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Oral relaxin maintains intestinal blood flow in a rat model of NEC
Journal of Pediatric Surgery 49 (2014) 961–965
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Oral relaxin maintains intestinal blood flow in a rat model of NEC☆, ☆☆, ★ Paul J. Matheson a, b, 1, Sarah K. Walker b, 1, Alexandra C. Maki b, Saad P. Shaheen a, R. Neal Garrison a, b, Cynthia D. Downard c,⁎ a b c
Robley Rex Veterans Affairs Medical Center at Louisville, KY Hiram C. Polk, Jr., M.D. Department of Surgery, University of Louisville, Louisville, KY Division of Pediatric Surgery, Hiram C. Polk, Jr. M.D. Department of Surgery, University of Louisville, Louisville, KY
a r t i c l e
i n f o
Article history: Received 21 January 2014 Accepted 27 January 2014 Key words: Necrotizing enterocolitis Intestinal vasoconstriction Relaxin Intestinal blood flow
a b s t r a c t Purpose: Intestinal vasoconstriction is a critical step in development of necrotizing enterocolitis (NEC). Relaxin (RLXN), a hormone found in breast milk but absent from formula, is a potent vasodilator. We hypothesized that relaxin-supplemented feeds with an NEC protocol would decrease NEC severity and increase intestinal blood flow. Methods: Timed-pregnant Sprague–Dawley rats were randomly assigned to CONTROL, NEC, NEC + 1xRLXN, or NEC + All Feeds RLXN, and all but CONTROL underwent NEC protocol. NEC + 1xRLXN and NEC + All Feeds RLXN groups were fed relaxin-supplemented formula with the last feed or every feed. At 48 h of life, intestinal blood flow was measured at baseline and after application of 2.5% Delflex® solution. Results: The addition of relaxin to NEC group feeds (1x or All Feeds) improved the degree of ileal injury. Ileal blood flow was decreased in the NEC pups compared to the CONTROLS, but the addition of relaxin to one feed increased baseline ileal blood flow in the NEC group compared to NEC alone. Furthermore, the addition of relaxin to ALL feeds significantly increased baseline ileal blood flow. Conclusion: Pups who received relaxin with all feeds had substantially increased ileal perfusion compared to control pups. Our data suggest that relaxin supplementation maintains intestinal blood flow and results in less histologic NEC. © 2014 Elsevier Inc. All rights reserved.
Necrotizing enterocolitis (NEC) predominantly affects premature infants, and those who are formula fed are 6 to 10 times more likely to develop NEC compared to similar infants who receive breast milk [1]. While the benefits of breast feeding are widely touted, the components specific to breast milk and absent from formula, especially those with effects on intestinal development and immune protection, are subject to investigation as potential therapeutic targets in NEC. Relaxin (RLXN) is a 6 kDa hormone of pregnancy that leads to relaxation of the pubic symphisis [2]. Relaxin is also a potent vasodilator which is responsible for the relative volume expanded state of pregnancy and is associated with decreased renal vascular resistance during pregnancy [3]. The cardiovascular effects of relaxin have made it a novel treatment in heart failure, and current phase three clinical trials of recombinant relaxin have shown improved ☆ Dr. Matheson contributed to the project design, execution (animal care and study), data collection, data analysis, and performed final critical revision of the manuscript. ☆☆ Dr. Walker contributed to the project design, execution (animal care and study), data collection, and provided the initial draft of the manuscript. ★ Supported by James R. Petersdorf Fund of Norton Healthcare and Kosair Charities. ⁎ Corresponding author at: Division of Pediatric Surgery, Hiram C. Polk, Jr., M.D. Department of Surgery, University of Louisville, 315 E. Broadway, Ste 565, Louisville, KY 40202. E-mail address: [email protected] (C.D. Downard). 1 Drs. Matheson and Walker have contributed equally to this study. http://dx.doi.org/10.1016/j.jpedsurg.2014.01.032 0022-3468/© 2014 Elsevier Inc. All rights reserved.
outcomes, decreased mortality, and decreased end organ damage in this clinical scenario [4–6]. We have previously shown that enteral relaxin supplementation given for 24 h increases intestinal blood flow in an animal model of NEC [7]. The purpose of this project is to 1) evaluate relaxin as a preventative agent for NEC, 2) to analyze its utility as a rescue therapy when NEC is suspected, and 3) to evaluate its effect on intestinal perfusion when used in conjunction with direct peritoneal resuscitation. 1. Materials and methods The research protocol was approved by the Institutional Animal Care and Use Committee, Biohazard Safety Committee, and Research and Development Committee prior to studies. Four timed pregnant Sprague–Dawley dams (Harlan, Indianapolis, IN) were maintained in an AAALAC-approved Veterinary Medical Unit at the Robley Rex VA Medical Center in Louisville, KY for at least one week prior to delivery of pups. Dams were acclimated on a 12 h light–dark cycle and were allowed rat chow and water ad libitum. The rat pups were randomized to group by litter for inclusion in the CONTROL group (n = 12) which were vaginally delivered and dam fed, or experimentally-induced NEC groups (3 groups, n = 11-13 per group) which were delivered by Caesarian section 12 h prematurely under carbon dioxide anesthesia.
