Intestine and brain TLR-4 modulation following N-Acetyl-Cysteine treatment in NEC rodent model

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Ross, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1684513/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 May, 2023 Read the published version in Scientific Reports → Version 2 posted 11 You are reading this latest preprint version Show more versions Abstract Objective: Necrotizing enterocolitis (NEC) brain injury is mediated through Toll-like receptor 4 (TLR4) on the intestinal epithelium and on brain microglia. We sought to determine whether postnatal and/or prenatal NAC can modify NEC associated intestinal and brain TLR4 expression and brain glutathione levels in a rat model of NEC. Study Design: Newborn Sprague-Dawley rats were randomized into 3 groups: Control (n=33); NEC (n=32) subjected to hypoxia and formula feeding; NEC-NAC (n=34) received NAC (300mg/kg IP) in addition to NEC conditions. Two additional groups included pups of dams who were treated once daily with NAC (300mg/kg IV) for the last 3 days of pregnancy: NAC-NEC (n=33) or NAC-NEC-NAC (n=36) with additional postnatal NAC. Pups were sacrificed on the fifth day, ileum and brains harvested for TLR-4 and glutathione protein levels. Results: NEC offspring had significantly increased brain and ileum TLR-4 protein levels compared to control (brain 2.5 + 0.6 vs 0.88 + 0.12 U; ileum 0.24 + 0.04 vs 0.09 + 0.01 U, p<0.05). NAC administered only to dams (NAC-NEC) significantly decreased NEC offspring brain (1.53 + 0.41 vs 2.5 + 0.6 U; p<0.05) and ileum (0.12 + 0.03 vs 0.24 + 0.04 U; p<0.05) TLR-4 protein levels compared to NEC. The same pattern was demonstrated when NAC was administered only or also postnatally. The decrease in offspring brain glutathione levels observed under NEC condition was reversed with all NAC treatment groups. Conclusion: NAC could reverse the increase in ileum and brain TLR-4 levels and the decrease in brain glutathione levels associated with NEC in a rat model, and thus may protect from NEC associated brain injury. necrotizing enterocolitis brain injury TLR-4 N-Acetyl-Cysteine Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Necrotizing enterocolitis (NEC) is a leading cause of preterm infant morbidity and mortality (Mihi and Good 2019 ). In the USA, more than 3000 neonates are diagnosed with NEC annually (Neu 1996 ); 7% of infants weighing less than 1500 gram are affected by NEC (Hunter et al. 2008 ) with mortality as high as 30% (Blakely et al. 2005 ). NEC is both an infectious and inflammatory disease that can affect only part or the entire gastrointestinal tract, predominantly found in the region of the terminal ileum (Balance et al. 1990). NEC etiology is considered multifactorial, a consequence of intestinal immaturity, microbial dysbiosis, and an exuberant inflammatory response. Toll-like receptor 4 (TLR-4), which plays a critical role in the induction of protective host innate immune response, is an important factor in normal gut development (Mihi and Good 2019 ). Recent evidence suggests that TLR-4 is expressed at higher levels in the premature intestine and is more abundant in the intestine of infants with NEC (Nanthakumar et al. 2011 ; Gomart et al., 2021 ). The excessive inflammatory response observed in NEC is explained in part due to over-expression and the exaggerated activity of TLR-4 in the preterm intestine (Nanthakumar et al. 2011 ). The attachment of ligand to TLR-4 initiates a cascade of events that result in phosphorylation of kappa-B inhibitor in the cytosol and activation of NFKB P65, which translocates into the nucleus where it activates and regulates the transcription of genes related to inflammatory responses (Anderson 2000 ). In addition to intestinal injury in NEC, several studies report significant severe neurodevelopmental disability in NEC survivors (Niño 2018). Recently it was demonstrated in NEC model that a brain injury was associated with decreased brain glutathione levels (Niño 2018). However, the mechanisms associated with brain injury in NEC have not been fully elucidated N-Acetyl Cysteine (NAC) is a known anti-inflammatory and anti-oxidant agent used widely for paracetamol intoxication, that is safe to use during pregnancy (class B) (Riggs et al. 1989 ; Beloosesky et al. 2005). Its anti-oxidant activity results from its conversion into metabolites that are capable of stimulating glutathione synthesis and promoting detoxification, and by its inherent function as a scavenger of free oxygen radicals. TLR4 signaling plays a central role in the induction of NEC through the modulation of the epithelial cell barrier and regulation of the innate and adaptive immune responses. We hypothesized that modulation of TLR-4 effects could reduce the risk of NEC. In the present study, we sought to determine the protective effect of NAC on TLR-4 protein levels in fetal intestine and fetal brain, and to determine its effect on fetal brain glutathione levels in an established rat model of NEC. Methods Pregnant Sprague-Dawley (SD) rats were obtained at day 11 of gestation and allowed to acclimate for 7 days before initiating the experiments (Fig. 1 ). The SD rats were maintained in light-controlled facilities with access to water and food ad libitum throughout the study, at ambient temperature (25°C). The first group of pregnant rats (nine dams) received no treatment during pregnancy and delivered spontaneously. After birth, the pups were divided into three groups. Group 1 – Control: 33 pups remained with their mothers (three dams) and were nursed at room temperature. Sixty-six pups from six dams were separated from the dams and divided into Groups 2 and 3, as described below. Group 2 – NEC: 32 pups were transferred to an incubator (Ohio Medical Products, Madison, WI, USA), where they were fed thrice daily by gavage of 0.2 mL clean formula consisting of 15 g of Similac 60/40 (Abbott Nutritional, Columbus, OH, USA) in 75 mL of Esbilac canine milk replacement (PetAg Inc., Hampshire, IL, USA). The pups also received daily hypoxia exposure for four days with 5% O 2 and 95% N 2, and intraperitoneal injections of saline three times daily (for 10 minutes) following each hypoxia exposure. Group 3 -- NEC-NAC: 34 pups were exposed to the same NEC conditions as pups in Group 2, with the addition of NAC treatment (N-acetyl cysteine, SIGMA, reconstituted in water, 300 mg/kg intraperitoneal) thrice daily, following each 4-day hypoxia exposure. To explore the possible protective effect of NAC during pregnancy, a second group of six pregnant rats received intravenous injections of NAC (300 mg/kg) once daily from gestational day 18 until delivery. After birth, the pups were divided into Groups 4 and 5 as described below. Group 4 -- NAC-NEC: 33 pups were separated from their dams and kept under the same conditions as Group 2 (NEC) above. Group 5 -- NAC-NEC-NAC: 36 pups were separated from their dams and kept under the same conditions as Group 3 (NEC-NAC). In all studies, pups were euthanized on the fifth day of life. Sample collection Pups from the five groups were anesthetized with isoflurane and decapitated on their fifth day of life. Brains were harvested and frozen immediately in liquid nitrogen for further processing and analysis. The intestine was removed, and the distal 3 cm of terminal ileum were harvested and immediately frozen in liquid nitrogen for further processing and analysis. Processing and analysis were performed on the mixture of intracellular and cellular components of the distal terminal ileum. Brain and terminal ileum TLR-4 protein levels were determined by Western blot, and brain Glutathione levels determined by ELISA. Western blot analysis Preparations of cell lysates and Western blot cells or tissues were lysed in RIPA buffer (phosphate-buffered saline containing 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], 1 mmol/L Na3VO4, 4 mm phenylmethylsulfonyl fluoride and 0.05% [w/v] apro-tinin). Insoluble proteins were discarded by high-speed centrifugation at 2000 g for 10 min at 4°C. Small volumes of lysate were taken to measure total protein concentration, using absorbance at 280 nm in triplicate by Nanodrop. Determination of the protein concentration samples was also compared with BSA standards, ensuring the standard was diluted in the same buffer as the samples, and a 50-μg sample was loaded into each well of a 12% SDS-PAGE acrylamide