Intestinal Microcirculatory Flow Alterations in Necrotizing Enterocolitis ...

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Vasoconstriction of the neonatal intestinal microvasculature is a central mechanistic event in development of necrotizing enterocolitis. We hypothesized that ...
Intestinal Microcirculatory Flow Alterations in Necrotizing Enterocolitis are Improved by Direct Peritoneal Resuscitation ALEXANDRA C. MAKI, M.D., PAUL J. MATHESON, PH.D., JESSICA A. SHEPHERD, B.S., R. NEAL GARRISON, M.D., CYNTHIA D. DOWNARD, M.D., M.M.SC.

From the Robley Rex Veterans Affairs Medical Center, and the Department of Surgery, and Division of Pediatric Surgery, University of Louisville, Louisville, Kentucky Vasoconstriction of the neonatal intestinal microvasculature is a central mechanistic event in development of necrotizing enterocolitis. We hypothesized that topical treatment of the intestine with dialysate fluid would ameliorate the vasoconstriction in necrotizing enterocolitis (NEC). NEC was induced in experimental groups. Control animals were delivered vaginally and dam-fed (control group). Neonatal pups underwent laser Doppler flow study of the terminal ileum to determine real-time blood flow in the intestinal microvasculature. After baseline flow was determined, dialysis solution was added to the peritoneal cavity and alterations in microcirculation were recorded. Baseline ileal blood flow in the control group was significantly higher than in NEC rat pups at 48 hours post delivery (P \ 0.05), but not at 24 hours (P 5 NS). Ileal blood flow increased in all groups after adding dialysate (P \ 0.05), improving ileal blood flow in the 48-hour NEC group and reaching the baseline level of the 48-hour control group (P \ 0.05). Our data shows blood flow to be higher in 48-hour controls as compared with 24-hour controls suggesting a timedependency in the development of intestinal vasoregulatory processes. All groups had an increase in blood flow with dialysate treatment. This may represent a novel initial therapy to improve intestinal ischemia in human necrotizing enterocolitis.

(NEC) is a devastating intestinal disease that affects approximately 7 per N cent of premature infants born weighing between 500 ECROTIZING ENTEROCOLITIS

and 1500 grams.1 It is characterized by intestinal hypoperfusion and bowel loss, potentially leading to short gut syndrome or death. Surgical intervention is required in an estimated 30 per cent of NEC cases and in this population there is a 50 per cent mortality rate.2 Current investigations focus on the potential for earlier, safe interventions, which could save intestinal length and lives. Direct peritoneal resuscitation (DPR) has been studied as an adjunct resuscitative treatment in addition to standard intravenous crystalloid and blood transfusion in a hemorrhagic shock animal model. This research has demonstrated that the microcirculation of the small Presented at the Annual Scientific Meeting and Postgraduate Course Program, Southeastern Surgical Congress, Birmingham, AL, February 11–14, 2012. Supported by James R. Peterdorf Fund of Norton Healthcare. Address correspondence and reprint requests to Cynthia D. Downard, M.D., M.M.Sc., Division of Pediatric Surgery, Department of Surgery, University of Louisville, 315 East Broadway, Suite 565, Louisville, KY 40202. E-mail: [email protected].

intestine constricts after initial conventional resuscitation. This vasoconstriction has been shown to reverse to baseline when peritoneal dialysis fluid is topically applied to the intestine.3 Recently, we have demonstrated that experimental necrotizing enterocolitis is associated with an abnormality in intestinal microcirculation, with NEC-affected animals having significantly smaller vessels than the control groups.4 Given positive experience with DPR improving intestinal microvascular flow in adult animals with hemorrhagic shock, we wanted to evaluate the potential for improvement of intestinal blood flow with peritoneal dialysate, DelflexÒ (Fresenius, Orem, UT), in an NEC model. We hypothesized that direct application of dialysate solution would ameliorate the intestinal vasoconstriction seen in this animal model of NEC. Methods

Animals were maintained in a facility approved by the American Association for the Accreditation of Laboratory Animal Care. The research protocol was approved by the Institutional Animal Care and Use Committee and Biohazard Safety Committee at the Robley Rex Veterans Administration Medical Center,

