The Role of Breast Milk Neurotrophin Levels in Infantile Colic Pathogenesis: A Cross-Sectional Case-Control Study

Abstract

Objective:

Immaturity of the digestive tract and enteric nervous system is a widely accepted theory for infantile colic (IC) etiopathogenesis. The study aimed to show whether neurotrophins that are necessary for normal functioning and development of the gastrointestinal system have a role in the pathogenesis of IC.

Materials and Methods:

The IC group (n = 75) comprising the mothers of infants with IC and the control group (n = 75) were included to this cross-sectional case-control study. Brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and nerve growth factor (NGF) levels of breast milk samples were evaluated by immunosorbent analysis method.

Results:

The mean age of infants with IC was 7.3 ± 2.8 weeks, while the mean age of the control group was 8.1 ± 2.9 weeks (p = 0.110). No significant difference was found between the breast milk BDNF, GDNF, CNTF, and NGF levels of two groups (p = 0.941, p = 0.510, p = 0.533, p = 0.839, respectively).

Conclusions:

This is the first report comparing the neurotrophin levels of the breast milk samples taken from the mothers of infants with and without IC. The study demonstrated that breast milk neurotrophin levels of the mothers did not differ significantly between the infants with and without IC.

Introduction

Infantile colic (IC) was first defined as “crying for more than 3 hours and 3 days and over more than 3 weeks, in otherwise healthy and well-fed infants” by Wessel et al. in 1954.¹ Currently, it is accepted as a functional gastrointestinal disorder of infants according to the Rome IV classification.² IC prevalence has been reported in a wide range depending on the diagnostic criteria selected or the population analyzed in previous studies. As a public health concern, IC prevalence ranges between 2% and 73%, with a median of 17.7%.³ Despite more than six decades of research, pathogenesis of IC is still poorly understood. Several theories have been proposed to explain the pathogenesis including neurodevelopmental, environmental, gastrointestinal, and behavioral mechanisms.⁴ Functional and structural immaturity of the digestive tract and enteric nervous system (ENS) is one of the widely accepted theories since it has a transient course during the first months of life.^5,6

Neurotrophins, which are essential polypeptides for the development, survival, and lifelong modulation of the neurons in the central nervous system and peripheral nervous system, also play an important role in ENS maturation and plasticity.⁷ These factors including brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), and neurotrophin-3 are expressed in the different cells and tissues of the gastrointestinal tract including epithelial cells, lamina propria cells, enteric neurons, enteric glial cells, Schwann cells, immune cells in the lamina propria, enteric plexuses, intermuscular basal lamina, and along circular and longitudinal smooth muscle cells,^7–9 and mediate differentiation of neural crest-derived precursors of the ENS prenatally as well as regulating neuroplasticity, neuroprotection, sensation, motility via modulation of neurotransmitter synthesis and synaptic functions postnatally.^9,10

Influence of these neurotrophic factors on the maturation of gastrointestinal tract and ENS is highly complex. Most data about the role of neurotrophins on the postnatal gastrointestinal tract and ENS maturation come from preclinical and animal studies. Their roles in regulation of gastrointestinal motility, colonic mucosal barrier function, visceral hypersensitivity, postnatal maturation of the ENS, and axonal and neuronal plasticity in the ENS have been shown in previous preclinical and animal studies so far.^11–15 Moreover, these neurotrophins have recently been shown to be found in breast milk.^16,17

Based on evidence of the role of this target-derived neuronal growth factor family in the development and maintenance of ENS, and regulation of motility of the gastrointestinal tract, we hypothesized that neurotrophins might have a role in the pathogenesis of IC which results from structural immaturity of the digestive tract and ENS.

Materials and Methods

Study design and patients

The study was designed as a randomized cross-sectional, case-control study conducted on 150 mothers and their breastfed infants younger than 3 months of age followed by the pediatrics outpatient clinics of our hospital during a period of 6 months between March 2020 and March 2021.

