ca2+-mediated generation of nitric oxide and peroxynitrite contribute to damage depending on the severity of the insult and other factors such as tissue redox state.Activation of lipases and proteases,including those involved in proinflammatory cytokine cascades,also contribute to excitotoxic injury triggered by hypoxia-ischemia[12].
Excitotoxicity appears to be even more intimately involved in the pathogenesis of cell destruction from hypoxic ischemia in the developing brain than in the adult. Mitochondria handle multiple oxidation reactions that can yield highly toxic oxygen free radicals under conditions of oxidative stress.They are major buffers of intracellular Ca2+ and can become overloaded by cytoplasmic Ca2+ flooding secondary to opening of voltage-sensitive Ca2+ channels.
Voltage-sensitive calcium and sodium channels are important controllers of excitatory neurotransmitter released in neurons.Recent results suggest that selective inhibitors of these channels can diminish extracellular glutamate concentration during cerebral ischemia and potentially modulate an important source of excitotoxic cell death[13~15].
Animal experiments have indicated that calcium antagonists administered after cerebral ischemia are effective in reducing infarct volume and lead to improvements in neurological outcome. Calcium antagonists may act as neuroprotective drugs by diminishing the influx of calcium ions through voltage-sensitive calcium channels[16].They showed a neuroprotective effect against hypoxic-ischemic injury in the newborn animals when given prophylactically[17~19]. They have been used in treating adult cerebrovascular diseases for years.
FNZ is classified as a type IV voltage-dependent calcium channel antagonist and a vasodilator.It is regarded as a wide-spectrum Ca2+ channel antagonist.It is reported to be a selective blocker of pathological Ca2+ influx with no or only low affinity for physiological slow Ca2+ channels[20]. FNZ also exhibits considerable affinity for Na+ channels[21].
FNZ is selectively concentrated in brain.This may be because FNZ 's difluoropiperazine structure allows it to penetrate the blood brain barrier easily and reach a relatively high level in the brain compared with serum over a prolonged period[22]. FNZ is reported to produce only slight decreases of systemic arterial blood pressure.
The calcium antagonistic effect of FNZ is related to environmental tissue pH.When pH is 5,this effect presents dose-dependent. During the brain ischemic injury，anaerobic glycolysis increases and acidity substances accumulate in the tissue, so FNZ can exert its supreme effects.
FNZ also has other pharmacologic properties,e.g.free radical scavenging[23], a potent inhibitor of adenosine uptake which may enhance reactive hyperemia in the cerebral vasculature during the post-hypoxic recovery period, reduce the susceptibility of neurons to excitotoxic transmitters,etc.These factors all contribute to neuroprotection.
LTG is a novel anticonvulsant with efficacy in patients with partial seizures or generalized tonic clonic seizures.
The protective effect of LTG was not based solely on its ability to prevent glutamate release.LTG shows voltage-dependent inhibition of Na+ currents, stabilizes the presynaptic neuronal membrane,thus preventing the release of excitatory neurotransmitters,and inhibits sustained repetitive neuronal damage.
It is the blockade of voltage-sensitive sodium channels by LTG inhibits postischemic sodium influx, thereby facilitating membrane repolarization，ameliorating intracellular edema formation,preventing reversal of the sodium/glutamate and sodium/hydrogen transporter, reducing cerebral synaptic activity,thus helping cells maintain more stable and more negative membrane potentials. Also LTG may act as a free radical scavenger because of its unsaturated ring structure.
In addition,LTG also inhibits calcium currents in corticostriatal and hippocampal neurons, which suggests its action at high voltage release-coupled calcium channels[24~26]. LTG also decreases cerebral metabolic rate, which suggests its another mechanism of protection for neurons[27].
Our study found that the hypoxic-ischemic insult in late pregnant rats could lead to fetal rats brain damage and the concentration of NSE,S-100 and CK-BB in newborn rats serum increased.Preventive administered with FNZ,LTG before hypoxic-ischemic operation,the concentrations of serum NSE,S-100 and CK-BB at different time point in FNZ group, LTG group, FNZ+ LTG group were distinctively lower than the control group.From the results,we can see that the combination of FNZ and LTG can reduce Ca2+ influx markedly,enhance the effect of single drug and strengthen the neuroprotective effect.The best time for sampling is 24~48 h after the hypoxic-ischemic operation.
