Clinical determinants and impact of hemorrhagic lesions on intestinal pathology in preterm infants with surgical necrotizing enterocolitis

1 Introduction

Necrotizing enterocolitis (NEC) is inflammatory bowel necrosis seen in 5–10% of very low birth weight infants [1]. The disease remains a leading cause of morbidity and mortality, surgical intervention, and prolonged medical needs in the surviving infants [2–8]. Infants with advanced NEC usually undergo bowel resection after an exploratory laparotomy, and the remaining intestine is managed with primary anastomoses and/or intestinal stoma.

The resected bowel typically shows coagulation necrosis, inflammation, interstitial hemorrhages, and reparative changes on the pathology examination. The severity of these pathological abnormalities may correlate with the disease’s severity and the pace of progression and may help predict clinical outcomes [9, 10]. For example, our previous study reported worse clinical outcomes for infants with incomplete resected margins in neonates with surgical necrotizing enterocolitis [11]. In addition, we have recently reported an association between bowel hemorrhage (OR 7.79 [95% CI: 2.19–27.72]; p = 0.002) and Grade 3-4 white matter brain injury in preterm infants with surgical NEC [12].

We have previously reported that neonates with fulminant NEC (death within 48 hours of NEC onset) frequently developed thrombocytopenia, lymphopenia, neutropenia, and leukopenia. Neonates who had also received platelet transfusions before and red blood cell transfusions after the onset of NEC were associated with a higher likelihood of developing fulminant disease [13]. This suggests active bleeding at the intestinal tissue level contributing to subsequent intra-vascular volume depletion, causing hypotension and shock. In clinical practice, occult or frank blood in stools as a presenting symptom is seen in less than 10% of infants with NEC. In the current literature, there is no study analyzing the clinical impact and factors linked with intestinal hemorrhage at the histopathological level in preterm infants with surgical necrotizing enterocolitis. This study aims to determine the clinical and histopathological predictors of intestinal hemorrhage after injury secondary to NEC and its correlation with clinical outcomes in preterm infants.

2 Methods

After Institutional Review Board approval, this retrospective study was conducted at the University of Mississippi Medical Center (UMMC) in Jackson, Mississippi. Our center is a regional referral center for infants with surgical NEC. A detailed review of the medical records identified 193 patients who underwent exploratory laparotomy for advanced NEC (NEC Bell stage III) [14] between January 2000 and December 2018. After excluding 15 infants due to incomplete clinical data and 30 with confounding disorders such as congenital heart disease, intestinal atresia, and spontaneous intestinal perforation (SIP), we identified 148 infants as eligible for this study.

2.2 Pathology

2.2.1 Sample assessment

Hematoxylin & eosin (H&E)-stained sections of surgically resected intestine were evaluated by a team of a board-certified gastrointestinal pathologist and a senior pathology trainee. The trained grossing personnel determined the tissue sections were taken from the representative most affected area of the intestine. See Fig. 1.

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Fig. 1

The H&E stain (20X) of resected intestinal tissue showing grade 4 hemorrhage. In infants with grades 1-2, the hemorrhage was limited to mucosa and submucosa. The arrow indicates areas of hemorrhage.

2.2.2 Pathology Definitions

We evaluated the resected bowel for histopathological evidence of NEC, including coagulative necrosis, inflammation, hemorrhages, and reparative changes. Coagulative necrosis in intestinal cells was defined by the loss of hematoxylin staining of the nuclei and diminished cytoplasmic staining with eosin but with relatively preserved, ‘ghost-like’ crypt-villus histoarchitecture. Inflammation was noted as infiltration by inflammatory cells such as monocytes and neutrophils. Hemorrhages, marked by the extravasation of blood cells, were recorded in various layers of the intestine. Reparative changes included microscopic evidence of neovascularization, increased fibroblasts or myofibroblasts, and epithelial regeneration. The severity of NEC was assessed by the depth to which these histopathological changes (coagulative necrosis, inflammation, hemorrhages, and reparative changes) were seen: Grade 1 was limited to the mucosa, Grade 2 changes extended to the submucosa, Grade 3 to the muscularis, and Grade 4 change was transmural. Histopathological changes were also evaluated based on percentage of tissue involved (low = <30%, medium = 30–60%, and high = >60%).

