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Cerebrospinal fluid and serum vascular endothelial growth factor and nitric oxide levels in newborns with hypoxic ischemic encephalopathy
Brain & Development 26 (2004) 283–286 www.elsevier.com/locate/braindev
Original article
Cerebrospinal fluid and serum vascular endothelial growth factor and nitric oxide levels in newborns with hypoxic ischemic encephalopathy Ebru Ergenekona,*, Kıvılcım Gu¨cu¨yenerb, Deniz Erbas¸c, Sema Arald, Esin Koc¸a, Yıldız Atalaya a
b
Department of Newborn, Gazi University, School of Medicine, Besevler, Ankara, Turkey Department of Pediatric Neurology, Gazi University, School of Medicine, Besevler, Ankara, Turkey c Department of Physiology, Gazi University, School of Medicine, Besevler, Ankara, Turkey d Research Department, Duzen Laboratory, Ankara, Turkey Received 29 August 2002; received in revised form 1 August 2003; accepted 4 August 2003
Abstract Excitatory amino acids, cytokines and nitric oxide (NO) have been studied in the etiology and pathogenesis of hypoxic ischemic encephalopathy (HIE) of the newborn. Vascular endothelial growth factor (VEGF) is a known mediator of angiogenesis and has been shown to induce vascular proliferation and permeability via NO-mediated mechanism during hypoxia. The objective of this study was to investigate the cerebrospinal fluid and serum VEGF and NO levels in different stages of HIE and the correlation between the two mediators. Cerebrospinal fluid (CSF) and serum samples of 19 newborns with HIE and 13 controls were obtained within the first 24 h of life and kept at 2 70 8C until the time of measurement. NO levels were determined by Sievers NOA by chemiluminescence method and VEGF levels were measured by the enzyme-linked immunosorbent assay double sandwich method. The NO levels in CSF were higher than the control and mild HIE group in newborns with moderate to severe HIE, and VEGF levels in CSF were higher in the mild HIE group compared to controls but similar in the moderate to severe HIE group compared to mild HIE and control patients. There was no difference between groups with regard to serum NO or VEGF levels, and no correlation was observed between NO and VEGF levels both in CSF and serum samples. Depending on the severity of the hypoxic insult the stimulus for NO production by VEGF may have variable effects on endothelial cells which may give rise to the current results. q 2003 Elsevier B.V. All rights reserved. Keywords: Hypoxia; Nitric oxide; Vascular endothelial growth factor; Newborn
1. Introduction Hypoxic ischemic encephalopathy (HIE) of the newborn has been studied extensively in various animal models and in humans, to investigate the pathogenesis and treatment options. Most of the animal research has pointed the role of excitatory amino acids, cytokines and nitric oxide (NO) determining the extent of the pathology [1 –4]. In a previous study on asphyxiated newborns we showed increased levels of cerebrospinal fluid (CSF) NO levels with increasing severity of HIE during the first 24 – 72 h of the hypoxic * Corresponding author. Yesilyurt Sokak No: 19/9, Cankaya 06690, Ankara, Turkey. Fax: þ 90-312-215-0143. E-mail address: [email protected] (E. Ergenekon). 0387-7604/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0387-7604(03)00166-9
insult [5]. Concerning vascular endothelial growth factor (VEGF) there is a wide spectrum of research on endothelial cells, tumors, angiogenesis and hypoxia [6 –11]. VEGF has been shown to induce vascular permeability and endothelial cell proliferation via NO by stimulating endothelial NO synthase (eNOS), and hypoxia has been shown to promote VEGF synthesis [12]. These results have raised questions about the role of VEGF in HIE and the increase of NO in newborns with HIE. The objective of this study was to measure VEGF and NO levels in CSF and serum in newborns with different stages of HIE during the first 24 h of the hypoxic ischemic insult, firstly to investigate whether NO is increased during the first 24 h or not, and secondly to investigate whether VEGF is also increased during the same time period.
