B. Biesterveld1, N. Heinzerling2, R. Rentea2, S. Welak3,4, K. Fredrich2, D. Gourlay2,5 1Medical College Of Wisconsin,Milwaukee, WI, USA 2Medical College Of Wisconsin,Surgery,Milwaukee, WI, USA 3Medical College Of Wisconsin,Pediatrics,Milwaukee, WI, USA 4Children’s Hospital Of Wisconsin,Neonatology,Milwaukee, WI, USA 5Children’s Hospital Of Wisconsin,Pediatric Surgery,Milwaukee, WI, USA
Introduction: Necrotizing enterocolitis (NEC) is the most common surgical emergency of the neonate. Previously, we demonstrated intestinal alkaline phosphatase (IAP) activity to be decreased in a neonatal rat model of NEC. Furthermore, enteral IAP supplementation before the onset of NEC prevented NEC development, reduced intestinal inflammation and maintained gut barrier function. It is not known if IAP given after NEC onset can reverse the course of the disease. We hypothesized that enteral IAP given after induction of NEC would not be able to reverse intestinal injury.
Methods: Sprague Dawley pups were delivered 1-day preterm followed by NEC induction with LPS added to formula and hypoxia exposure. All pups received NEC stressors on days 0 and 1. They were subsequently divided into 4 groups: continued NEC stressors (NEC), continued NEC stressors with enteral IAP (NEC+IAP), withdrawal of NEC stressors then formula fed (NEC then FF) or withdrawal of NEC stressors then formula fed with supplemental IAP (NEC then IAP) on days 2,3. Control pups were spontaneously delivered and dam-fed. All pups were then sacrificed on day 4. NEC severity was scored based on H&E stained terminal ileum sections, and AP activity was measured using a colorimetric assay. IAP and IL-6 expression were measured using RT-PCR.
Results: Intestinal AP activity in the NEC group was significantly decreased 0.18 U/mg compared to controls of 0.57 U/mg (p<0.001). Discontinuation of NEC stressors after 2 days (NEC then FF) was associated with increased AP activity to 0.36 U/mg (p<0.01 vs. NEC). IAP supplementation in matched groups, NEC+IAP and NEC then IAP, did not impact AP activity. IAP mRNA expression followed a similar trend as activity, with significantly decreased IAP expression in NEC which significantly increased with withdrawal of NEC stressors on day 2. Similarly, the NEC group had significantly worse mean injury score (2.25 vs. 0.67 in control (p<0.01). Discontinuation of NEC stressors on day 2 resulted in improved intestinal injury scores to 1.14 compared to continued NEC stressors (p<0.01). While, IAP supplementation (NEC then IAP) significantly decreased IL-6 expression two-fold compared to NEC (p<0.05), it did not decrease the intestinal injury when compared to matched groups in NEC vs. NEC+IAP (p=0.50) and NEC then FF vs. NEC then IAP (p=0.54).
Conclusion: This is the first work to demonstrate that the current clinical treatment, to remove the source of NEC, improves intestinal damage and this is associated with a return of IAP expression and activity. When used as a rescue treatment after NEC onset, IAP decreased intestinal inflammation though did not impact the severity of histologic injury. Given the often rapid progression of NEC, and our previous work demonstrating the ability of early IAP supplementation to prevent NEC development, this data supports the use of IAP as a preventive therapy given to all those identified as at risk early in life.