gel. After transferring and blocking to nitrocellulose membranes, the blots were incubated overnight with primary antibodies against the target protein, at 4°C. The antibodies were diluted in blocking buffer according to the manufacturer’s recommendations. Following incubation with primary antibodies and rinsing, the blots were incubated for 1 hour at room temperature with HRP-conjugated secondary antibody, according to the manufacturer’s recommended ratio. Antibodies recognizing the TLR-4 ( cat#NB100-56566 novus) were used in combination with a donkey anti-mouse horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA, USA). Following incubation with the diluted secondary antibody and washing with TBST the membrane was incubated with the peroxide solution according to the manufactory orders andenhanced to chemiluminescent (ECL) development solution (ab133406)by exposing to X-ray. Actin was used as a reference protein. Densitometric analysis was used to determine differences in protein expression. Densitometry analysis To compare target protein expression levels among samples on the same blot or across blots, we normalized the bands by loading controls (housekeeping protein actin). To get an objective measure of the signal generated on Western blot, a densitometer was used to scan the blot or film, and imaging software was used to compare signals. The quantification reflects the ratio of each protein band relative to the control (actin). Films were subsequently imaged with ChemiDoc MP using the white light conversion screen and the silver stain (visible stain) application. The Band Analysis tools of IMAGELAB, software version 4.1 (Bio-Rad), were used to select and determine the back-ground-subtracted density of the bands in all the gels and blots. ELISA determinations Offspring brain glutathione level was determined with a HT Glutathione Assay kit, catalog number: 7511-100-K (Bio-Techne Ltd., Minneapolis, MN, USA), according to the manufacturer’s instructions. Statistical analysis Offspring brain and terminal ileum TLR-4 protein levels were compared among pups from the different groups (5 brain samples and 5 terminal ileum from each group). Offspring brain glutathione protein levels were compared among pups from the different groups. All results were expressed as means±SD using one-way analysis of variance followed by post hoc tests for pairwise comparisons (Holm-Sidak method). Differences were significant at P< 0.05. Sigma Stat software, version 4.0 was used to perform the statistical analysis. Ethical approval The protocols and procedures were approved by the Institutional Animal Care Committee at the Rappaport Faculty of Medicine (Protocol number: IL001-01-2016). This study was carried out in strict accordance with the recommendations of the Israeli National Institute of Health guide for care and use of laboratory animals. Guidelines for the care and use of the animals approved by the local institution were also followed. All efforts were made to minimize animal suffering. The study was supervised by a veterinarian on a daily basis. The pain suffering of the rats was categorized as low. Results Brain and ileum TLR-4 NEC offspring had significantly increased brain and ileum TLR-4 protein levels as compared to control group (brain 2. 5 + 0. 6 vs. 0.88 + 0.1 2 U; ileum 0.24 + 0.04 vs. 0.09 + 0.02 U, respectively, p<0.05). NAC, administered only to pregnant dams (NAC-NEC), significantly decreased NEC offspring brain (1. 53 + 0. 41 vs. 2. 5 + 0. 6 U, p<0.05) and ileum (0.12 + 0.03 vs. 0.2 + 0.01 U, p<0.05) TLR-4 protein levels as compared to NEC offspring. The same pattern of decreased brain and ileum TLR-4 protein levels was demonstrated when NAC was administered only to the offspring (NEC-NAC) (brain 1. 03 + 0. 32 vs. 2. 5 + 0. 6 U; ileum 0.09 + 0.01 vs. 0.24 + 0.04 U, p<0.05).NAC administered to both dams and offspring (NAC-NEC-NAC) resulted in decreased in TLR-4 in brains (0.68+0.11 vs. 2.3 + 0.25 U; p<0.05) and ileum (0.07+0.02 vs. 0.24 + 0.04 U; p<0.05) as compared to NEC group (Figs. 2 and 3). Brain glutathione level NEC offspring demonstrated decreased brain glutathione levels as compared to control offspring (0.023 + 0.001 vs. 0.036 + 0.001 pmol/ml, respectively, p<0.05). NAC administered only to pregnant dams (NAC-NEC) significantly increased offspring glutathione levels as compared to NEC offspring (0.03+0.001vs. 0.023 + 0.001 pmol/ml, respectively, p<0.05). The same pattern was observed when NAC was administered only to offspring (NEC-NAC; 0.038+0.001 vs. 0.203 + 0.001 pmol/ml) or when NAC was administered to both dams and offspring (NAC-NEC-NAC; 0.031+0.001 vs. 0.203 + 0.001 pmol/ml, respectively, p<0.05) (Fig. 4). Discussion In previous studies using this NEC model, we and others (Kelly et al. 2004; Sulistyo et al. 2018) demonstrated features of NEC with severe bowel damage in pups, inflammatory changes and increased oxidative stress, accompanied with offspring brain injury. This model is based on factors that are recognized to contribute to the development of human NEC, including intestinal immaturity, hypoxia, and artificial hyperosmolar formula. Dams are removed from the pups soon after birth to avoid breastfeeding, which is known to be protective against NEC. We demonstrated, in our animal NEC model, that offspring with NEC had increased ileum TLR-4 protein levels as well as increased TLR-4 levels in the fetal brain and decreased fetal brain glutathione levels as compared to control. NAC administered to dams during pregnancy or to offspring under NEC conditions decreased offspring ileum and brain TLR-4 protein levels while increasing brain glutathione levels as compared to NEC offspring. In recent years exaggerated TLR-4 levels and increased activity in the immature intestine of preterm neonates has emerged as an inciting event in the pathophysiology of NEC (Mihi and Good, 2019). High TLR-4 activity in epithelial cells results in the initiation of an exaggerated immune response with increased production of pro inflammatory cytokines and destruction of the mucosal barrier (Mihi and Good, 2019). The exact reason for increased expression of TLR-4 in enterocytes of infants with NEC has not been fully elucidated, though Soliman et al. (2010) suggested that platelet-activating factor is responsible for TLR-4 over expression. TLR-4 also contributes to bacterial translocation across the mucosal barrier, which facilitates the development of the severe illness observed during NEC (Neal et al. 2006). Yazji further demonstrated the importance of TLR-4 in the pathogenesis of NEC, showing that a specific deletion of the TLR4 locus in endothelial cells in a mouse model of NEC was associated with a significant reduction of NEC severity when compared to endothelial TLR-4 sufficient animals (Azji et al. 2013). Our novel finding that NAC administered to either dams or offspring decreased TLR-4 protein levels in offspring ileum is of great importance. These findings are consistent with reports of decreased NEC observed in previous studiesfollowing NAC treatment (Zmora et al. 2020; Zmora et al. 2021). We demonstrated recently that NAC treatment was associated with decreased inflammatory response and attenuated activation of NFKBin a rodent NEC model. It is uncertain where NAC attenuates the cascade of the inflammatory response. Following attachment and activation of TLR-4, a cascade of event occurs through the MyD88 dependent pathway that involves activation of the IRAK family of kinases, in which TAK1 activates the downstream kinase IKK in the final step, which in turn phosphorylates the NF-κB inhibitor IκBα, leading to ubiquitin-dependent IκBα degradation and NF-κB activation. This cascade results in transcription of inflammatory genes, including those encoding TNF-α, IL-1β, IL-6, IL-12p40, and cyclooxygenase (Liu et al. 2017). In our current study, we demonstrated that the attenuation of the inflammatory response is at least in part connected to modulation of the TLR-4 levels in the ileum and brain upstream of the cascade. A recent study by Niño et al. (2018) demonstrated a gut-brain signaling axis in an NEC mouse model in which activation of intestinal TLR-4 signaling led to release of high-mobility group box 1 (HMGB1) in the intestine that, in turn, promoted microglial TLR-4 activation in the brain and neurological dysfunction. The brain injury that accompanies NEC was directly associated with intestine