G Model
ADVMS 87 1–5 Advances in Medical Sciences xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Advances in Medical Sciences journal homepage: www.elsevier.com/locate/advms 1 2 3 4 5 6 7 8 9 10
Original Research Article
Small intestinal lactoferrin and calprotectin levels in different stages of necrotizing enterocolitis in a rat model Sag˘lam a,*, Vural Kesik a, Bahadır C¸alıs¸kan b, Tuncer C¸aycı c, Mehmet Ag˘ıllı c, Nuri Yig˘it d, Oguzhan Babacan a, Nadir Korkmazer a, Erman Atas a, Mustafa Gulgun a
Q1 Celal
a
Department of Pediatrics, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey Department of Pediatric Surgery, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey Department of Biochemistry, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey d Department of Pathology, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 February 2014 Accepted 20 February 2015 Available online xxx
Background and aim: Necrotizing enterocolitis (NEC) is a severe disease of mostly premature infants with high morbidity and mortality rates. There is no reliable biomarker for detecting newborns at risk for NEC development. We aimed to investigate small intestinal lactoferrin (LF) and calprotectin (CAL) levels as predictors and indicators of disease severity in an experimental newborn rat model. Methods: Newborn pups were randomly divided into two groups, NEC and control. The NEC group pups were decapitated on the second, third and fourth days of the experiment for an assessment of the different stages of NEC. In the study group, hypoxia-reoxygenation model used to induce NEC. As biochemical parameters, small intestinal LF and CAL levels were measured with an enzyme-linked immunosorbent assay technique and intestinal injury scoring was evaluated as a pathologic parameter. Results: Small intestinal levels of both LF and CAL increased in the second and the third day groups, but began to decrease by the fourth day. The first, second and third day levels of LF and CAL were higher than controls. The intestinal injury scores of all NEC groups were significantly higher than the control group. Conclusion: Small intestinal lactoferrin and calprotectin were good markers for demonstrating NEC. However, instead of spot testing, monitoring the levels of these markers may be more informative. ß 2015 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Medical University of Bialystok.
Keywords: Small intestinal biomarkers Calprotectin Lactoferrin Necrotizing enterocolitis
11 12
1. Introduction
13 14 15 16 17 18 19 20 21 22 23
Necrotizing enterocolitis (NEC) is one of the most common gastrointestinal (GI) emergencies afflicting predominantly premature infants in neonatal intensive care units (NICU) [1]. The disease is often observed in premature infants with low birth-weight and gestational age. Low APGAR scores, chorioamnionitis, exchange transfusion, prolonged rupture of membranes, congenital heart disease, and neural tube defects are among other predisposing factors [2]. Its incidence varies between 5% and 10% among infants born at less than 32 weeks of gestation and with a birth weight of less than 1500 g [3]. The mortality of NEC ranges from 10% to 50% and survivors suffer from significant morbidity, such as long-term requirement
* Corresponding author at: Gulhane Military Medical Academy, Department of Pediatrics, Etlik 06018, Ankara, Turkey. Tel.: +90 312 3044394; fax: +90 312 3044381. E-mail address: [email protected] (C. Sag˘lam).
for parenteral nutrition, malabsorption and malnutrition related to short bowel syndrome, and neurodevelopmental, neurosensory, and functional disabilities [4]. Despite prematurity, enteral feeding, intestinal hypoxia-ischemia, and bacterial colonization have been hypothesized to cause NEC. Besides being difficult to treat, there is no effective preventative strategy of NEC [5]. The diagnosis of NEC is based on modified Bell criteria proposed by Walsh and Kleigman, who developed the criteria with systemic, abdominal and radiologic findings to more clearly classify NEC [6]. There is no diagnostic laboratory findings, but only supportive in diagnosis of NEC [7]. The presenting signs are nonspecific, such as feeding intolerance, abdominal distention or tenderness, occult or gross blood in the stool, lethargy, apnea, respiratory distress, or poor perfusion that resembles diseases like sepsis and respiratory distress syndrome [8]. Thus, detecting infants at risk for NEC or those in early stages of the disease would offer opportunities for early intervention. The fecal proteins calprotectin (FCAL) and lactoferrin (FLF), which are derived from neutrophils, have the potential to be ideal
http://dx.doi.org/10.1016/j.advms.2015.02.004 1896-1126/ß 2015 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Medical University of Bialystok.
Please cite this article in press as: Sag˘lam C, et al. Small intestinal lactoferrin and calprotectin levels in different stages of necrotizing enterocolitis in a rat model. Adv Med Sci (2015), http://dx.doi.org/10.1016/j.advms.2015.02.004
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
G Model
ADVMS 87 1–5 C. Sag˘lam et al. / Advances in Medical Sciences xxx (2015) xxx–xxx
2
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
markers of intestinal inflammation because their measurement is non-invasive, easy to obtain, and inexpensive. Measurement can be done in small intestinal contents/feces with less than 5 g of stool in the case of intestinal inflammation [9]. Fecal calprotectin, a calcium- and zinc-binding neutrophilic cytosolic protein, is found in proportion to the degree of inflammation present, is resistant to colonic bacterial degradation, and is stable in stools for up to 1 week at room temperature. Fecal lactoferrin is an iron-binding protein that is similar to fecal calprotectin regarding that it is released upon neutrophil activation and is also resistant to proteolysis in the feces for up to 1 week [9]. Both of the fecal biomarkers have been previously established as screening tests for use in diagnosing inflammatory bowel disease in children (Crohn’s disease, ulcerative colitis, etc.) [7,9,10] and are now taking on an expanding role in disease monitoring and relapse detection. They have also been evaluated as potential diagnostic tools for NEC [11–18]. This research has concluded that the utility of these biomarkers for early diagnosis and assessment of resolution of NEC needs to be studied in wider NEC series. In this study, we were able to investigate small intestinal LF and CAL levels in different stages of NEC as predictors and indicators of disease severity in an experimental newborn rat model and their correlation with intestinal injury score.