In previous study, ways to treat rats with drugs are through veins or abdominal cavity.In our study,we do this through the rats' mouth.The results are more close to the clinic.The aim of our study is to investigate the neuroprotective effects of FNZ and LTG in fetal rats with hypoxic-ischemic damage.Thus provide some new clues to HIE.
Funding:Shenzhen Bureau of Science and Technology,Project No.200405204.
REFERENCES
1. Berger R, Lehmann T, KAarcher J,et al.Low dose flunarizine protects the fetal brain from ischemic injury in sheep.Pediar Res, 1998, 44(3):272-282.
2. Halonen T, et al. Effect of lamotrigine treatment on status epilepticus induced neuronal damage and memory impairment in rat.Epilepsy Res, 2001,46(3): 205-23.
3. Binienda Z.A fetal rat model of acute perinatal ischemia-hypoxia. Ann N Y Acad Sci,1995,72:28-38.
4. Bjelke B, Anderson k, Ogren SO. Asphycitic lesions proliferation of tyrosine hydroxylase-immunoreactive nerve cell bodies in the rat substantia nigra and functional changes in dopamine neurotransmission.Brain Res,1991,543:1-9.
5. Kristna TJ, Christer A, Anareas H,et al. S100 Protein in serum as a prognostic marker for cerebral injury in term newborn infants with hypoxic ischemic encephalopathy.Pediar Res, 2004,55:406-412.
6. Nicole N, Wolfgang K, Hae- KK, et al. Early Biochemical Indicators of Hypoxic-Ischemic Encephalopathy after Birth Asphyxia.Pediar Res,2001,49:502-506.
7. Kristina T-J, Christer A, Andreas H,et al.S100 Protein in Serum as a Prognostic Marker for Cerebral Injury in Term Newborn Infants with Hypoxic Ischemic Encephalopathy. Pediar Res,2004,55:406-412.
8. Levene MI, Evans DJ, Mason S,et al. An international network for evaluating neuroprotective therapy after severe birth asphyxia. Semin Perinatol, 1999,23(3):226-233.
9.Vannucci RC.Experimental biology of cerebral hypoxia-ischemia: relation to perinatal brain damage. Pediatr Res,1990,27:317-326.
10. Johnston MV,Trescher WH,Ishida A,et al.Novel treatments after experimental brain injury.Semin Neonatol, 2000,5:75-86.
11. Choi DW, Rothman SM. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death.Ann Rev Neurosci, 1990,13: 171-182.
12. Johnston MV, Trescher WH, Ishida A,et al. Neurobiology of Hypoxic-Ischemic Injury in the Developing Brain.Pediar Res, 2001,49:735-741.
13. Shuaib A,Mahmood RH,Wishart T,Kanthan R,et al.Neuroprotective effects of lamotrigine in global ischemia in gerbils: a histological, in vivo microdialysis and behavioral study.Brain Res, 1995,702:199-206.
14. Bacher A, Zornow MH. Lamotrigine inhibits extracellular glutamate accumulation during transient global cerebral ischemia in rabbits. Anesthesiology, 1997,86:459-463.
15. Conroy BP, Black D, Lin CY, et al.Lamotrigine attenuates cortical glutamate release during global cerebral ischemia in pigs on cardiopulmonary bypass. Anesthesiology, 1999,90:844-854.
16. Horn J,Limburg M. Calcium antagonists for ischemic stroke: a systematic review.Stroke, 2001,32:570-576.
17. Gunn AJ, Mydlar T,Bennet L,et al.The neuroprotective actions of a calcium channel antagonist, flunarizine, in the infants rat. Pediar Res, 1989,25(6):573-576.
18. Chunas PD,Delbigio MR,Drake JM, et al.A comparison of the protective of cerebral hypoxia-ischemia.Neurosurg, 1993,79(3):414-420.
19. Gunn AJ,Williams CE,Mallard EC, et al. Flunarizine, a calcium channel antagonist, is partially prophylactically neuroprotective in hypoxic-ischemic encephalopathy in the fetal sheep.Pediar Res, 1994,35(6):657-663.