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E. Ergenekon et al. / Brain & Development 26 (2004) 283–286
2. Patients and methods
2.3. Statistics
Term newborns with gestational age $ 37 weeks were included in the study group based on the following criteria for HIE:
VEGF and NO levels in CSF and serum of controls and newborns with different stages of HIE were compared by using the Mann – Whitney U-test and P , 0:05 was considered significant. Results are expressed as mean ^ SD unless stated otherwise. The correlation between NO and VEGF levels in CSF and serum were determined by Pearson correlation test and P , 0:05 was considered significant.
Antenatal evidence of fetal distress Umbilical artery pH , 7 Apgar score at 5th minute , 7 Postnatal resuscitation with positive pressure ventilation The staging for HIE was made based on Sarnat and Sarnat at the worst stage of the newborn [13]. Patients with congenital anomalies, documented infections or history of prolonged rupture of membranes . 18 h were excluded. Term newborns admitted to the neonatal intensive care unit requiring spinal tap as part of the septic work-up with culture (2 ) CSF samples were considered as controls. The study was approved by the hospital ethics committee and parental consent was obtained for inclusion. CSF and serum samples (1 ml each) were obtained within the first 24 h of life and kept at 2 70 8C until the time of measurement. 2.1. No Measurements The amount of total NO in the samples was determined by a modification in the procedure defined by Brahman and Hendrix [14] using the purge system of Sievers Instruments model 280 NOA based on chemiluminescence. A saturated solution of (vanadium chloride) VCl3 in 1 M HCl was prepared and filtered before use. Five milliliters of this reagent was added to purge the vessel with nitrogen for 5 –10 min before use. The purge vessel was equipped with a cold water condenser and a water jacket to permit heating of the reagent to 95 8C using a circulating water bath. The HCl vapors were removed by a gas bubbler containing 15 ml of 1 M NaOH. The gas flow rate into the chemiluminescence detector was controlled by using a needle valve adjusted to yield a constant cell pressure. Samples and standards were injected into the purge vessel to react with the VCl3 reagent which converted nitrate, nitrite and S-nitroso compounds to NO which was detected by ozone-induced chemiluminescence.
3. Results Nineteen patients with HIE and 13 control patients were included in the study. Twelve patients had stage 1 HIE, six stage 2 HIE, and one stage 3 HIE. The last two groups were pooled together and expressed as stage 2 þ 3 HIE. Gestational age and birth weight were similar in the three groups (controls, grade 1 HIE and stage 2 þ 3 HIE) (Table 1). Ten of the 12 patients in the grade 1 HIE group had renal involvement and three also had pulmonary and cardiovascular dysfunction. All the patients in the stage 2 þ 3 HIE group had renal involvement together with pulmonary, cardiac and hepatic dysfunction in three of them. All patients with grade 1 HIE and five with grade 2 þ 3 HIE had EEG and cranial magnetic resonance imaging (MRI) done, and one patient with grade 2 þ 3 HIE had cranial ultrasound performed. One patient in the same group did not have any radiological workup (died). Of the 12 patients with stage 1 HIE, one had abnormal paroxysmal activity on EEG and hemorrhage on right putamen on MRI. Of the five patients with stage 2 þ 3 HIE, one had abnormal MRI with signal change at basal ganglia suggestive of hypoxic insult, one had goiter as an incidental finding, and two had abnormal EEG findings defined as suppressed baseline activity. All of the patients in this group had seizure activity within the first 24 h. CSF NO levels were similar in controls and stage 1 HIE patients (8.05 ^ 2.91 mmol/l and 8.48 ^ 1.60 mmol/l, respectively). CSF NO levels in patients with moderate to severe HIE (stage 2 þ 3) were significantly higher than that of mild HIE (stage 1) and control patients (11.25 ^ 1.82 mmol/l). CSF VEGF levels were significantly higher in patients with mild HIE compared to control patients (12.70 ^ 2.25 pg/ml and 11.10 ^ 1.97 pg/ml, respectively). However, CSF VEGF levels were similar in the moderate to severe HIE
2.2. VEGF measurements VEGF levels were measured by an enzyme-linked immunosorbent assay method by sandwich enzyme immunoassay (Oncogene Research Products Cat. no. Q1A51). The method used was designed to measure VEGF 165 and VEGF 121 isoforms which are the isoforms of VEGF found in body fluids [9,15].