The IC group (n = 75) comprising the mothers of infants with IC and the healthy control group (n = 75) were included. Patients with IC were diagnosed according to the revised Rome IV criteria as follows: (I) The infant should be younger than 5 months of age. (II) Caregivers should report recurrent and prolonged episodes of crying, fussing, or irritability that have no obvious cause and cannot be prevented or resolved by caregivers. (III) There should be no evidence of failure to thrive, fever, or illness with clinical grounds.² The inclusion criteria were (1) being diagnosed with IC according to the revised Rome IV criteria²; (2) term “delivery” (≥37 gestational weeks at birth); (3) being exclusively breastfed; (4) having no perinatal risk factors such as preterm birth history, birth weight of small-for-gestational-age¹⁸; (5) not being given any medication for colic. The infants whose mothers had gestational hypertension, diabetes, infection, fever, chronic or neurological diseases, and those with a major medical problem including acute infection or gastroesophageal reflux as determined by a pediatrician were excluded (CONSORT 2010 Flow Diagram).

Written informed consent was obtained from infants' parents or legal guardian following a detailed explanation of the objectives and protocol. The study was conducted in accordance with the ethical principles stated in the “Declaration of Helsinki,” and the study protocol was approved by the institutional ethics committee (Decision number and date: 2019/04, October 9, 2019).

Measurement of breast milk neurotrophin levels

A breast milk sample of 20 mL was collected from all the participants and centrifuged at 10,000 g for 30 minutes at 4°C. The upper-fat layer and cell sediment were eliminated, and the remaining liquid part was isolated for each sample. The liquid part obtained was stored at −80°C until analysis. Breast milk BDNF, GDNF, CNTF, and NGF concentrations were measured by using enzyme-linked immunosorbent assay (ELISA) method. Sensitivity, intra-assay, and inter-assay coefficient variation of CNTF ELISA kit (Cat. No. E1202Hu; Bioassay Technology Laboratory, Shanghai, China) were 1.57 ng/L, <8.0%, and <10.0%, respectively. For BDNF ELISA kit (Cat. No. E1302Hu; Bioassay Technology Laboratory, Shanghai, China) were 0.023 ng/mL, <8.0%, and <10.0%, respectively. Sensitivity, intra-assay, and inter-assay coefficient variation of NGF ELISA kit (Cat. No. E-EL-H1205; Elabscience Biotechnology Co., Ltd, TX) were 9.38 pg/mL, <5.66%, and <5.76%, respectively. GDNF ELISA kit (Cat. No. E-EL-H1495; Elabscience Biotechnology Co., Ltd) were 0.19 ng/mL, <5.66%, and <6.60%, respectively.

Statistical analysis

Data were analyzed using the software IBM Statistical Package for Social Science (SPSS), version 28 (IBM Corp., Armonk, NY). Frequency (n) and percentage (%) were calculated for the categorical data, whereas mean and standard deviation (SD) were calculated for the continuous variables. Normality assumptions were assessed before using parametric tests. Chi-square test was used to compare categorical data. In the comparison of continuous variables of two independent groups; the Mann–Whitney U test was used for the dependent variable that was not normally distributed, and independent t test was used for the dependent variable that was normally distributed. p-Value <0.05 was considered significant.

Results

The study was carried out on the IC group consisting of 75 infants and the control group consisting of 75 healthy infants. The mean age of infants with IC was 7.3 ± 2.8 weeks, while the mean age of the control group was 8.1 ± 2.9 weeks (p = 0.11). There was no significant difference between the breast milk BDNF, GDNF, CNTF, and NGF levels of two groups (p = 0.941, p = 0.510, p = 0.533, p = 0.839, respectively). Age, gender characteristics of the infants with IC and the control group, and neurotrophin levels of the breast milk samples obtained from mothers of two groups were summarized in Table 1.

Table 1.