20. Sukriti Nag. Protective effect of flunarizine on blood-brain barrier permeability alterations in acutely hypertensive rats.Stroke, 1991,22:1265-1269.
21. Osborne NN, Wood JPM, Cupido A, et al.Topical flunarizine reduces IOP and protects the retina against ischemia-excitotoxicity. Invest Ophthalmol Vis Sci, 2002,43:1456-1464.
22. Silverstein FS, Buchanan K, Hudson C,et al.Flunarizine limits hypoxia-ischemia induced morphologic injury in immature rat brain.Stroke, 1986,17:477-482.
23. Phillis JW, Delong RE, Towner JK.The effects of lidoflazine and flunarizine on cerebral reactive hyperemia.Eur J of Pharm, 1985,112:323-329.
24. Grunze H,Wegerer JV,Greene R,et al.Modulation of calcium and potassium currents by lamotri gine. Neuropsychobiol,1998,38:131-138.
25. Xie X,Hagan RM. Cellular and molecular actions of lamotrigine: possible mechanisms of efficacy in bipolar disorder. Neuropsychobiol, 1998,38:119-130.
26. Stefani A,Spadoni F,Sinicalchi A,et al.Lamotrigine inhibits Ca currents in cortical neurons:functional implications.Eur J Pharmacol, 1996,307:113-116.
27. Richard J,Traystman, Judith A, et al. Anticonvulsant Lamotrigine Administered on Reperfusion Fails To Improve Experimental Stroke Outcomes.Stroke, 2001,32:783.
(Editor Jaque)(HE Li, LU Chang-dong, DEN)
Department of Pediatric, Affiliated Fourth People's Hospital,Guangdong Medical College, Shenzhen,Guangdong Province 518033，China
Correspondence to HE Li,Department of Pediatric, Affiliated Fourth People's Hospital,Guangdong Medical College, Shenzhen,Guangdong Province 518033，China
E-mail:fhlily 2002@ yahoo.com.cn
[Abstract] Objective To investigate the neuroprotective effects of flunarizine(FNZ),lamotrigine(LTG) and the combination of two drugs on hypoxic-ischemic brain damage in fetal rats.Methods 40 cases of pregnant Wistar rats(pregnancy for 20 days) were randomly divided into FNZ group, LTG group, FNZ+ LTG group and control group. Drugs were administered through feeding tube 3 days before the hypoxic-ischemic operation once a day. The dosages to the first three groups respectively were: FNZ 0.5 mg/kg·d，LTG 10 mg/kg·d，FNZ 0.5 mg/kg·d + LTG 10 mg/kg·d. Three hours after the third dose was administered,8 cases of pregnant rats of each group were operated for fetal rats hypoxic-ischemic brain damage model,2 cases for control.5 fetal rats were taken out of each pregnant rat.0.3 ml serum were taken from fetal rats 6,12,24,48 h respectively after the birth. The concentration of neuro specific enolase(NSE),and S-100 protein(S-100) in serum was detected by enzyme immunoassay.The concentration of brain-specific creatine kinase (CK-BB) in serum was detected by electrochemistry radiation method.Results The concentrations of serum NSE,S-100 protein and CK-BB of hypoxic-ischemic fetal rats were markedly higher than that of no ischemic group. The concentrations of serum NSE,S-100 and CK-BB protein in FNZ group, LTG group, FNZ+ LTG group were distinctively lower than that of control group, there were significant difference between drug groups and control group,and between combined drugs group with single drug group(P<0.01).Conclusions Preventive antenatal oral FNZ and LTG has positive neuroprotective effects on intrauterine hypoxic-ischemic brain damage. The effects of the combination of two drugs are much better.
[Key words] flunarizine;lamotrigine;hypoxic-ischemic brain damage;neuro specific enolase; S-100 protein;brain-specific creatine kinase
INTRODUCTION
The diphenylpiperazine flunarizine(FNZ) is classified as a type IV voltage-dependent calcium channel antagonist. It has been widely used in adult ischemic cerebrovascular diseases.Small doses of FNZ was effective on hypoxic-ischemic brain damage in newborn animals[1].Lamotrigine(LTG) is a powerful antiepileptic drug. Study shows that LTG and its ramifications possesses the neuroprotective effects[2].