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The CONTROL group was time-matched at post-delivery hour of life to the NEC groups. As previously reported, experimental NEC was induced by formula feeds via gastric gavage every 4–5 h, intermittent hypoxia (100% nitrogen gas for 60 s) and hypothermia (4 °C for 10 min) every 12 h, and a single gastric dose of lipopolysaccharide at 12 h of life (SigmaAldrich, St. Louis, MO) [8–10]. The formula feeds consisted of 20 g Similac 60/40 (Ross Pediatrics, Columbus, OH) dissolved in 100 mL Esbilac (Pet-Ag, New Hampshire, IL) puppy formula. Feeds were started at 0.1 mL/feed and were advanced to 0.15 mL/feed at 24 h of life and 0.2 mL/feed at 48 h of life. These feeds were calculated to supply approximately 200 kcal/kg/day caloric intake. The experimental protocol is outlined in Fig. 1. At birth, NEC pups were randomized to the following groups: 1) NEC alone (n = 11), 2) NEC with a single oral supplemental dose of rat relaxin (0.25 ng/feed, Sigma-Aldrich, St. Louis, MO) in the final feed prior to blood flow experiments (n = 12, NEC + 1xRLXN), or 3) NEC with oral supplemental doses of relaxin (0.25 ng/0.1 mL) in all formula feeds (n = 13, NEC + All Feeds RLXN). CONTROL animals were dam fed and received no relaxin supplementation. At 48 h of life, the pups were weighed and anesthetized with isoflurane with induction of 3.5% and maintenance of 1.0% on 1 L oxygen per minute. All animals were separated from their littermates and underwent laparotomy for the study of ileal blood flow by laser Doppler flowmetry (Periflux system, Perimed AB, Järfälla, Sweden). Gross appearance of the intestines was recorded for signs of necrosis, hemorrhage, gaseous distension, or perforation. Any blood or stool that was present was flushed from the peritoneum with prewarmed saline (37.0 °C). Body temperature was maintained at 37 °C by feedback controller. A 7-site integrating flow probe was placed over the terminal ileum and organ blood flow was recorded. Correct positioning of the flow probe was verified visually at each time point for the duration of the experiment. Warmed saline was dripped on the peritoneum and the animals were allowed to equilibrate for 20 min prior to initiation of the flow protocol. In each animal, laser Doppler
perfusion was recorded in the ileum at two baseline time points 10 min apart. If flow was stable (less than 10% difference) during that baseline period the study was continued. No pups were excluded for unstable blood flow at baseline. After the completion of the baseline period, a 2.5% peritoneal dialysis solution prewarmed to 37.0 °C was dripped in the peritoneum and flow was recorded for an additional 10 min to evaluate if the alteration in intestinal blood flow seen with relaxin supplementation works against or in concert with our previously demonstrated changes in intestinal blood flow seen with the addition of peritoneal dialysis solution [11]. At the completion of the laser Doppler flowmetry studies, the degree of bladder distension in the pups was observed and recorded. The grading scale used was: 0 when the bladder appeared completely empty, 1 when the bladder diameter was N 0 and b 1 mm, 2 when bladder distension was N 1 and b 2, and 3 when bladder distension was N 2 mm. In addition, ileum samples were obtained, and placed in 10% neutral buffered formalin for 16 h and then placed in 70% ethanol for hematoxylin and eosin (H&E) staining. Histopathology scores for signs of NEC were obtained from a pathologist masked to group and experimental protocols. The samples were graded as follows: Grade 0, normal or no damage; Grade 1, epithelial cell lifting or separation; Grade 2, sloughing of epithelial cells to mid villus level; Grade 3, necrosis of entire villus; or Grade 4, transmural necrosis [12]. All data are expressed as mean ± standard error of the mean (SEM). Differences between groups (CONTROL, NEC, NEC + 1xRLXN, and NEC + All Feeds RLXN) and time points (baseline1, baseline2, DPR1 min, DPR5 min, and DPR10 min) were determined by two-way analysis of variance (ANOVA) using SigmaPlot for Windows 11.1.0.102 (Systat Software, Inc., San Jose, CA). Differences between baseline intestinal perfusion, bladder distension, body weights, or histopathologic scores were determined by one-way ANOVA. The null hypothesis was rejected a priori at P b 0.05. When differences were found using ANOVA, the post hoc Tukey–Kramer honestly significant difference test was applied.
Fig. 1. Experimental timeline. Experimental timeline. DPR, 2.5% Delflex; BL, baseline time point; NBF, 10% neutral buffered formalin; RLXN, rat relaxin-1 at 0.25 ng/feed.
P.J. Matheson et al. / Journal of Pediatric Surgery 49 (2014) 961–965
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2. Results A summary of baseline values of body weight, bladder distension score, and ileal blood flow for each group is shown in Table 1. These data show that despite significant caloric delivery in the formula feeds which were designed to match caloric delivery in the dam feeds, the body weights in the NEC groups were significantly lower at 48 h of life compared to the time-matched CONTROL group. Bladder distension was significantly higher in the CONTROL group than in the NEC groups, suggesting improved hydration in the dam fed CONTROLs. A single supplemental oral dose of relaxin improved hydration status compared to NEC alone, and supplemental oral relaxin in all feeds further improved hydration status. Fig. 2 shows that with regard to histopathologic grading for signs of NEC, the CONTROL animals were all graded as zero by a pathologist masked to group and experimental protocol. In addition, none of the CONTROL animals had any visual signs of NEC when gross condition of the intestines was recorded after the laparotomy. The NEC Grade ranged from 1 to 4 in the NEC alone group which was significantly worse than the CONTROL group (2.04 ± 0.19 versus 0.0 ± 0.0, P b 0.05). The single oral dose of relaxin in the NEC + 1xRLXN group improved NEC Grade which ranged from 0 to 2 (1.12 ± 0.12, P b 0.05 versus NEC), and relaxin added to all feeds further improved NEC Histopathology Grade which ranged from 0 to 1 (0.46 ± 0.14, P b 0.05 versus all groups). Fig. 3 shows the changes in ileal perfusion over time as evaluated by laser Doppler flowmetry. Baseline blood flow was decreased in the NEC group compared to the CONTROL group (58.2 ± 2.2 versus 35.9 ± 1.1 PU, P b 0.05). The addition of a single oral relaxin supplement in the NEC + 1xRLXN group significantly improved ileal baseline blood flow compared to the NEC group (35.9 ± 1.1 versus 62.9 ± 1.7 PU, P b 0.05), as did oral relaxin supplements in all feeds (35.9 ± 1.1 versus 84.5 ± 2.6 PU, P b 0.05). The addition of DPR to the NEC group improved blood flow to levels near the baseline levels in the CONTROL group. The addition of a single oral relaxin dose improved blood flow to the CONTROL levels and the addition of relaxin to all feeds improved ileal perfusion to levels higher than CONTROL, which remained elevated to CONTROL levels during the addition of DPR.
3. Discussion
Fig. 2. Histologic grading by group. NEC Histopathology Grades in CONTROL, NEC, NEC + 1x RLXN, and NEC + All Feeds groups. CONTROLs had no signs of NEC (all graded zero), while the NEC alone group had significant NEC grading ranging from 1 to 4 (none scored as normal). A single oral supplement of RLXN prevented the ileal damage observed in the NEC alone group, and RLXN supplements in all feeds further improved ileal condition. *P b 0.05 versus CONTROL, †P b 0.05 versus NEC alone, and ‡P b 0.05 versus NEC + 1x RLXN.
