Endothelial Dysfunction and Intestinal Barrier Injury in Preterm Infants with Perinatal Asphyxia

Abstract

Perinatal asphyxia is one of the most frequent causes of perinatal morbidity, accounting for approximately 23% of neonatal deaths worldwide. Fetuses that suffer from hypoxia-ischemia are at high risk of developing multiorgan dysfunction, including the gut. Hypoxie-induced gut injury may result in adverse clinical outcomes, such as feeding intolerance and necrotizing enterocolitis. Increased permeability and subsequently an enhanced entry of bacteria and endotoxins into the systemic circulation can contribute to endotoxin aggression and further trigger numerous diseases. The aim of study is to investigate the effect of perinatal asphyxia on the integrity of the intestinal barrier and the state of antiendotoxin immunity. The study included preterm neonates exposed to perinatal asphyxia, who were comparable with non-asphyxiated infants. The concentrations of intestinal mucosa barrier injury markers (intestinal fatty acid binding protein, liver fatty acid protein, lipopolysaccharide binding protein), neurospecific proteins (neurospesific enolase, NR-2 antibodies), and also endothelial dysfunction markers (endothelin-1, nitric oxide) were determined in serum of included neonates on day of 1 and 7. The high risk of intestinal mucosal injury in newborn exposed to perinatal asphyxia decreases the level of antiendotoxic immunity and should be considered as an unfavorable factor for sepsis.

1. Introduction

The main pathophysiological change that develops in intestinal ischemia is damage to the epithelial integrity of the mucosa, leading to bacterial translocation, release of pro- and anti-inflammatory cytokines, activation of innate immunity dominated by pro-inflammatory effects, with the final manifestation of the process as intestinal perforation or fulminant sepsis [1, 2]. The progression of intestinal ischemia is combined with a paradoxical direction of oxygen flow in the villi from tissues to blood, which contributes to their hypoxia and can result in necrosis starting from the tops of the villi of the intestinal mucosa. In this case, enterocytes exfoliate from their own plate and into the intestinal lumen. One of the promising methods for assessing damage to intestinal epithelial cells is the determination of endogenous proteins of enterocytes in blood serum or urine [3]. These are low molecular weight proteins (14–15 kDa), fatty acid-binding proteins (FABPs), and bile acid-binding proteins. The intestinal form of FABP (I-FABP) is tissue-specific for the gut. I-FABP is localized to the tops of the villi in enterocytes and enters circulation when they are destroyed and is excreted from the body by the kidneys in a single passage, resulting in a half-life of 11 min. The concentration of this protein in the blood of healthy individuals does not usually exceed 2 ng/mL, but this increases rapidly after an episode of acute intestinal ischemia. Reisinger et al. found that the content of I-FABP in human and sheep intestinal villi depends on gestational age, and an increase in I-FABP content in blood occurs in parallel with gestational age, allowing its use as a marker to indicate intestinal maturity [4]. Given the particular importance of hypoxia in the pathogenesis of ulcerative necrotizing enterocolitis (NEC), the determination of I-FABP content is typically carried out to diagnose patients and determine the severity of this condition. In particular, in a study by Guthmann, concentrations of I-FABP and hepatic FABP (L-FABP) were determined in healthy preterm newborns and in preterm infants with ulcerative NEC. An increase in the I-FABP concentration was found in end-stage ulcerative NEC, while the concentration of L-FABP was significantly increased only in newborns with suspected ulcerative NEC. Tentatively, the identified changes may be useful for the early diagnosis of ulcerative NEC in newborns [5]. Mannoia K. et al. found high concentrations of I-FABP in urine collected in the first 60 h of life from preterm infants who subsequently developed ulcerative NEC with the onset of enteral nutrition. The authors proposed the use of this protein for early ulcerative NEC detection in newborns who are at high risk of developing it, allowing for timely preventive measures [6].