At present,seldom report are about the application of FNZ and LTG in intrauterine hypoxic-ischemic brain damage.In this study, we treated late pregnant rats with FNZ,LTG, observed the variation of serum neuro specific enolase(NSE), S-100 protein(S-100),brain-specific creatine kinase(CK-BB) after the hypoxic-ischemic brain damage operation.The aim of this study is to investigate the neuroprotective effects of preventive antenatal taking medicine and new methods in preventing neonatal hypoxic-ischemic encephalopathy(HIE).
MATERIALS AND METHODS
Materials
40 cases of pregnant Wistar rats(pregnancy for 20 days,offered by Experimental Animal Center in Guangzhou Medical College) were randomly divided into FNZ group, LTG group, FNZ+ LTG group and control group. Drugs were administered through feeding tube 3 days before the hypoxic-ischemic operation once a day. The dosages to the first three groups respectively were: FNZ 0.5 mg/kg·d(capsula,5 mg/cap, XIAN-Janssen pharmaceutical Ltd.)，LTG 10 mg/kg·d(tablet,25 mg/tablet,GlaxoWellcome Inc.)，FNZ 0.5 mg/kg·d+LTG 10 mg/kg·d.
Preparation for Animal Model[3,4]
Three hours after the third dose,8 cases of pregnant rats from each group were operated for fetal rats hypoxic-ischemic brain damage model.2 cases for control.Under inhaled aerosol anaesthesia, abdominal midline incision were made, unilateral uterine artery was ligated.10 minutes later,uterus incision was made.5 fetal rats were taken out from each pregnant rat and were kept warm.
Measurement of Serum NSE,S-100,CK-BB
10 newborn rats were taken 6,12,24,48 h after birth respectively.At each time,plasma were collected by cutting rats' heads and centrifuged at 3000 rpm for 3 min. 0.3 ml serum samples were collected and stored at -30 ℃ for analysis. The concentration of neuro specific enolase(NSE ),S-100 protein(S-100) in serum was detected by enzyme immunoassay.The concentration of brain-specific creatine kinase (CK-BB) in serum was detected by electrochemistry radiation method. All procedures were performed according to the manufacturer's instructions.
Statistical Analysis
The data were expressed as x±s. The SPSS 11.0 for windows was used. One-way ANOVA was used to evaluate the differences between the groups. Correlation analysis was also made. The P value < 0.01 was considered statistically significant.
RESULTS
The concentrations of serum NSE,S-100 and CK-BB of hypoxic-ischemic fetal rats were markedly higher than that of no ischemic group. The concentrations of serum NSE,S-100 and CK-BB in FNZ group, LTG group, FNZ+ LTG group were distinctively lower than that of control group, there were significant difference between drug groups and control group and between combined drugs group with single drug group(P<0.01)(Table 1,2,3).
Table 1 Fetal Rat Serum NSE at Different Times after Ischemia (μg/ml，x±s，n＝10)
Compared with control group *P<0.01,compared with FNZ+LTG △P<0.01
Table 2 Fetal Rat Serum S-100 at Different Times after Ischemia (μg/ml，x±s，n＝10)
Compared with control group *P<0.01,compared with FNZ+LTG △P<0.01
Table 3 Fetal Rat Serum CK-BB at Different Times after Ischemia (μg/ml，x±s，n＝10)
Compared with control group *P<0.01,compared with FNZ+LTG △P<0.01
DISCUSSION
Several biochemical markers in cerebrospinal fluid have been associated with cerebral complications in infants after perinatal asphyxia.Recent investigations suggest that serum NSE,S-100 and CK-BB are capable of identifying patients with a risk of developing neonatal hypoxic-ischemic encephalopathy(HIE)[5].
NSE is the neuronal form of the intracytoplasmic glycolytic enolase. It is found in neuronal cell bodies, axons, and neuroendocrine cells.S-100 is a dimeric acidic calcium-binding protein constituting a major component of the cytosol of various cell types which predominantly present in astrocytes and Schwann cells[6]. CK-BB is found in both neurons and astrocytes.