tions. Its effect on the intestinal microvasculature has been appreciated when Bigazzi and colleagues demonstrated that topical relaxin application causes vasodilation in the mesocaecum of adult rats [13]. The hormone relaxin acts via g-protein coupled receptors RXFP1 and RXFP2, and we have previously demonstrated that both are present in the neonatal rat intestine of control and NEC rat pups [14]. We have also shown that oral relaxin supplementation in an animal NEC model increases intestinal blood flow at 24 h of life [7]. In this series of experiments, neonatal animals that underwent the NEC protocol but received relaxin with all feeds (NEC + All Feeds
Compromised intestinal blood flow is a critical event in development of NEC [10]. Numerous reports suggest that formula feeding is a definite risk factor for NEC, therefore a logical therapeutic target for either preventing or treating NEC would be a structure present in breast milk and missing from formula which might affect intestinal blood flow. Relaxin is a 6 kDa hormone most often associated with pelvic girdle relaxation in pregnancy. Its vasoactive properties have been recently described, and it is a potent vasodilator even at picomolar concentraTable 1 Body weight, bladder distention, and baseline perfusion. Group
Body Weight, g
Bladder Distension Score
Baseline ileal blood flow, PU
CONTROL, n = 12 NEC along, n = 11 NEC + RLXN, n = 12 NEC + All Feeds, n = 13
8.3 ± 0.2
2.9 ± 0.1
58.2 ± 2.2
5.2 ± 0.1*
1.2 ± 0.1*
35.9 ± 1.1*
4.8 ± 0.1*
1.7 ± 0.1*
62.9 ± 1.7*†
5.4 ± 0.1*‡
2.2 ± 0.1*†‡
84.5 ± 2.6*†‡
Summary of body weights, bladder distension scores, and ileal blood flow baseline values. * P b 0.05 versus CONTROL, †P b 0.05 versus NEC, ‡P b 0.05 versus NEC + 1x RLXN by one-way ANOVA and Tukey-Kramer honestly significant difference test. Abbreviations: PU, perfusion units; g, grams; RLXN, rat relaxin-1 at 0.25 ng/feed; N, number of rats per group; and NEC, necrotizing enterocolitis.
Fig. 3. Ileal perfusion over time. Ileum blood flow as assessed by laser Doppler Flowmetry. The flow in the NEC group was significantly lower than the time-matched CONTROL group and the addition of DPR to the peritoneum of the NEC animals increased ileal perfusion to near CONTROL baseline levels. The addition of a single oral dose of RLXN to the NEC + 1x RLXN group improved ileal blood flow, both at baseline and in DPR stimulated conditions. The addition of RLXN to all of the feeds in the NEC + All Feeds group further improved ileal perfusion compared to NEC alone or NEC + 1x RLXN. *P b 0.05 versus CONTROL, †P b 0.05 versus NEC alone, and ‡P b 0.05 versus NEC + 1x RLXN.
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RLXN) had substantially increased ileal perfusion compared to not only NEC animals but also CONTROL animals at baseline. In addition, animals that were subjected to the NEC protocol but only received relaxin with one feed four hours prior to sacrifice (NEC + 1xRLXN) had baseline intestinal blood flow greater than NEC animals, but more closely approximated the blood flow in CONTROL animals. Interestingly, the NEC + All Feeds RLXN animals had very limited response to treatment with 2.5% Delflex®, which has previously been shown to increase intestinal blood flow in our animal model of NEC [11]. This suggests that continual relaxin supplementation maximally dilates the intestinal microvasculature, and further increase in flow is not possible. NEC animals who received relaxin supplementation with only one feed (NEC + 1xRXLN) had substantially increased ileal perfusion compared to baseline NEC animals, supporting that even transient relaxin supplementation in formula feeds affects intestinal blood flow. This series of experiments was designed to simulate use of relaxin not only as a preventative treatment for those at risk for NEC (NEC + All Feeds RLXN group) but also as a rescue therapy for those already diagnosed with NEC (NEC + 1xRLXN group). The NEC + 1xRLXN animals who had undergone the NEC protocol identical to the NEC pups had increased baseline ileal blood flow compared to the NEC pups. Moreover, the NEC + 1xRLXN pups had less clinically apparent NEC than the NEC alone group even after a single dose of relaxin, citing a possibility for arrest of progression of NEC with relaxin supplementation. The NEC model we use has been shown to produce 50% of grade 2 NEC by 48 h and 75% at 96 h [7]. In our study, the histology scores of the NEC + 1xRLXN group fall between the baseline NEC group and the NEC + All Feeds RLXN group, suggesting that improvement in histologic-apparent cellular injury can be achieved with the increased blood flow obtained with a single dose of relaxin. Relaxin has been demonstrated in rats, dogs, pigs, and humans with levels present in the corpora luteum helping maintain pregnancy, prepare the uterus for delivery, and promote breast and nipple development for nursing offspring [15–27]. Mammary tissue is the source of relaxin in nursing animals and is thought to be important for specific growth and development using a lactocrine hypothesis [19,28]. We confirmed this in rat mothers and also demonstrated that there were no detectable levels of relaxin in pups who did not receive supplementation, similar to previous studies with pigs and dogs [16–18,23]. Prior to clinical treatment of infants with NEC with relaxin supplementation, we plan to evaluate the effect of intermittent and continuous relaxin supplementation in the NEC protocol at further time points. Human use of relaxin has been associated with bleeding complications such as metromenorrhagia and epistaxis [29,30]. We have noted gastrointestinal hemorrhage in our initial studies with relaxin supplementation in our animal model at higher doses, therefore precise dose–response will need to be evaluated prior to clinical application in human infants (unpublished data). Relaxin is a potent vasodilatory hormone which is in clinical trials in other diseases, has oral bioavailability, and is a hormone associated with pregnancy which is missing in the intestine of formula fed infants. We believe that the vasodilatory effects of relaxin are potentially responsible for the decreased incidence of NEC in breast fed infants, and enteral supplementation of relaxin could either prevent or treat the intestinal hypoperfusion seen in NEC. Our data support the intestinal vascular effects of continuous and intermittent enteral relaxin supplementation and encourage further investigation as a potential new preventative and rescue therapy for NEC. Acknowledgments We would like to acknowledge the technical assistance of Jessica A. Shepherd, Amy J. Matheson, and Samuel A. Matheson. References [1] Lucas A, Cole TJ. Breast milk and neonatal necrotising enterocolitis. Lancet 1990;336:1519–23.