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Louisville, Kentucky. All experiments were performed in Sprague-Dawley rats delivered from timed-pregnant dams (Harlan, Indianapolis, IN) by C-section or unassisted vaginal delivery. Dams were acclimated on a 12-hour light-dark cycle for at least 1 week before delivery and were allowed access to standard rat chow and water ad libitum. Rat pups were randomized by litter to either have experimentally-induced necrotizing enterocolitis (NEC group) or to serve as dam-fed controls (control group). Experimental NEC was induced by previously published methods.4, 5 In brief, NEC rat pups were delivered by caesarian section under carbon dioxide anesthesia at gestation day 21.5. After delivery, NEC pups were stressed twice daily by asphyxia (exposure to 100% nitrogen gas for 60 seconds) followed by cold exposure (4°C for 10 minutes). In addition, at 12-hours post delivery, each NEC pup was administered lipopolysaccharide (Sigma, St. Louis, MO) by gavagefeeding to facilitate NEC development. NEC pups were gavage-fed six times/day with a cows-milk based rat milk substitute at a rate that provided approximately 200 kcal/kg/day and that consisted of 20 g Similac 60/ 40 (Ross Pediatrics, Columbus, OH) dissolved into 100 mL Esbilac (Pet-Ag, New Hampshire, IL). Feeds were started at 0.1 mL/feed and were advanced as tolerated by 0.05 mL every 24 hours to a maximum of 0.2 mL/ feed by 48 hours post delivery. NEC pups were housed in a temperature controlled incubator and all experimental measures were performed at least 12 hours after the last asphyxia/cold stress exposure. Control group pups were allowed to deliver vaginally, were housed in standard caging with their dams, and were fed ad libitum by nursing of maternal milk. Their time of birth was defined as Day 0, Hour 0. At 48 hours post delivery, NEC and control group pups were separated from their littermates and underwent laparotomy for the study of laser Doppler blood flow of the terminal ileum using standard techniques with PeriFlux system (Perimed AB, Ja¨rfa¨lla, Sweden). All pups were anesthetized by inhaled isoflurane before surgical manipulation at an induction dose of 3.5 per cent and maintenance dose of 2 per cent on 1 L/minute oxygen. Body temperature was maintained at 37.0 ± 0.5°C by feedback controller and was recorded throughout the experimental protocol. A right paramedian incision in the abdomen was made and

FIG. 1.

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gross appearance of the intestines was recorded (necrosis, hemorrhage, gaseous distension, or perforation). Any gross blood or stool was flushed from the peritoneum with prewarmed saline (37.0°) and a 7-site integrating flow probe was placed over the terminal ileum and correct placement was verified by visual inspection over the duration of the experiment. Figure 1 shows the experimental timeline. Animals were randomized to the following experimental groups: 1) control group, 24 hours; 2) control group, 48 hours; 3) NEC group, 24 hours; and 4) NEC group, 48 hours. Pups were allowed to equilibrate for 20 minutes before the start of the experimental protocol. In each group, laser Doppler perfusion was evaluated at baseline for 10 minutes before the addition of any test substances to the peritoneal space. The baseline blood flow was termed stable when consecutive readings over 10 minutes were within 5 per cent of each other. After the baseline period, prewarmed (37.0°) direct peritoneal resuscitation of 2.5 per cent DelflexÒ was then added to the peritoneal space. Ileal laser Doppler blood flow was measured at 1 minute, 5 minutes, and 10 minutes. At the completion of the experimental protocol, the degree of bladder distension was noted and recorded according to the following scale: 0 when the bladder appeared completely empty; 1 when bladder diameter was > 0 mm and # 1 mm; 2 when > 1 mm and # 2 mm; and 3 when > 2 mm. Animal weights were recorded at the initiation and conclusion of the protocol. All data are expressed as mean ± standard error of the mean. Differences between comparison groups (i.e., NEC vs control group) and time points (i.e., baseline1, baseline2, DPR1, DPR5, and DPR10) were determined by two-way analysis of variance (ANOVA) using SPSS 19 (IBM, Armonk, NY). Differences between baseline intestinal perfusion levels for all groups, bladder distension, and body weights were determined by one-way ANOVA. The null hypothesis was rejected a priori at P < 0.05. When differences were found using ANOVA, the post hoc Tukey-Kramer honestly significant difference test was applied. Results

A summary of baseline values for body weight, laser Doppler blood flow in the terminal ileum, and bladder

Experimental timeline. DPR, 2.5 per cent DelflexÒ; BL, baseline time point; NBF, 10 per cent neutral buffered formalin.