Intestinal mucosa and submucosa layers are characterized by an intensive blood supply, which is due to their high demand for oxygen to support the processes of hydrolysis and absorption of substances as well as the constant need to renew enterocytes exfoliating into the intestinal cavity [7]. At the same time, the consistency of the protective properties of the mucous barrier of the large intestine is determined by the level of expression of epithelial mucins and their properties. Active expression of mucins by the epithelium of the gastrointestinal tract (GIT) is accompanied by the formation of a high-molecular viscoelastic layer, which is a protective barrier between the surface of the mucous membrane and the cavity contents of the GIT. According to modern concepts, MUC2, MUC4, MUC3, and MUC17 are synthesized both in the small intestine and in the proximal and distal parts of the large intestine. The localization of mucins in the unchanged mucosa of the GIT coincides with the distribution of trefoil peptides expressed by goblet cells. Intestinal trefoil factor (ITF) together with mucins performs protective and regenerative functions, participating in the formation of the gastrointestinal barrier. Due to its compact structure, ITF is extremely resistant to acid attack, proteolytic degradation, and thermal degradation. Excessive synthesis of this substance occurs at sites of GIT damage, such as peptic ulcers or damage caused be inflammatory bowel disease. Patients with these pathologies have an elevated level of ITF in the blood serum. Lin J. et al., studying the level of ITF in neonatal meconium, found no difference in ITF content depending on birth weight [8]. Louis N.A. et al. argued that hypoxia has an inducing effect on the expression of MUC3 and ITF in the intestinal mucosa, performing a protective function in oxygen-deficient conditions [9].

It has been proven that the violation of intestinal perfusion is accompanied by an increase in intestinal permeability, which may be due to the extensive death of the intestinal mucosa. The hyperoxia that occurs during the period of reperfusion, stimulating the processes of free radical oxidation and neutrophil chemotaxis, exacerbates inflammatory processes in the intestinal wall [10]. Another reason for the death of the intestinal mucosa may be severe endotoxemia. Endotoxins (lipopolysaccharides (LPS)), by stimulating the release of inflammatory mediators from macrophages and neutrophils, ultimately lead to an imbalance of pro- and anti-inflammatory responses, the occurrence of excessive systemic inflammation, and damage to the intestinal mucosal barrier [11].

Increased intestinal permeability describes an increase in the ability of substances to pass through the intestinal wall. Several non-invasive methods of examining intestinal permeability have been described. These methods can be divided into the following three groups: active direct, passive, and indirect. The “active” assessment of barrier function is based on the hypothesis that under physiological conditions, orally administered large molecules are unable to cross the intestinal wall. In the case of increased permeability, such samples cross the intestinal barrier and enter the bloodstream with subsequent detection in the urine after renal excretion. Absorption tests of water-soluble non-ionized compounds, such as lactulose, mannitol, and ethylenediaminetetraacetic acid, are most commonly used for this purpose. The “passive” assessment of the state of the intestinal barrier is based on the hypothesis that the intracavitary components of the intestine are translocated into the bloodstream with a decrease in its barrier function. Determination of the level of LPS of the outer membrane of Gram-negative bacteria (endotoxins) as well as measurement of the fermentation product D-lactate are the most commonly used tests for monitoring passive permeability. An indirect way to assess the translocation of bacterial products is to measure serum LPS-binding protein (LBP) levels and antibody levels in the endotoxin cortex.

LBP got its name from its ability to form a complex with LPS. LPS, which is the main component of the cell wall of Gram-negative microorganisms, is found in small amounts in the blood of healthy organisms and helps to maintain the protective mechanisms of innate immunity. LBP initiates the launch of a complex response cascade of the receptor complex with LPS, which consists of the recognition, binding, and transport of LPS and the enhancement of the infection danger signal [12]. The cascade of successive LPS-LRP reactions with receptor structures that recognize pathogen-associated samples initiates an intracellular signaling cascade, which, in turn, activates the nuclear transcription factor NF-kB with increased expression of pro-inflammatory mediators and rapid elimination of the infectious agent from the body. With excessive intake of LPS into the bloodstream and insufficient antiendotoxin protection, endotoxin aggression can develop. Synthesis of LBP mainly occurs in the liver with release into the blood after glycosylation with a molecular weight of 58–60 kDa [13].

Juli M. Richter et al. in experimental studies on rats established a dose-dependent effect of LBP on the processes of neonatal enterocyte migration and wound healing of the intestinal wall induced by LPS. Studies have found that a high concentration of LBP, as opposed to a normal concentration, suppresses the release of cytokines and improves survival after exposure of rats to LPS from Escherichia coli [14].

Present chapter describes the results of prospective clinical trial about the role of perinatal asphyxia in formation of intestinal barrier dysfunction. The study conducted by the neonatology research group of Azerbaijan Medical University and involved 240 preterm newborns with the risk of perinatal hypoxic encephalopathy with a gestational age of 32–36 weeks. Group 1 included newborns who experienced asphyxia during childbirth, and Group 2 included newborns with chronic intrauterine hypoxia but a relatively favorable course during the intranatal period. The Control group consisted of apparently healthy premature newborns. The levels of intestinal ischemia and neuronal injury makers were detected with standard ELISA method.