[2] Hisaw FL. Experimental relaxation of the pubic ligament of the guinea pig. Proc Soc Exp Biol 1926;23:661–3. [3] Conrad KP. Unveiling the vasodilatory actions and mechanisms of relaxin. Hypertension 2010;56:2–9. [4] King A. Heart failure. Promising data for serelaxin. Nat Rev Cardiol 2013;10:3. [5] Metra M, Cotter G, Davison BA, et al. Effect of serelaxin on cardiac, renal, and hepatic biomarkers in the Relaxin in Acute Heart Failure (RELAX-AHF) development program: correlation with outcomes. J Am Coll Cardiol 2013;61:196–206. [6] Teerlink JR, Cotter G, Davison BA, et al. Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF): a randomised, placebocontrolled trial. Lancet 2013;381:29–39. [7] Maki AM, Matheson PJ, Shepherd JA, et al. Enteral relaxin supplementation ameliorates necrotizing enterocolitis associated intestinal hypoperfusion. Presented at the American Academy of Pediatrics Section on Surgery, New Orleans, LA, October 19-21; 2012. [8] Barlow B, Santulli TV. Importance of multiple episodes of hypoxia or cold stress on the development of enterocolitis in an animal model. Surgery 1975;77:687–90. [9] Barlow B, Santulli TV, Heird WC, et al. An experimental study of acute neonatal enterocolitis—the importance of breast milk. J Pediatr Surg 1974;9:587–95. [10] Downard CD, Grant SN, Matheson PJ, et al. Altered intestinal microcirculation is the critical event in the development of necrotizing enterocolitis. J Pediatr Surg 2011;46:1023–8. [11] Maki AC, Matheson PJ, Shepherd JA, et al. Intestinal microcirculatory flow alterations in necrotizing enterocolitis are improved by direct peritoneal resuscitation. Am Surg 2012;78:803–7. [12] Feng J, El-Assal ON, Besner GE. Heparin-binding epidermal growth factor-like growth factor decreases the incidence of necrotizing enterocolitis in neonatal rats. J Pediatr Surg 2006;41:144–9. [13] Bigazzi M, Del Mese A, Petrucci F, et al. The local administration of relaxin induces changes in the microcirculation of the rat mesocaecum. Acta Endocrinol (Copenh) 1986;112:296–9. [14] Walker SM, Maki AC, Matheson PJ, et al. Complex regulation of intestinal relaxin receptor RXFP-1 levels is time-dependent and altered by necrotizing enterocolitis in rat pups. Presented at the American Academy of Pediatrics Section on Surgery, New Orleans, LA, October 19-21; 2012. [15] Eddie LW, Sutton B, Fitzgerald S, et al. Relaxin in paired samples of serum and milk from women after term and preterm delivery. Am J Obstet Gynecol 1989;161:970–3. [16] Goldsmith LT, Lust G, Steinetz BG. Transmission of relaxin from lactating bitches to their offspring via suckling. Biol Reprod 1994;50:258–65. [17] Steinetz BG, Williams AJ, Lust G, et al. Transmission of relaxin and estrogens to suckling canine pups via milk and possible association with hip joint laxity. Am J Vet Res 2008;69:59–67. [18] Bagnell CA, Steinetz BG, Bartol FF. Milk-borne relaxin and the lactocrine hypothesis for maternal programming of neonatal tissues. Ann N Y Acad Sci 2009;1160:152–7. [19] Frankshun AL, Ho TY, Reimer DC, et al. Characterization and biological activity of relaxin in porcine milk. Reproduction 2011;141:373–80. [20] Frankshun AL, Ho TY, Steinetz BG, et al. Biological activity of relaxin in porcine milk. Ann N Y Acad Sci 2009;1160:164–8. [21] Masters RA, Crean BD, Yan W, et al. Neonatal porcine endometrial development and epithelial proliferation affected by age and exposure to estrogen and relaxin. Domest Anim Endocrinol 2007;33:335–46. [22] Yan W, Ryan PL, Bartol FF, et al. Uterotrophic effects of relaxin related to age and estrogen receptor activation in neonatal pigs. Reproduction 2006;131:943–50. [23] Yan W, Wiley AA, Bathgate RA, et al. Expression of LGR7 and LGR8 by neonatal porcine uterine tissues and transmission of milk-borne relaxin into the neonatal circulation by suckling. Endocrinology 2006;147:4303–10. [24] Sherwood OD, Chang CC, Bevier GW, et al. Radioimmunoassay of plasma relaxin levels throughout pregnancy and at parturition in the pig. Endocrinology 1975;97:834–7. [25] Sherwood OD, Crnekovic VE, Gordon WL, et al. Radioimmunoassay of relaxin throughout pregnancy and during parturition in the rat. Endocrinology 1980;107:691–8. [26] Sherwood OD, Nara BS, Welk FA, et al. Relaxin levels in the maternal plasma of pigs before, during, and after parturition and before, during, and after suckling. Biol Reprod 1981;25:65–71. [27] Hwang JJ, Lee AB, Fields PA, et al. Monoclonal antibodies specific for rat relaxin. V. Passive immunization with monoclonal antibodies throughout the second half of pregnancy disrupts development of the mammary apparatus and, hence, lactational performance in rats. Endocrinology 1991;129:3034–42. [28] Steinetz BG, Horton L, Lasano S. The source and secretion of immunoactive relaxin in rat milk. Exp Biol Med (Maywood) 2009;234:562–5. [29] Seibold JR, Clements PJ, Furst DE, et al. Safety and pharmacokinetics of recombinant human relaxin in systemic sclerosis. J Rheumatol 1998;25:302–7. [30] Seibold JR, Korn JH, Simms R, et al. Recombinant human relaxin in the treatment of scleroderma. A randomized, double-blind, placebo-controlled trial. Ann Int Med 2000;132:871–9.
Discussion Discussant Dr. Joel Shilyansky, (Iowa City, IA): Can you compare relaxin to breast milk as far as its effects in this kind of model, and is relaxin the main component of breast milk that improves outcomes with NEC?
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Response Dr. Walker: There are other components of breast milk that are absent from formula. We were most interested in relaxin because of its vaso-active properties since our lab works on the micro perfusion of the intestine. Discussant Dr. Shilyansky: Can I ask a controversial question. Why not just give the breast milk as opposed to small components of it.
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hyperemia, post-prandial increase in blood flow outside of the model of NEC, and if so how is that affected by relaxin? Response Dr. Walker: Most of our experimentation is done in about four hours after the last feeding. We feed the last time at 6:00 AM and then perform our experiments at 10:00 AM. To my knowledge we have not looked at blood flow directly after feeding. However, that is an excellent point to investigate.
Response Dr. Walker: Breast milk supplementation would be ideal, especially breast milk in infants. However, we are also looking at this as a possible rescue therapy as well, and I'm not sure that just a single dose of breast milk would be as preventative in improving intestinal blood flow.
Discussant Dr. Shilyansky: Just to follow up are there inhibitors of relaxin? Perhaps if you gave an inhibitor of Relaxin along with breast milk you might prevent the effect of breast milk and that would be more supportive of your hypothesis.
