Department of Pediatric Surgery, Medical University of Warsaw, Marszałkowska 24, Warszawa 00576, PolandPoland
Department of Histology and Embryology, Center of Biostructure Research, Medical University of Warsaw, Chałubińskiego 5, Warszawa 02004, PolandPoland
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Purpose: The purpose of the study was to analyze the pro-inflammatory cytokines (TNF, IL-1, IL-6 and HNP) profile in the peritoneal fluid in preterm infants with intestinal perforation due to Necrotizing enterocolitis (NEC) or Spontaneous Intestinal Perforation (SIP) treated with percutaneous peritoneal drainage. Methods: 6 infants with intestinal perforation due to NEC or SIP treated with peritoneal drain age were analyzed prospectively. Samples of peritoneal fluid were drawn for 36 hours after drain placement. Concentrations of cytokines: TNF, IL-1, IL-6 and Human Neutrophil Peptide (HNP-1) were determined using enzyme like immunoassay technique. Results: The peritoneal fluid concentrations of TNF were 0-11,963 pg/mL (mean 960±2.249 pg/mL), IL-1 were 0-10,390 pg/mL (mean 968±2.219 pg/mL), IL-6 were 0.3-1.660 ng/mL (mean 184±335 ng/mL), HNP were 0.04-18.36 μg/mL (mean 3.18±4.32 μg/mL). Peritoneal fluid TNF level>1.000 pg/mL, IL-1>500-1.000 pg/mL and IL-6>200-500 ng/mL were associated with fatal outcome. Conclusions: In analyzed material the pattern of pro-inflammatory cytokines concentrations in peritoneal fluid after intestinal perforation in preterm infants was not identified. The highest peritoneal fluid TNF, IL-1 and IL-6 concentrations were related to fatal outcome in the most premature infants with unspecified extent of intestinal necrosis.
Keywords: Peritoneal drainage; Intestinal perforation; Tumor necrosis factor; Interleukin 1; Interleukin 6; Human neutrophil peptide (HNP-1)
Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are common gastrointestinal diseases in the preterm infants – in 1-3% of Neonatal Intensive Care Unit (NICU) patients.1 Traditional surgical management of perforated intra-abdominal viscus includes laparotomy with debridement of the offending intestinal segment and formation of stomas.2 In 1975, Marshall introduced peritoneal drainage as a way to stabilize and improve Page 48 the systemic status of premature infants with intestinal perforation due to NEC or SIP before formal laparotomy. 3 In 1977 Ein proved significant impact of bedside peritoneal drainage on premature infants’survival.4 Experience in utilizing peritoneal drainage in numerous centers is growing; however its mecha nism of action remains unknown.
Inflammation ongoing within intestinal wall tissues in NEC or SIP activates inflammatory cascade and releases numerous pro-inflammatory cytokines, which have also systemic effect.5 The number of cytokines and their role in the pathogenesis of hemodynamic instability in patients with NEC or SIP are still undefined.6 Elevated plasma levels of Tumor Necrosis Factor (TNF), Interleukin 1 (IL-1), Interleukin 6 (IL-6) and Interleu kin18 (IL-18) in infants with NEC reflect their local production in damaged intestinal tissue.7,8 Relation with symptoms of hemodynamic instability in infants with NEC treated with traditional laparotomy and pro-inflammatory mediators like TNF and IL-6 (which were similar to those in bacterial sepsis) was confirmed in many studies. 8 Correlation between cytokines (TNF, IL-1, IL-6) concentrations and severity of systemic inflammation was reported in many in vitro and in vivo studies.9,10 The role of inflammatory response mechanism in NEC and SIP pathogenesis was defined in animal model studies, in which intraperitoneal injection of anti-TNF antibodies resulted in increased survival of rat infants with NEC.11 The evaluation of serum cytokines profile requires multiple drawing of blood samples, which is unacceptable in very low birth weight infants. The use of peritoneal fluid obtained during abdominal drainage to determine cytokines concentration is non-invasive method compared to evaluation in serum. Data concerning peritoneal fluid concentrations of pro-inflammatory cytokines in NEC is uncommon. 6 Whereas data concerning cytokines profile in the peritoneal fluid in NEC or SIP newborns is not available.
The aim of the study was to assess the pro-inflamma tory cytokines profile in the peritoneal fluid as a result of bowel perforation in newborns with NEC or SIP treated with bedside peritoneal drainage.
During last three-year period, 10 infants with suspected intestinal perforation related to NEC or SIP were treated with bedside peritoneal drainage. Only 6 patients were prospectively analyzed (3 males and 3 females) as in 3 cases consents weren’t obtained, in 1 case intestinal perforation wasn’t confirmed by radiological examination.