66
2. Materials and methods
67
2.1. Animals and experimental design
68 69 70 71 72 73 74 75 76
The study protocol was approved by the Animal Care and Use Committee of Gulhane Military Medical School (Ankara, Turkey). Forty-six Sprague-Dawley neonatal rats, originating from five different litters, were used in four separate groups in the experiment. Newborn pups were randomly divided into one of the following four groups: NEC (assigned to three groups; G1, G2, G3) and control (group C). Group C rats (controls; n = 7) were left with their mothers, breast fed ad libitum, and not submitted to stress. In the NEC groups, newborn rats were collected from their
mothers immediately after birth to prevent suckling of maternal milk and kept at 37 8C in a humidified incubator.
77 78
2.2. Necrotizing enterocolitis procedure
79
All the pups in NEC groups were gavage fed 0.2 mL of special rodent formula using a 24 gauge thickness silicon catheter 3 times a day (Fig. 1A). A hyperosmolar rat milk substitute was prepared using 15 g Similac 60/40 (Abbott-Turkey) in 75-ml Puppy-milk canine milk replacement (Beaphar-bogena, B.V. Sedel, Netherland). To induce clinical and pathological signs of NEC, rat pups from all three NEC groups (G1, for 48 h; G2, for 72 h; G3, for 96 h) were stressed twice daily with asphyxia by breathing 100% CO2 gas for 5 min followed by cold stress at 4 8C for 5 min, and 100% O2 inhalation for 5 min in a sealed airtight plastic container.
80 81 82 83 84 85 86 87 88 89
2.3. Sample collection
90
All rats were killed on the day as previously described for each group (for G1 [n = 9], G2 [n = 7], G3 [n = 6], and C groups, at the end of the 2nd day, 3rd day, and 4th day, respectively) via decapitation. The abdomen was opened and the whole intestine were inspected for evidence of NEC, such as intestinal discoloration, fragility, weakness of tissue integrity, edema, intestinal hemorrhage, ileal distension, pneumatosis intestinalis, perforation, and necrosis, and fixed length of intestinal specimens such as a 2 cm section of the distal ileum and a 3 cm to the ileocaecal valve were harvested for histopathologic and biochemical evaluation, respectively. Tissue specimens were flushed with cold saline solution and fixed in 10% buffered formalin for histopathologic evaluation. Materials from all of the newborn rats included in the study were collected from the resected intestinal segments. Tissue specimens were taken away from 3 cm-fixed length to the ileocaecal valve placed in 0.5 mL of saline for providing standardization procedure in the measurements of the biomarkers. Then all materials was frozen in liquid nitrogen and stored at 80 8C for biochemical examination of small intestinal lactoferrin and calprotectin.
91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
Fig. 1. (A) A 24 gauge-thickness silicon catheter was used to feed the pups, (B) Blackly abdomen with hyperemia, and distension, (C) Dark purple/black discolorated intestines, (D) Dilated intestines as a result of extensive gas.
Please cite this article in press as: Sag˘lam C, et al. Small intestinal lactoferrin and calprotectin levels in different stages of necrotizing enterocolitis in a rat model. Adv Med Sci (2015), http://dx.doi.org/10.1016/j.advms.2015.02.004
G Model
ADVMS 87 1–5 C. Sag˘lam et al. / Advances in Medical Sciences xxx (2015) xxx–xxx
111
2.4. Histopathologic evaluation
112 113 114 115 116 117 118 119 120 121 122 123
Samples of intestinal tissues from a fixed length (2 cm section of the distal ileum) were fixed in 70% ethanol, processed, embedded in paraffin, and microtome sectioned at 4–6 mm. The slides were counterstained with hematoxylin–eosin (H&E) and examined under a light microscope for histological evaluation of NEC. Histological changes in the ileum were scored by a blinded pathologist and graded as follows: 1 = normal, intact villous epithelium with normal histology; 2 = mild villous edema, with epithelial sloughing confined to tips of the villi; 3 = mild midvillous necrosis; 4 = moderate midvillous necrosis, with crypts still readily detectable; and 5 = severe necrosis of entire villi with complete absence of epithelial structures [9,10].
124
2.5. Small intestinal lactoferrin and calprotectin measurements
125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142
Each intestinal material was tested for concentrations of LF and CAL with an enzyme-linked immunosorbent assay (BI˙O-TEK, Synergy HT; ELISA) according to the manufacturer’s instructions (Rat Lactoferrin (LTF/LF) ELI˙SA kit, Hangzhou Eastbiopharm Co. Ltd. Hangzhou, China, Cat. No: CK-E11678 for LF; Rat Calprotectin (CAL) ELI˙SA kit, Hangzhou Eastbiopharm Co. Ltd. Hangzhou, China, Cat. No: CK-E90475 for CAL). Briefly, intestinal samples were suspended in diluent-buffer. After homogenization, and if necessary, centrifugation, the homogenates (supernatants) were transferred to microplates for detection with antibodies specific for the respective inflammatory markers. Anti-FLF and Anti-FCAL antibody, each conjugated with peroxidase, were used for development. For each well, the optical density was measured at 450 nm with a microplate ELISA reader (BI˙O-TEK, Synergy HT, USA). The results of the tests samples were calculated from the standard curve and expressed as micrograms per milliliter for LF and as nanograms per milliliter for CAL. The intra-assay and the inter-assay coefficient variations of each kit were 10% and 12%, respectively.