NSE,S-100,CK-BB in serum or cerebrospinal fluid varies constantly after brain damage.They may be released during hypoxia or asphyxia.Measurement of their concentration may aid the early clinical diagnosis of hypoxic ischemic stress and susceptibility to brain damage during the perinatal period. Also,it is beneficial to the evaluation of curative effect and prognosis.
Birth asphyxia remains a considerable problem in perinatal medicine with an incidence around 0.6%~0.8% of births[7].In addition to pulmonary, renal, and cardiac dysfunction, HIE develops in one third of asphyxiated newborns.The early prediction of outcome is necessary to identify infants with a higher risk for brain damage for neuroprotective interventions aiming to limit the extent of brain injury[8].
The complicated pathogenesis of hypoxic-ischemic brain injury in the fetus and neonate has been understood considerably over the last two decades related to both clinical and laboratory observations[9,10].Though it is contributed some factors,a fundamental process believed to be responsible for hypoxic-ischemic damage to neurons is called excitotoxicity which refers to cell death mediated by excessive stimulation of extracellular excitatory amino acid receptors[11].
Normally these receptors mediate physiological excitatory effects of the dicarboxylic acid glutamate,one of the most ubiquitous and versatile neurotransmitters in the brain. When excessively stimulated by combinations of elevated synaptic levels of glutamate and membrane depolarization associated with ischemia,channels associated with these receptors allow a lethal flood of Ca2+ and sodium to enter neurons.Direct effects of Ca2+ flooding and Ca2+-mediated generation of nitric oxide and peroxynitrite contribute to damage depending on the severity of the insult and other factors such as tissue redox state.Activation of lipases and proteases,including those involved in proinflammatory cytokine cascades,also contribute to excitotoxic injury triggered by hypoxia-ischemia[12].
Excitotoxicity appears to be even more intimately involved in the pathogenesis of cell destruction from hypoxic ischemia in the developing brain than in the adult. Mitochondria handle multiple oxidation reactions that can yield highly toxic oxygen free radicals under conditions of oxidative stress.They are major buffers of intracellular Ca2+ and can become overloaded by cytoplasmic Ca2+ flooding secondary to opening of voltage-sensitive Ca2+ channels.
Voltage-sensitive calcium and sodium channels are important controllers of excitatory neurotransmitter released in neurons.Recent results suggest that selective inhibitors of these channels can diminish extracellular glutamate concentration during cerebral ischemia and potentially modulate an important source of excitotoxic cell death[13~15].
Animal experiments have indicated that calcium antagonists administered after cerebral ischemia are effective in reducing infarct volume and lead to improvements in neurological outcome. Calcium antagonists may act as neuroprotective drugs by diminishing the influx of calcium ions through voltage-sensitive calcium channels[16].They showed a neuroprotective effect against hypoxic-ischemic injury in the newborn animals when given prophylactically[17~19]. They have been used in treating adult cerebrovascular diseases for years.
FNZ is classified as a type IV voltage-dependent calcium channel antagonist and a vasodilator.It is regarded as a wide-spectrum Ca2+ channel antagonist.It is reported to be a selective blocker of pathological Ca2+ influx with no or only low affinity for physiological slow Ca2+ channels[20]. FNZ also exhibits considerable affinity for Na+ channels[21].
FNZ is selectively concentrated in brain.This may be because FNZ 's difluoropiperazine structure allows it to penetrate the blood brain barrier easily and reach a relatively high level in the brain compared with serum over a prolonged period[22]. FNZ is reported to produce only slight decreases of systemic arterial blood pressure.
The calcium antagonistic effect of FNZ is related to environmental tissue pH.When pH is 5,this effect presents dose-dependent. During the brain ischemic injury，anaerobic glycolysis increases and acidity substances accumulate in the tissue, so FNZ can exert its supreme effects.
FNZ also has other pharmacologic properties,e.g.free radical scavenging[23], a potent inhibitor of adenosine uptake which may enhance reactive hyperemia in the cerebral vasculature during the post-hypoxic recovery period, reduce the susceptibility of neurons to excitotoxic transmitters,etc.These factors all contribute to neuroprotection.
LTG is a novel anticonvulsant with efficacy in patients with partial seizures or generalized tonic clonic seizures.