Chronic intrauterine hypoxia was determined on the basis of dopplerographic and cardiotocographic examination of the fetus. Asphyxia was diagnosed according to the guidelines of the American Academy of Pediatrics based on an Apgar score of <7 taken in the first 30 min of life [15]. Gestational age was determined by the date of the last menstruation of the mother, ultrasound examination of fetometric parameters. The severity of hypoxic–ischemic encephalopathy was determined based on the Sarnat score [16]. An ultrasound examination of the brain was performed on the third day of life using transducers with a frequency of 5 and 7.5 MHz. The degree of intraventricular hemorrhage was determined according to the Papile classification [17]. The exclusion criteria were a gestational age of <32 weeks, congenital malformations, and manifest forms of TORCH infections.

2. The impact of perinatal asphyxia on the intestinal barrier function

Involvement in the pathological process of the GIT is a logical outcome of severe hypoxic injury [18]. The causes of damage to the GIT are hemodynamic disorders, including regional disorders that involve a decrease in blood circulation in the mesenteric arteries in the first few minutes of life in newborns who have undergone asphyxia [19, 20]. The urinary concentration of the intestinal ischemia marker in newborns exposed to acute asphyxia in labor at 7–10 days old is significantly different from that of both healthy infants and newborns of Group 2 (р_1–3 = 0.05: reliability of the difference for 1–3 days, р_7–10 = 0.005: reliability of the difference for 7–10 days) (Figure 1).

Figure 1.

The levels of serum LBP (A, ng/ml) and urine IFABP (B, ng/ml) of newborns in asphyxia in 1–3 and 7–10 days of life (DOL).

Due to damage to the intestinal mucosa, failure of defense mechanisms, and overgrowth of Gram-negative intestinal flora, colonizing bacteria enter the mesenteric lymph nodes and systemic circulation. Impaired gut barrier function, even in the absence of bacteremia, leads to portal and systemic endotoxemia, which triggers a hypermetabolic and immunoinflammatory response. As can be seen from Figure 1, in Group 2 newborns, ischemia of the intestinal wall was accompanied by a high concentration of a marker of antiendotoxin immunity. In infants in Group 1, the level of LBP was significantly low compared to that in Group 2 infants, which indicated the failure of immune defense mechanisms in acute asphyxia that developed against the background of chronic intrauterine hypoxia (р_1–3 = 0.04: reliability of the difference for 1–3 days, р_7–10 = 0.042: reliability of the difference for 7–10 days). It should be noted that Juli M. Richter et al. indicated an improvement in the processes of regeneration of the intestinal epithelium susceptible to LPS attack after intraperitoneal administration of LBP at high concentrations [14]. The physiological increase in the level of this marker in newborns of the Control group was due to intestinal colonization, contact with bacteria, components of the colostrum, or postpartum maturation of the liver of newborns [21, 22].

As shown in Figure 2, the concentration of ITF, which stabilizes intestinal mucus and attenuates damage to the intestinal barrier, in newborns of Group 1 exceeded that of healthy newborns and infants of Group 2 (37.3 ± 9.0 ng/mL: subgroup asphyxia +, 15.58 ± 3.7 ng/mL: subgroup asphyxia −, р_1–3 = 0.05: reliability of the difference for 1–3 days). Despite the decrease in the concentration of this marker in dynamics, the concentration continued to exceed the indicators of the other two groups (20.1 ± 2.5 ng/mL: subgroup asphyxia +, 7.2 ± 4.8 ng/mL: subgroup asphyxia −, р_7–10 = 0/064: reliability of the difference for 7–10 days). According to Nancy A. Louis, the expression of HIF-1 under hypoxic conditions and early reperfusion after ischemia triggers a physiological response characterized by the activation of functional mucus proteins, such as trefoil factor and P glycoprotein, aimed at preventing inflammatory processes in the intestine [9, 23].

Figure 2.

The levels of ITF (A, ng/ml), MUC2 (B, ng/ml), and LFABP (C, ng/ml) in peripheral blood of newborns in asphyxia in 1–3 and 7–10 days of life (DOL).