Discussant Dr. Brad Warner, (St. Louis, MO): Congratulations that's a beautiful study. I had some questions. Do you see using your model where you are gauging blood flow? Do you see a post-prandial
Response Dr. Walker: There is currently no inhibitor for relaxin per se clinically available, but that would be an excellent study in the small model.
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Oral relaxin maintains intestinal blood flow in a rat model of NEC☆, ☆☆, ★ Paul J. Matheson a, b, 1, Sarah K. Walker b, 1, Alexandra C. Maki b, Saad P. Shaheen a, R. Neal Garrison a, b, Cynthia D. Downard c,⁎ a b c
Robley Rex Veterans Affairs Medical Center at Louisville, KY Hiram C. Polk, Jr., M.D. Department of Surgery, University of Louisville, Louisville, KY Division of Pediatric Surgery, Hiram C. Polk, Jr. M.D. Department of Surgery, University of Louisville, Louisville, KY
a r t i c l e
i n f o
Article history: Received 21 January 2014 Accepted 27 January 2014 Key words: Necrotizing enterocolitis Intestinal vasoconstriction Relaxin Intestinal blood flow
a b s t r a c t Purpose: Intestinal vasoconstriction is a critical step in development of necrotizing enterocolitis (NEC). Relaxin (RLXN), a hormone found in breast milk but absent from formula, is a potent vasodilator. We hypothesized that relaxin-supplemented feeds with an NEC protocol would decrease NEC severity and increase intestinal blood flow. Methods: Timed-pregnant Sprague–Dawley rats were randomly assigned to CONTROL, NEC, NEC + 1xRLXN, or NEC + All Feeds RLXN, and all but CONTROL underwent NEC protocol. NEC + 1xRLXN and NEC + All Feeds RLXN groups were fed relaxin-supplemented formula with the last feed or every feed. At 48 h of life, intestinal blood flow was measured at baseline and after application of 2.5% Delflex® solution. Results: The addition of relaxin to NEC group feeds (1x or All Feeds) improved the degree of ileal injury. Ileal blood flow was decreased in the NEC pups compared to the CONTROLS, but the addition of relaxin to one feed increased baseline ileal blood flow in the NEC group compared to NEC alone. Furthermore, the addition of relaxin to ALL feeds significantly increased baseline ileal blood flow. Conclusion: Pups who received relaxin with all feeds had substantially increased ileal perfusion compared to control pups. Our data suggest that relaxin supplementation maintains intestinal blood flow and results in less histologic NEC. © 2014 Elsevier Inc. All rights reserved.
Necrotizing enterocolitis (NEC) predominantly affects premature infants, and those who are formula fed are 6 to 10 times more likely to develop NEC compared to similar infants who receive breast milk [1]. While the benefits of breast feeding are widely touted, the components specific to breast milk and absent from formula, especially those with effects on intestinal development and immune protection, are subject to investigation as potential therapeutic targets in NEC. Relaxin (RLXN) is a 6 kDa hormone of pregnancy that leads to relaxation of the pubic symphisis [2]. Relaxin is also a potent vasodilator which is responsible for the relative volume expanded state of pregnancy and is associated with decreased renal vascular resistance during pregnancy [3]. The cardiovascular effects of relaxin have made it a novel treatment in heart failure, and current phase three clinical trials of recombinant relaxin have shown improved ☆ Dr. Matheson contributed to the project design, execution (animal care and study), data collection, data analysis, and performed final critical revision of the manuscript. ☆☆ Dr. Walker contributed to the project design, execution (animal care and study), data collection, and provided the initial draft of the manuscript. ★ Supported by James R. Petersdorf Fund of Norton Healthcare and Kosair Charities. ⁎ Corresponding author at: Division of Pediatric Surgery, Hiram C. Polk, Jr., M.D. Department of Surgery, University of Louisville, 315 E. Broadway, Ste 565, Louisville, KY 40202. E-mail address: [email protected] (C.D. Downard). 1 Drs. Matheson and Walker have contributed equally to this study. http://dx.doi.org/10.1016/j.jpedsurg.2014.01.032 0022-3468/© 2014 Elsevier Inc. All rights reserved.
outcomes, decreased mortality, and decreased end organ damage in this clinical scenario [4–6]. We have previously shown that enteral relaxin supplementation given for 24 h increases intestinal blood flow in an animal model of NEC [7]. The purpose of this project is to 1) evaluate relaxin as a preventative agent for NEC, 2) to analyze its utility as a rescue therapy when NEC is suspected, and 3) to evaluate its effect on intestinal perfusion when used in conjunction with direct peritoneal resuscitation. 1. Materials and methods The research protocol was approved by the Institutional Animal Care and Use Committee, Biohazard Safety Committee, and Research and Development Committee prior to studies. Four timed pregnant Sprague–Dawley dams (Harlan, Indianapolis, IN) were maintained in an AAALAC-approved Veterinary Medical Unit at the Robley Rex VA Medical Center in Louisville, KY for at least one week prior to delivery of pups. Dams were acclimated on a 12 h light–dark cycle and were allowed rat chow and water ad libitum. The rat pups were randomized to group by litter for inclusion in the CONTROL group (n = 12) which were vaginally delivered and dam fed, or experimentally-induced NEC groups (3 groups, n = 11-13 per group) which were delivered by Caesarian section 12 h prematurely under carbon dioxide anesthesia.