Comparison of Age Characteristics and Breast Milk Neurotrophin Levels of the Subjects With and Without Infantile Colic

		Infantile colic group (n = 75)	Control group (n = 75)	p
Age (week)	Mean ± SD	7.3 ± 2.8	8.1 ± 2.9	0.110
Age (week)	Range	3–12.5	3–12.8	0.110
Sex	Male (%)	41 (54.7)	35 (46,7)	0.160
Sex	Female (%)	34 (45.3)	40 (53,3)	0.160
BDNF (ng/mL)	Mean ± SD	0.8 ± 0.6	0.8 ± 0.5	0.941
CNTF (ng/L)	Mean ± SD	63.8 ± 28.7	60.9 ± 28	0.533
GDNF (ng/mL)	Mean ± SD	30.3 ± 1.6	30.5 ± 1.5	0.510
NGF (pg/mL)	Mean ± SD	42 ± 56.8	40.4 ± 42.2	0.839

BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; GDNF, glial cell-derived neurotrophic factor; NGF, nerve growth factor; SD, standard deviation.

Discussion

This study reports the breast milk neurotrophin levels of the mothers of infants with and without IC. Our results revealed no significant difference between the levels of these neurotrophins (BDNF, GDNF, CNTF, and NGF) in breast milk samples of two groups. To our knowledge, this is the first report investigating the association between the breast milk neurotrophin levels and IC.

BDNF and its receptor “tropomyosin-related kinase B” have been shown to increase gastrointestinal motility by enhancing neuronal response to neurotransmitters 5-Hydroxytryptamine and substance P in the ENS.¹¹ It has also been shown that BDNF could regulate colonic mucosal barrier function and affect gut microbiota in mice.¹² Fu et al.¹³ showed that mechanical stress-induced elevation in BDNF levels increase the neuronal excitability of afferent neurons leading to distention-associated visceral hypersensitivity in the gut of rats.

Moreover, the signaling of GDNF through its receptor “Ret” has a crucial role not only in the migration, proliferation, and maturation of ENS precursor cells during embryonic development but also in postnatal maturation of the ENS.^14,15 It has been revealed that GDNF increases survival of neurons and numbers of neurites resulting in both structural and functional plasticity in neurons and axons in the ENS.¹⁵ A study by Zhang et al.¹⁹ showed that GDNF reduces epithelial permeability and mucosal inflammation through downregulation of proinflammatory cytokines during inflammation in vivo. Since increased epithelial permeability may cause abnormal immune response to food antigens and bacteria, reducing effect of GDNF on epithelial permeability could inhibit several pathologies such as IC, Crohn's disease, or inflammatory bowel disease.^20–22 Meir et al.²³ demonstrated that GDNF enhances maturation of epithelial functions in immature epithelial cells of the intestinal crypts through the cAMP/protein kinase A and p38 MAPK signaling pathways. Interestingly, continuous abdominal massage has been found to upregulate the expression of GDNF significantly in rats with irritable bowel syndrome in a recent study.²⁴

CNTF and NGF have also several functions in development and maintenance of the ENS.^25,26 As a member of the cytokine family, CNTF has been proposed to be an emergency factor rather than to be vital for the ENS since it is released only following tissue injury.²⁵ In a previous study, CNTF knockout mice was found to have not only normal motor neurons at birth but also progressive loss with age, supporting the idea of its protective role from neuronal injury.²⁵ However, mice lacking CNTF receptor exhibited profound motor neuron deficits at birth and had a fatal massive dilation of the gut in the postnatal period, demonstrating the vital role of CNTF receptor in the development of ENS.²⁶ NGF has been shown to be involved in the regulation of neurotransmitter expression of enteric neurons.²⁷ Previous animal studies showed that NGF plays a role in the visceral hypersensitivity via afferent nerves in conditions including chronic stress, inflammation, and mechanic obstruction.^28,29 von Boyen et al.³⁰ revealed that NGF increased visceral sensitivity and also improved bowel inflammation in rats.

Only a few clinical studies investigating the effects of neurotrophins on the gastrointestinal tract and ENS maturation have been published up to now. The limited data are available on the levels of BDNF in human breast milk. The first study demonstrating BDNF level in breast milk and pointing to its long-term effects was carried out by Li et al.¹⁶ Coulie et al.³¹ revealed that recombinant BDNF dose-dependently accelerated colonic transit and increased stool frequency in humans. In a study by Dangat et al.,³² higher levels of BDNF in breast milk samples of the women with preeclampsia have been found.