3 Results

One hundred and forty-eight infants were included in the analysis. Eighty-one infants (81/148, 54.7%) had severe-grade (Grade 3-4) hemorrhage, and sixty-seven infants (67/148, 45.2%) had mild to moderate intestinal hemorrhage.

Our study reports clinical correlates of intestinal hemorrhage in preterm infants with surgical NEC. The infants with severe hemorrhage (Grade 3-4) had significantly lower exposure to antenatal steroids, had higher gestational age, lost more bowel length, had higher mean CRP levels two weeks following the onset of NEC, and had a lower frequency of intestinal failure in the bivariate analysis compared to preterm infants with mild to moderate hemorrhage. However, factors such as exposure to antenatal steroids, lymphocyte count on the onset and 24 hours following NEC diagnosis, and tissue inflammation remained significant on multivariable logistic regression modeling.

In this cohort, infants with severe intestinal hemorrhage received less antenatal steroids, most likely increasing the risk of intestinal hemorrhage in preterm infants. Randomized control trials and meta-analysis have shown that maternal administration of a single course of antenatal corticosteroids is commonly associated with an overall reduction in neonatal death and morbidity, including respiratory distress syndrome, intraventricular hemorrhage, NEC, and neonatal intensive care unit admissions [15]. The effects of glucocorticoids on vascular health and dysfunction may be mediated in part by the activation of endothelial nitric oxide synthase (eNOS) [16].

Infants with severe intestinal bleeding had a median gestation age of 28.5 weeks vs. 26.5 weeks, pointing towards gestational age-specific changes in the vascular structure and responses to various stimuli and functions. The endothelial glycocalyx (EG) has been recognized as an essential determinant of vascular integrity and health. This delicate structure of proteoglycans, glycoproteins, glycosaminoglycans, and associated plasma proteins covers the endothelial cells of the whole vasculature. It is involved in important vascular functions such as the regulation of vessel tone, permeability, cell-endothelial interaction, and hemostasis, as reviewed by Cosgun et al. [17]. EG maturity is gestational and postnatal age-dependent [18].

In this cohort, infants with intestinal hemorrhage had significantly higher coagulative necrosis than other groups. The intestinal vascular resistance is determined by the balance between vasodilatation by nitric oxide and endothelin (ET-1) mediated vasoconstrictor inputs [19]. Furthermore, studies have shown that vascular endothelial growth factor (VEGF) and the VEGF receptor-2 signaling pathway play a vital role in intestinal mucosal microvasculature [20]. In addition, endothelial TLR4 activation is required for NEC development, which impairs intestinal perfusion [21] via inhibition of the vasodilator molecule endothelial nitric oxide synthase (eNOS).

Animals with NEC (rat pups) demonstrated significantly smaller inflow and premucosal arterioles than control animals. The rat pups with NEC also had an altered intestinal arteriolar flow with a distinct “stop-and-go” pattern, suggesting severe vascular dysfunction compared to control animals [22]. The experimental animals also demonstrated an increased expression of inflammatory mediators. In our cohort, we noticed a higher severity of inflammation in infants with intestinal hemorrhage, suggesting a positive correlation between the degree of inflammation and vascular dysfunction leading to intestinal bleed. These studies underline that ischemia and intestinal microcirculation play an important role in NEC pathogenesis; however, it is still unclear whether vascular dysfunction is a primary initiator or a secondary response to intestinal injury, which needs further study.