143
2.6. Statistical analysis
144 145 146 147 148
Software SPSS 13.0 for Windows was used in all statistical tests. Dual comparisons among the groups were performed by Mann– Whitney U test. All data are expressed as mean SD, their minimum–maximum ranges. When P was less than 0.05, the difference was considered statistically significant.
149
3. Results
150 151 152 153 154 155 156 157
Twenty-nine pups survived until the end of the study. During the NEC procedure, in the NEC groups (total, n = 39) seventeen pups (43.6%) died as a result of aspiration due to the difference in practice of feeding process by gavage between practitioners such as the catheter was inserted into the trachea instead of the stomach, accidentally. There was no loss in the control group during the study because of that the control group were breastfeded.
158
3.1. Macroscopic and microscopic examination of the GI tract
159 160 161 162 163 164 165 166
Though all pups in the control group had a normal macroscopic appearance of the GI tract without any discoloration and distension of the abdomen and the intestines, we observed all the pups in the NEC groups had abdomens with varying degrees of hyperemia, distension, and had dark purple/black discolored intestines that were also dilated as a result of common gas. Results of macroscopic visual evaluation were recorded, and pictures were taken using a digital camera (Fig. 1B–D).
3
Table 1 Scores of histological analyses of intestinal segments of neonatal rats (mean SD). Groups
G1 (n:9)
G2 (n:7)
G3 (n:6)
Score
2.11 0.33* (2.0–3.0)
2.28 0.48# (2.0–3.0)
2.16 0.40 (2.0–3.0)
* #
a
C (n:7) a
1.0 0 (1.0–1.0)
p = 0.001: G1 vs. control (C) group. p = 0.001: G2 vs. control (C) group. p = 0.001: G3 vs. control (C) group.
Histological analyses of intestinal segments were performed using a scoring system from 1 to 5 to determine the severity of NEC, as previously described. All of the intestinal samples collected from the control group rats showed no visual pathological changes in the structure resembling the development of NEC (Stage 1). After exclusion of 4 intestinal tissue samples of the G1 group due to autolysis, histological findings in most pups of all three NEC groups showed significant pathological intestinal changes in the structure, described as minimal (Stage 2) and mild (Stage 3) necrosis (Table 1). Comparison of the degree of intestinal histopathology between the NEC and the control groups revealed that all the rats in the NEC groups (G1, G2, G3) had a statistically significant difference (p = 0.001, p = 0.001, p = 0.001, respectively). But when we compared NEC groups with each other in terms of the degree of intestinal histopathology, we did not observe any statistically significant difference (p > 0.05) (Table 1).
167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182
3.2. Small intestinal lactoferrin and calprotectin levels
183
Small intestinal LF and CAL levels were measured using ELISA kits. The mean values and the linear graph visualizing the level changes of both markers in the different groups are shown in Table 2, and Fig. 2, respectively. When we compared LF and CAL levels of the G1 and G3 groups with the control group, we did not observe any statistically significant difference (G1 vs. the control group, p = 0.229; G3 vs. the control group, p = 0.445, respectively). Conversely, there was a statistically significant difference between G2 and the control group (G2 vs. the control group, p = 0.026). When we compared the levels of markers between each other of NEC groups, we did not observe any statistically significant difference (p > 0.05) (Table 2).
184 185 186 187 188 189 190 191 192 193 194 195
4. Discussion
196
Necrotizing enterocolitis is a severe problem of premature infants. The initial presentation of the disease is nonspecific and is often hard to distinguish from other gastrointestinal diseases and sepsis. High morbidity and mortality rates urge us to find new biomarkers for early detection of the disease; there is not a reliable
197 198 199 200 201
Table 2 Results of small intestinal lactoferrin and calprotectin levels (mean SD). Groups
CAL (ng/mL)
LF (mg/mL)
G1 (n:9)
19.52 11.32* (4.96–36.30) 24.74 11.56# (1.49–38.21) a 18.14 10.53 (6.44–32.24) 12.81 5.74 (6.11–19.61)
14.56 8.28* (3.92–26.84) 18.39 8.42# (1.36–28.02) a 13.56 7.75 (4.93–23.95) 9.62 4.24 (4.67–14.63)
G2 (n:7) G3 (n:6) C (n:7)
Abbreviations: CAL: calprotectin LF: lactoferrin. * p = 0.229: G1 vs. control (C) group. # p = 0.026: G2 vs. control (C) group. a p = 0.445: G3 vs. control (C) group.
Please cite this article in press as: Sag˘lam C, et al. Small intestinal lactoferrin and calprotectin levels in different stages of necrotizing enterocolitis in a rat model. Adv Med Sci (2015), http://dx.doi.org/10.1016/j.advms.2015.02.004
G Model
ADVMS 87 1–5 4
C. Sag˘lam et al. / Advances in Medical Sciences xxx (2015) xxx–xxx
Fig. 2. Linear graph visualizing the changes of the CAL and LF levels in the different groups.