At the same time, the level of secreted mucin does not increase in response to hypoxia (Figure 2); on the contrary, in newborns exposed to acute hypoxia, the level of Mucin-2 (MUC2), although not significantly, was slightly lower than that in the other two groups (14.83 ± 9.0 ng/mL: subgroup asphyxia +, 16.82 ± 0.7 ng/mL: subgroup asphyxia −, р_1–3 = 0.16: reliability of the difference for 1–3 days, 10.58 ± 1.4 ng/mL: subgroup asphyxia +, 16.67 ± 1.4 ng/mL: subgroup asphyxia −, р_7–10 = 0.06: reliability of the difference for 7–10 days).

The level of plasma L-FABP in newborns prone to asphyxia was 1.5 times lower than that in newborns of Group 2 (Figure 2) and did not differ from that of the Control group (1.94 ± 0.5 ng/mL: subgroup asphyxia +, 2.94 ± 0.5 ng/mL: subgroup asphyxia − for 1–3 days, 1.34 ± 0.2 ng/mL: subgroup asphyxia +, 2.15 ± 0.3 ng/mL: subgroup asphyxia − for 7–10 days). In general, on days 1–3 and 7–10, no significant differences in relation to this indicator were found between the studied groups (р_1–3 = 0.172: value of difference for 1–3 days, р_7–10 = 0.098: reliability of the difference for 7–10 days).

Despite chronic hypoxia and weakness of autoregulation in the GIT and kidneys compared to other vascular pools, the liver, due to its blood supply from two sources, is better protected from hypoxia and ischemia. Thus, newborns with a low birth weight prone to acute birth asphyxia are characterized by a high detection rate of organ and systemic disorders of posthypoxic origin. The obtained results showed that perinatal hypoxia initiates processes leading to an increase in the permeability of cell membranes, neuronal death due to necrosis or apoptosis, disruption of the integrity of the blood–brain barrier structure, and entry of brain antigens into systemic circulation, stimulating the immune system to produce antibrain antibodies. Disorders of the systemic and peripheral circulation, disorders of oxygen uptake, and delivery to tissues accompanying perinatal asphyxia develop a number of pathophysiological and pathobiochemical cascades that lead to secondary damage to the intestines and kidney parenchyma.

3. Endothelial dysfunction and the formation of the functional status of the intestinal mucosal barrier in newborns with a low birth weight prone to perinatal hypoxia

Endothelial dysfunction against the background of hypoxia and ischemia is accompanied by central nervous system lesions as well as dysfunctions of peripheral organs and systems [24, 25]. Visceral hypoperfusion is accompanied by the activation of indigenous microflora, damaging the immature intestinal barrier. Loss of the intestinal barrier can lead, due to the resorption of endotoxins, bacteria, and other substances, to systemic inflammatory reaction syndrome, remote organ damage, and multiple organ failure [26]. Considering the above, the purpose of this study was to determine the effect of endothelial dysfunction on the levels of markers, reflecting the functional state of the digestive system in newborns with a low birth weight with perinatal hypoxia.

Neurons and glial cells, whose need for energy supply is higher than that of all other cells of the body, are the first to suffer under conditions of oxygen deficiency. In this study, the biochemical marker of the destruction of nerve cells and the permeability of the blood–brain barrier in newborns of Group 1 and 2 significantly exceeded the indicators of the Control group on days 1–3 and 7–10 of life (Figure 3). The highest values of this marker are typical for newborns prone to acute birth asphyxia. Degradation of NMDA receptors as a result of neurotoxicity, assessed by the level of antibodies to NR2, in newborns of Group 1 significantly differed from the indicators of the Control group for both the first and second measurements, although a significant difference between Groups 1 and 2 was not found. This may be due, on the one hand, to the peculiarities of the immune response of immature newborns, which significantly predominate in the group subject to acute birth asphyxia. On the other hand, a longer period of time is required for the formation of ischemia foci and the accumulation of antibodies to glutamate receptors. The dynamics of the content of vasoregulatory markers in the peripheral blood in the studied groups are also shown in Figure 3. Both Group 1 and 2 were characterized by high concentrations of NO, while a significant difference in relation to this indicator was found between Group 1 and the Control group. Despite the level of endothelin-1 being initially high in both groups subject to chronic intrauterine hypoxia, in the group of newborns with acute asphyxia, the content of this marker sharply decreased toward the end of the perinatal period.

Figure 3.

The blood concentrations of NR2 (A, ng/ml), NSE (B, ng/ml), NO (C, ng/ml), and ET-1 (D, ng/ml) in infants with hypoxic–ischemic encephalopathy in 1–3 and 7–10 days of life (DOL). *Significance of difference in comparison with control infants; significance of difference in comparison with second group.