Another study showed the increased risk of pediatric gastrointestinal comorbidities such as necrotizing enterocolitis in offspring from mothers with preeclampsia.³³ Previous studies have also reported reduced BDNF and BDNF receptor levels in infantile hypertrophic pyloric stenosis and Hirschprung disease.^34,35 Moreover, reduced BDNF expression has been found to be related to inflammation.³⁶ In a previous study, higher fecal calprotectin levels were detected in infants comparing to children older than 1 year suggesting the inflammatory microenvironment in infantile period.³⁷ Pärtty et al. reported that IC was associated with low-grade inflammation.³⁸ Besides inflammation of the gastrointestinal tract, upregulated expression of BDNF in the visceral sensory pathways in infancy, suggests that BDNF may be involved in visceral hypersensitivity.^9,13 Increased expression of GDNF has also been found in functional dyspepsia as a repair mechanism for disrupted epithelium.³⁹

Based on accumulating evidence supporting the roles of neurotrophins on postnatal ENS maturation, gut motility, and visceral hypersensitivity, we had hypothesized that neurotrophins found in breast milk might be involved in the pathogenesis of IC. However, we found no significant difference between the breast milk neurotrophin levels of the infants with and without IC. Another possible factor affecting the development of IC may be that intestinal permeability to the neurotrophins might be affected by other factors. It is unknown how much of the factors are reabsorbed by the intestinal epithelium in infantile period. On the other hand, the mucosal barrier of the infant gut allows macromolecular transport since the epithelium of the gastrointestinal tract is still immature in the infants.⁴⁰

Conclusions

No significant difference between the breast milk neurotrophin levels of the infants with and without IC has been found in this study. The link between breast milk neurotrophins and ENS may be important for a better understanding of the etiopathogenesis of IC. In the future, more clinical studies are needed to prove functional roles of these breast milk neurotrophins for the postnatal maturation of ENS and gut as well as the pathogenesis of IC.

Footnotes

Authors' Contributions

P.G.: Conceptualization, analysis, supervision, reviewing, and editing. E.B.Y.: Data curation and methodology. G.B.: Methodology, software, investigation, and writing original draft. A.K.: Conceptualization and methodology. F.D.A.: Conceptualization and validation. N.O.D.: Conceptualization, methodology, and supervision. I.K.: Visualization, investigation, and validation. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Supplementary Material

References

Wessel

, Cobb

, Jackson

, et al. Paroxysmal fussing in infancy, sometimes called colic. Pediatrics, 1954; 14:421–435; doi: 10.1542/peds.14.5.421

Zeevenhooven

, Koppen

, Benninga

. The new Rome IV criteria for functional gastrointestinal disorders in infants and toddlers. Pediatr Gastroenterol Hepatol Nutr, 2017; 20(1):1–13; doi: 10.5223/pghn.2017.20.1.1

Vandenplas

, Abkari

, Bellaiche

, et al. Prevalence and health outcomes of functional gastrointestinal symptoms in infants from birth to 12 months of age. J Pediatr Gastroenterol Nutr, 2015; 61:531–537; doi: 10.1097/MPG.0000000000000949

Zeevenhooven

, Browne

, L'Hoir

, et al. Infant colic: Mechanisms and management. Nat Rev Gastroenterol Hepatol, 2018; 15:479–496; doi: 10.1038/s41575-018-0008-7

Daelemans

, Peeters

, Hauser

, et al. Recent advances in understanding and managing infantile colic. F1000Res, 2018; 7:F1000 Faculty Rev-1426; doi: 10.12688/f1000research.14940.1

Sarasu

, Narang

, Shah

. Infantile colic: An update. Indian Pediatr, 2018; 55:979–987; PMID: 29941700.

von Boyen

, Reinshagen

, Steinkamp

, et al. Enteric nervous plasticity, and development: Dependence on neurotrophic factors. J Gastroenterol, 2002; 37:583–588; doi: 10.1007/s005350200093

Lake

, Heuckeroth

. Enteric nervous system development: Migration, differentiation, and disease. Am J Physiol Gastrointest Liver Physiol, 2013; 305:G1–G24; doi: 10.1152/ajpgi.00452.2012

Liu

. Neurotrophic factors in enteric physiology and pathophysiology. Neurogastroenterol Motil, 2018; 30:e13446; doi: 10.1111/nmo.13446

10.