Laparotomy is preferred method of treatment of bowel perforation due to NEC or SIP in author’s department. However, it cannot be performed in infants with symptoms of hemodynamic instability. In these rare cases peritoneal drainage is used as initial treatment before formal laparotomy.
All analyzed patients were born prematurely (gestational age: 23-29 weeks, mean 25.5±2.4 weeks), with birth weight 580-1.300 g (mean 823±263 g) and with asphyxia (mean APGAR score at first minute of life 3.5±1.7). Mechanical ventilation after birth was required in 5 infants. Intraventricular hemorrhages (IVH) were revealed in all patients, in 4 children IVH grade 4.
Intestinal perforation was confirmed by radiological examination in all infants.
Peritoneal drain was placed between 6-21 days of age (mean 12.5±5.9 days). During drain placement 4 infants presented symptoms of metabolic acidosis (pH arterial blood: 7.07- 7.285, mean 7.17±0.03). All patients required ventilation supporting: 4 infants were ventilated in SIMV mode, remaining 2 in HFO mode. Saturation (SaO2) was 87-96% (mean 92.5±3%), Heart Rate (HR) was 147-206 per minute (mean 173±20) and Mean Arterial Pressure (MAP) was 16.78 mm Hg (mean 37.5±19.3 mm Hg) at the moment of drain placement. 4 infants required administration of at least one catecholamine, in one patient diuretic therapy was necessary (Table 1).
In four cases drain was placed in local NICU due to patient’s hemodynamic instability. Drain placement was per formed under sterile conditions, sedation and local anesthesia in all patients. Peritoneal fluid contained gas in 5 cases and meconium was observed in 4 of them. 3 infants died (in day 1., 2. and 5. after drain placement), 2 of them presented clinical symptoms of NEC, one of SIP confirmed in X-ray and ultrasound examinations, however autopsy wasn’t performed. 3 patients survived and underwent laparotomy 19-48 hours after the drain place ment. SIP was confirmed in 2 patients and segmentary NEC in Patient GA birth weight Apgar SCORE IVH pH SaO2 MAP NEC/SIP survive p1 26 940 6 IV 7,195 87 36 SIP + p2 28 930 4 II 7 94 29 NEC + p3 23 580 1 IV 7,12 94 29 SIP died p4 23 590 2 IV 7,166 94 16 NEC died p5 24 600 3 IV 7,285 96 78 NEC died p6 29 1300 5 III 7,28 90 37 SIP + Table 1: Baseline characteristics of the patients treated with peritoneal drainage. Page 49 one child during surgical intervention. In all survived patients resection of perforated bowel were performed and stomas were formed (Table 1).
Samples of peritoneal fluid were drawn every 3 hours during initial 12 hours followed by every 6 hours later on. Collection of samples was continued for 36 hours after drain placement. Drawn peritoneal fluid in portions of 0.2-1.0 mL was centrifuged and stored at -86°C.
Quantikine ELISA kits R&D (Mineapolis, MN, USA) were used to determine TNF, IL-1 and IL-6 concentrations using enzyme-linked immunoassay technique. Concentration of Human Neutrophil Peptide (HNP) was measured using Human Neutrophil Peptide 1-3 ELISA kit Hycult Biotechnology (Uden, Netherlands). Concentrations of cytokines: TNF, IL-1, IL-6 and HNP were determined in all collected samples.
Juxtaposition of peritoneal fluid volume of all collected samples and outcome of treatment with bedside peritoneal drainage is shown in the Table 2.
Concentrations of TNF in peritoneal fluid were 0-11,963 pg/mL (mean 960±2,249 pg/mL). The highest concentrations were observed in patient 4, who symptoms of NEC presented and died in day 2, after drain placement. The lowest concentrations were observed in patient 1 in whom SIP was confirmed during laparotomy (Table 3).
IL-1 Concentrations of IL-1 in peritoneal fluid were 0-10,390 pg/mL (mean 968±2,219 pg/mL). The highest concentrations were observed in patient 4. The lowest concentrations were observed in patient 2 in whom segmentary NEC was confirmed during surgical intervention after 24 hours treatment with bedside peritoneal drainage (Table 4).
Table 2. Sample volume summary and treatment outcome.
Table 3. Concentrations of TNF, diagnosis and survival in patients treated with peritoneal drainage.