202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249
laboratory test to detect either newborns at risk for NEC or those in early stages of the disease. A rapid increase in small intestinal LF and CAL levels and also increased intestinal injury scores were detected in ongoing stages of NEC after the fourth day of the model (fourth day was reported as the day with the most intense injury in most studies). From this point of view, LF and CAL levels in small intestinal contents were increased in the early stages of NEC, and in the stages of proven NEC there was a decline in the levels of these markers. Results considered that there was a rapid response of these markers to ischemic and hyperosmolar food injury in our study. It is generally thought that there are multiple preparatory factors such as the followings: preterm birth [8], hyperosmolar enteral feeding [19], dysfunctional intestinal motility [20], reduced intestinal blood flow [21], microbe-induced pro-inflammatory epithelial signaling [22], and enteroinvasive microbes [23]. From this point of view, we suggested that intestinal injury due to the reduced intestinal blood flow and hyperosmolar enteral feeding is the ‘‘First Hit’’ in starting the disease. In our study, we showed that LF and CAL levels began to rise, just at that point. If a ‘‘Second Hit’’, as we mentioned above, increases the injury, necrosis climbs due to significant inflammation in response to colonization. Laforgia et al. investigated FCAL levels in the first passed meconium in 84 term and 47 preterm infants. Mean FCAL level was 145.2 78.5 mg kg 1 in meconium and negatively correlated with gestational age, birth weight, and 5-min Apgar score. They hypothesized that the migration of neutrophils into the gut lumen is increased in perinatal asphyxia because of ischemic changes in the intestinal mucosa [18]. Both of these biomarkers have been previously validated as accurate markers of inflammatory bowel disease in children (Crohn’s disease, ulcerative colitis, etc.) [7,9,10] and were also investigated in the diagnosis of NEC [11–18]. There are few studies in newborns [11–15,17,18] about the role of FCAL in diagnosing NEC, but only one [16] investigated FLF in NEC. While an increased level of FCAL was detected in some of these studies, others revealed no significant elevation in newborns with NEC compared to patients without NEC. FCAL levels in preterm infants with a diagnosis of NEC were significantly increased compared with controls [11,15]. A prospective study, in a population of 59 very low birth weight infants (with a mean gestational age of 27.2 2.6 weeks and a mean birth weight of 939 273 g), which is analyzing weekly FCAL levels from birth revealed that levels above 2000 mg/g are a useful but not an early marker of NEC (seven of them were eventually diagnosed with NEC) [17]. In a brief report by Thuijls et al., 14 of 35 neonates suspected of NEC developed NEC. Median FCAL levels were significantly higher
in neonates suspected of NEC who finally developed NEC than in those with other final diagnoses (589.2 and 69.6 mg/g feces, respectively). The ideal cut-off value for discrimination of neonates with NEC from others was determined to be 286.2 mg/g feces for FCAL. They reported that FCAL had a high diagnostic accuracy (sensitivity of 93% and specificity of 86%) and clinically relevant likelihood ratios (LRs) (positive LRs of 12.29, and negative LRs of 0.15) for an early diagnosis of NEC [13]. Reisinger et al. investigated FCAL levels in 62 neonates with clinical suspicion of NEC (29 of them were eventually diagnosed with NEC). The median gestational age was 215 (175–289) days, and the median birth weight was 1328 (585–3570) grams. There were significant differences in gestational age, birth weight, or sex between the groups. Median FCAL levels in the NEC group were significantly higher than those in the non-NEC group (402.2 and 79.6 mg/g feces), respectively [14]. Selimoglu et al. analyzed FCAL and FLF as diagnostic tools for NEC for 14 newborns with a control group of 63 newborns. There was no significant difference in FCAL and FLF levels between NEC and control groups. The authors suggested that these two biomarkers were not useful in the early diagnosis of the disease, but FCAL could be able to confirm the diagnosis only in the late stages of NEC [16]. The discrepancies between the results and the inclusion of a small number of patients were considered as limitations of these studies. Besides theses markers were elevated in all previous studies, studies also showed that they were affected from multiple factors such as gestational age, birth weight, gender, and Apgar score. Due to not having strong clues regarding of which factor will effect or cause to NEC, monitoring their levels may be more effective rather than spot testing in the diagnosis of NEC. Therefore, in our study, we analyzed small intestinal levels of LF and CAL in the different stages of NEC. We showed that levels of both of these markers increased in the second and the third day groups, but tended to decrease on the fourth day. Although the results of day 2, day 3, day 4 were higher than control and were increased from the beginning of the first day, statistical significance was only determined on the third day in our experiment. The groups represent the day of NEC. Thus G1 represents the cases that exposed one day to the NEC conditions and so the early phase of NEC. The intestinal injury scores of all NEC groups were significantly higher than the control group. In conclusion, we found that the levels of two markers increased in the early stages of NEC and these results correlated with intestinal injury scores. Despite the importance of ischemic intestinal injury and intestinal immaturity as main factors involved in the pathogenesis of NEC, as we mentioned them as ‘‘First Hit’’ above, we hypothesized that a ‘‘Second Hit’’ is mandatory for the development of NEC. From this point of view, monitoring the levels of these markers may give more accurate results in diagnosing NEC. Further studies with a higher number of cases are essential for monitoring the levels of these fecal markers in NEC.
250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300
Conflict of interests
301
None declared.
302
Financial disclosure
303
None declared.
304
References [1] Neu J, Weiss MD. Necrotizing enterocolitis: pathophysiology and prevention. JPEN J Parenter Enteral Nutr 1999;23(5 Suppl.):S13–7. [2] Ostlie DJ, et al. Necrotizing enterocolitis in full-term infants. J Pediatr Surg Q2 2003;38(7):1039–42.