Violation of the vasoregulatory function of the vascular endothelium, manifested by a low level of endothelin-1 against the background of a high concentration of NO, in chronic intrauterine hypoxia complicated by acute birth asphyxia, being a causative factor in reducing blood pressure, leads to tissue ischemia. Visceral hypoperfusion in children with perinatal asphyxia is the leading cause of abdominal complications. Ischemia triggers a vicious circle of damage to the gastrointestinal mucosa and serves as a trigger for the formation of multiple organ failure [27]. The level of the intestinal ischemia marker I-FABP in newborns of Group 1 and 2 did not significantly exceed that of the Control group in the first days of life; however, it almost doubled in dynamics and differed significantly from the level of healthy infants (Figure 4).

Figure 4.

The blood concentrations of IFABP (A, ng/ml), LFABP (B, ng/ml), and LBP (C, ng/ml) in infants with hypoxic–ischemic encephalopathy in 1–3 and 7–10 days of life (DOL). *Significance of difference in comparison with control infants; significance of difference in comparison with group 1.

Due to damage to the intestinal mucosa, failure of defense mechanisms, and overgrowth of Gram-negative intestinal flora, colonizing bacteria penetrate the mesenteric lymph nodes and systemic circulation (bacterial translocation) [28]. Violation of the intestinal barrier function, even in the absence of bacteremia, leads to portal and systemic endotoxemia, which serves as a trigger for a hypermetabolic and immunoinflammatory response [29]. The level of plasma L-FABP in newborns subject to asphyxia was 1.5 times lower than that in newborns of Group 2 and did not differ from that in the Control group. In general, on days 1–3 and 7–10, no significant differences in relation to this indicator were found between the studied groups. Despite chronic hypoxia and weakness of autoregulation in the GIT and liver compared to other vascular pools, the liver, due to its blood supply being from two sources, is better protected from hypoxia and ischemia. As can be seen in Figure 4, in newborns of Group 2, ischemia of the intestinal wall was accompanied by a high concentration of the marker of antiendotoxin immunity. In children in Group 1, the level of LBP was significantly lower than that in Group 2, indicating the failure of immune defense mechanisms in acute asphyxia that developed against the background of chronic intrauterine hypoxia.

Juli M. Richter et al. indicated an improvement in the processes of regeneration of the intestinal epithelium susceptible to LPS attack after intraperitoneal administration of LBP at high concentrations [14]. The physiological increase in the level of this marker in newborns of the Control group was due to intestinal colonization, contact with bacteria, colostrum components, or postpartum maturation of the liver [30]. The concentration of ITF, which stabilizes intestinal mucus and attenuates damage to the intestinal barrier, in newborns of Group 1 exceeded that of healthy newborns and infants in Group 2 (Figure 5). This difference was significant only for the Control group. Despite the decrease in this marker in dynamics, its content continued to exceed the indicators of the other two groups. According to Nancy A. Louis, HIF1 expression under hypoxic conditions and early reperfusion after ischemia trigger a physiological response characterized by the activation of functional mucus proteins, such as trefoil factor and P glycoprotein, aimed at preventing inflammatory processes in the intestine [23, 31]. At the same time, the level of secreted mucin does not increase in response to hypoxia. On the contrary, in this study, in newborns exposed to acute hypoxia, the level of MUC2, although not significantly, was somewhat lower than that of the other two groups.

Figure 5.

The blood concentrations of ITF (A, ng/ml) and MUC-2 (B, ng/ml) in infants with hypoxic–ischemic encephalopathy in 1–3 and 7–10 days of life (DOL). *Significance of difference in comparison with control infants.

Thus, newborns with a low birth weight subject to acute birth asphyxia are characterized by a high detection rate of organ and systemic disorders of posthypoxic origin. The results obtained showed that perinatal hypoxia initiated processes leading to an increase in the permeability of cell membranes, neuron death due to necrosis and/or apoptosis, disruption of the integrity of the blood–brain barrier structure, and entry of brain antigens into the systemic circulation, stimulating the immune system to produce antibrain antibodies. Involvement in the pathological process of the GIT is a logical outcome of severe hypoxic injury. The cause of damage to the digestive system is hemodynamic disturbances as a result of endothelial dysfunction, including regional dysfunction with a decrease in blood circulation in the mesenteric arteries in the first minutes of life in newborns who have experienced asphyxia. Disorders of the systemic and peripheral circulation as well as disorders of oxygen uptake and delivery to tissues accompanying perinatal asphyxia result in the development of a number of pathophysiological and pathobiochemical cascades, leading to secondary damage to the GIT.