Table 1 Gestational age and birth weight of controls and patients (mean ^ SD)
Gestational age (weeks) Birth weight (g)
Controls (n ¼ 13)
Grade 1 HIE (n ¼ 12)
Grade 2 þ 3 HIE (n ¼ 7)
39.1 ^ 1.1 3137 ^ 443
39.3 ^ 1.3 3327 ^ 471
39 ^ 1.3 3323 ^ 461
E. Ergenekon et al. / Brain & Development 26 (2004) 283–286
285
Table 2 CSF NO and VEGF values in controls and patients with HIE
NO (mmol/l) VEGF (pg/ml)
Controls (n ¼ 13)
Grade 1 HIE (n ¼ 12)
Grade 2 þ 3 HIE (n ¼ 7)
8.05 ^ 2.91 (7.52) 11.10 ^ 1.97 (10.90)
8.48 ^ 1.60 (839) 12.70 ^ 2.25 (12.10)***
11.25 ^ 1.82 (10.96)*,** 12.51 ^ 3.31 (11.20)
Values are mean ^ SD (median). *High NO values compared to controls, P , 0:05; **high NO values compared to Grade 1 HIE, P , 0:05; ***high VEGF values compared to controls, P , 0:05.
group compared to that of the mild HIE and control group (12.51 ^ 3.31 pg/ml) (Table 2). Serum NO and VEGF determinations were made only in HIE patients, and no difference was observed between the levels in patients with mild HIE and moderate to severe HIE (Table 3). There was no correlation between VEGF and NO levels.
4. Discussion Nitric oxide has been investigated in thousands of studies regarding various disease states on a very wide spectrum from hypoxia –ischemia to septic shock, or from diabetes to chronic lung disease of prematurity [16 –20]. Recently VEGF has been discovered as a potent stimulator of NO synthesis from endothelium [15,21]; it stimulates endothelial nitric oxide synthase (eNOS), which in turn causes vasodilatation and increased vascular permeability [12]. This has been complicated by reports showing that VEGF is up-regulated by NO particularly the inducible form, indicating a positive feedback between these factors [22]. To complicate matters further, Ghiso et al. have shown suppression of hypoxia-associated VEGF gene expression by NO via a cyclic GMP-induced mechanism [23]. The time period required to cause an increase in NO levels by VEGF is reported to be 4 h in rat mesenteric circulation [21]. On the other hand NO produced via inducible NOS has a positive effect on VEGF production, as shown during wound repair [22]. Furthermore, Leker et al., in their permanent ischemia model; had found VEGF to be significantly increased with slowly increased eNOS expression in the same anatomical regions until the 7th day after ischemia, possibly protecting the penumbral tissue from additional damage [24]. Since the aim of this study was to investigate the early effects of hypoxia on VEGF and NO production, the serum and CSF samples were collected within the first 24 h of hypoxia– ischemia, which is the time period when endothelial and neuronal NO, both dependent on constitutive NOS, are increased. In the mild HIE group elevated CSF VEGF levels were observed despite CSF NO levels being similar to controls; this may be due to the timing of the sampling where there was not enough elapse of time for the NO levels to increase in response to VEGF together with the slow response of neurons or endothelial cells to mild hypoxia in terms of NO production.
On the other hand, in the moderate to severe HIE group CSF NO levels, both due to a higher degree of hypoxia and the induced seizure activity [25], were higher than mild HIE and control patients whereas CSF VEGF levels were similar to the mild HIE and the control group, although the levels were close both in mild and moderate to severe HIE groups. We considered that this finding could be explained as follows. The intensity of hypoxia– ischemia in the moderate to severe HIE group might have caused a rapid and sharp increase in CSF VEGF levels, resulting again in a sharp increase in NO levels high enough to suppress VEGF production and therefore, the VEGF levels might have been similar in all groups of HIE. The time of the sampling might have caught VEGF on the rise during mild HIE when NO had not yet increased and on the decline during moderate to severe HIE due to excess NO, depending on the severity of the insult. There was no significant difference between serum NO and VEGF levels in different stages of HIE, pointing to the fact that the real chain of events during hypoxia– ischemia takes place in the brain, although VEGF levels of mild HIE patients were higher than the moderate to severe group paralleling the CSF levels. We were not able to show a direct correlation between VEGF and NO levels either in CSF or in serum, which could be due to the complex interaction between the two as explained above. In conclusion, we believe that our study gives rise to other questions on mechanisms of brain injury during hypoxia– ischemia of the neonate, some of which require further research based on a large number of patients with different times of sampling of these mediators to show their deleterious and protective roles. However, considering the difficulty of including a sufficient number of patients, particularly with severe HIE, a multicentered approach would be more reasonable to reach more concrete and reliable conclusions if rapid answers are needed on the subject.
Table 3 Serum NO and VEGF levels in newborns with HIE
NO (mmol/l) VEGF (pg/ml)
Grade 1 HIE (n ¼ 12)
Grade 2 þ 3 HIE (n ¼ 7)
17.93 ^ 8.82 (14.61) 342.34 ^ 315.89 (196.00)
17.70 ^ 6.62 (16.02) 313.38 ^ 300.12 (234.00)
Values are mean ^ SD (median).
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E. Ergenekon et al. / Brain & Development 26 (2004) 283–286
Acknowledgements The authors would like to thank Professor Dr. Yahya Laleli and Du¨zen Laboratories for VEGF assays.
References [1] Du Plessis AJ, Johnson VM. Hypoxic-ischemic brain injury in the newborn. Cellular mechanisms and potential strategies for neuroprotection. Clin Perinatol 1997;24:627–57. [2] Fellman V, Raivivo OK. Reperfusion injury as the mechanism of brain damage after perinatal asphyxia. Pediatr Res 1996;41: 599–606. [3] Moskowitz AM, Dalkara T. Nitric oxide and cerebral ischemia. In: Siesjo BK, Tadeusz W, editors. Cellular and molecular mechanics of ischemic brain damage. Philadelphia: Lippincott-Raven; 1996. p. 365–9. [4] Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. Trends Neurosci 1999;20:132 –9. [5] Ergenekon E, Gu¨cu¨yener K, Erbas¸ D, Ezgu¨ SF, Atalay Y. Cerebrospinal fluid and serum nitric oxide levels in asphyxiated newborns. Biol Neonate 1999;76:200–7. [6] Papapetropoulos A, Garcia-Cardena G, Madri AJ, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 1999;100:3131–9. [7] Yi N, May K, Braas K, Osol O. Pregnancy augments uteroplacental and vascular endothelial growth factor gene expression and vasodilator effects. Am J Physiol 1997;273:H938–44. [8] Plate KR. Mechanisms of angiogenesis in the brain. J Neuropathol Exp Neurol 1999;58:313–20. [9] Neufeld O, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999; 13:9–22. [10] Martin J. Learning. Clin Exp Allergy 2000;30:33– 6. [11] Boulumie A, Schini-Kerth VB, Busse R. Vascular endothelial growth factor up-regulates nitric oxide synthase expression in endothelial cells. Cardiovasc Res 1999;41:773–80. [12] Tilton RG, Chang KC, LeJeune WS, Stephan CC, Brock TA, Williamson JR. Role for nitric oxide in hyperpermeability and hemodynamic changes induced by intravenous VEGF. Invest Ophthalmol Vis Sci 1999;40:689 –96.
[13] Sarnat HB, Sarnat MS. Neonatal encephalopathy following fetal distress. A clinical and electroencephalographic study. Arch Neurol 1976;33:696–705. [14] Brahman RS, Hendrix SA. Nanograms nitrite and nitrate in environmental and biological materials by vanadium (III) reduction with chemiluminescence detection. Anal Chem 1989;61:2715 –8. [15] Ahmed A, Dunk C, Kniss D, Wilkes M. Role of VEOF receptor-l (Flt1) in mediating calcium-dependent nitric oxide release and limiting DNA synthesis in human trophoblast cells. Lab Invest 1997;76: 779 –91. [16] Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 1991;43: 109–42. [17] Dalkara T, Moskowitz AM. Neurotoxic and neuroprotective roles of nitric oxide in cerebral ischemia. Int Rev Neurobiol 1989;40:319 –36. [18] Chowdhary S, Townend JN. Role of nitric oxide in the regulation of cardiovascular autonomic control. Clin Sci 1999;97:5–17. [19] Doughty L, Cardillo JA, Kaplan S, Janosky J. Plasma nitrite and nitrate concentrations and multiple organ failure in pediatric sepsis. Crit Care Med 1998;26:157–62. [20] Vyas IR, Currie AE, Shuker DE, Field DJ, Kotecha S. Concentration of nitric oxide products in bronchoalveolar lavage fluid obtained from infants who develop chronic lung disease of prematurity. Arch Dis Child Fetal Neonatal Ed 1999;81:F217–20. [21] Scalia R, Booth G, Lefer DJ. Vascular endothelial growth factor attenuates leucocyte–endothelium interaction during acute endothelial dysfunction: essential role of endothelium-derived nitric oxide. FASEB J 1999;13:1039 –46. [22] Frank S, Stallmeyer B, Kampfer H, Kolb N, Pfeilschifter J. Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair. FASEB J 1999;13:2002–14. [23] Ghiso N, Rohan RM, Amano S, Garland R, Adamis AP. Suppression of hypoxia-associated vascular endothelial growth factor gene expression by nitric oxide via cGMP. Invest Ophthalmol Vis Sci 1999;40:1033–9. [24] Leker RR, Teichner A, Ovadia H, Keshet E, Reinherz E, Ben-Hur T. Expression of endothelial nitric oxide synthase in the ischemic penumbra: relationship to expression of neuronal nitric oxide synthase and vascular endothelial growth factor. Brain Res 2001;909:1–7. [25] Takei Y, Takashima S, Ohyu J, Takami T, Miyajima T, Hoshika A. Effects of nitric oxide synthase inhibition on the cerebral circulation and brain damage during kainic acid induced seizures in newborn rabbits. Brain Dev 1999;21:253–9.
Original article
Cerebrospinal fluid and serum vascular endothelial growth factor and nitric oxide levels in newborns with hypoxic ischemic encephalopathy Ebru Ergenekona,*, Kıvılcım Gu¨cu¨yenerb, Deniz Erbas¸c, Sema Arald, Esin Koc¸a, Yıldız Atalaya a
b
Department of Newborn, Gazi University, School of Medicine, Besevler, Ankara, Turkey Department of Pediatric Neurology, Gazi University, School of Medicine, Besevler, Ankara, Turkey c Department of Physiology, Gazi University, School of Medicine, Besevler, Ankara, Turkey d Research Department, Duzen Laboratory, Ankara, Turkey Received 29 August 2002; received in revised form 1 August 2003; accepted 4 August 2003
Abstract Excitatory amino acids, cytokines and nitric oxide (NO) have been studied in the etiology and pathogenesis of hypoxic ischemic encephalopathy (HIE) of the newborn. Vascular endothelial growth factor (VEGF) is a known mediator of angiogenesis and has been shown to induce vascular proliferation and permeability via NO-mediated mechanism during hypoxia. The objective of this study was to investigate the cerebrospinal fluid and serum VEGF and NO levels in different stages of HIE and the correlation between the two mediators. Cerebrospinal fluid (CSF) and serum samples of 19 newborns with HIE and 13 controls were obtained within the first 24 h of life and kept at 2 70 8C until the time of measurement. NO levels were determined by Sievers NOA by chemiluminescence method and VEGF levels were measured by the enzyme-linked immunosorbent assay double sandwich method. The NO levels in CSF were higher than the control and mild HIE group in newborns with moderate to severe HIE, and VEGF levels in CSF were higher in the mild HIE group compared to controls but similar in the moderate to severe HIE group compared to mild HIE and control patients. There was no difference between groups with regard to serum NO or VEGF levels, and no correlation was observed between NO and VEGF levels both in CSF and serum samples. Depending on the severity of the hypoxic insult the stimulus for NO production by VEGF may have variable effects on endothelial cells which may give rise to the current results. q 2003 Elsevier B.V. All rights reserved. Keywords: Hypoxia; Nitric oxide; Vascular endothelial growth factor; Newborn
1. Introduction Hypoxic ischemic encephalopathy (HIE) of the newborn has been studied extensively in various animal models and in humans, to investigate the pathogenesis and treatment options. Most of the animal research has pointed the role of excitatory amino acids, cytokines and nitric oxide (NO) determining the extent of the pathology [1 –4]. In a previous study on asphyxiated newborns we showed increased levels of cerebrospinal fluid (CSF) NO levels with increasing severity of HIE during the first 24 – 72 h of the hypoxic * Corresponding author. Yesilyurt Sokak No: 19/9, Cankaya 06690, Ankara, Turkey. Fax: þ 90-312-215-0143. E-mail address: [email protected] (E. Ergenekon). 0387-7604/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0387-7604(03)00166-9
insult [5]. Concerning vascular endothelial growth factor (VEGF) there is a wide spectrum of research on endothelial cells, tumors, angiogenesis and hypoxia [6 –11]. VEGF has been shown to induce vascular permeability and endothelial cell proliferation via NO by stimulating endothelial NO synthase (eNOS), and hypoxia has been shown to promote VEGF synthesis [12]. These results have raised questions about the role of VEGF in HIE and the increase of NO in newborns with HIE. The objective of this study was to measure VEGF and NO levels in CSF and serum in newborns with different stages of HIE during the first 24 h of the hypoxic ischemic insult, firstly to investigate whether NO is increased during the first 24 h or not, and secondly to investigate whether VEGF is also increased during the same time period.
284
E. Ergenekon et al. / Brain & Development 26 (2004) 283–286
2. Patients and methods
2.3. Statistics
Term newborns with gestational age $ 37 weeks were included in the study group based on the following criteria for HIE:
VEGF and NO levels in CSF and serum of controls and newborns with different stages of HIE were compared by using the Mann – Whitney U-test and P , 0:05 was considered significant. Results are expressed as mean ^ SD unless stated otherwise. The correlation between NO and VEGF levels in CSF and serum were determined by Pearson correlation test and P , 0:05 was considered significant.
Antenatal evidence of fetal distress Umbilical artery pH , 7 Apgar score at 5th minute , 7 Postnatal resuscitation with positive pressure ventilation The staging for HIE was made based on Sarnat and Sarnat at the worst stage of the newborn [13]. Patients with congenital anomalies, documented infections or history of prolonged rupture of membranes . 18 h were excluded. Term newborns admitted to the neonatal intensive care unit requiring spinal tap as part of the septic work-up with culture (2 ) CSF samples were considered as controls. The study was approved by the hospital ethics committee and parental consent was obtained for inclusion. CSF and serum samples (1 ml each) were obtained within the first 24 h of life and kept at 2 70 8C until the time of measurement. 2.1. No Measurements The amount of total NO in the samples was determined by a modification in the procedure defined by Brahman and Hendrix [14] using the purge system of Sievers Instruments model 280 NOA based on chemiluminescence. A saturated solution of (vanadium chloride) VCl3 in 1 M HCl was prepared and filtered before use. Five milliliters of this reagent was added to purge the vessel with nitrogen for 5 –10 min before use. The purge vessel was equipped with a cold water condenser and a water jacket to permit heating of the reagent to 95 8C using a circulating water bath. The HCl vapors were removed by a gas bubbler containing 15 ml of 1 M NaOH. The gas flow rate into the chemiluminescence detector was controlled by using a needle valve adjusted to yield a constant cell pressure. Samples and standards were injected into the purge vessel to react with the VCl3 reagent which converted nitrate, nitrite and S-nitroso compounds to NO which was detected by ozone-induced chemiluminescence.
3. Results Nineteen patients with HIE and 13 control patients were included in the study. Twelve patients had stage 1 HIE, six stage 2 HIE, and one stage 3 HIE. The last two groups were pooled together and expressed as stage 2 þ 3 HIE. Gestational age and birth weight were similar in the three groups (controls, grade 1 HIE and stage 2 þ 3 HIE) (Table 1). Ten of the 12 patients in the grade 1 HIE group had renal involvement and three also had pulmonary and cardiovascular dysfunction. All the patients in the stage 2 þ 3 HIE group had renal involvement together with pulmonary, cardiac and hepatic dysfunction in three of them. All patients with grade 1 HIE and five with grade 2 þ 3 HIE had EEG and cranial magnetic resonance imaging (MRI) done, and one patient with grade 2 þ 3 HIE had cranial ultrasound performed. One patient in the same group did not have any radiological workup (died). Of the 12 patients with stage 1 HIE, one had abnormal paroxysmal activity on EEG and hemorrhage on right putamen on MRI. Of the five patients with stage 2 þ 3 HIE, one had abnormal MRI with signal change at basal ganglia suggestive of hypoxic insult, one had goiter as an incidental finding, and two had abnormal EEG findings defined as suppressed baseline activity. All of the patients in this group had seizure activity within the first 24 h. CSF NO levels were similar in controls and stage 1 HIE patients (8.05 ^ 2.91 mmol/l and 8.48 ^ 1.60 mmol/l, respectively). CSF NO levels in patients with moderate to severe HIE (stage 2 þ 3) were significantly higher than that of mild HIE (stage 1) and control patients (11.25 ^ 1.82 mmol/l). CSF VEGF levels were significantly higher in patients with mild HIE compared to control patients (12.70 ^ 2.25 pg/ml and 11.10 ^ 1.97 pg/ml, respectively). However, CSF VEGF levels were similar in the moderate to severe HIE
2.2. VEGF measurements VEGF levels were measured by an enzyme-linked immunosorbent assay method by sandwich enzyme immunoassay (Oncogene Research Products Cat. no. Q1A51). The method used was designed to measure VEGF 165 and VEGF 121 isoforms which are the isoforms of VEGF found in body fluids [9,15].
Table 1 Gestational age and birth weight of controls and patients (mean ^ SD)
Gestational age (weeks) Birth weight (g)
Controls (n ¼ 13)
Grade 1 HIE (n ¼ 12)
Grade 2 þ 3 HIE (n ¼ 7)
39.1 ^ 1.1 3137 ^ 443
39.3 ^ 1.3 3327 ^ 471
39 ^ 1.3 3323 ^ 461
E. Ergenekon et al. / Brain & Development 26 (2004) 283–286
285
Table 2 CSF NO and VEGF values in controls and patients with HIE
NO (mmol/l) VEGF (pg/ml)
Controls (n ¼ 13)
Grade 1 HIE (n ¼ 12)
Grade 2 þ 3 HIE (n ¼ 7)
8.05 ^ 2.91 (7.52) 11.10 ^ 1.97 (10.90)
8.48 ^ 1.60 (839) 12.70 ^ 2.25 (12.10)***
11.25 ^ 1.82 (10.96)*,** 12.51 ^ 3.31 (11.20)
Values are mean ^ SD (median). *High NO values compared to controls, P , 0:05; **high NO values compared to Grade 1 HIE, P , 0:05; ***high VEGF values compared to controls, P , 0:05.
group compared to that of the mild HIE and control group (12.51 ^ 3.31 pg/ml) (Table 2). Serum NO and VEGF determinations were made only in HIE patients, and no difference was observed between the levels in patients with mild HIE and moderate to severe HIE (Table 3). There was no correlation between VEGF and NO levels.