The protective effect of LTG was not based solely on its ability to prevent glutamate release.LTG shows voltage-dependent inhibition of Na+ currents, stabilizes the presynaptic neuronal membrane,thus preventing the release of excitatory neurotransmitters,and inhibits sustained repetitive neuronal damage.
It is the blockade of voltage-sensitive sodium channels by LTG inhibits postischemic sodium influx, thereby facilitating membrane repolarization，ameliorating intracellular edema formation,preventing reversal of the sodium/glutamate and sodium/hydrogen transporter, reducing cerebral synaptic activity,thus helping cells maintain more stable and more negative membrane potentials. Also LTG may act as a free radical scavenger because of its unsaturated ring structure.
In addition,LTG also inhibits calcium currents in corticostriatal and hippocampal neurons, which suggests its action at high voltage release-coupled calcium channels[24~26]. LTG also decreases cerebral metabolic rate, which suggests its another mechanism of protection for neurons[27].
Our study found that the hypoxic-ischemic insult in late pregnant rats could lead to fetal rats brain damage and the concentration of NSE,S-100 and CK-BB in newborn rats serum increased.Preventive administered with FNZ,LTG before hypoxic-ischemic operation,the concentrations of serum NSE,S-100 and CK-BB at different time point in FNZ group, LTG group, FNZ+ LTG group were distinctively lower than the control group.From the results,we can see that the combination of FNZ and LTG can reduce Ca2+ influx markedly,enhance the effect of single drug and strengthen the neuroprotective effect.The best time for sampling is 24~48 h after the hypoxic-ischemic operation.
In previous study, ways to treat rats with drugs are through veins or abdominal cavity.In our study,we do this through the rats' mouth.The results are more close to the clinic.The aim of our study is to investigate the neuroprotective effects of FNZ and LTG in fetal rats with hypoxic-ischemic damage.Thus provide some new clues to HIE.
Funding:Shenzhen Bureau of Science and Technology,Project No.200405204.
REFERENCES
1. Berger R, Lehmann T, KAarcher J,et al.Low dose flunarizine protects the fetal brain from ischemic injury in sheep.Pediar Res, 1998, 44(3):272-282.
2. Halonen T, et al. Effect of lamotrigine treatment on status epilepticus induced neuronal damage and memory impairment in rat.Epilepsy Res, 2001,46(3): 205-23.
3. Binienda Z.A fetal rat model of acute perinatal ischemia-hypoxia. Ann N Y Acad Sci,1995,72:28-38.
4. Bjelke B, Anderson k, Ogren SO. Asphycitic lesions proliferation of tyrosine hydroxylase-immunoreactive nerve cell bodies in the rat substantia nigra and functional changes in dopamine neurotransmission.Brain Res,1991,543:1-9.
5. Kristna TJ, Christer A, Anareas H,et al. S100 Protein in serum as a prognostic marker for cerebral injury in term newborn infants with hypoxic ischemic encephalopathy.Pediar Res, 2004,55:406-412.
6. Nicole N, Wolfgang K, Hae- KK, et al. Early Biochemical Indicators of Hypoxic-Ischemic Encephalopathy after Birth Asphyxia.Pediar Res,2001,49:502-506.
7. Kristina T-J, Christer A, Andreas H,et al.S100 Protein in Serum as a Prognostic Marker for Cerebral Injury in Term Newborn Infants with Hypoxic Ischemic Encephalopathy. Pediar Res,2004,55:406-412.
8. Levene MI, Evans DJ, Mason S,et al. An international network for evaluating neuroprotective therapy after severe birth asphyxia. Semin Perinatol, 1999,23(3):226-233.
9.Vannucci RC.Experimental biology of cerebral hypoxia-ischemia: relation to perinatal brain damage. Pediatr Res,1990,27:317-326.
10. Johnston MV,Trescher WH,Ishida A,et al.Novel treatments after experimental brain injury.Semin Neonatol, 2000,5:75-86.
11. Choi DW, Rothman SM. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death.Ann Rev Neurosci, 1990,13: 171-182.
12. Johnston MV, Trescher WH, Ishida A,et al. Neurobiology of Hypoxic-Ischemic Injury in the Developing Brain.Pediar Res, 2001,49:735-741.
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(Editor Jaque)(HE Li, LU Chang-dong, DEN)