TLR-4 activation. The authors demonstrated that intestinal injury led to activation of TLR-4 on brain microglial cells resulting in accumulation of reactive oxygen species, loss of oligodendrocyte premature cells, dysmyelination, and cognitive impairment. They further demonstrated the importance of TLR-4 in mediating the brain injury in their NEC model, by generating mice lacking TLR-4 in microglia. The mice lacking microglia TLR-4 were significantly protected from NEC associated brain injury. In their model, loss of myelin required TLR-4 mediated microglial activation and resulted in ROS generation. The intestine brain axis was confirmed in their NEC model, as HMGB1 deficient mice showed significantly reduced microglial activation, while the administration of intranasal anti-HMGB1 antibody to a wild-type mouse model of NEC resulted in reduced microglial activation (Niño et al. 2018). In the present study, we demonstrated for the first time that under NEC conditions there is a significant increase in TLR-4 protein levels in the offspring brain. This finding may explain reports that brain injury associated with NEC is both more severe and more difficult to treat than brain injuries that develop in premature infants who do not develop NEC. Our findings that NEC condition increased TLR-4 levels in the intestine and brain was reversed by NAC treatment, may explain the pathophysiology of NAC protective effect in both ileum and brain. We further demonstrated that the decrease in brain TLR-4 following NAC was associated with increase in brain antioxidant glutathione levels, potentially enabling the brain to cope with increased oxidative stress and thus to decrease the brain injury. N-acetyl cysteine, a known anti-inflammatory and anti-oxidative agent, is considered safe for use during pregnancy (class B) (Zmora et al. 2021). NAC’s therapeutic properties stem from its action on the cystine-glutamate antiporter system and as an antioxidant to regulate the neuroinflammatory response (Dean et al. 2011; Durieux et al. 2015). Glutamate is implicated in fetal brain injury. Overexposure to glutamate and subsequent excess intracellular calcium influx, termed excitotoxicity, destroys neurons both in vivo and in vitro (Park et al. 2010). NAC may exert therapeutic benefit through decreasing synaptic glutamate release, thus mitigating subsequent excitotoxic neurological damage (Dean et al. 2011). Interestingly, we demonstrated that NAC was effective even when administered during pregnancy to dams long before exposing the offspring to NEC conditions. Our novel finding that maternal NAC administered long before delivery of the pups may protect the offspring from NEC features in both intestine and brain is supported by a human study that demonstrated rapid transfer of NAC from the mother to the fetus through the placenta, with umbilical cord concentrations frequently exceeding maternal concentrations (Wiest et al. 2014). Buhimshi et al. (2003) demonstrated in a maternal inflammation model in mice, that maternal inflammation resulted in oxidative stress associated with maternal and fetal liver glutathione (GSH) precursor depletion, while maternal NAC restored both maternal and fetal oxidative balance and increased liver GSH levels in both dams and fetuses. Maternal NAC administration may be of benefit for newborns at increased risk of NEC, as it attenuates the inflammatory and oxidative responses. The strength of our study is that we used an established rat model of NEC that replicates the key clinical features of NEC. This allowed us to assess the brain and intestine and to study the mechanisms mediating the development of NEC and NEC- associated brain injury. There are some limitations to our study. It is an animal study with a relatively small sample size and little clinically correlated data. Also, we studied the offspring at 5 days of age which might be too early a stage in brain development. Further studies should be performed at later stages of gestation with larger numbers. In conclusion, we demonstrated that NAC could prevent an increase in TLR-4 protein levels in both ileum and brain in an established NEC model, while increasing brain glutathione levels. This study extends our understanding of the mechanisms associated with NEC injury and its prevention and may help develop new strategies to cope with offspring injury associated with NEC. Declarations Author Contributions: All authors contributed to the study conception and design. Material preparation, experiment, and data collection were performed by Osnat Zmora, Ron Beloosesky, Ola Gutzeit Yuval Ginsberg, and Nizar Khatib. Analyses were performed by Zeev Wiener and Michael Ross. The first draft of the manuscript was written by Osnat Zmora, Ron Beloosesky, and Michael Ross. All authors commented on previous versions of the manuscript, read, and approved the final manuscript. Conflicts of Interest: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Funding : The authors have no relevant financial or non-financial interests to disclose. The study was carried out in compliance with the ARRIVE guidelines Data availability : All data generated or analyzed during this study are included in this published article [and its supplementary information files]. References Anderson KV (2000) Toll signaling pathways in the innate immune response. Curr Opin Immunol 12:13–9. https://doi.org/10.1016/s0952-7915(99)00045-x . Azji I, Sodhi CP, Lee EK, Good M, Egan CE, Afrazi A, Neal MD, Jia H, Lin J, Ma C, Branca MF, Prindle T, Richardson WM, Ozolek J, Billiar TR, Binion DG, Gladwin MT, Hackam DJ (2013) Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS-NO-nitrite signaling. Proc Natl Acad Sci U S A 110:9451–6. https://doi.org/ 10.1073/pnas.1219997110 . Ballance WA, Dahms BB, Shenker N, Kliegman RM (1990) Pathology of neonatal necrotizing enterocolitis: a ten-year experience. J Pediatr 117:S6–13. https://doi.org/ 10.1016/s0022-3476(05)81124-2 . Beloosesky R, Gayle DA, Amidi F, Nunez SE, Babu J, Desai M, Ross MG (2006) N-acetyl-cysteine suppresses amniotic fluid and placenta inflammatory cytokine responses to lipopolysaccharide in rats. Am J Obstet Gynecol 194:268–73. https://doi.org/10.1016/j.ajog.2005.06.082 . Blakely ML, Lally KP, McDonald S, Brown RL, Barnhart DC, Ricketts RR, Thompson WR, Scherer LR, Klein MD, Letton RW, Chwals WJ, Touloukian RJ, Kurkchubasche AG, Skinner MA, Moss RL, Hilfiker ML; NEC Subcommittee of the NICHD Neonatal Research Network (2005) Postoperative outcomes of extremely low birth-weight infants with necrotizing enterocolitis or isolated intestinal perforation: a prospective cohort study by the NICHD Neonatal Research Network. Ann Surg 241:984–9; discussion 989–94. https://doi.org/10.1097/01.sla.0000164181.67862.7f . Buhimschi IA, Buhimschi CS, Weiner CP (2003) Protective effect of N-acetylcysteine against fetal death and preterm labor induced by maternal inflammation. Am J Obstet Gynecol 188:203–8. https://doi.org/10.1067/mob.2003.112 . Dean O, Giorlando F, Berk M (2011) N-acetylcysteine in psychiatry: current therapeutic evidence and potential mechanisms of action. J Psychiatry Neurosci 36:78–86. https://doi.org/10.1503/jpn.100057 . Durieux AM, Fernandes C, Murphy D, Labouesse MA, Giovanoli S, Meyer U, Li Q, So PW, McAlonan G (2015) Targeting Glia with N-Acetylcysteine Modulates Brain Glutamate and Behaviors Relevant to Neurodevelopmental Disorders in C57BL/6J Mice. Front Behav Neurosci 9:343. https://doi.org/10.3389/fnbeh.2015.00343 . Gomart A, Vallée A, Lecarpentier Y (2021) Necrotizing Enterocolitis: LPS/TLR4-Induced Crosstalk Between Canonical TGF-β/Wnt/β-Catenin Pathways and PPARγ. Front Pediatr 9:713344. https://doi.org/10.3389/fped.2021.713344 . Hunter CJ, Upperman JS, Ford HR, Camerini V (2008) Understanding the; susceptibility of the premature infant to necrotizing enterocolitis (NEC). Pediatr Res 63:117–23. https://doi.org/10.1203/PDR.0b013e31815ed64c . Kelly N, Friend K, Boyle P, Zhang XR, Wong C, Hackam DJ, Zamora R, Ford HR, Upperman JS (2004) The role of the glutathione antioxidant system in gut barrier failure in a rodent model of experimental necrotizing enterocolitis. Surgery 136:557–66. https://doi.org/10.1016/j.surg.2004.05.034 . Liu T, Zhang L, Joo D, Sun SC (2017) NF-κB signaling in inflammation. Signal Transduct Target Ther 2:17023–. https://doi.org/10.1038/sigtrans.2017.23 . Mihi B, Good M (2019) Impact of Toll-Like Receptor 4 Signaling