Please cite this article in press as: Sag˘lam C, et al. Small intestinal lactoferrin and calprotectin levels in different stages of necrotizing enterocolitis in a rat model. Adv Med Sci (2015), http://dx.doi.org/10.1016/j.advms.2015.02.004
305 306 307 308 309
G Model
ADVMS 87 1–5 C. Sag˘lam et al. / Advances in Medical Sciences xxx (2015) xxx–xxx
310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331
[3] Llanos AR, et al. Epidemiology of neonatal necrotising enterocolitis: a population-based study. Paediatr Perinat Epidemiol 2002;16(4):342–9. [4] Salhab WA, et al. Necrotizing enterocolitis and neurodevelopmental outcome in extremely low birth weight infants
ADVMS 87 1–5 Advances in Medical Sciences xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Advances in Medical Sciences journal homepage: www.elsevier.com/locate/advms 1 2 3 4 5 6 7 8 9 10
Original Research Article
Small intestinal lactoferrin and calprotectin levels in different stages of necrotizing enterocolitis in a rat model Sag˘lam a,*, Vural Kesik a, Bahadır C¸alıs¸kan b, Tuncer C¸aycı c, Mehmet Ag˘ıllı c, Nuri Yig˘it d, Oguzhan Babacan a, Nadir Korkmazer a, Erman Atas a, Mustafa Gulgun a
Q1 Celal
a
Department of Pediatrics, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey Department of Pediatric Surgery, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey Department of Biochemistry, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey d Department of Pathology, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 February 2014 Accepted 20 February 2015 Available online xxx
Background and aim: Necrotizing enterocolitis (NEC) is a severe disease of mostly premature infants with high morbidity and mortality rates. There is no reliable biomarker for detecting newborns at risk for NEC development. We aimed to investigate small intestinal lactoferrin (LF) and calprotectin (CAL) levels as predictors and indicators of disease severity in an experimental newborn rat model. Methods: Newborn pups were randomly divided into two groups, NEC and control. The NEC group pups were decapitated on the second, third and fourth days of the experiment for an assessment of the different stages of NEC. In the study group, hypoxia-reoxygenation model used to induce NEC. As biochemical parameters, small intestinal LF and CAL levels were measured with an enzyme-linked immunosorbent assay technique and intestinal injury scoring was evaluated as a pathologic parameter. Results: Small intestinal levels of both LF and CAL increased in the second and the third day groups, but began to decrease by the fourth day. The first, second and third day levels of LF and CAL were higher than controls. The intestinal injury scores of all NEC groups were significantly higher than the control group. Conclusion: Small intestinal lactoferrin and calprotectin were good markers for demonstrating NEC. However, instead of spot testing, monitoring the levels of these markers may be more informative. ß 2015 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Medical University of Bialystok.
Keywords: Small intestinal biomarkers Calprotectin Lactoferrin Necrotizing enterocolitis
11 12
1. Introduction
13 14 15 16 17 18 19 20 21 22 23
Necrotizing enterocolitis (NEC) is one of the most common gastrointestinal (GI) emergencies afflicting predominantly premature infants in neonatal intensive care units (NICU) [1]. The disease is often observed in premature infants with low birth-weight and gestational age. Low APGAR scores, chorioamnionitis, exchange transfusion, prolonged rupture of membranes, congenital heart disease, and neural tube defects are among other predisposing factors [2]. Its incidence varies between 5% and 10% among infants born at less than 32 weeks of gestation and with a birth weight of less than 1500 g [3]. The mortality of NEC ranges from 10% to 50% and survivors suffer from significant morbidity, such as long-term requirement
* Corresponding author at: Gulhane Military Medical Academy, Department of Pediatrics, Etlik 06018, Ankara, Turkey. Tel.: +90 312 3044394; fax: +90 312 3044381. E-mail address: [email protected] (C. Sag˘lam).
for parenteral nutrition, malabsorption and malnutrition related to short bowel syndrome, and neurodevelopmental, neurosensory, and functional disabilities [4]. Despite prematurity, enteral feeding, intestinal hypoxia-ischemia, and bacterial colonization have been hypothesized to cause NEC. Besides being difficult to treat, there is no effective preventative strategy of NEC [5]. The diagnosis of NEC is based on modified Bell criteria proposed by Walsh and Kleigman, who developed the criteria with systemic, abdominal and radiologic findings to more clearly classify NEC [6]. There is no diagnostic laboratory findings, but only supportive in diagnosis of NEC [7]. The presenting signs are nonspecific, such as feeding intolerance, abdominal distention or tenderness, occult or gross blood in the stool, lethargy, apnea, respiratory distress, or poor perfusion that resembles diseases like sepsis and respiratory distress syndrome [8]. Thus, detecting infants at risk for NEC or those in early stages of the disease would offer opportunities for early intervention. The fecal proteins calprotectin (FCAL) and lactoferrin (FLF), which are derived from neutrophils, have the potential to be ideal
http://dx.doi.org/10.1016/j.advms.2015.02.004 1896-1126/ß 2015 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Medical University of Bialystok.
Please cite this article in press as: Sag˘lam C, et al. Small intestinal lactoferrin and calprotectin levels in different stages of necrotizing enterocolitis in a rat model. Adv Med Sci (2015), http://dx.doi.org/10.1016/j.advms.2015.02.004
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
G Model
ADVMS 87 1–5 C. Sag˘lam et al. / Advances in Medical Sciences xxx (2015) xxx–xxx
2
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
markers of intestinal inflammation because their measurement is non-invasive, easy to obtain, and inexpensive. Measurement can be done in small intestinal contents/feces with less than 5 g of stool in the case of intestinal inflammation [9]. Fecal calprotectin, a calcium- and zinc-binding neutrophilic cytosolic protein, is found in proportion to the degree of inflammation present, is resistant to colonic bacterial degradation, and is stable in stools for up to 1 week at room temperature. Fecal lactoferrin is an iron-binding protein that is similar to fecal calprotectin regarding that it is released upon neutrophil activation and is also resistant to proteolysis in the feces for up to 1 week [9]. Both of the fecal biomarkers have been previously established as screening tests for use in diagnosing inflammatory bowel disease in children (Crohn’s disease, ulcerative colitis, etc.) [7,9,10] and are now taking on an expanding role in disease monitoring and relapse detection. They have also been evaluated as potential diagnostic tools for NEC [11–18]. This research has concluded that the utility of these biomarkers for early diagnosis and assessment of resolution of NEC needs to be studied in wider NEC series. In this study, we were able to investigate small intestinal LF and CAL levels in different stages of NEC as predictors and indicators of disease severity in an experimental newborn rat model and their correlation with intestinal injury score.