Table 4. Concentrations of IL-1, diagnosis and survival in patients treated with peritoneal drainage.
time (h) follow-up patient 0 3 6 9 12 18 24 30 36 p1 5 6 3,5 4 2 4 2 3,5 1,7 laparotomy in 48. hours p2 18 4 1 4 0 0 laparotomy p3 3,2 1,2 1 0 1,2 0 0 death p4 5 1 0.5 0 1,2 2,5 death p5 0,5 1 1 3 1,5 5 8 3 1,5 death in day 5.
p6 18 2,5 0 1 1,5 5,5 laparotomy - Volume (mL) patient time (h) NEC/SIP survive 0 3 6 9 12 18 24 30 36 p1 0 16 0 22 27 0 0 0 40 SIP + p2 70 128 53 90 NEC + p3 1465 3903 5499 721 SIP died p4 11963 2934 512 320 132 NEC died p5 0 222 20 216 2752 1096 67 38 36 NEC died p6 716 458 354 414 302 SIP + patient time (h) NEC/SIP survive 0 3 6 9 12 18 24 30 36 p1 0 0 11 28 28 165 193 185 238 SIP + p2 90 16 48 34 NEC + p3 1467 1100 982 449 SIP died p4 10390 7834 5456 2139 565 NEC died p5 0 13 10 102 741 1190 363 101 105 NEC died p6 475 212 93 31 27 SIP +
IL-6 Ranges of IL-6 concentrations were 0.3-1,660 ng/mL (mean 184±335 ng/mL). The highest concentrations were observed in patient 4 and the lowest in patient 6, who underwent laparotomy in 19th hour after drain placement and SIP was confirmed (Table 5).
Concentrations of peritoneal fluid HNP were 0.04- 18.36 μg/mL (mean 3.18±4.32 μg/mL). The highest concentrations were observed in patient 6 and the lowest in patient 2 (Table 6).
Any particular tendency for TNF, IL-1, IL-6 and HNP wasn’t observed throughout drainage duration. Furthermore the maximum concentrations were noted in different time points for each patient.
Inflammation ongoing within intestinal wall tissues in NEC or SIP releases numerous mediators including the most pro-inflammatory cytokines like: TNF, IL-1 and IL-6. These cytokines released in the intestine, implicated in the hepatic production of acute-phase proteins, in the enhancement of T-cell proliferation and in the promotion of B-cell antibody secretion. Pro-inflammatory cytokines also stimulate macrophages and neutrophils to produce large quantities of reactive and cytotoxic species of oxygen and nitrogen, which are important element of inflammatory response. However, these cytotoxic compounds may also cause substantial host tissue damage and multiple organ dysfunction, especially when their production remains unchecked when the host defense system is immature, as commonly seen in stressed neonates in the NICU. 5 In these cases cytokines and cytotoxic species like Nitrogen Oxide (NO) could affect hemodynamic instability in preterm infants. Symptoms of progressive multiple organ dysfunction described in patients with NEC or SIP result from inflammatory damage in intestinal tissue but could be also consequence of large production of proinflammatory mediators which have systemic effect.
Additionally intestinal inflammation with secondary failure of the gut barrier expedites bacterial translocation from the gut into the systemic circulation and releases human neutrophil peptides (HNP) from neutrophil granules. Furthermore, antimicrobial activity HNP participates in the regulation of chemotaxis and triggering of the inflammatory reactions. Correlation of elevated HNP in peritoneal fluid with pro-inflammatory cytokines like TNF, IL-6 and IL-8 was described in patients with endometriosis.12 The inflammatory process within intestinal wall due to NEC or SIP concerns also the capillaries and may result in leaking of plasma cytokines into the peritoneum enhancing the levels of peritoneal fluid cytokines. The study comparing the cytokines profile in peritoneal fluid and serum requires multiple drawing of blood samples, which is unacceptable in very low birth weight infants.
In our study, the infants who died during treatment with peritoneal drainage had lower gestational age, birth weight and Apgar scores than infants who survived and underwent subsequent laparotomy. Each patient who died required mechanical ventilation in the first minute after birth and every patient had IVH IV° detected. Differences in clinical condition weren’t observed (Table 1).
patient time (h) NEC/SIP survive 0 3 6 9 12 18 24 30 36 p1 88,6 89,1 97,8 106,5 154,1 67,1 33,3 54,8 SIP + p2 95 90 233 32,9 NEC + p3 508 90 30 25,1 SIP died p4 1022 1660 24 39 NEC died p5 0,3 9,2 12,8 53,3 396 732 248 120,3 109,6 NEC died p6 5 12 35 6 2 SIP + patient time (h) NEC/SIP survive 0 3 6 9 12 18 24 30 36 p1 0,35 1,03 0,19 0,3 0,64 0,19 0,46 0,24 SIP + p2 0,21 0,18 0,21 0,24 NEC + p3 10,46 6,47 6,11 1,17 SIP died p4 11,8 10,81 0,04 2,6 1,3 NEC died p5 0,16 0,43 1,01 3,16 2,2 2,1 1,13 NEC died p6 18,36 7,04 8,03 2,85 3,6 SIP + Table 5: Concentrations of IL-6, diagnosis and survival in patients treated with peritoneal drainage.