The infants with intestinal hemorrhage had moderate to high neovascularization and fibroblasts in the intestinal submucosa, suggesting reparative changes in the intestine following the coagulative intestinal necrosis secondary to intestinal ischemia, vascular dysfunction, and inflammation. The inflammatory response following tissue injury plays a vital role in tissue repair [23]. Studies have shown that two-thirds of the leukocyte infiltrate in NEC consists of macrophages and play a crucial role in the pathogenesis of necrosis, inflammation, hemorrhage, and repair in necrotizing enterocolitis [10, 24]. The data has shown that macrophages in neonates with NEC have suppressed TGF-β signaling and augmented nuclear factor-kappa B (NF-κB) activation and cytokine production in areas of tissue damage and high bacterial load [25]. In addition, the macrophages promote healing by removing inflammatory cells, such as neutrophils, by actively ingesting them [24]. In our cohort, infants with severe hemorrhage (Grade 3-4) had significantly lower WBC count, absolute neutrophil counts (ANC), higher lymphocyte percentages up to 48 hours, and elevated CRP up to 2 weeks following the onset of NEC.

In this cohort, infants were thrombocytopenic following NEC onset. Platelets play an important role in controlling hemorrhage within the body. Several previous studies [26–29] have reported thrombocytopenia in surgical NEC. In our previous report, we recorded significant thrombocytopenia in fulminant and non-fulminant NEC (73.0 [41.8; 166] and 68.0 [33.8; 101] surgical non-fulminant) lasting 48 and 96 hours, respectively, and noticed recovery generally after four days [13]. The exact mechanism causing thrombocytopenia in these cases of NEC is still uncertain, but consumptive disorders are likely and include the formation of microthrombi in the diseased intestine [30] and platelet activation from bacterial products leading to aggregation in the microvasculature [31]. Coagulative parameters such as prothrombin time (PT), PT international normalized ratio, activated partial thromboplastin time (APTT), fibrinogen, and platelet count at the time of NEC diagnosis were independently associated with surgical NEC [32]. Our study lacks data on the coagulation parameters.

Our study also has several limitations. First, a single-center, retrospective experience may reduce the study’s generalizability. The retrospective study design affects the completeness of each patient’s available data. Secondly, the small sample size coupled with multiple factors and outcomes yields a high probability of Type I errors from multiple comparisons. The study is observational and descriptive; causality cannot be established with or without formal adjustment for multiple comparisons. Thirdly, our study lacks data on coagulation parameters (PT/INR). We acknowledge the proper determination of each histology category by biomarkers would be ideal, but we did not perform those determinations, which is an entirely different study that would require appreciable funding. In this study, we focused on H &E evaluation of intestinal pathology and its correlation with clinical outcomes in infants with surgical NEC. Finally, the infant cohort was predominantly African American, reflecting the racial distribution in Mississippi.

The study’s strengths are the data describing the clinical correlates of severe intestinal hemorrhage and the association of histopathological and hematological factors from a consecutive single-center cohort in a comprehensive manner.

In conclusion, 54.7% of infants had a severe intestinal hemorrhage in preterm infants with surgical NEC. Those with severe intestinal hemorrhage likely had lower exposure to antenatal steroids, a higher grade of inflammation, and higher lymphocyte counts following NEC onset on multi regression modeling. There is a need for further clinical, animal, and translational studies to evaluate the effect of gestational age, intestinal inflammation, and hematological factors on an intestinal hemorrhage. There is also a need for investigating and discovering biomarkers of vascular injury in preterm infants with surgical NEC. In the future, larger, prospective multi-center studies that include additional clinical detail (e.g., mesenteric perfusion using NIRS) and laboratory predictors such as hematological and stool biomarkers may inform earlier recognition of intestinal bleed in preterm infants with surgical NEC.

Author Contribution

P.M.G designed the study, P.M.G., MD, RT, MO, CM, HS; NV, WBH collected and analyzed the data. All the authors contributed to and approved the manuscript.