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distension score for NEC and control groups at 24 hours and 48 hours post birth is shown in Table 1. A total of 10 rat pups were in the 24-hour control group and 12 pups in the 48-hour control group. Sixteen pups underwent the NEC protocol for 24 hours before study, and 12 pups were assigned to the 48-hour NEC group. There were no differences between control groups in bladder distension (not significant, NS) or body weight (NS). Baseline intestinal blood flow was significantly higher in the 48-hour post birth control group compared with the 24-hour post birth control group (P < 0.05). In the NEC groups, there were no differences in baseline intestinal blood flow (NS), bladder distension (NS), or body weight (NS) between the 24-hour and 48-hour post delivery NEC groups. Body weight at the completion of the feeding protocols was significantly less (P < 0.05) in the two NEC groups compared with the two control groups. Also, bladder distension, which was used as a measure of hydration status, was much lower in the NEC groups compared with the control groups (P < 0.05). The addition of DPR to the peritoneal space in all groups increased intestinal blood flow compared with baseline levels for each group (Fig. 2). As noted above, baseline ileal blood flow in controls was significantly higher than in NEC rat pups at 48 hours post delivery (P < 0.05) but not at 24 hours (P 4 NS). The placement of the hypertonic dialysate solution into the peritoneum increased blood flow by 114.9 per cent ± 18.9 in the 24-hour control group, 63.2 per cent ± 7.5 in the 48-hour control group, 11.9 per cent ± 17.6 in the 24-hour NEC group and 52.4 per cent ± 12.4 in the 48hour post delivery NEC group (all P < 0.05). In the 48hour NEC group DPR significantly improved ileal blood flow, reaching baseline levels of the 48-hour control group (P < 0.05).

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Discussion

These results demonstrate that ileal blood flow is significantly decreased in NEC animal compared with controls at 48 hours of life, and that topically applied dialysate fluid increases blood flow of the ileal microcirculation in NEC animals to control baseline levels. This was consistent in all groups tested. Presumably, the control baseline flow of 60 perfusion units is sufficient for normal animal pups to survive to adulthood. Given our finding that topically applied peritoneal dialysate increases intestinal blood flow and may ameliorate the intestinal hypoperfusion in NEC, we feel it could be an important adjunct to the current management of this disease. NEC has been shown to be a direct consequence of intestinal microvascular constriction.6 This study demonstrates that DPR increases intestinal blood flow in an animal model of NEC. This effect is virtually instantaneous and our results reflected this with a baseline flow of 60 perfusion units at 1 minute after DPR application in NEC animals at 48 hours of life. This immediate action along with peritoneal dialysate fluid being commercially available makes it an attractive option for treatment. Various formulations of DelflexÒ peritoneal dialysis solution are clinically available, including 1.5 per cent, 2.5 per cent, and 4.25 per cent dextrose concentrations. We chose 2.5 per cent DelflexÒ because of our previous experience with it in the hemorrhagic shock animal model and initial clinical studies.7, 8 The vasoactive elements of the DPR solution are thought to be glucose, hyperosmolarity, low pH, and perhaps glucose degradation products produced in the heat sterilization of glucose solutions.9 Translating direct peritoneal

TABLE 1. Summary of Baseline Values, as Well as the Bladder Distension Scores, Done at the Completion of the Studies for all Groups Bladder Distension Score

Ileum Blood Flow, PU

Group

N

Body Weight, g

24-hours control 48-hours control 24-hours NEC 48-hours NEC

10

9.10 ± 0.07

2.90 ± 0.06y

37.3 ± 1.4

12

8.32 ± 0.05

2.83 ± 0.06

58.6 ± 1.5y

16

5.03 ± 0.04*

2.56 ± 0.05*

38.5 ± 1.0

12

5.01 ± 0.05*

1.17 ± 0.09*y

35.9 ± 1.1*

* P < 0.05 versus timed CONTROL group. y P < 0.05 versus 24-hours (CONTROL or NEC) by one way ANOVA and Tukey-Kramer honestly significant difference test. PU, perfusion units.

FIG. 2. Intestinal blood flow in the terminal ileum was measured by laser Doppler flowmeter with seven-site integrating flow probe. DPR significantly increased blood flow in all groups. Baseline (BL) blood flow in the NEC groups was significantly lower compared with the baseline blood flow in the control groups. * P < 0.05 versus baseline level. y P < 0.05 versus control. z P < 0.05 versus NEC by 2-way ANOVA and Tukey-Kramer honestly significant difference test.