in Necrotizing Enterocolitis: The State of the Science. Clin Perinatol 46:145–157. https://doi.org/10.1016/j.clp.2018.09.007 . Nanthakumar N, Meng D, Goldstein AM, Zhu W, Lu L, Uauy R, Llanos A, Claud EC, Walker WA (2011) The mechanism of excessive intestinal inflammation in necrotizing enterocolitis: an immature innate immune response. PLoS One 6:e17776. https://doi.org/10.1371/journal.pone.0017776 . Neal MD, Leaphart C, Levy R, Prince J, Billiar TR, Watkins S, Li J, Cetin S, Ford H, Schreiber A, Hackam DJ (2006) Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier. J Immunol 176:3070–9. https://doi.org/10.4049/jimmunol.176.5.3070 . Neu J (1996) Necrotizing enterocolitis: the search for a unifying pathogenic theory leading to prevention. Pediatr Clin North Am 43:409–32. https://doi.org/10.1016/s0031-3955(05)70413-2 . Niño DF, Zhou Q, Yamaguchi Y, Martin LY, Wang S, Fulton WB, Jia H, Lu P, Prindle T Jr, Zhang F, Crawford J, Hou Z, Mori S, Chen LL, Guajardo A, Fatemi A, Pletnikov M, Kannan RM, Kannan S, Sodhi CP, Hackam DJ (2018) Cognitive impairments induced by necrotizing enterocolitis can be prevented by inhibiting microglial activation in mouse brain. Sci Transl Med 10:eaan0237. https://doi.org/10.1126/scitranslmed.aan0237 . Park E, Lee GJ, Choi S, Choi SK, Chae SJ, Kang SW, Park HK (2010) Correlation between extracellular glutamate release and neuronal cell death in an eleven vessel occlusion model in rat. Brain Res 1342:160–6. https://doi.org/10.1016/j.brainres.2010.04.054 . Riggs BS, Bronstein AC, Kulig K, Archer PG, Rumack BH (1989) Acute acetaminophen overdose during pregnancy. Obstet Gynecol 74:247–53. Soliman A, Michelsen KS, Karahashi H, Lu J, Meng FJ, Qu X, Crother TR, Rabizadeh S, Chen S, Caplan MS, Arditi M, Jilling T (2010) Platelet-activating factor induces TLR4 expression in intestinal epithelial cells: implication for the pathogenesis of necrotizing enterocolitis. PLoS One 5:e15044. https://doi.org/10.1371/journal.pone.0015044 . Sulistyo A, Rahman A, Biouss G, Antounians L, Zani A (2018) Animal models of necrotizing enterocolitis: review of the literature and state of the art. Innov Surg Sci. 3:87–92. https://doi.org/10.1515/iss-2017-0050 . Wiest DB, Chang E, Fanning D, Garner S, Cox T, Jenkins DD (2014) Antenatal pharmacokinetics and placental transfer of N-acetylcysteine in chorioamnionitis for fetal neuroprotection. J Pediatr 165:672-7.e2. https://doi.org/10.1016/j.jpeds.2014.06.044 . Zmora O, Gutzeit O, Segal L, Boulos S, Millo Z, Ginsberg Y, Khatib N, Dabbah-Assad F, Fainaru O, Weiner Z, Beloosesky R (2020) Prophylactic antenatal N-Acetyl Cysteine administration combined with postnatal administration can decrease mortality and injury markers associated with necrotizing enterocolitis in a rat model. PLoS One 15:e0233612. https://doi.org/10.1371/journal.pone.0233612 . Zmora O, Gutzeit O, Segal L, Boulos S, Millo Z, Ginsberg Y, Khatib N, Fainaru O, Ross MG, Weiner Z, Beloosesky R (2021) Maternal N-acetyl-cysteine prevents neonatal brain injury associated with necrotizing enterocolitis in a rat model. Acta Obstet Gynecol Scand 100:979–987. https://doi.org/ 10.1111/aogs.14054 . Additional Declarations No competing interests reported. Supplementary Files RAWDATABRAINSR.pptx rawdataILEUMSR.pptx Cite Share Download PDF Status: Published Journal Publication published 22 May, 2023 Read the published version in Scientific Reports → Version 2 posted Editorial decision: Major revision 05 Jan, 2023 Reviews received at journal 04 Jan, 2023 Reviewers agreed at journal 28 Dec, 2022 Reviews received at journal 08 Dec, 2022 Reviewers agreed at journal 05 Dec, 2022 Reviewers agreed at journal 03 Dec, 2022 Reviewers invited by journal 25 Oct, 2022 Editor assigned by journal 25 Oct, 2022 Editor invited by journal 23 Jun, 2022 Submission checks completed at journal 23 Jun, 2022 First submitted to journal 15 Jun, 2022 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1684513","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2022-05-31 16:07:09","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"articleType":"Article","associatedPublications":[],"authors":[{"id":118845242,"identity":"6af993d0-10ed-49bf-871a-524ab2278234","order_by":0,"name":"Ron Beloosesky","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYHACNgaGgho5EOvAA+K1GBwzBmtJIEELc2IDiEmUFvP23mMPPhiwpc8PO/wQaIudnG4DAS0yZ86lG84wkMndeDvNAKgl2djsAAEtEhI5ZtI8Bmy5G2cngLQcSNxGlJY/BszphrPTP5CgBej9BHnpHGJt4TljbthjcMxwg3ROwYEEA2L8wt5j9uBHRY28/Oz0zR8+VNjJEdQCBwZglQbEKgcB+QZSVI+CUTAKRsGIAgAS0kBXej7rhAAAAABJRU5ErkJggg==","orcid":"","institution":"Rappaport Faculty of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ron","middleName":"","lastName":"Beloosesky","suffix":""},{"id":118845243,"identity":"11089d3a-49a0-4a91-a4ee-22475325c738","order_by":1,"name":"Ola Gutzeit","email":"","orcid":"","institution":"Rambam Health Care Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ola","middleName":"","lastName":"Gutzeit","suffix":""},{"id":118845244,"identity":"bc596c3d-0460-43f1-a977-b3ebfacadee1","order_by":2,"name":"Yuval Ginsberg","email":"","orcid":"","institution":"Rambam Health Care Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuval","middleName":"","lastName":"Ginsberg","suffix":""},{"id":118845245,"identity":"1da64705-fd93-4753-b2cc-87f6c926656c","order_by":3,"name":"Nizar Khatib","email":"","orcid":"","institution":"Rambam Health Care Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nizar","middleName":"","lastName":"Khatib","suffix":""},{"id":118845246,"identity":"4038f3e7-5755-4c12-852b-482346e996c7","order_by":4,"name":"Michael G. Ross","email":"","orcid":"","institution":"Harbor-UCLA Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michael","middleName":"G.","lastName":"Ross","suffix":""},{"id":118845247,"identity":"c29072ee-718e-4b25-b7d3-4f6509222b0f","order_by":5,"name":"Weiner Zeev","email":"","orcid":"","institution":"Rambam Health Care Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weiner","middleName":"","lastName":"Zeev","suffix":""},{"id":118845248,"identity":"eb2abd3d-8f4e-4aca-a462-ad5adafdfabe","order_by":6,"name":"Osnat zmora","email":"","orcid":"","institution":"Tel Aviv University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Osnat","middleName":"","lastName":"zmora","suffix":""}],"badges":[],"createdAt":"2022-05-23 11:14:23","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-1684513/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1684513/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-35019-5","type":"published","date":"2023-05-22T20:57:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":23631722,"identity":"e8c3ee41-ba87-4e3c-907a-c9a91449dc23","added_by":"auto","created_at":"2022-07-08 13:34:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22867,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of the different study groups. IV, intravenous; NAC, N-acetyl cysteine; NEC, necrotizing enterocolitis; IP, intraperitoneal; NS, normal saline\u003c/p\u003e","description":"","filename":"FIG1S.R.png","url":"https://assets-eu.researchsquare.com/files/rs-1684513/v2/318e8506e49fd375f70f5420.png"},{"id":23631725,"identity":"d2de7b09-d3ca-485a-ab39-e2d50c42138a","added_by":"auto","created_at":"2022-07-08 13:34:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20952,"visible":true,"origin":"","legend":"\u003cp\u003eOffspring brain glutathione protein levels in Groups 1-5\u003c/p\u003e\u003cp\u003eNEC pups had significantly decreased brain glutathione levels as compared with control pups; NAC to pregnant dams, offspring, or both dams and offspring significantly increased offspring brain glutathione levels as compared with NEC pups. CTL, NEC, NEC-NAC, NAC-NEC and NAC-NEC-NAC. \u003c/p\u003e\u003cp\u003e*Significant difference (P\u0026lt;0.05) from NEC. #Significant difference (P\u0026lt;0.05) from NAC-NEC. ~Significant difference (P\u0026lt;0.05) from NAC-NEC-NAC. NAC, N-acetyl cysteine; NEC, and necrotizing enterocolitis.