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The CONTROL group was time-matched at post-delivery hour of life to the NEC groups. As previously reported, experimental NEC was induced by formula feeds via gastric gavage every 4–5 h, intermittent hypoxia (100% nitrogen gas for 60 s) and hypothermia (4 °C for 10 min) every 12 h, and a single gastric dose of lipopolysaccharide at 12 h of life (SigmaAldrich, St. Louis, MO) [8–10]. The formula feeds consisted of 20 g Similac 60/40 (Ross Pediatrics, Columbus, OH) dissolved in 100 mL Esbilac (Pet-Ag, New Hampshire, IL) puppy formula. Feeds were started at 0.1 mL/feed and were advanced to 0.15 mL/feed at 24 h of life and 0.2 mL/feed at 48 h of life. These feeds were calculated to supply approximately 200 kcal/kg/day caloric intake. The experimental protocol is outlined in Fig. 1. At birth, NEC pups were randomized to the following groups: 1) NEC alone (n = 11), 2) NEC with a single oral supplemental dose of rat relaxin (0.25 ng/feed, Sigma-Aldrich, St. Louis, MO) in the final feed prior to blood flow experiments (n = 12, NEC + 1xRLXN), or 3) NEC with oral supplemental doses of relaxin (0.25 ng/0.1 mL) in all formula feeds (n = 13, NEC + All Feeds RLXN). CONTROL animals were dam fed and received no relaxin supplementation. At 48 h of life, the pups were weighed and anesthetized with isoflurane with induction of 3.5% and maintenance of 1.0% on 1 L oxygen per minute. All animals were separated from their littermates and underwent laparotomy for the study of ileal blood flow by laser Doppler flowmetry (Periflux system, Perimed AB, Järfälla, Sweden). Gross appearance of the intestines was recorded for signs of necrosis, hemorrhage, gaseous distension, or perforation. Any blood or stool that was present was flushed from the peritoneum with prewarmed saline (37.0 °C). Body temperature was maintained at 37 °C by feedback controller. A 7-site integrating flow probe was placed over the terminal ileum and organ blood flow was recorded. Correct positioning of the flow probe was verified visually at each time point for the duration of the experiment. Warmed saline was dripped on the peritoneum and the animals were allowed to equilibrate for 20 min prior to initiation of the flow protocol. In each animal, laser Doppler
perfusion was recorded in the ileum at two baseline time points 10 min apart. If flow was stable (less than 10% difference) during that baseline period the study was continued. No pups were excluded for unstable blood flow at baseline. After the completion of the baseline period, a 2.5% peritoneal dialysis solution prewarmed to 37.0 °C was dripped in the peritoneum and flow was recorded for an additional 10 min to evaluate if the alteration in intestinal blood flow seen with relaxin supplementation works against or in concert with our previously demonstrated changes in intestinal blood flow seen with the addition of peritoneal dialysis solution [11]. At the completion of the laser Doppler flowmetry studies, the degree of bladder distension in the pups was observed and recorded. The grading scale used was: 0 when the bladder appeared completely empty, 1 when the bladder diameter was N 0 and b 1 mm, 2 when bladder distension was N 1 and b 2, and 3 when bladder distension was N 2 mm. In addition, ileum samples were obtained, and placed in 10% neutral buffered formalin for 16 h and then placed in 70% ethanol for hematoxylin and eosin (H&E) staining. Histopathology scores for signs of NEC were obtained from a pathologist masked to group and experimental protocols. The samples were graded as follows: Grade 0, normal or no damage; Grade 1, epithelial cell lifting or separation; Grade 2, sloughing of epithelial cells to mid villus level; Grade 3, necrosis of entire villus; or Grade 4, transmural necrosis [12]. All data are expressed as mean ± standard error of the mean (SEM). Differences between groups (CONTROL, NEC, NEC + 1xRLXN, and NEC + All Feeds RLXN) and time points (baseline1, baseline2, DPR1 min, DPR5 min, and DPR10 min) were determined by two-way analysis of variance (ANOVA) using SigmaPlot for Windows 11.1.0.102 (Systat Software, Inc., San Jose, CA). Differences between baseline intestinal perfusion, bladder distension, body weights, or histopathologic scores were determined by one-way ANOVA. The null hypothesis was rejected a priori at P b 0.05. When differences were found using ANOVA, the post hoc Tukey–Kramer honestly significant difference test was applied.
Fig. 1. Experimental timeline. Experimental timeline. DPR, 2.5% Delflex; BL, baseline time point; NBF, 10% neutral buffered formalin; RLXN, rat relaxin-1 at 0.25 ng/feed.
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2. Results A summary of baseline values of body weight, bladder distension score, and ileal blood flow for each group is shown in Table 1. These data show that despite significant caloric delivery in the formula feeds which were designed to match caloric delivery in the dam feeds, the body weights in the NEC groups were significantly lower at 48 h of life compared to the time-matched CONTROL group. Bladder distension was significantly higher in the CONTROL group than in the NEC groups, suggesting improved hydration in the dam fed CONTROLs. A single supplemental oral dose of relaxin improved hydration status compared to NEC alone, and supplemental oral relaxin in all feeds further improved hydration status. Fig. 2 shows that with regard to histopathologic grading for signs of NEC, the CONTROL animals were all graded as zero by a pathologist masked to group and experimental protocol. In addition, none of the CONTROL animals had any visual signs of NEC when gross condition of the intestines was recorded after the laparotomy. The NEC Grade ranged from 1 to 4 in the NEC alone group which was significantly worse than the CONTROL group (2.04 ± 0.19 versus 0.0 ± 0.0, P b 0.05). The single oral dose of relaxin in the NEC + 1xRLXN group improved NEC Grade which ranged from 0 to 2 (1.12 ± 0.12, P b 0.05 versus NEC), and relaxin added to all feeds further improved NEC Histopathology Grade which ranged from 0 to 1 (0.46 ± 0.14, P b 0.05 versus all groups). Fig. 3 shows the changes in ileal perfusion over time as evaluated by laser Doppler flowmetry. Baseline blood flow was decreased in the NEC group compared to the CONTROL group (58.2 ± 2.2 versus 35.9 ± 1.1 PU, P b 0.05). The addition of a single oral relaxin supplement in the NEC + 1xRLXN group significantly improved ileal baseline blood flow compared to the NEC group (35.9 ± 1.1 versus 62.9 ± 1.7 PU, P b 0.05), as did oral relaxin supplements in all feeds (35.9 ± 1.1 versus 84.5 ± 2.6 PU, P b 0.05). The addition of DPR to the NEC group improved blood flow to levels near the baseline levels in the CONTROL group. The addition of a single oral relaxin dose improved blood flow to the CONTROL levels and the addition of relaxin to all feeds improved ileal perfusion to levels higher than CONTROL, which remained elevated to CONTROL levels during the addition of DPR.
