Low Abdominal NIRS Values and Elevated Plasma Intestinal Fatty Acid-Binding Protein in a Premature Piglet Model of Necrotizing Enterocolitis

<div><p>To identify early markers of necrotizing enterocolitis (NEC), we hypothesized that continuous abdominal near-infrared spectroscopy (A-NIRS) measurement of splanchnic tissue oxygen saturation and intermittent plasma intestinal fatty-acid binding protein (<i>p</i>I-FABP) measured every 6 hours can detect NEC prior to onset of clinical symptoms. Premature piglets received parenteral nutrition for 48-hours after delivery, followed by enteral feeds every three hours until death or euthanasia at 96-hours. Continuous A-NIRS, systemic oxygen saturation (SpO₂), and heart rate were measured while monitoring for clinical signs of NEC. Blood samples obtained at 6-hour intervals were used to determine <i>p</i>I-FABP levels by ELISA. Piglets were classified as fulminant-NEC (f-NEC), non-fulminant-NEC (nf-NEC) and No-NEC according to severity of clinical and histologic features. Of 38 piglets, 37% (n=14) developed nf-NEC, 18% (n=7) developed f-NEC and 45% (n=17) had No-NEC. There were significant differences in baseline heart rate (p=0.008), SpO₂ (p<0.001) and A-NIRS (p<0.001) among the three groups. A-NIRS values of NEC piglets remained lower throughout the study with mean for f-NEC of 69±3.8%, 71.9±4.04% for nf-NEC, and 78.4±1.8% for No-NEC piglets (p<0.001). A-NIRS <75% predicted NEC with 97% sensitivity and 97% specificity. NEC piglets demonstrated greater variability from baseline in A-NIRS than healthy piglets (10.1% vs. 6.3%; p=0.04). Mean pI-FABP levels were higher in animals that developed NEC compared to No-NEC piglets (0.66 vs. 0.09 ng/mL;p<0.001). In f-NEC piglets, pI-FABP increased precipitously after feeds (0.04 to 1.87 ng/mL;p<0.001). <i>p</i>I-FABP levels increased in parallel with disease progression and a value >0.25ng/mL identified animals with NEC (68% sensitivity and 90% specificity). NIRS is a real-time, non-invasive tool that can serve as a diagnostic modality for NEC. In premature piglets, low A-NIRS in the early neonatal period and increased variability during initial feeds are highly predictive of NEC, which is then confirmed by rising plasma I-FABP levels. These modalities may help identify neonates with NEC prior to clinical manifestations of disease.</p></div

Baseline measurements of piglets by NEC severity.

<p>NEC—necrotizing enterocolitis; HR—heart rate; bpm—beats per minute; SpO2—oxygen saturation; A-NIRS—abdominal near infrared spectroscopy.</p><p>Baseline measurements of piglets by NEC severity.</p

Continuous Abdominal NIRS-Tissue Oxygen Content of Hemoglobin (StO₂) Measurements.

<p>Abdominal NIRS data stratified by NEC severity groups demonstrated that f-NEC piglets had significantly lower A-NIRS values than both the nf-NEC and No-NEC groups at baseline, and then both NEC groups maintained significantly lower A-NIRS values than No-NEC piglets throughout the majority of the study. <i>‡ = p<0</i>.<i>05</i>, <i>compared to Non-Fulminant NEC; * = p<0</i>.<i>05</i>, <i>compared to No-NEC</i>.</p

Intestinal Fatty Acid Binding Protein (I-FABP) in Plasma.

<p>Plasma I-FABP values stratified by NEC severity groups demonstrating a precipitous rise in the f-NEC group shortly after the initiation of feeds and a more gradual rise in the nf-NEC group that paralleled clinical disease progression. The No-NEC piglets never had an appreciable rise in their serum I-FABP.</p

Premature Pig Model of NEC—Study Design.

<p>This is a graphic representation of our premature piglet model of NEC demonstrating timing of parenteral and enteral nutrition.</p

Premature Pig Model of NEC—Gross and Histological Examination.

<p>A, Gross examination of the abdominal viscera of healthy No-NEC piglet. B, Gross examination of piglet with non-fulminant NEC shows inflamed and congested small bowel with areas of focal necrosis. C, Gross examination of piglet with fulminant NEC shows diffuse necrosis throughout the entire bowel. D, Histologic examination of the normal small intestine of a No-NEC piglet. E, Histologic examination of the small intestine of a piglet with non-fulminant NEC demonstrating moderate mucosal injury, blunting of villi and separation of the basement membrane. F, Histologic examination of the small intestine of a piglet with fulminant NEC demonstrating severe mucosal injury and diffuse pneumatosis.</p

Continuous Systemic Oxygen Saturation (SpO₂) Measurements.

<p>Systemic oxygen saturation data stratified by NEC severity groups demonstrating that f-NEC piglets were significantly more hypoxic at baseline and for the first 18 hours of life until the normalized at 21 hours of life, and nf-NEC were only more hypoxic than No-NEC piglets during the first 3 hours of life. <i>‡ = p<0</i>.<i>05</i>, <i>compared to Non-Fulminant NEC; * = p<0</i>.<i>05</i>, <i>compared to No-NEC</i>.</p

Proximal Jejunum Intestinal Fatty Acid Binding Protein (I-FABP) Abundance.

<p>Western blot data of the proximal jejunum specimens demonstrating that No-NEC piglets had significantly’ higher densitometry readings than NEC piglets.</p

Serum Amyloid A (SAA) in Plasma.

<p>Plasma SAA values stratified by NEC severity groups demonstrating a gradual rise in all piglets, particularly after the initiation of enteral feeds, but no significant differences between the three experimental groups.</p

Continuous Heart Rate Measurements.

<p>Heart rate data stratified by NEC severity groups demonstrating an overall increasing trend in heart rates throughout the study period and that f-NEC and nf-NEC piglets were significantly more tachycardic during the first 24 hours of life than No-NEC piglets. <i>‡ = p<0</i>.<i>05</i>, <i>compared to Non-Fulminant NEC; * = p<0</i>.<i>05</i>, <i>compared to No-NEC</i>.</p