\u003c/p\u003e","description":"","filename":"FIG2S.R.png","url":"https://assets-eu.researchsquare.com/files/rs-1684513/v2/5c909837334353f1639eca05.png"},{"id":23631896,"identity":"88e9b667-fb89-45a5-853a-ba451bfb6d64","added_by":"auto","created_at":"2022-07-08 13:39:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":50617,"visible":true,"origin":"","legend":"\u003cp\u003eOffspring brain TLR-4 protein levels in Groups 1-5 \u003c/p\u003e\u003cp\u003eNEC pups had significantly increased brain TLR-4 levels as compared with control pups; NAC to pregnant dams, offspring, or both dams and offspring significantly decreased offspring LLR-4 levels as compared with NEC pups (CTL, NEC, NEC-NAC, NAC-NEC and NAC-NEC-NAC). \u003c/p\u003e\u003cp\u003e*Significant difference (P\u0026lt;0.05) from NEC. #Significant difference (P\u0026lt;0.05) from NAC-NEC. ^Significant difference (P\u0026lt;0.05) from NEC-NAC. NAC, N-acetyl cysteine; NEC, necrotizing enterocolitis; TLR-4, Toll-like receptor 4.\u003c/p\u003e","description":"","filename":"FIG3S.R.png","url":"https://assets-eu.researchsquare.com/files/rs-1684513/v2/f49018992d0863023af1334a.png"},{"id":23631897,"identity":"e43d0fa0-ef49-46a2-ab70-3c8594836a5a","added_by":"auto","created_at":"2022-07-08 13:39:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49040,"visible":true,"origin":"","legend":"\u003cp\u003eOffspring ileal TLR-4 protein levels in Groups 1-5\u003c/p\u003e\u003cp\u003eNEC pups had significantly increased ileal TLR-4 levels as compared with control pups; NAC to pregnant dams, offspring, or both dams and offspring significantly decreased offspring LLR-4 levels as compared with NEC pups (CTL, NEC, NEC-NAC, NAC-NEC and NAC-NEC-NAC). \u003c/p\u003e\u003cp\u003e*Significant difference (P\u0026lt; .05) from NEC. #Significant difference (P\u0026lt;0.05) from NAC-NEC. NAC, N-acetyl cysteine; NEC, necrotizing enterocolitis; TLR-4, Toll-like receptor 4.\u0026nbsp;\u003c/p\u003e","description":"","filename":"FIG4S.R.png","url":"https://assets-eu.researchsquare.com/files/rs-1684513/v2/dba7455477da463dbcb375ca.png"},{"id":44729699,"identity":"e1c458ef-17a6-49db-8704-e036f26910cb","added_by":"auto","created_at":"2023-10-16 21:20:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":489586,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1684513/v2/c3191395-4128-4d4b-9810-1eb111bc32fd.pdf"},{"id":23631724,"identity":"6ade0e1f-451d-4505-9532-aa87924743d2","added_by":"auto","created_at":"2022-07-08 13:34:22","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":201236,"visible":true,"origin":"","legend":"","description":"","filename":"RAWDATABRAINSR.pptx","url":"https://assets-eu.researchsquare.com/files/rs-1684513/v2/5c9a46372b36a85cc1a0ec67.pptx"},{"id":23631727,"identity":"5e35cd2b-aba3-40b7-b9fb-4705e1bcd319","added_by":"auto","created_at":"2022-07-08 13:34:22","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":273818,"visible":true,"origin":"","legend":"","description":"","filename":"rawdataILEUMSR.pptx","url":"https://assets-eu.researchsquare.com/files/rs-1684513/v2/b86b2945576958a305e97f70.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intestine and brain TLR-4 modulation following N-Acetyl-Cysteine treatment in NEC rodent model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNecrotizing enterocolitis (NEC) is a leading cause of preterm infant morbidity and mortality (Mihi and Good \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the USA, more than 3000 neonates are diagnosed with NEC annually (Neu \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1996\u003c/span\u003e); 7% of infants weighing less than 1500 gram are affected by NEC (Hunter et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) with mortality as high as 30% (Blakely et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNEC is both an infectious and inflammatory disease that can affect only part or the entire gastrointestinal tract, predominantly found in the region of the terminal ileum (Balance et al. 1990). NEC etiology is considered multifactorial, a consequence of intestinal immaturity, microbial dysbiosis, and an exuberant inflammatory response.\u003c/p\u003e \u003cp\u003eToll-like receptor 4 (TLR-4), which plays a critical role in the induction of protective host innate immune response, is an important factor in normal gut development (Mihi and Good \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Recent evidence suggests that TLR-4 is expressed at higher levels in the premature intestine and is more abundant in the intestine of infants with NEC (Nanthakumar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Gomart et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The excessive inflammatory response observed in NEC is explained in part due to over-expression and the exaggerated activity of TLR-4 in the preterm intestine (Nanthakumar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe attachment of ligand to TLR-4 initiates a cascade of events that result in phosphorylation of kappa-B inhibitor in the cytosol and activation of NFKB P65, which translocates into the nucleus where it activates and regulates the transcription of genes related to inflammatory responses (Anderson \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to intestinal injury in NEC, several studies report significant severe neurodevelopmental disability in NEC survivors (Ni\u0026ntilde;o 2018). Recently it was demonstrated in NEC model that a brain injury was associated with decreased brain glutathione levels (Ni\u0026ntilde;o 2018). However, the mechanisms associated with brain injury in NEC have not been fully elucidated\u003c/p\u003e \u003cp\u003eN-Acetyl Cysteine (NAC) is a known anti-inflammatory and anti-oxidant agent used widely for paracetamol intoxication, that is safe to use during pregnancy (class B) (Riggs et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Beloosesky et al. 2005). Its anti-oxidant activity results from its conversion into metabolites that are capable of stimulating glutathione synthesis and promoting detoxification, and by its inherent function as a scavenger of free oxygen radicals.\u003c/p\u003e \u003cp\u003eTLR4 signaling plays a central role in the induction of NEC through the modulation of the epithelial cell barrier and regulation of the innate and adaptive immune responses. We hypothesized that modulation of TLR-4 effects could reduce the risk of NEC. In the present study, we sought to determine the protective effect of NAC on TLR-4 protein levels in fetal intestine and fetal brain, and to determine its effect on fetal brain glutathione levels in an established rat model of NEC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003ePregnant Sprague-Dawley (SD) rats were obtained at day 11 of gestation and allowed to acclimate for 7 days before initiating the experiments (Fig.\u0026nbsp;\u003ca href=\"https://obgyn.onlinelibrary.wiley.com/doi/10.1111/aogs.14054#aogs14054-fig-0001\"\u003e1\u003c/a\u003e). The SD rats were maintained in light-controlled facilities with access to water and food ad libitum throughout the study, at ambient temperature (25\u0026deg;C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe first group of pregnant rats (nine dams) received no treatment during pregnancy and delivered spontaneously. After birth, the pups were divided into three groups.\u003c/p\u003e\n\u003cp\u003eGroup 1 \u0026ndash; Control: 33 pups remained with their mothers (three dams) and were nursed at room temperature. Sixty-six pups from six dams were separated from the dams and divided into Groups 2 and 3, as described below.\u003c/p\u003e\n\u003cp\u003eGroup 2 \u0026ndash; NEC: 32 pups were transferred to an incubator (Ohio Medical Products, Madison, WI, USA), where they were fed thrice daily by gavage of 0.2 mL clean formula consisting of 15 g of Similac 60/40 (Abbott Nutritional, Columbus, OH, USA) in 75 mL of Esbilac canine milk replacement (PetAg Inc., Hampshire, IL, USA). The pups also received daily hypoxia exposure for four days with 5% O\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand 95% N\u003csub\u003e2,\u0026nbsp;\u003c/sub\u003eand intraperitoneal injections of saline three times daily (for 10 minutes) following each hypoxia exposure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGroup 3 -- NEC-NAC: 34 pups were exposed to the same NEC conditions as pups in Group 2, with the addition of NAC treatment (N-acetyl cysteine, SIGMA, reconstituted in water, 300 mg/kg intraperitoneal) thrice daily, following each 4-day hypoxia exposure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo explore the possible protective effect of NAC during pregnancy, a second group of six pregnant rats received intravenous injections of NAC (300 mg/kg) once daily from gestational day 18 until delivery. After birth, the pups were divided into Groups 4 and 5 as described below.