, Deng

, Liu

, et al. CNTF-STAT3-IL-6 axis mediates neuroinflammatory cascade across Schwann cell-neuron-microglia. Cell Rep, 2020; 31:107657; doi: 10.1016/j.celrep.2020.107657

11.

Boesmans

, Gomes

, Janssens

, et al. Brain-derived neurotrophic factor amplifies neurotransmitter responses and promotes synaptic communication in the enteric nervous system. Gut, 2008; 57:314–322; doi: 10.1136/gut.2007.131839

12.

, Cai

, Yan

. Brain-derived neurotrophic factor preserves intestinal mucosal barrier function and alters gut microbiota in mice. Kaohsiung J Med Sci, 2018; 34:134–141; doi: 10.1016/j.kjms.2017.11.002

13.

, Lin

, Winston

, et al. Role of brain-derived neurotrophic factor in the pathogenesis of distention-associated abdominal pain in bowel obstruction. Neurogastroenterol Motil, 2018; 30:e13373; doi: 10.1111/nmo.13373

14.

Uesaka

, Nagashimada

, Yonemura

, et al. Diminished Ret expression compromises neuronal survival in the colon and causes intestinal aganglionosis in mice. J Clin Invest, 2008; 118:1890–1898; doi: 10.1172/JCI34425

15.

Rodrigues

, Li

, Nair

, et al. Glial cell line-derived neurotrophic factor is a key neurotrophin in the postnatal enteric nervous system. Neurogastroenterol Motil, 2011; 23:e44–e56; doi: 10.1111/j.1365-2982.2010.01626.x

16.

, Xia

, Zhang

, et al. S100B protein, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in human milk. PLoS One, 2011; 6:e21663; doi: 10.1371/journal.pone.0021663

17.

Fichter

, Klotz

, Hirschberg

, et al. Breast milk contains relevant neurotrophic factors and cytokines for enteric nervous system development. Mol Nutr Food Res, 2011; 55:1592–1596; doi: 10.1002/mnfr.201100124

18.

Milidou

, Søndergaard

, Jensen

, et al. Gestational age, small for gestational age, and infantile colic. Paediatr Perinat Epidemiol, 2014; 28(2):138–145; doi: 10.1111/ppe.12095

19.

Zhang

, He

, Li

, et al. Glial-derived neurotrophic factor regulates intestinal epithelial barrier function and inflammation and is therapeutic for murine colitis. J Pathol, 2010; 222:213–222; doi: 10.1002/path.2749

20.

Arrieta

, Bistritz

, Meddings

. Alterations in intestinal permeability. Gut, 2006; 55:1512–1520; doi: 10.1136/gut.2005.085373

21.

Aijaz

, Balda

, Matter

. Tight junctions: Molecular architecture and function. Int Rev Cytol, 2006; 248:261–298; doi: 10.1016/S0074-7696(06)48005-0

22.

Hollander

, Vadheim

, Brettholz

, et al. Increased intestinal permeability in patients with Crohn's disease and their relatives. A possible etiologic factor. Ann Intern Med, 1986; 105:883–885; doi: 10.7326/0003-4819-105-6-883

23.

Meir

, Flemming

, Burkard

, et al. Glial cell line-derived neurotrophic factor promotes barrier maturation and wound healing in intestinal epithelial cells in vitro. Am J Physiol Gastrointest Liver Physiol, 2015; 309:613–624; doi: 10.1152/ajpgi.00357.2014

24.

, Luo

, Liu

, et al. Abdominal massage reduces visceral hypersensitivity via regulating GDNF and PI3K/AKT signal pathway in a rat model of irritable bowel syndrome. Evid Based Complement Alternat Med, 2020; 2020:3912931; doi: 10.1155/2020/3912931

25.

Sleeman

, Anderson

, Lambert

, et al. The ciliary neurotrophic factor and its receptor, CNTFR alpha. Pharm Acta Helv, 2000; 74:265–272; doi: 10.1016/s0031-6865(99)00050-3

26.