Table 6. Concentrations of HNP, diagnosis and survival in patients treated with peritoneal drainage.
The analysis presented revealed higher concentrations of TNF, IL-1 and IL-6 in patients who died comparing to patients who survived, although correlation between cytokines concentrations and treatment outcome wasn’t defined.
The highest concentrations of TNF, IL-1 and IL-6 were observed in patient 4, who presented clinical and radiological symptoms of NEC and died in second day after drain placement. He was born in the 23rd gestational week as extremely low birth weight infant with asphyxia. The autopsy wasn’t performed and therefore correlation of cytokines concentration and the extent of intestinal damage couldn’t be determined.
The lowest concentrations were observed in patient 1 for TNF, in patient 2 for IL-1 and HNP and in patient 6 for IL-6. All of these patients survived and underwent laparotomy. Dur ing surgical intervention SIP in patients 1 and 6 and segmentary NEC in patient 2 was confirmed. Their gestational week, birth weight and Apgar score were higher than in patient 4.
In our study peritoneal fluid TNF level>1,000 pg/mL, IL-1>500-1,000 pg/mL and IL-6>200-500 ng/mL were associated with fatal outcome. Similar differences were described by Morecroft in analysis of 18 infants with NEC. In his study plasma IL-6 levels were significantly higher in infants with Bell stage III disease compared to infants with Bell II stage disease (3,127 pg/mL vs. 127 pg/mL), nonetheless there was no discernible pattern in plasma TNF concentration in both groups.13 In another study of 24 infants with NEC, Morecroft described plasma IL-6 levels tended to be higher in infants who died compared to survivors (2,835 pg/mL vs. 1,023 pg/mL).9 The maximum rather than decreasing concentrations of these cytokines would have prognostic value in analyzed mate rial. Tendency of cytokines concentrations as well as time point of maximum concentration weren’t relevant. In presented analysis, HNP concentration seems not to have any value in children with NEC or SIP.
Furthermore, our study revealed higher mean perito neal fluid concentrations of TNF, IL-1 and IL-6 comparing to the mean plasma concentrations of these cytokines in patients with bacterial Gram-positive sepsis. The results presented by de Bont were: TNF 560 pg/mL, IL-1 18 pg/mL, IL-6 79.7 ng/mL comparing to 960 pg/mL, 968 pg/mL, 184 ng/mL in our mate rial, respectively.14 The results of peritoneal fluid cytokines levels demonstrated in this study are considerably higher than plasma cytokines levels in patients with NEC described in literature. Sharma described data of 27 children- mean plasma concentrations of cytokines in NEC which were accordingly: for TNF: 277 pg/ mL vs. 1,147 pg/mL, for IL-1: 499 pg/mL vs. 1,622 pg/mL and for IL-6: 316 pg/mL vs. 286,900 pg/mL in presented NEC patients.5 Higher peritoneal fluid level of these cytokines compared to plasma levels may suggest an advantage of their local production in damaged intestinal tissues.
The analysis of cytokine profiles presented in this study is an attempt to explain beneficial effect of using bedside peritoneal drainage in newborn with bowel perforation as a result of NEC or SIP. Regardless this is a pilot study, an evacuation of cytokines with peritoneal fluid seems to be not the only factor effecting beneficially (gradual decrease of cytokines concentra tions wasn’t observed clearly). The relationship between cytokines concentration and the degree of infants immaturity as well as the extent of intestinal damage remains unknown.
This study was approved by Local Ethics Committee.
In analyzed material the pattern of pro-inflammatory cytokines concentrations in peritoneal fluid after intestinal per foration in preterm infants was not identified.
The highest peritoneal fluid TNF, IL-1 and IL-6 concentrations were related to fatal outcome in the most premature infants with unspecified extent of intestinal necrosis.
CONFLICTS OF INTEREST: None.
We hereby confirm that all patients’ parents participating in study “The TNF, IL-1, IL-6 and HNP peritoneal fluid concentrations in premature infants treated with peritoneal drainage for intestinal perforation- preliminary study” were consented relevantly and all signed consent forms are retained.