\u003c/p\u003e\n\u003cp\u003eGroup 4 -- NAC-NEC: 33 pups were separated from their dams and kept under the same conditions as Group 2 (NEC) above.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGroup 5 -- NAC-NEC-NAC: 36 pups were separated from their dams and kept under the same conditions as Group 3 (NEC-NAC).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn all studies, pups were euthanized on the fifth day of life.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePups from the five groups were anesthetized with isoflurane and decapitated on their fifth day of life. Brains were harvested and frozen immediately in liquid nitrogen for further processing and analysis. The intestine was removed, and the distal 3 cm of terminal ileum were harvested and immediately frozen in liquid nitrogen for further processing and analysis. Processing and analysis were performed on the mixture of intracellular and cellular components of the distal terminal ileum. Brain and terminal ileum TLR-4 protein levels were determined by Western blot, and brain Glutathione levels determined by ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWestern blot analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreparations of cell lysates and Western blot cells or tissues were lysed in RIPA \u0026nbsp;buffer \u0026nbsp; (phosphate-buffered saline \u0026nbsp; containing \u0026nbsp;1% Nonidet \u0026nbsp;P-40, \u0026nbsp; 0.1% \u0026nbsp;sodium \u0026nbsp;dodecyl \u0026nbsp; sulfate \u0026nbsp;[SDS], \u0026nbsp;1 mmol/L Na3VO4, 4 mm phenylmethylsulfonyl fluoride and 0.05% [w/v] apro-tinin). Insoluble proteins were discarded by high-speed centrifugation at 2000 g for 10 min at 4\u0026deg;C. Small volumes of lysate were taken to measure total protein concentration, using absorbance at 280 nm in triplicate by Nanodrop. Determination of the protein concentration samples was also compared with BSA standards, ensuring the standard was diluted in the same buffer as the samples, and a 50-\u0026mu;g sample was loaded into each well of a 12% SDS-PAGE acrylamide gel. After transferring and blocking to nitrocellulose membranes, the blots were incubated overnight with primary antibodies against the target protein, at 4\u0026deg;C. The antibodies were diluted in blocking buffer according to the manufacturer\u0026rsquo;s recommendations.\u003c/p\u003e\n\u003cp\u003eFollowing incubation with primary antibodies and rinsing, the blots were incubated for 1 hour at room temperature with HRP-conjugated secondary antibody, according to the manufacturer\u0026rsquo;s recommended ratio. Antibodies recognizing the TLR-4 ( cat#NB100-56566 novus) were used in combination with a donkey anti-mouse horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA, USA).\u0026nbsp;Following incubation with the diluted secondary antibody and washing with TBST\u0026nbsp;the membrane was incubated with the peroxide solution according to the manufactory orders andenhanced to chemiluminescent (ECL) development solution (ab133406)by exposing to X-ray.\u0026nbsp;Actin was used as a reference protein. Densitometric analysis was used to determine differences in protein expression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDensitometry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo compare target protein expression levels among samples on the same blot or across blots, we normalized the bands by loading controls (housekeeping protein actin). To get an objective measure of the signal generated on Western blot, a densitometer was used to scan the blot or film, and imaging software was used to compare signals. The quantification reflects the ratio of each protein band relative to the control (actin). Films were subsequently imaged with ChemiDoc MP using the white light conversion screen and the silver stain (visible stain) application. \u0026nbsp;The Band Analysis tools of IMAGELAB, software version 4.1 (Bio-Rad), were used to select and determine the back-ground-subtracted density of the bands in all the gels and blots.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA determinations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOffspring brain glutathione level was determined with a HT Glutathione Assay kit, catalog number: 7511-100-K (Bio-Techne Ltd., Minneapolis, MN, USA), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOffspring brain and terminal ileum TLR-4 protein levels were compared among pups from the different groups (5 brain samples and 5 terminal ileum from each group). Offspring brain glutathione protein levels were compared among pups from the different groups. All results were expressed as means\u0026plusmn;SD using one-way analysis of variance followed by post hoc tests for pairwise comparisons (Holm-Sidak method). Differences were significant at P\u0026lt; 0.05. Sigma Stat software, version 4.0 was used to perform the statistical analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocols and procedures were approved by the Institutional Animal Care Committee at the Rappaport Faculty of Medicine (Protocol number: IL001-01-2016). This study was carried out in strict accordance with the recommendations of the Israeli National Institute of Health guide for care and use of laboratory animals. Guidelines for the care and use of the animals approved by the local institution were also followed. All efforts were made to minimize animal suffering. The study was supervised by a veterinarian on a daily basis. The pain suffering of the rats was categorized as low.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eBrain and ileum TLR-4\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNEC offspring had significantly increased brain and ileum TLR-4 protein levels as compared to control group (brain 2.\u003cspan dir=\"RTL\"\u003e5\u003c/span\u003e\u003cu\u003e+\u003c/u\u003e0.\u003cspan dir=\"RTL\"\u003e6\u003c/span\u003e vs. 0.88\u003cu\u003e+\u003c/u\u003e0.1\u003cspan dir=\"RTL\"\u003e2\u003c/span\u003e U; ileum 0.24\u003cu\u003e+\u003c/u\u003e0.04 vs. 0.09\u003cu\u003e+\u003c/u\u003e0.02 U, respectively, p\u0026lt;0.05). NAC, administered only to pregnant dams (NAC-NEC), significantly decreased NEC offspring brain (1.\u003cspan dir=\"RTL\"\u003e53\u003c/span\u003e\u003cu\u003e+\u003c/u\u003e0.\u003cspan dir=\"RTL\"\u003e41\u003c/span\u003e vs. 2.\u003cspan dir=\"RTL\"\u003e5\u003c/span\u003e\u003cu\u003e+\u003c/u\u003e0.\u003cspan dir=\"RTL\"\u003e6\u003c/span\u003e U, p\u0026lt;0.05) and ileum (0.12\u003cu\u003e+\u003c/u\u003e0.03 vs. 0.2\u003cu\u003e+\u003c/u\u003e0.01 U, p\u0026lt;0.05) TLR-4 protein levels as compared to NEC offspring. The same pattern of decreased brain and ileum TLR-4 protein levels was demonstrated when NAC was administered only to the offspring (NEC-NAC) (brain 1.\u003cspan dir=\"RTL\"\u003e03\u003c/span\u003e\u003cu\u003e+\u003c/u\u003e0.\u003cspan dir=\"RTL\"\u003e32\u003c/span\u003e vs. 2.\u003cspan dir=\"RTL\"\u003e5\u003c/span\u003e\u003cu\u003e+\u003c/u\u003e0.\u003cspan dir=\"RTL\"\u003e6\u003c/span\u003e U; ileum 0.09\u003cu\u003e+\u003c/u\u003e0.01 vs. 0.24\u003cu\u003e+\u003c/u\u003e0.04 U, p\u0026lt;0.05).NAC administered to both dams and offspring (NAC-NEC-NAC) resulted in decreased in TLR-4 in brains \u0026nbsp;(0.68+0.11 vs. 2.3\u003cu\u003e+\u003c/u\u003e0.25 U; p\u0026lt;0.05) and ileum (0.07+0.02 vs. 0.24\u003cu\u003e+\u003c/u\u003e0.04 U; p\u0026lt;0.05) as compared to NEC group (Figs. 2 and 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBrain glutathione level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNEC offspring demonstrated decreased brain glutathione levels as compared to control offspring (0.023\u003cu\u003e+\u003c/u\u003e0.001 vs. 0.036\u003cu\u003e+\u003c/u\u003e0.001 pmol/ml, respectively, p\u0026lt;0.05). NAC administered only to pregnant dams (NAC-NEC) significantly increased offspring glutathione levels as compared to NEC offspring (0.03+0.001vs. 0.023\u003cu\u003e+\u003c/u\u003e0.001 pmol/ml, respectively, p\u0026lt;0.05). The same pattern was observed when NAC was administered only to offspring (NEC-NAC; 0.038+0.001 vs. 0.203\u003cu\u003e+\u003c/u\u003e0.001 pmol/ml) or when NAC was administered to both dams and offspring (NAC-NEC-NAC; 0.031+0.001 vs. 0.203\u003cu\u003e+\u003c/u\u003e0.001 pmol/ml, respectively, p\u0026lt;0.05) (Fig. 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn previous studies using this NEC model, we and others (Kelly et al. 2004; Sulistyo et al. 2018) demonstrated features of NEC with severe bowel damage in pups, inflammatory changes and increased oxidative stress, accompanied with offspring brain injury. This model is based on factors that are recognized to contribute to the development of human NEC, including intestinal immaturity, hypoxia, and artificial hyperosmolar formula. Dams are removed from the pups soon after birth to avoid breastfeeding, which is known to be protective against NEC. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe demonstrated, in our animal NEC model, that offspring with NEC had increased ileum TLR-4 protein levels as well as increased TLR-4 levels in the fetal brain and decreased fetal brain glutathione levels as compared to control. NAC administered to dams during pregnancy or to offspring under NEC conditions decreased offspring ileum and brain TLR-4 protein levels while increasing brain glutathione levels as compared to NEC offspring.\u003c/p\u003e\n\u003cp\u003eIn recent years exaggerated TLR-4 levels and increased activity in the immature intestine of preterm neonates has emerged as an inciting event in the pathophysiology of NEC (Mihi and Good, 2019). High TLR-4 activity in epithelial cells results in the initiation of an exaggerated immune response with increased production of pro inflammatory cytokines and destruction of the mucosal barrier (Mihi and Good, 2019). \u0026nbsp;The exact reason for increased expression of TLR-4 in enterocytes of infants with NEC has not been fully elucidated, though Soliman et al. (2010) suggested that platelet-activating factor is responsible for TLR-4 over expression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTLR-4 also contributes to bacterial translocation across the mucosal barrier, which facilitates the development of the severe illness observed during NEC (Neal et al. 2006). Yazji further demonstrated the importance of TLR-4 in the pathogenesis of NEC, showing that a specific deletion of the TLR4 locus in endothelial cells in a mouse model of NEC was associated with a significant reduction of NEC severity when compared to endothelial TLR-4 sufficient animals (Azji et al. 2013). \u0026nbsp;Our novel finding that NAC administered to either dams or offspring decreased TLR-4 protein levels in offspring ileum is of great importance. These findings are consistent with reports of decreased NEC observed in previous studiesfollowing NAC treatment (Zmora et al. 2020;\u0026nbsp;Zmora et al. 2021).\u003c/p\u003e\n\u003cp\u003eWe demonstrated recently that NAC treatment was associated with decreased inflammatory response and attenuated activation of NFKBin a rodent NEC model. It is uncertain where NAC attenuates the cascade of the inflammatory response. Following attachment and activation of TLR-4, a cascade of event occurs through the MyD88 dependent pathway that involves activation of the IRAK family of kinases, in which TAK1 activates the downstream kinase IKK in the final step, which in turn phosphorylates the NF-\u0026kappa;B inhibitor I\u0026kappa;B\u0026alpha;, leading to ubiquitin-dependent I\u0026kappa;B\u0026alpha; degradation and NF-\u0026kappa;B activation. This cascade results in transcription of inflammatory genes, including those encoding TNF-\u0026alpha;, IL-1\u0026beta;, IL-6, IL-12p40, and cyclooxygenase (Liu et al. 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our current study, we demonstrated that the attenuation of the inflammatory response is at least in part connected to modulation of the TLR-4 levels in the ileum and brain upstream of the cascade. A recent study by Ni\u0026ntilde;o et al. (2018) demonstrated a gut-brain signaling axis in an NEC mouse model in which activation of intestinal TLR-4 signaling led to release of high-mobility group box 1 (HMGB1) in the intestine that, in turn, promoted microglial TLR-4 activation in the brain and neurological dysfunction. The brain injury that accompanies NEC was directly associated with intestine TLR-4 activation. The authors demonstrated that intestinal injury led to activation of TLR-4 on brain microglial cells resulting in accumulation of reactive oxygen species, loss of oligodendrocyte premature cells, dysmyelination, and cognitive impairment. They further demonstrated the importance of TLR-4 in mediating the brain injury in their NEC model, by generating mice lacking TLR-4 in microglia. The mice lacking microglia TLR-4 were significantly protected from NEC associated brain injury. In their model, loss of myelin required TLR-4 mediated microglial activation and resulted in ROS generation. The intestine brain axis was confirmed in their NEC model, as HMGB1 deficient mice showed significantly reduced microglial activation, while the administration of intranasal anti-HMGB1 antibody to a wild-type mouse model of NEC resulted in reduced microglial activation (Ni\u0026ntilde;o et al. 2018).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study, we demonstrated for the first time that under NEC conditions there is a significant increase in TLR-4 protein levels in the offspring brain. This finding may explain reports that brain injury associated with NEC is both more severe and more difficult to treat than brain injuries that develop in premature infants who do not develop NEC. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur findings that NEC condition increased TLR-4 levels in the intestine and brain was reversed by NAC treatment, may explain the pathophysiology of NAC protective effect in both ileum and brain. \u0026nbsp;We further demonstrated that the decrease in brain TLR-4 following NAC was associated with increase in brain antioxidant glutathione levels, potentially\u0026nbsp;enabling the brain to cope with increased oxidative stress\u0026nbsp;and thus to decrease the brain injury. N-acetyl cysteine, a known anti-inflammatory and anti-oxidative agent, is considered safe for use during pregnancy (class B) (Zmora et \u0026nbsp;al. 2021). NAC\u0026rsquo;s therapeutic properties stem from its action on the cystine-glutamate antiporter system and as an antioxidant to regulate the neuroinflammatory response (Dean et al. 2011; Durieux et al. 2015).\u0026nbsp;Glutamate is implicated in fetal brain injury. Overexposure to glutamate and subsequent excess intracellular calcium influx, termed excitotoxicity, destroys neurons both in vivo and in vitro (Park et al. 2010). NAC may exert therapeutic benefit through decreasing synaptic glutamate release, thus mitigating subsequent excitotoxic neurological damage (Dean et al. 2011).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, we demonstrated that NAC was effective even when administered during pregnancy to dams long before exposing the offspring to NEC conditions.\u0026nbsp;\u0026nbsp;Our novel finding that maternal NAC administered long before delivery of the pups may protect the offspring from NEC features in both intestine and brain is supported by a human study that demonstrated rapid transfer of NAC from the mother to the fetus through the placenta, with umbilical cord concentrations frequently exceeding maternal concentrations (Wiest et al. 2014). Buhimshi et al. (2003) demonstrated in a maternal inflammation model in mice, that maternal inflammation resulted in oxidative stress associated with maternal and fetal liver glutathione (GSH) precursor depletion, while maternal NAC restored both maternal and fetal oxidative balance and increased liver GSH levels in both dams and fetuses. Maternal NAC administration may be of benefit for newborns at increased risk of NEC, as it attenuates the inflammatory and oxidative responses.\u003c/p\u003e\n\u003cp\u003eThe strength of our study is that we used an established rat model of NEC that replicates the key clinical features of NEC. This allowed us to assess the brain and intestine and to study the mechanisms mediating the development of NEC and NEC- associated brain injury. There are some limitations to our study. It is an animal study with a relatively small sample size and little clinically correlated data. Also, we studied the offspring at 5 days of age which might be too early a stage in brain development. \u0026nbsp;Further studies should be performed at later stages of gestation with larger numbers.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we demonstrated that NAC could prevent an increase in TLR-4 protein levels in both ileum and brain in an established NEC model, while increasing brain glutathione levels. This study extends our understanding of the mechanisms associated with NEC injury and its prevention and may help develop new strategies to cope with offspring injury associated with NEC.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eAll authors contributed to the study conception and design. Material preparation, experiment, and data collection were performed by Osnat Zmora, Ron Beloosesky, Ola Gutzeit Yuval Ginsberg, and Nizar Khatib. Analyses were performed by Zeev Wiener and Michael Ross. The first draft of the manuscript was written by Osnat Zmora, Ron Beloosesky, and Michael Ross. All authors commented on previous versions of the manuscript, read, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e:\u0026nbsp;The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eThe study was carried out in compliance with the ARRIVE guidelines\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: All data generated or analyzed during this study are included in this published article [and its supplementary information files].\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnderson KV (2000) Toll signaling pathways in the innate immune response. Curr Opin Immunol 12:13\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0952-7915(99)00045-x\u003c/span\u003e\u003cspan address=\"10.1016/s0952-7915(99)00045-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzji I, Sodhi CP, Lee EK, Good M, Egan CE, Afrazi A, Neal MD, Jia H, Lin J, Ma C, Branca MF, Prindle T, Richardson WM, Ozolek J, Billiar TR, Binion DG, Gladwin MT, Hackam DJ (2013) Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS-NO-nitrite signaling. Proc Natl Acad Sci U S A 110:9451\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/ 10.1073/pnas.1219997110\u003c/span\u003e\u003cspan address=\" 10.1073/pnas.1219997110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBallance WA, Dahms BB, Shenker N, Kliegman RM (1990) Pathology of neonatal necrotizing enterocolitis: a ten-year experience. J Pediatr 117:S6\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/ 10.1016/s0022-3476(05)81124-2\u003c/span\u003e\u003cspan address=\" 10.1016/s0022-3476(05)81124-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeloosesky R, Gayle DA, Amidi F, Nunez SE, Babu J, Desai M, Ross MG (2006) N-acetyl-cysteine suppresses amniotic fluid and placenta inflammatory cytokine responses to lipopolysaccharide in rats. Am J Obstet Gynecol 194:268\u0026ndash;73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ajog.2005.06.082\u003c/span\u003e\u003cspan address=\"10.1016/j.ajog.2005.06.082\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlakely ML, Lally KP, McDonald S, Brown RL, Barnhart DC, Ricketts RR, Thompson WR, Scherer LR, Klein MD, Letton RW, Chwals WJ, Touloukian RJ, Kurkchubasche AG, Skinner MA, Moss RL, Hilfiker ML; NEC Subcommittee of the NICHD Neonatal Research Network (2005) Postoperative outcomes of extremely low birth-weight infants with necrotizing enterocolitis or isolated intestinal perforation: a prospective cohort study by the NICHD Neonatal Research Network. Ann Surg 241:984\u0026ndash;9; discussion 989\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/01.sla.0000164181.67862.7f\u003c/span\u003e\u003cspan address=\"10.1097/01.sla.0000164181.67862.7f\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuhimschi IA, Buhimschi CS, Weiner CP (2003) Protective effect of N-acetylcysteine against fetal death and preterm labor induced by maternal inflammation. Am J Obstet Gynecol 188:203\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1067/mob.2003.112\u003c/span\u003e\u003cspan address=\"10.1067/mob.2003.112\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDean O, Giorlando F, Berk M (2011) N-acetylcysteine in psychiatry: current therapeutic evidence and potential mechanisms of action. J Psychiatry Neurosci 36:78\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1503/jpn.100057\u003c/span\u003e\u003cspan address=\"10.1503/jpn.100057\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDurieux AM, Fernandes C, Murphy D, Labouesse MA, Giovanoli S, Meyer U, Li Q, So PW, McAlonan G (2015) Targeting Glia with N-Acetylcysteine Modulates Brain Glutamate and Behaviors Relevant to Neurodevelopmental Disorders in C57BL/6J Mice. Front Behav Neurosci 9:343. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fnbeh.2015.00343\u003c/span\u003e\u003cspan address=\"10.3389/fnbeh.2015.00343\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGomart A, Vall\u0026eacute;e A, Lecarpentier Y (2021) Necrotizing Enterocolitis: LPS/TLR4-Induced Crosstalk Between Canonical TGF-β/Wnt/β-Catenin Pathways and PPARγ. Front Pediatr 9:713344. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fped.2021.713344\u003c/span\u003e\u003cspan address=\"10.3389/fped.2021.713344\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHunter CJ, Upperman JS, Ford HR, Camerini V (2008) Understanding the; susceptibility of the premature infant to necrotizing enterocolitis (NEC). Pediatr Res 63:117\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1203/PDR.0b013e31815ed64c\u003c/span\u003e\u003cspan address=\"10.1203/PDR.0b013e31815ed64c\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKelly N, Friend K, Boyle P, Zhang XR, Wong C, Hackam DJ, Zamora R, Ford HR, Upperman JS (2004) The role of the glutathione antioxidant system in gut barrier failure in a rodent model of experimental necrotizing enterocolitis. Surgery 136:557\u0026ndash;66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.surg.2004.05.034\u003c/span\u003e\u003cspan address=\"10.1016/j.surg.2004.05.034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu T, Zhang L, Joo D, Sun SC (2017) NF-κB signaling in inflammation. Signal Transduct Target Ther 2:17023\u0026ndash;. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/sigtrans.2017.23\u003c/span\u003e\u003cspan address=\"10.1038/sigtrans.2017.23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMihi B, Good M (2019) Impact of Toll-Like Receptor 4 Signaling in Necrotizing Enterocolitis: The State of the Science. Clin Perinatol 46:145\u0026ndash;157. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.clp.2018.09.007\u003c/span\u003e\u003cspan address=\"10.1016/j.clp.2018.09.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNanthakumar N, Meng D, Goldstein AM, Zhu W, Lu L, Uauy R, Llanos A, Claud EC, Walker WA (2011) The mechanism of excessive intestinal inflammation in necrotizing enterocolitis: an immature innate immune response. PLoS One 6:e17776. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0017776\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0017776\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeal MD, Leaphart C, Levy R, Prince J, Billiar TR, Watkins S, Li J, Cetin S, Ford H, Schreiber A, Hackam DJ (2006) Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier. J Immunol 176:3070\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4049/jimmunol.176.5.3070\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.176.5.3070\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeu J (1996) Necrotizing enterocolitis: the search for a unifying pathogenic theory leading to prevention. Pediatr Clin North Am 43:409\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0031-3955(05)70413-2\u003c/span\u003e\u003cspan address=\"10.1016/s0031-3955(05)70413-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi\u0026ntilde;o DF, Zhou Q, Yamaguchi Y, Martin LY, Wang S, Fulton WB, Jia H, Lu P, Prindle T Jr, Zhang F, Crawford J, Hou Z, Mori S, Chen LL, Guajardo A, Fatemi A, Pletnikov M, Kannan RM, Kannan S, Sodhi CP, Hackam DJ (2018) Cognitive impairments induced by necrotizing enterocolitis can be prevented by inhibiting microglial activation in mouse brain. Sci Transl Med 10:eaan0237. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/scitranslmed.aan0237\u003c/span\u003e\u003cspan address=\"10.1126/scitranslmed.aan0237\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark E, Lee GJ, Choi S, Choi SK, Chae SJ, Kang SW, Park HK (2010) Correlation between extracellular glutamate release and neuronal cell death in an eleven vessel occlusion model in rat. Brain Res 1342:160\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.brainres.2010.04.054\u003c/span\u003e\u003cspan address=\"10.1016/j.brainres.2010.04.054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiggs BS, Bronstein AC, Kulig K, