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UDC 616.8-005-053.35:612.273.2:616.15-07
DYNAMICS OF MATRIX METALLOPROTEINASIS IN BLOOD OF NEWBORN CHILDREN IN THE EARLY NEONATAL PERIOD FOR DIAGNOSTICS OF HYPOXIC DISORDERS OF THE CENTRAL NERVOUS SYSTEM
Perinatal Center (Clinical) of Altai Krai, Barnaul Altai State Medical University, Barnaul
T.N. Chugunova, Yu.V. Korenovsky, Yu.F. Lobanov
Newborns with hypoxic lesions of the central nervous system are characterized by increased concentration of matrix metalloproteinases-1,-9 and reduced levels of tissue inhibitor of metalloproteinases-1 in plasma of umbilical cord blood in comparison with the healthy children. On the 3rd-4th day, the increase of these concentrations proceeds in plasma of venous blood, while in the healthy new-born - they decrease.
Key words: newborns, hypoxia, matrix metalloproteinases-1,-9 and tissue inhibitor of metalloproteinases-1.
Currently available information on the clinical and functional status of newborn infants in the early neonatal period with perinatal hypoxia and the role of matrix metalloproteinases (MMPs) and systemic metabolic changes in its violation are fragmentary, methodologically heterogeneous and contradictory [1, 2].
The purpose of this work was to study the dynamics of matrix metalloproteinases in the blood of newborn infants in the early neonatal period for the diagnosis of hypoxic lesions of the central nervous system.
Materials and methods
The work is based on the results of a survey of 124 children, 74 of whom were born prematurely. The main group consisted of 25 children with hypoxic-ischemic lesions of the central nervous system (CNS), a comparison group of 25 children with hemorrhagic CNS lesion, and the control group consisted of 24 conditionally healthy preterm infants. In full-term children, the main group consisted of 25 children with cerebral ischemia of I-II degree, the comparison group - 25 conditionally healthy children.
In this study, clinical, ultrasound (neurosonog-raphy) and laboratory methods (enzyme-linked immunoassays of the concentrations of matrix metalloproteinases 1, 9 (MMP-1 and MMP-9) and tissue inhibitor of matrix metalloproteinases (TIMP-1) were used. Neurosonography of newborn children was carried out on the apparatus Vivi-i (General Electrics, USA), the study was carried out using linear 12v and convective 8v sensors, a common technique was used: through a large fontanel and temporal bone in different planes. The enzyme immunoassay was performed by means of black Raybio® ELH-MMP1-001, RayBio® ELH-MMP9-001 and RayBio® ELH-TIMP1-001 (RayBiotech, USA). Concentrations of MMP-1, MMP-9 and TIMP-1 were determined
in the umbilical plasma and venous blood plasma on the 3-4th day of life. To determine the relative changes in the matrix metalloproteinase system, the MMP-1/TIMP-1 and MMP-9/TIMP-1 concentration relations were determined. The clinical picture of hypoxic damage to the central nervous system in newborn children is non-specific, therefore, signs of CNS involvement were registered in the form of oppression or excitation syndromes. Statistical analysis of the results of the study was carried out using SigmaPlot 11. The reliability of the intergroup differences was assessed according to the Mann-Whitney U criterion, the difference in parameters in dynamics was determined by the Wilcoxon test.
Results and discussion
Taking into account the latest data on the important role of the extracellular proteolytic system in the regulation of remodeling of the connective, in particular, nervous tissue [4, 5], we conducted a prospective complex study of the clinical status in the early neonatal period in newborn children with parallel determination of the specific profile of the matrix metalloproteinase system in new-borns. In the course of the study, there were determined significantly higher enzyme indices of the proteolytic system in premature infants (Table 1) in the 1st and 2nd groups in the umbilical cord blood plasma compared to children who do not have hypoxic damage to the central nervous system. In premature children of the 2nd group with hypoxic-hemorrhagic CNS lesion in the cord blood plasma, the concentration of MMP-1 was 0.38 ± 0.012 ng/ml, which significantly (p<0.01) exceeded the data in the 3rd group (0, 28 ± 0.016 ng/ml), but was comparable with the data in the 1st group (0.32 ± 0.012 ng/ml). There is also a significant increase (p<0.01) of MMP-9 concentration in the 1st (104.0 ± 4.8 ng/ml) and the 2nd groups (118.4 ± 8.75 ng/ml) in comparison from the 3rd (92.23 ± 2.08 ng/
ml), the data of the 1st and 2nd groups are statistically comparable. However, TIMP-1 (30.7 ± 1.51 ng/ ml) in the 3rd group in umbilical cord blood plasma was significantly higher (p<0.01) than in the 1st and 2nd groups (23.5 ± 1.47 ng/ml and 24.4 ± 2.31 ng/ml, p>0.05). The data presented indicate an increase in the proteolytic activity of matrix metal-loproteinases in neonates with perinatal hypoxia. Confirmation of this phenomenon is a significant increase in the ratio of cord blood plasma concentrations of MMP-9/TIMP-1 in the 1st and 2nd groups in comparison with the 3rd (p <0.01) and an increase in MMP-1/TIMP-1 ratios in the 1st and 2nd groups in comparison with the 3rd without considerable significance (p> 0.05). Changes in the levels of matrix metalloproteinases and their tissue inhibitor TIMP-1, previously noted by other investigators [6, 7] by hypoxia are explained by increased production of matrix metalloproteinases by connective tissue cells at the level of transcription and translation [8]. From the point of view of pathogenesis, these data indicate a possible relationship between perinatal hypoxic damage to the central nervous system and the activation of the system of matrix metalloproteinases, which is traced in our study.
Thus, we found that the greatest activity of the proteolytic system in umbilical cord plasma was observed in premature infants with hypoxic lesions of the central nervous system, especially
in children of the 2nd group with hypoxic-hemor-rhagic CNS lesions.
According to recent studies [5, 9, 10], it is known that changes in the matrix metalloproteinase system after the primary stimulus (hypoxia, cytokines) may occur with some delay. Taking into account this peculiarity of the central nervous system damage, by perinatal hypoxia, we evaluated the dynamics of these indices in comparison with the initial level at birth. There was a higher (p<0.01) concentration of MMP-1 on the 3rd-4th day of life in plasma of venous blood in children of the 1st (0.4 ± 0.02 ng/ml) and the 2nd group (0 , 4 ± 0.02 ng/ml) compared with the 3rd (0.3 ± 0.02 ng/ml), the data in the 1st and 2nd groups had no statistical differences. The greatest activity of MMP-9 on the 3rd-4th day was observed in children of the 2nd group (188.6 ± 14.31 ng/ml), the concentration of which was significantly higher (p <0.01 and p<0.05) than in children 1st (108,9 ± 4,99 ng/ml) and the 3rd group (74,43 ± 3,69 ng/ml). In the 1st and 2nd groups, at the same time, the concentration of TIMP-1 (26.9 ± 1.82 ng/ml 43.8 ± 3,18 ng/ml) compared with the 3rd (16.96 ± 1.51 ng/ml), and between the 1st and 2nd groups (p <0.05), a statistical difference was also recorded. The data presented indicate damage to cell membranes with an increase in the blood-brain barrier permeability.
Table 1
Dynamics of indices of the proteolytic system of matrix metalloproteinases on the 3rd-4th day of life in premature
infants, M±m
Premature infants with hypoxic-ischemic lesions of the central nervous system 1st group (n = 25) Premature infants with hypoxic-hemorrhagic CNS lesions 2nd group (n = 25) Healthy premature babies 3rd group (n = 24)
Index umbilical cord plasma plasma of venous blood on the 3rd-4th day of life umbilical cord plasma plasma of venous blood on the 3rd-4th day of life umbilical cord plasma plasma of venous blood on the 3rd-4th day of life
MMP-1, ng/ml 0,310,01*! 0,4±0,02 0,4±0,01** 0,4±0,02** 0,3±0,02 0,3±0,02
MMP-9, ng/ml 104,0±4,8* 108,9±4,99* 118,4±8,75**! 188,6±14,31** 92,2±2,08 74,43±3,69
TIMP-1, ng/ml 23,5±1,47* 26,9±1,82*# 24,4±2,31**! 43,8±3,18** 30,7±1,511 16,96±1,51
MMP-1/ TIMP-1 0,02±0,001 0,015±0,001 0,02±0,004! 0,011±0,001** 0,01±0,001! 0,017±0,001
MMP-9/ TIMP-1 5,2±0,35* 4,3±0,33# 4,8±0,47** 4,5±0,49 3,2±0,17i 4,94±0,44
Note: according to the Mann-Whitney U criterion, * - significance of differences (p<0.05) when comparing the 1st and 2nd groups, ** - significance of differences (p<0.05) when comparing the 2nd and 3rd (p <0.05), # - significance of differences (p<0,05) when comparing the 1st and 3rd groups on the 3rd day of life, 1 - significance of differences (p <0.05) when comparing the data obtained in the umbilical cord plasma and venous blood on the 3rd-4th day of life in groups (according to the Wilcoxon test).
There was investigated the dynamics of matrix metalloproteinase indices in newborns with perinatal hypoxic CNS lesion in the early neonatal period and without it. In our study, the continued activation of MMP-1 in the 1st and 2nd groups and the decrease in the concentration in the 3rd
group without significant differences (p> 0.05) were noted in comparison with the baseline data. When comparing the concentration of MMP-9 on the 3rd-4th day from the baseline, an increase was clearly seen without significance in children of the 1st group (p>0.05), whereas a significant increase (p
<0.001) in the concentration of MMP-9 in children of the 2nd group, but significant (p <0.05) decrease in the third group were noted. The presented data demonstrated that the process of central nervous system damage in children of the 2nd group with hypoxic-hemorrhagic lesion continues, which indicates a persisting immunopathological process at the cell-tissue level in this category of children. Activity of the ongoing damage process on the 3rd-4th day in children of the 2nd group was confirmed by significant (p<0.05) increase in TIMP-1 concentration in plasma of venous blood.
In children of the 1st group, the concentration of TIMP-1 also continued to increase without significance in comparison with the baseline, but a statistically significant (p <0.001) decrease in concentration was noted in children of the 3rd group. Consequently, the greater the MMP concentration, the greater the need for its inhibitor. A comparative analysis of the parameters of the system of matrix metalloproteinases in term infants (Table 2) at birth showed that in full-term children with hypoxic CNS lesions, MMP-1 concentrations were comparable to healthy in cord blood plasma and amounted to 0.31±0.01 ng/ml and 0.28±0.015 ng/ml. However, the concentration of MMP-9 in the children of the 1st group was 100.8±6.0 ng/ml and was significantly (p<0.05) higher than the data of the second group -77.5±3.6 ng/ml, at the same time, the concentration of their tissue inhibitor TIMP-1 was lower (25. ±1.5
and the system of matrix metalloproteinases in our study (Table 3), we found that there was no significant difference between MMP-1 concentrations in full-term and premature babies in umbilical cord blood plasma (p=0.675, p=0.148 ). At the same time, an increase in the concentration of MMP-1 in plasma of venous blood on the 3rd-4th day of life in premature infants with oppressive syndrome is markedly significant (p=0.012), while in the term infants the decrease is due to excitation syndrome (p=0.443). Table 3 shows the continued activation of MMP-9 in premature infants
ng/ml versus 27.3±1.3 ng/ml, p> 0.05). This indirectly indicates the presence of disorders in newborns with perinatal hypoxia - increased proteolytic activity of matrix metalloproteinases [12,13,14]. Confirmation of this phenomenon is an increase in ratios in the cord blood plasma of MMP-1/TIMP-1 concentrations (0.013±0.001 ng/ml and 0.012±0.0009 ng/ml) and MMP-9/TIMP-1 in the 1st group (4,45±0.46 ng/ml and 3.17±0.29 ng/ml), but there was no significant difference in the relationship between the groups. From the point of view of patho-genesis, these data indicate a possible link between perinatal CNS damage and activation of the matrix metalloproteinase system.
On the 3-4th day of life in our study, as in preterm infants, there continued the activation of the system of matrix metalloproteinases. The concentration of MMP-1 (0.32±0.02 ng/ml versus 0.25±0.001 ng/ml, p>0.05) continues to increase, but without significant differences in the groups, the MMP-9 concentration in the cord blood plasma is reliably higher in children in the 1st group (104.6±11.0 vs. 73.1±3.8, p<0.05). At the same time, the concentration of TIMP-1 in the 1st group significantly increased, together with a decrease in the 2nd group (23.3±2.7 ng/ml vs. 15. ±0.7 ng/ml, p<0.05). However, there were no significant differences in MMP-1/ TIMP-1 and MMP-9/TIMP-1 (p> 0.05) correlation groups, as on the 1st day.
(110.11±4.838 ng/ml and 143.88±10.333 ng/ml, p=0.002 and 117.89±19.669 ng/ml and 178.56±28.468 ng/ml, p=0.023) with different syndromes, however, prolonged activation is observed only in the syndrome of depression (from 122,34±9,606 ng/ml to 145,51±20,321ng/ml, p=0.292). In the cord blood plasma, the concentration of TIMP-1 in children of the study groups had no significant differences in syndromes. And in plasma of venous blood, a significant increase in the concentration of TIMP-1 (23.94±1.454 and 34.88±2.394, p<0.001, 24.04±3.499 and 28.23±4.621, p=0.023). At full-term
Table 2
М±m Dynamics of the system of matrix metalloproteinases on the 3rd-4th day of life in full-term children, M m
Main group (n=25) Control group (n=25)
Index umbilical cord plasma plasma of venous blood on the 3rd-4th day of life umbilical cord plasma plasma of venous blood on the 3rd-4th day of life
MMP-1, ng/ml 0,31±0,01 0,32±0,02 0,28±0,001 0,25±0,01
MMP-9, ng/ml 100,8±29,43 104,6±11,0 77,52±16,224 73,1±3,8
TIMP-1, ng/ml 25,8±1,5 23,3±2,7 27,3±1,7* 15,7±0,7
MMP-1/ TIMP-1 0,013±0,001 0,016±0,002 0,0115±0,0009 0,017±0,001
MMP-9/ TIMP-1 4,45±0,46 4,82±0,41 3,17±0,29 4,72±0,28
*- р<0,05
Estimating the relationship between syndromes
on the 3rd-4th day with significant differences
children with excitation syndrome on the 3rd day, significant decrease in TIMP-1 concentration (p=0.016) was observed in comparison with the initial one. However, in preterm infants with
excitatory syndrome on the 3rd day of life in venous blood plasma, the TIMP-1 concentration was significantly higher in comparison with full-term blood (p<0.001).
Table 3
Interrelation of syndromes and proteolytic system of matrix metalloproteinases, M±m
Premature infants Mature infants
Index suppression excitation suppression suppression
syndrome syndrome syndrome syndrome
MMP-1
1st day 0,35±0,0096 0,34±0,030 0,34±0,021 0,29±0,018
3rd-4th day 0,397±0,0134* 0,32±0,0263 0,39±0,033* 0,27±0,016
р=0,012 р=0,574 р=0,121 р=0,443
MMP-9
1st day 110,11±4,838 117,89±19,669 122,34±9,606 86,38±4,743
3rd-4'h day 143,88±10,333* 178,56±28,468 145,51±20,321* 77,34±4,407
р=0,002 р=0,022 р=0,292 р=0,614
TIMP-1
1st day 23,94±1,454 24,043±3,499 25,480±2,585 25,987±1,887
3rd-4'h day 34,88±2,394 28,23±4,621** 34,64±4,363* 15,67±1,398
р<0,001 р=0,023 р=0,079 р=0,016
Note. According to the Mann-Whitney U criterion: * - significant differences (p<0.05) when comparing the syndromes of suppression and excitation in the groups of full-term or premature infants, ** - significant difference (p <0.05) when comparing the excitation syndrome in premature and full-term infants. According to Wilcoxon test, the initial data were compared with the data on the 3rd-4th day of life.
Thus, the maximum activity of MMP-1 was observed in children in the groups with the syndrome of oppression on the 3rd day, and also on the 3rd day there was an increase in the concentration of MMP-9, but the highest level was observed in premature infants with the syndrome of excitation. The minimal concentration of tissue inhibitor MMP (TIMP-1) was in term infants with excitation syndrome on the 3rd day.
Conclusions
1. In children with hypoxic lesions of the central nervous system, increased concentrations of matrix metalloproteinases (MMP-1, -9) and a decreased level of TIMP-1 in umbilical cord blood plasma are observed in comparison with healthy children.
2. On the 3rd-4th day of life in term and preterm infants with hypoxic lesions of the central nervous system in plasma of venous blood, there is an increase in the concentration of MMP-1, MMP-9 and TIMP-1 in comparison with baseline data, whereas in healthy newborns they are decreasing.
3. A significant increase in the concentrations of matrix metalloproteinases (MMP-1, -9) and tissue inhibitor (TIMP-1) is observed on the 3rd-4th day of life in plasma of venous blood in premature infants under oppressive syndrome.
References
1. Bednarek N., Svedin P., Garnotel R. et.al. Increased MMP-9 and TIMP-1 in mouse neonatal brain and plasma and in human neonatal plasma after hypoxia-ischemia: a potential marker of neonatal encephalopathy. Pediatr Res. 2012; 71(1):63-70.
2. Perlman J.M. Intrapartum hypoxic-ischemic cerebral injury and subsequent cerebral palsy: medicolegal issues. Pediatrics. 1997; 99: 851.
Classification of perinatal lesions of the nervous system in newborns: Method. Recommendations. Moscow: Ros. Ass. specialists in perinatal medicine; 2000.
3. Yan C., Boyd D. D. Regulation of matrix metalloproteinase gene expression. Cell Physiol. 2007; 211: 19-26.
4. Yang Y., Hill J.W., Rosenberg G.A. Multiple roles of metalloproteinases in neurological disorders. Prog. Mol. Biol. Transl. Sci. 2011; 99: 241-263.
5. Sunagawa S., Ichiyama T., Honda R. et.al. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in perinatal asphyxia. Brain & Development. 2009; 31: 588-593.
6. Tong W., Zhang L. Fetal hypoxia and programming of matrix metalloproteinases. Drug Discovery Today. 2012; 17(3/4): 124-134.
7. Dejonckheere E., Vandenbroucke R.E., Libert C. Matrix metalloproteinases as drug targets in ischemia/reperfusion injury. Drug Discov Today. 2011; 16(17-18):762-78.
8. Sandoval K.E. ,Witt K,A. Blood-brain barrier tight junction permeability and ischemic stroke. Neurobiol Dis. 2008; 32: 200-219.
9. Ries C. Cytokine functions of TIMP-1. Cell Mol. Life Sci. 2013; 71(4): 659-672.
10. Rosell A., Cuadrado E., Ortega-Aznar A. et al. MMP-9-positive neutrophil infiltration is associated to blood-brain barrier breakdown and basal lamina type IV collagen degradation during hemorrhagic transformation after human ischemic stroke. Al - Stroke. 2008; 39: 1121-1126.
11. Wang C.X., Shuaib A. Critical role of mi-crovasculature basal lamina in ischemic brain injury. Prog Neurobiol. 2007; 83: 140-148.
Contacts
Corresponding author: Korenovsky Yuri Vladi-mirovich, Candidate of Medical Sciences, Associate Professor, Head of the Department of General and Biological Chemistry, Clinical Laboratory Diagnostics, Altai State Medical University, Barnaul.
656038, Barnaul, ul. Papanitsev, 126. Tel.: (3852) 566938. E-mail: timidin@gmail.com
Chugunova Tatyana Nikolaevna, Candidate of Medical Science, Deputy Chief Physician for pediatric work of the "Perinatal Center (Clinical) of Altai Krai", Barnaul. 656038, Barnaul, ul. Papanitsev, 126. Тел.: (3852) 566938. E-mail: chugunova.t@inbox.ru
Lobanov Yuri Fedorovich, Doctor of Medical Sciences, Professor, Head of the Department of Propaedeutics of Children's Diseases, Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40 Tel.: (3852) 400883. E-mail: luf@list.ru