66
2. Materials and methods
67
2.1. Animals and experimental design
68 69 70 71 72 73 74 75 76
The study protocol was approved by the Animal Care and Use Committee of Gulhane Military Medical School (Ankara, Turkey). Forty-six Sprague-Dawley neonatal rats, originating from five different litters, were used in four separate groups in the experiment. Newborn pups were randomly divided into one of the following four groups: NEC (assigned to three groups; G1, G2, G3) and control (group C). Group C rats (controls; n = 7) were left with their mothers, breast fed ad libitum, and not submitted to stress. In the NEC groups, newborn rats were collected from their
mothers immediately after birth to prevent suckling of maternal milk and kept at 37 8C in a humidified incubator.
77 78
2.2. Necrotizing enterocolitis procedure
79
All the pups in NEC groups were gavage fed 0.2 mL of special rodent formula using a 24 gauge thickness silicon catheter 3 times a day (Fig. 1A). A hyperosmolar rat milk substitute was prepared using 15 g Similac 60/40 (Abbott-Turkey) in 75-ml Puppy-milk canine milk replacement (Beaphar-bogena, B.V. Sedel, Netherland). To induce clinical and pathological signs of NEC, rat pups from all three NEC groups (G1, for 48 h; G2, for 72 h; G3, for 96 h) were stressed twice daily with asphyxia by breathing 100% CO2 gas for 5 min followed by cold stress at 4 8C for 5 min, and 100% O2 inhalation for 5 min in a sealed airtight plastic container.
80 81 82 83 84 85 86 87 88 89
2.3. Sample collection
90
All rats were killed on the day as previously described for each group (for G1 [n = 9], G2 [n = 7], G3 [n = 6], and C groups, at the end of the 2nd day, 3rd day, and 4th day, respectively) via decapitation. The abdomen was opened and the whole intestine were inspected for evidence of NEC, such as intestinal discoloration, fragility, weakness of tissue integrity, edema, intestinal hemorrhage, ileal distension, pneumatosis intestinalis, perforation, and necrosis, and fixed length of intestinal specimens such as a 2 cm section of the distal ileum and a 3 cm to the ileocaecal valve were harvested for histopathologic and biochemical evaluation, respectively. Tissue specimens were flushed with cold saline solution and fixed in 10% buffered formalin for histopathologic evaluation. Materials from all of the newborn rats included in the study were collected from the resected intestinal segments. Tissue specimens were taken away from 3 cm-fixed length to the ileocaecal valve placed in 0.5 mL of saline for providing standardization procedure in the measurements of the biomarkers. Then all materials was frozen in liquid nitrogen and stored at 80 8C for biochemical examination of small intestinal lactoferrin and calprotectin.
91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
Fig. 1. (A) A 24 gauge-thickness silicon catheter was used to feed the pups, (B) Blackly abdomen with hyperemia, and distension, (C) Dark purple/black discolorated intestines, (D) Dilated intestines as a result of extensive gas.
Please cite this article in press as: Sag˘lam C, et al. Small intestinal lactoferrin and calprotectin levels in different stages of necrotizing enterocolitis in a rat model. Adv Med Sci (2015), http://dx.doi.org/10.1016/j.advms.2015.02.004
G Model
ADVMS 87 1–5 C. Sag˘lam et al. / Advances in Medical Sciences xxx (2015) xxx–xxx
111
2.4. Histopathologic evaluation
112 113 114 115 116 117 118 119 120 121 122 123
Samples of intestinal tissues from a fixed length (2 cm section of the distal ileum) were fixed in 70% ethanol, processed, embedded in paraffin, and microtome sectioned at 4–6 mm. The slides were counterstained with hematoxylin–eosin (H&E) and examined under a light microscope for histological evaluation of NEC. Histological changes in the ileum were scored by a blinded pathologist and graded as follows: 1 = normal, intact villous epithelium with normal histology; 2 = mild villous edema, with epithelial sloughing confined to tips of the villi; 3 = mild midvillous necrosis; 4 = moderate midvillous necrosis, with crypts still readily detectable; and 5 = severe necrosis of entire villi with complete absence of epithelial structures [9,10].
124
2.5. Small intestinal lactoferrin and calprotectin measurements
125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142
Each intestinal material was tested for concentrations of LF and CAL with an enzyme-linked immunosorbent assay (BI˙O-TEK, Synergy HT; ELISA) according to the manufacturer’s instructions (Rat Lactoferrin (LTF/LF) ELI˙SA kit, Hangzhou Eastbiopharm Co. Ltd. Hangzhou, China, Cat. No: CK-E11678 for LF; Rat Calprotectin (CAL) ELI˙SA kit, Hangzhou Eastbiopharm Co. Ltd. Hangzhou, China, Cat. No: CK-E90475 for CAL). Briefly, intestinal samples were suspended in diluent-buffer. After homogenization, and if necessary, centrifugation, the homogenates (supernatants) were transferred to microplates for detection with antibodies specific for the respective inflammatory markers. Anti-FLF and Anti-FCAL antibody, each conjugated with peroxidase, were used for development. For each well, the optical density was measured at 450 nm with a microplate ELISA reader (BI˙O-TEK, Synergy HT, USA). The results of the tests samples were calculated from the standard curve and expressed as micrograms per milliliter for LF and as nanograms per milliliter for CAL. The intra-assay and the inter-assay coefficient variations of each kit were 10% and 12%, respectively.
143
2.6. Statistical analysis
144 145 146 147 148
Software SPSS 13.0 for Windows was used in all statistical tests. Dual comparisons among the groups were performed by Mann– Whitney U test. All data are expressed as mean SD, their minimum–maximum ranges. When P was less than 0.05, the difference was considered statistically significant.
149
3. Results
150 151 152 153 154 155 156 157
Twenty-nine pups survived until the end of the study. During the NEC procedure, in the NEC groups (total, n = 39) seventeen pups (43.6%) died as a result of aspiration due to the difference in practice of feeding process by gavage between practitioners such as the catheter was inserted into the trachea instead of the stomach, accidentally. There was no loss in the control group during the study because of that the control group were breastfeded.
158
3.1. Macroscopic and microscopic examination of the GI tract
159 160 161 162 163 164 165 166
Though all pups in the control group had a normal macroscopic appearance of the GI tract without any discoloration and distension of the abdomen and the intestines, we observed all the pups in the NEC groups had abdomens with varying degrees of hyperemia, distension, and had dark purple/black discolored intestines that were also dilated as a result of common gas. Results of macroscopic visual evaluation were recorded, and pictures were taken using a digital camera (Fig. 1B–D).
3
Table 1 Scores of histological analyses of intestinal segments of neonatal rats (mean SD). Groups
G1 (n:9)
G2 (n:7)
G3 (n:6)
Score
2.11 0.33* (2.0–3.0)
2.28 0.48# (2.0–3.0)
2.16 0.40 (2.0–3.0)
* #
a
C (n:7) a
1.0 0 (1.0–1.0)
p = 0.001: G1 vs. control (C) group. p = 0.001: G2 vs. control (C) group. p = 0.001: G3 vs. control (C) group.
Histological analyses of intestinal segments were performed using a scoring system from 1 to 5 to determine the severity of NEC, as previously described. All of the intestinal samples collected from the control group rats showed no visual pathological changes in the structure resembling the development of NEC (Stage 1). After exclusion of 4 intestinal tissue samples of the G1 group due to autolysis, histological findings in most pups of all three NEC groups showed significant pathological intestinal changes in the structure, described as minimal (Stage 2) and mild (Stage 3) necrosis (Table 1). Comparison of the degree of intestinal histopathology between the NEC and the control groups revealed that all the rats in the NEC groups (G1, G2, G3) had a statistically significant difference (p = 0.001, p = 0.001, p = 0.001, respectively). But when we compared NEC groups with each other in terms of the degree of intestinal histopathology, we did not observe any statistically significant difference (p > 0.05) (Table 1).
167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182
3.2. Small intestinal lactoferrin and calprotectin levels
183
Small intestinal LF and CAL levels were measured using ELISA kits. The mean values and the linear graph visualizing the level changes of both markers in the different groups are shown in Table 2, and Fig. 2, respectively. When we compared LF and CAL levels of the G1 and G3 groups with the control group, we did not observe any statistically significant difference (G1 vs. the control group, p = 0.229; G3 vs. the control group, p = 0.445, respectively). Conversely, there was a statistically significant difference between G2 and the control group (G2 vs. the control group, p = 0.026). When we compared the levels of markers between each other of NEC groups, we did not observe any statistically significant difference (p > 0.05) (Table 2).
184 185 186 187 188 189 190 191 192 193 194 195
4. Discussion
196
Necrotizing enterocolitis is a severe problem of premature infants. The initial presentation of the disease is nonspecific and is often hard to distinguish from other gastrointestinal diseases and sepsis. High morbidity and mortality rates urge us to find new biomarkers for early detection of the disease; there is not a reliable
197 198 199 200 201
Table 2 Results of small intestinal lactoferrin and calprotectin levels (mean SD). Groups
CAL (ng/mL)
LF (mg/mL)
G1 (n:9)
19.52 11.32* (4.96–36.30) 24.74 11.56# (1.49–38.21) a 18.14 10.53 (6.44–32.24) 12.81 5.74 (6.11–19.61)
14.56 8.28* (3.92–26.84) 18.39 8.42# (1.36–28.02) a 13.56 7.75 (4.93–23.95) 9.62 4.24 (4.67–14.63)
G2 (n:7) G3 (n:6) C (n:7)
Abbreviations: CAL: calprotectin LF: lactoferrin. * p = 0.229: G1 vs. control (C) group. # p = 0.026: G2 vs. control (C) group. a p = 0.445: G3 vs. control (C) group.
Please cite this article in press as: Sag˘lam C, et al. Small intestinal lactoferrin and calprotectin levels in different stages of necrotizing enterocolitis in a rat model. Adv Med Sci (2015), http://dx.doi.org/10.1016/j.advms.2015.02.004
G Model
ADVMS 87 1–5 4
C. Sag˘lam et al. / Advances in Medical Sciences xxx (2015) xxx–xxx
Fig. 2. Linear graph visualizing the changes of the CAL and LF levels in the different groups.
202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249
laboratory test to detect either newborns at risk for NEC or those in early stages of the disease. A rapid increase in small intestinal LF and CAL levels and also increased intestinal injury scores were detected in ongoing stages of NEC after the fourth day of the model (fourth day was reported as the day with the most intense injury in most studies). From this point of view, LF and CAL levels in small intestinal contents were increased in the early stages of NEC, and in the stages of proven NEC there was a decline in the levels of these markers. Results considered that there was a rapid response of these markers to ischemic and hyperosmolar food injury in our study. It is generally thought that there are multiple preparatory factors such as the followings: preterm birth [8], hyperosmolar enteral feeding [19], dysfunctional intestinal motility [20], reduced intestinal blood flow [21], microbe-induced pro-inflammatory epithelial signaling [22], and enteroinvasive microbes [23]. From this point of view, we suggested that intestinal injury due to the reduced intestinal blood flow and hyperosmolar enteral feeding is the ‘‘First Hit’’ in starting the disease. In our study, we showed that LF and CAL levels began to rise, just at that point. If a ‘‘Second Hit’’, as we mentioned above, increases the injury, necrosis climbs due to significant inflammation in response to colonization. Laforgia et al. investigated FCAL levels in the first passed meconium in 84 term and 47 preterm infants. Mean FCAL level was 145.2 78.5 mg kg 1 in meconium and negatively correlated with gestational age, birth weight, and 5-min Apgar score. They hypothesized that the migration of neutrophils into the gut lumen is increased in perinatal asphyxia because of ischemic changes in the intestinal mucosa [18]. Both of these biomarkers have been previously validated as accurate markers of inflammatory bowel disease in children (Crohn’s disease, ulcerative colitis, etc.) [7,9,10] and were also investigated in the diagnosis of NEC [11–18]. There are few studies in newborns [11–15,17,18] about the role of FCAL in diagnosing NEC, but only one [16] investigated FLF in NEC. While an increased level of FCAL was detected in some of these studies, others revealed no significant elevation in newborns with NEC compared to patients without NEC. FCAL levels in preterm infants with a diagnosis of NEC were significantly increased compared with controls [11,15]. A prospective study, in a population of 59 very low birth weight infants (with a mean gestational age of 27.2 2.6 weeks and a mean birth weight of 939 273 g), which is analyzing weekly FCAL levels from birth revealed that levels above 2000 mg/g are a useful but not an early marker of NEC (seven of them were eventually diagnosed with NEC) [17]. In a brief report by Thuijls et al., 14 of 35 neonates suspected of NEC developed NEC. Median FCAL levels were significantly higher
in neonates suspected of NEC who finally developed NEC than in those with other final diagnoses (589.2 and 69.6 mg/g feces, respectively). The ideal cut-off value for discrimination of neonates with NEC from others was determined to be 286.2 mg/g feces for FCAL. They reported that FCAL had a high diagnostic accuracy (sensitivity of 93% and specificity of 86%) and clinically relevant likelihood ratios (LRs) (positive LRs of 12.29, and negative LRs of 0.15) for an early diagnosis of NEC [13]. Reisinger et al. investigated FCAL levels in 62 neonates with clinical suspicion of NEC (29 of them were eventually diagnosed with NEC). The median gestational age was 215 (175–289) days, and the median birth weight was 1328 (585–3570) grams. There were significant differences in gestational age, birth weight, or sex between the groups. Median FCAL levels in the NEC group were significantly higher than those in the non-NEC group (402.2 and 79.6 mg/g feces), respectively [14]. Selimoglu et al. analyzed FCAL and FLF as diagnostic tools for NEC for 14 newborns with a control group of 63 newborns. There was no significant difference in FCAL and FLF levels between NEC and control groups. The authors suggested that these two biomarkers were not useful in the early diagnosis of the disease, but FCAL could be able to confirm the diagnosis only in the late stages of NEC [16]. The discrepancies between the results and the inclusion of a small number of patients were considered as limitations of these studies. Besides theses markers were elevated in all previous studies, studies also showed that they were affected from multiple factors such as gestational age, birth weight, gender, and Apgar score. Due to not having strong clues regarding of which factor will effect or cause to NEC, monitoring their levels may be more effective rather than spot testing in the diagnosis of NEC. Therefore, in our study, we analyzed small intestinal levels of LF and CAL in the different stages of NEC. We showed that levels of both of these markers increased in the second and the third day groups, but tended to decrease on the fourth day. Although the results of day 2, day 3, day 4 were higher than control and were increased from the beginning of the first day, statistical significance was only determined on the third day in our experiment. The groups represent the day of NEC. Thus G1 represents the cases that exposed one day to the NEC conditions and so the early phase of NEC. The intestinal injury scores of all NEC groups were significantly higher than the control group. In conclusion, we found that the levels of two markers increased in the early stages of NEC and these results correlated with intestinal injury scores. Despite the importance of ischemic intestinal injury and intestinal immaturity as main factors involved in the pathogenesis of NEC, as we mentioned them as ‘‘First Hit’’ above, we hypothesized that a ‘‘Second Hit’’ is mandatory for the development of NEC. From this point of view, monitoring the levels of these markers may give more accurate results in diagnosing NEC. Further studies with a higher number of cases are essential for monitoring the levels of these fecal markers in NEC.
250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300
Conflict of interests
301
None declared.
302
Financial disclosure
303
None declared.
304
References [1] Neu J, Weiss MD. Necrotizing enterocolitis: pathophysiology and prevention. JPEN J Parenter Enteral Nutr 1999;23(5 Suppl.):S13–7. [2] Ostlie DJ, et al. Necrotizing enterocolitis in full-term infants. J Pediatr Surg Q2 2003;38(7):1039–42.
Please cite this article in press as: Sag˘lam C, et al. Small intestinal lactoferrin and calprotectin levels in different stages of necrotizing enterocolitis in a rat model. Adv Med Sci (2015), http://dx.doi.org/10.1016/j.advms.2015.02.004
305 306 307 308 309
G Model
ADVMS 87 1–5 C. Sag˘lam et al. / Advances in Medical Sciences xxx (2015) xxx–xxx
310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331
[3] Llanos AR, et al. Epidemiology of neonatal necrotising enterocolitis: a population-based study. Paediatr Perinat Epidemiol 2002;16(4):342–9. [4] Salhab WA, et al. Necrotizing enterocolitis and neurodevelopmental outcome in extremely low birth weight infants