4. Discussion Nitric oxide has been investigated in thousands of studies regarding various disease states on a very wide spectrum from hypoxia –ischemia to septic shock, or from diabetes to chronic lung disease of prematurity [16 –20]. Recently VEGF has been discovered as a potent stimulator of NO synthesis from endothelium [15,21]; it stimulates endothelial nitric oxide synthase (eNOS), which in turn causes vasodilatation and increased vascular permeability [12]. This has been complicated by reports showing that VEGF is up-regulated by NO particularly the inducible form, indicating a positive feedback between these factors [22]. To complicate matters further, Ghiso et al. have shown suppression of hypoxia-associated VEGF gene expression by NO via a cyclic GMP-induced mechanism [23]. The time period required to cause an increase in NO levels by VEGF is reported to be 4 h in rat mesenteric circulation [21]. On the other hand NO produced via inducible NOS has a positive effect on VEGF production, as shown during wound repair [22]. Furthermore, Leker et al., in their permanent ischemia model; had found VEGF to be significantly increased with slowly increased eNOS expression in the same anatomical regions until the 7th day after ischemia, possibly protecting the penumbral tissue from additional damage [24]. Since the aim of this study was to investigate the early effects of hypoxia on VEGF and NO production, the serum and CSF samples were collected within the first 24 h of hypoxia– ischemia, which is the time period when endothelial and neuronal NO, both dependent on constitutive NOS, are increased. In the mild HIE group elevated CSF VEGF levels were observed despite CSF NO levels being similar to controls; this may be due to the timing of the sampling where there was not enough elapse of time for the NO levels to increase in response to VEGF together with the slow response of neurons or endothelial cells to mild hypoxia in terms of NO production.
On the other hand, in the moderate to severe HIE group CSF NO levels, both due to a higher degree of hypoxia and the induced seizure activity [25], were higher than mild HIE and control patients whereas CSF VEGF levels were similar to the mild HIE and the control group, although the levels were close both in mild and moderate to severe HIE groups. We considered that this finding could be explained as follows. The intensity of hypoxia– ischemia in the moderate to severe HIE group might have caused a rapid and sharp increase in CSF VEGF levels, resulting again in a sharp increase in NO levels high enough to suppress VEGF production and therefore, the VEGF levels might have been similar in all groups of HIE. The time of the sampling might have caught VEGF on the rise during mild HIE when NO had not yet increased and on the decline during moderate to severe HIE due to excess NO, depending on the severity of the insult. There was no significant difference between serum NO and VEGF levels in different stages of HIE, pointing to the fact that the real chain of events during hypoxia– ischemia takes place in the brain, although VEGF levels of mild HIE patients were higher than the moderate to severe group paralleling the CSF levels. We were not able to show a direct correlation between VEGF and NO levels either in CSF or in serum, which could be due to the complex interaction between the two as explained above. In conclusion, we believe that our study gives rise to other questions on mechanisms of brain injury during hypoxia– ischemia of the neonate, some of which require further research based on a large number of patients with different times of sampling of these mediators to show their deleterious and protective roles. However, considering the difficulty of including a sufficient number of patients, particularly with severe HIE, a multicentered approach would be more reasonable to reach more concrete and reliable conclusions if rapid answers are needed on the subject.
Table 3 Serum NO and VEGF levels in newborns with HIE
NO (mmol/l) VEGF (pg/ml)
Grade 1 HIE (n ¼ 12)
Grade 2 þ 3 HIE (n ¼ 7)
17.93 ^ 8.82 (14.61) 342.34 ^ 315.89 (196.00)
17.70 ^ 6.62 (16.02) 313.38 ^ 300.12 (234.00)
Values are mean ^ SD (median).
286
E. Ergenekon et al. / Brain & Development 26 (2004) 283–286
Acknowledgements The authors would like to thank Professor Dr. Yahya Laleli and Du¨zen Laboratories for VEGF assays.
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