DeChiara

, Vejsada

, Poueymirou

, et al. Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth. Cell, 1995; 83:313–322; doi: 10.1016/0092-8674(95)90172-8

27.

Belai

, Aberdeen

, Burnstock

. Differential effect of immunosympathectomy on the expression of rat enteric neurotransmitters. Neurosci Lett, 1992; 139:157–160; doi: 10.1016/0304-3940(92)90541-e

28.

Chen

, Winston

, Sarna

. Neurological and cellular regulation of visceral hypersensitivity induced by chronic stress and colonic inflammation in rats. Neuroscience, 2013; 248:469–478; doi: 10.1016/j.neuroscience.2013.06.024

29.

Lin

, Fu

, Winston

, et al. Pathogenesis of abdominal pain in bowel obstruction: role of mechanical stress-induced upregulation of nerve growth factor in gut smooth muscle cells. Pain, 2017; 158:583–592; doi: 10.1097/j.pain.0000000000000797

30.

von Boyen

, Steinkamp

, Reinshagen

, et al. Nerve growth factor secretion in cultured enteric glia cells is modulated by proinflammatory cytokines. J Neuroendocrinol, 2006; 18:820–825; doi: 10.1111/j.1365-2826.2006.01478.x

31.

Coulie

, Szarka

, Camilleri

, et al. Recombinant human neurotrophic factors accelerate colonic transit and relieve constipation in humans. Gastroenterology, 2000; 119:41–50; doi: 10.1053/gast.2000.8553

32.

Dangat

, Kilari

, Mehendale

, et al. Higher levels of brain derived neurotrophic factor but similar nerve growth factor in human milk in women with preeclampsia. Int J Dev Neurosci, 2013; 31:209–213; doi: 10.1016/j.ijdevneu.2012.12.007

33.

Leybovitz-Haleluya

, Wainstock

, Sheiner

. Maternal preeclampsia, and the risk of pediatric gastrointestinal diseases of the offspring: A population-based cohort study. Pregnancy Hypertens, 2019; 17:144–147; doi: 10.1016/j.preghy.2019.06.005

34.

Hoehner

, Wester

, Påhlman

, et al. Alterations in neurotrophin and neurotrophin-receptor localization in Hirschsprung's disease. J Pediatr Surg, 1996; 31:1524–1529; doi: 10.1016/s0022-3468(96)90170-0

35.

Guarino

, Yoneda

, Shima

, et al. Selective neurotrophin deficiency in infantile hypertrophic pyloric stenosis. J Pediatr Surg, 2001; 36:1280–1284; doi: 10.1053/jpsu.2001.25795

36.

Mondelli

, Cattaneo

, Murri

, et al. Stress and inflammation reduce brain-derived neurotrophic factor expression in first-episode psychosis: A pathway to smaller hippocampal volume. J Clin Psychiatry, 2011; 72:1677–1684; doi: 10.4088/JCP.10m06745

37.

Olafsdottir

, Aksnes

, Fluge

, et al. Faecal calprotectin levels in infants with infantile colic, healthy infants, children with inflammatory bowel disease, children with recurrent abdominal pain and healthy children. Acta Paediatr, 2002; 91:45–50; doi: 10.1080/080352502753457932

38.

Pärtty

, Kalliomäki

, Salminen

, et al. Infantile colic is associated with low-grade systemic inflammation. J Pediatr Gastroenterol Nutr, 2017; 64:691–695; doi: 10.1097/MPG.0000000000001340

39.

Tanaka

, Tominaga

, Fujikawa

, et al. Concentration of glial cell line-derived neurotrophic factor positively correlates with symptoms in functional dyspepsia. Dig Dis Sci, 2016; 61:3478–3485; doi: 10.1007/s10620-016-4329-5

40.

Drozdowski

, Clandinin

, Thomson

. Ontogeny, growth, and development of the small intestine: Understanding pediatric gastroenterology. World J Gastroenterol, 2010; 16:787–799; doi: 10.3748/wjg.v16.i7.787