3. Discussion
Fig. 2. Histologic grading by group. NEC Histopathology Grades in CONTROL, NEC, NEC + 1x RLXN, and NEC + All Feeds groups. CONTROLs had no signs of NEC (all graded zero), while the NEC alone group had significant NEC grading ranging from 1 to 4 (none scored as normal). A single oral supplement of RLXN prevented the ileal damage observed in the NEC alone group, and RLXN supplements in all feeds further improved ileal condition. *P b 0.05 versus CONTROL, †P b 0.05 versus NEC alone, and ‡P b 0.05 versus NEC + 1x RLXN.
tions. Its effect on the intestinal microvasculature has been appreciated when Bigazzi and colleagues demonstrated that topical relaxin application causes vasodilation in the mesocaecum of adult rats [13]. The hormone relaxin acts via g-protein coupled receptors RXFP1 and RXFP2, and we have previously demonstrated that both are present in the neonatal rat intestine of control and NEC rat pups [14]. We have also shown that oral relaxin supplementation in an animal NEC model increases intestinal blood flow at 24 h of life [7]. In this series of experiments, neonatal animals that underwent the NEC protocol but received relaxin with all feeds (NEC + All Feeds
Compromised intestinal blood flow is a critical event in development of NEC [10]. Numerous reports suggest that formula feeding is a definite risk factor for NEC, therefore a logical therapeutic target for either preventing or treating NEC would be a structure present in breast milk and missing from formula which might affect intestinal blood flow. Relaxin is a 6 kDa hormone most often associated with pelvic girdle relaxation in pregnancy. Its vasoactive properties have been recently described, and it is a potent vasodilator even at picomolar concentraTable 1 Body weight, bladder distention, and baseline perfusion. Group
Body Weight, g
Bladder Distension Score
Baseline ileal blood flow, PU
CONTROL, n = 12 NEC along, n = 11 NEC + RLXN, n = 12 NEC + All Feeds, n = 13
8.3 ± 0.2
2.9 ± 0.1
58.2 ± 2.2
5.2 ± 0.1*
1.2 ± 0.1*
35.9 ± 1.1*
4.8 ± 0.1*
1.7 ± 0.1*
62.9 ± 1.7*†
5.4 ± 0.1*‡
2.2 ± 0.1*†‡
84.5 ± 2.6*†‡
Summary of body weights, bladder distension scores, and ileal blood flow baseline values. * P b 0.05 versus CONTROL, †P b 0.05 versus NEC, ‡P b 0.05 versus NEC + 1x RLXN by one-way ANOVA and Tukey-Kramer honestly significant difference test. Abbreviations: PU, perfusion units; g, grams; RLXN, rat relaxin-1 at 0.25 ng/feed; N, number of rats per group; and NEC, necrotizing enterocolitis.
Fig. 3. Ileal perfusion over time. Ileum blood flow as assessed by laser Doppler Flowmetry. The flow in the NEC group was significantly lower than the time-matched CONTROL group and the addition of DPR to the peritoneum of the NEC animals increased ileal perfusion to near CONTROL baseline levels. The addition of a single oral dose of RLXN to the NEC + 1x RLXN group improved ileal blood flow, both at baseline and in DPR stimulated conditions. The addition of RLXN to all of the feeds in the NEC + All Feeds group further improved ileal perfusion compared to NEC alone or NEC + 1x RLXN. *P b 0.05 versus CONTROL, †P b 0.05 versus NEC alone, and ‡P b 0.05 versus NEC + 1x RLXN.
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RLXN) had substantially increased ileal perfusion compared to not only NEC animals but also CONTROL animals at baseline. In addition, animals that were subjected to the NEC protocol but only received relaxin with one feed four hours prior to sacrifice (NEC + 1xRLXN) had baseline intestinal blood flow greater than NEC animals, but more closely approximated the blood flow in CONTROL animals. Interestingly, the NEC + All Feeds RLXN animals had very limited response to treatment with 2.5% Delflex®, which has previously been shown to increase intestinal blood flow in our animal model of NEC [11]. This suggests that continual relaxin supplementation maximally dilates the intestinal microvasculature, and further increase in flow is not possible. NEC animals who received relaxin supplementation with only one feed (NEC + 1xRXLN) had substantially increased ileal perfusion compared to baseline NEC animals, supporting that even transient relaxin supplementation in formula feeds affects intestinal blood flow. This series of experiments was designed to simulate use of relaxin not only as a preventative treatment for those at risk for NEC (NEC + All Feeds RLXN group) but also as a rescue therapy for those already diagnosed with NEC (NEC + 1xRLXN group). The NEC + 1xRLXN animals who had undergone the NEC protocol identical to the NEC pups had increased baseline ileal blood flow compared to the NEC pups. Moreover, the NEC + 1xRLXN pups had less clinically apparent NEC than the NEC alone group even after a single dose of relaxin, citing a possibility for arrest of progression of NEC with relaxin supplementation. The NEC model we use has been shown to produce 50% of grade 2 NEC by 48 h and 75% at 96 h [7]. In our study, the histology scores of the NEC + 1xRLXN group fall between the baseline NEC group and the NEC + All Feeds RLXN group, suggesting that improvement in histologic-apparent cellular injury can be achieved with the increased blood flow obtained with a single dose of relaxin. Relaxin has been demonstrated in rats, dogs, pigs, and humans with levels present in the corpora luteum helping maintain pregnancy, prepare the uterus for delivery, and promote breast and nipple development for nursing offspring [15–27]. Mammary tissue is the source of relaxin in nursing animals and is thought to be important for specific growth and development using a lactocrine hypothesis [19,28]. We confirmed this in rat mothers and also demonstrated that there were no detectable levels of relaxin in pups who did not receive supplementation, similar to previous studies with pigs and dogs [16–18,23]. Prior to clinical treatment of infants with NEC with relaxin supplementation, we plan to evaluate the effect of intermittent and continuous relaxin supplementation in the NEC protocol at further time points. Human use of relaxin has been associated with bleeding complications such as metromenorrhagia and epistaxis [29,30]. We have noted gastrointestinal hemorrhage in our initial studies with relaxin supplementation in our animal model at higher doses, therefore precise dose–response will need to be evaluated prior to clinical application in human infants (unpublished data). Relaxin is a potent vasodilatory hormone which is in clinical trials in other diseases, has oral bioavailability, and is a hormone associated with pregnancy which is missing in the intestine of formula fed infants. We believe that the vasodilatory effects of relaxin are potentially responsible for the decreased incidence of NEC in breast fed infants, and enteral supplementation of relaxin could either prevent or treat the intestinal hypoperfusion seen in NEC. Our data support the intestinal vascular effects of continuous and intermittent enteral relaxin supplementation and encourage further investigation as a potential new preventative and rescue therapy for NEC. Acknowledgments We would like to acknowledge the technical assistance of Jessica A. Shepherd, Amy J. Matheson, and Samuel A. Matheson. References [1] Lucas A, Cole TJ. Breast milk and neonatal necrotising enterocolitis. Lancet 1990;336:1519–23.