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resuscitation to clinical use in treatment of necrotizing enterocolitis, however, will require further studies to elucidate appropriate dosage and timing of the intervention. The ultimate goal would be salvage of intestine before onset of irreversible changes of intestinal vascular necrosis and sepsis. Interestingly, the baseline intestinal blood flow in control animals between the 24-hour and 48-hour groups were markedly different, with the 24-hour pups having equivalent flow to their NEC counterparts. Again, DPR did increase blood flow compared with baseline; however, it was still decreased compared with the 48-hour control group. We suspect that this is due to maturation of the microvasculature that occurs at sometime between the 24-hour and 48-hour time points in our neonatal NEC model. Intestinal microcirculation experiments have shown nearly instantaneous and prolonged vasodilation of inflow and premucosal arterioles and postmucosal and outflow venules in normal rats with the addition of dialysate.10 In addition, intestinal intravital videomicroscopy studies demonstrated that topical intraperitoneal DelflexÒ exposure can overcome shock-induced vasoconstriction in rats that have undergone resuscitated hemorrhagic shock protocols.11 This DPR-induced microvascular vasodilation produced a significant increase in both microvascular perfusion and whole organ blood flow in the small intestine.12 These findings suggest that DPR might be useful to improve intestinal perfusion in low flow shock states such as necrotizing enterocolitis. DPR also seems to alter the compartmental fluid shifts that occur during shock, which was again shown in a rodent model of resuscitated hemorrhagic shock.13 The addition of DelflexÒ to the conventional resuscitation strategy normalized total tissue water and water compartment distribution and prevented organ specific edema in the gut, lung, and muscle. These findings suggest that the use of intraperitoneal DelflexÒ might help normalize tissue fluid handling in shock states such as NEC. Another study of DPR in resuscitated hemorrhagic shock, which focused on mesenteric lymph production and composition, suggested that DPR minimizes intestinal injury and inflammation during shock.14 The addition of DPR to the conventional resuscitation regimen (i.e., returning shed blood plus two equal volumes of saline) normalized the mesenteric lymph flow rate, normalized proinflammatory cytokine levels in the lymph and serum, prevented the electrolyte derangement, and normalized the elevated liver enzyme levels associated with resuscitated hemorrhagic shock. This anti-inflammatory effect is an interesting benefit of DPR and one that may help ameliorate another potential cause of NEC. Nanthakumar and colleagues15 found that NEC is due in part to an

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excessive inflammatory response within the small intestine. This is secondary to immaturity of the immune system that leads to an upregulation of proinflammatory genes like, interleukin-8, without the benefit of immunomodulator genes, like Inhibitor Kappa B a nuclear transcription inhibitor that is upstream from the interleukin-8 gene. They found that these inhibitors have not yet been activated in premature enterocytes.15 Other areas of intestinal vasculature maturation have been extensively researched recently: these include the roles of Nitric Oxide Synthase; a vasodilator; Endothelin-1, a vasoconstrictor; and prostaglandins as a part of vascular maturity. Nowicki and colleagues16 looked at pathologic specimens from neonates diagnosed with NEC based on surgical specimens and found that endothelial nitric oxide sythase is not functional in these patients.16 Nowicki and colleagues17 have also studied Endothelin-1 in surgical specimens of NEC patients and found significantly higher levels of Endothelin-1 in surgical specimens from patients with NEC. Prostaglandins have a dual role in the intestinal vasculature where they both vasodilate and vasoconstrict. It is unclear what is their exact role in the vascular development of the neonatal intestine because of this dual nature. However, prostaglandin inhibitors such as indomethacin have been shown to have an association with spontaneous isolated intestinal perforation, as disease is often mistaken for NEC.18 In relation to prostaglandin synthesis, the Cycloxygenase-2 enzyme has also been shown to be a potent stimulator of vascular endothelial growth factor (VEGF). VEGF is a potent stimulator of endothelial cells that is essential for angiogenesis and vasculogenesis in the neonatal period.19 Vascular branching patterns of juvenile rat intestine arcades have also been studied in an attempt to determine what kind of growth and remodeling are occurring over time.20 Unthank et al. looked at rat pups at both 10 and 20 weeks of age, and noted no changes in the intestinal vascular branching pattern or density. From this observation they concluded that there was very little change in intestinal vasculature from juvenile to adult rats but instead there was an ongoing remodeling of the terminal vasculature.21 No studies to date have evaluated intestinal vascular maturation in the first week of life. Overall, it is clear that more research into the intestinal vascular maturation process is necessary. Mucosal immaturity may result in clinical disease states to which the newborn is specifically susceptible, such as NEC.22 Gut maturation is thought to evolve through five stages with the fourth and fifth occurring around the time of birth and the first few weeks of life. Wagner and colleagues22 compared and contrasted aspects of amniotic fluid, colostrum, breast milk, and

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formula over the stages of gut maturation. One theme that is prevalent in all three human-produced forms of nutrition is that they all provide trophic factors, including epidermal growth factor and VEGF that help mature the intestinal tract, whereas formula is lacking these factors. Identifying the key time points in intestinal maturation of both the mucosa and the vasculature would help identify ideal timing for using peritoneal resuscitation and other novel therapies. In summary, we have demonstrated that direct peritoneal resuscitation is a novel treatment that targets the underlying pathophysiology of necrotizing enterocolitis. This therapy is easily instituted with tools and formulations which are clinically available in the current Neonatal Intensive Care Unit environment. Before clinical implementation, however, it will be important to determine the optimal timing and concentration of DPR to best curtail the devastating effects of this disease. REFERENCES

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