[2] Hisaw FL. Experimental relaxation of the pubic ligament of the guinea pig. Proc Soc Exp Biol 1926;23:661–3. [3] Conrad KP. Unveiling the vasodilatory actions and mechanisms of relaxin. Hypertension 2010;56:2–9. [4] King A. Heart failure. Promising data for serelaxin. Nat Rev Cardiol 2013;10:3. [5] Metra M, Cotter G, Davison BA, et al. Effect of serelaxin on cardiac, renal, and hepatic biomarkers in the Relaxin in Acute Heart Failure (RELAX-AHF) development program: correlation with outcomes. J Am Coll Cardiol 2013;61:196–206. [6] Teerlink JR, Cotter G, Davison BA, et al. Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF): a randomised, placebocontrolled trial. Lancet 2013;381:29–39. [7] Maki AM, Matheson PJ, Shepherd JA, et al. Enteral relaxin supplementation ameliorates necrotizing enterocolitis associated intestinal hypoperfusion. Presented at the American Academy of Pediatrics Section on Surgery, New Orleans, LA, October 19-21; 2012. [8] Barlow B, Santulli TV. Importance of multiple episodes of hypoxia or cold stress on the development of enterocolitis in an animal model. Surgery 1975;77:687–90. [9] Barlow B, Santulli TV, Heird WC, et al. An experimental study of acute neonatal enterocolitis—the importance of breast milk. J Pediatr Surg 1974;9:587–95. [10] Downard CD, Grant SN, Matheson PJ, et al. Altered intestinal microcirculation is the critical event in the development of necrotizing enterocolitis. J Pediatr Surg 2011;46:1023–8. [11] Maki AC, Matheson PJ, Shepherd JA, et al. Intestinal microcirculatory flow alterations in necrotizing enterocolitis are improved by direct peritoneal resuscitation. Am Surg 2012;78:803–7. [12] Feng J, El-Assal ON, Besner GE. Heparin-binding epidermal growth factor-like growth factor decreases the incidence of necrotizing enterocolitis in neonatal rats. J Pediatr Surg 2006;41:144–9. [13] Bigazzi M, Del Mese A, Petrucci F, et al. The local administration of relaxin induces changes in the microcirculation of the rat mesocaecum. Acta Endocrinol (Copenh) 1986;112:296–9. [14] Walker SM, Maki AC, Matheson PJ, et al. Complex regulation of intestinal relaxin receptor RXFP-1 levels is time-dependent and altered by necrotizing enterocolitis in rat pups. Presented at the American Academy of Pediatrics Section on Surgery, New Orleans, LA, October 19-21; 2012. [15] Eddie LW, Sutton B, Fitzgerald S, et al. Relaxin in paired samples of serum and milk from women after term and preterm delivery. Am J Obstet Gynecol 1989;161:970–3. [16] Goldsmith LT, Lust G, Steinetz BG. Transmission of relaxin from lactating bitches to their offspring via suckling. Biol Reprod 1994;50:258–65. [17] Steinetz BG, Williams AJ, Lust G, et al. Transmission of relaxin and estrogens to suckling canine pups via milk and possible association with hip joint laxity. Am J Vet Res 2008;69:59–67. [18] Bagnell CA, Steinetz BG, Bartol FF. Milk-borne relaxin and the lactocrine hypothesis for maternal programming of neonatal tissues. Ann N Y Acad Sci 2009;1160:152–7. [19] Frankshun AL, Ho TY, Reimer DC, et al. Characterization and biological activity of relaxin in porcine milk. Reproduction 2011;141:373–80. [20] Frankshun AL, Ho TY, Steinetz BG, et al. Biological activity of relaxin in porcine milk. Ann N Y Acad Sci 2009;1160:164–8. [21] Masters RA, Crean BD, Yan W, et al. Neonatal porcine endometrial development and epithelial proliferation affected by age and exposure to estrogen and relaxin. Domest Anim Endocrinol 2007;33:335–46. [22] Yan W, Ryan PL, Bartol FF, et al. Uterotrophic effects of relaxin related to age and estrogen receptor activation in neonatal pigs. Reproduction 2006;131:943–50. [23] Yan W, Wiley AA, Bathgate RA, et al. Expression of LGR7 and LGR8 by neonatal porcine uterine tissues and transmission of milk-borne relaxin into the neonatal circulation by suckling. Endocrinology 2006;147:4303–10. [24] Sherwood OD, Chang CC, Bevier GW, et al. Radioimmunoassay of plasma relaxin levels throughout pregnancy and at parturition in the pig. Endocrinology 1975;97:834–7. [25] Sherwood OD, Crnekovic VE, Gordon WL, et al. Radioimmunoassay of relaxin throughout pregnancy and during parturition in the rat. Endocrinology 1980;107:691–8. [26] Sherwood OD, Nara BS, Welk FA, et al. Relaxin levels in the maternal plasma of pigs before, during, and after parturition and before, during, and after suckling. Biol Reprod 1981;25:65–71. [27] Hwang JJ, Lee AB, Fields PA, et al. Monoclonal antibodies specific for rat relaxin. V. Passive immunization with monoclonal antibodies throughout the second half of pregnancy disrupts development of the mammary apparatus and, hence, lactational performance in rats. Endocrinology 1991;129:3034–42. [28] Steinetz BG, Horton L, Lasano S. The source and secretion of immunoactive relaxin in rat milk. Exp Biol Med (Maywood) 2009;234:562–5. 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Discussion Discussant Dr. Joel Shilyansky, (Iowa City, IA): Can you compare relaxin to breast milk as far as its effects in this kind of model, and is relaxin the main component of breast milk that improves outcomes with NEC?
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Response Dr. Walker: There are other components of breast milk that are absent from formula. We were most interested in relaxin because of its vaso-active properties since our lab works on the micro perfusion of the intestine. Discussant Dr. Shilyansky: Can I ask a controversial question. Why not just give the breast milk as opposed to small components of it.
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hyperemia, post-prandial increase in blood flow outside of the model of NEC, and if so how is that affected by relaxin? Response Dr. Walker: Most of our experimentation is done in about four hours after the last feeding. We feed the last time at 6:00 AM and then perform our experiments at 10:00 AM. To my knowledge we have not looked at blood flow directly after feeding. However, that is an excellent point to investigate.
Response Dr. Walker: Breast milk supplementation would be ideal, especially breast milk in infants. However, we are also looking at this as a possible rescue therapy as well, and I'm not sure that just a single dose of breast milk would be as preventative in improving intestinal blood flow.
Discussant Dr. Shilyansky: Just to follow up are there inhibitors of relaxin? Perhaps if you gave an inhibitor of Relaxin along with breast milk you might prevent the effect of breast milk and that would be more supportive of your hypothesis.
Discussant Dr. Brad Warner, (St. Louis, MO): Congratulations that's a beautiful study. I had some questions. Do you see using your model where you are gauging blood flow? Do you see a post-prandial
Response Dr. Walker: There is currently no inhibitor for relaxin per se clinically available, but that would be an excellent study in the small model.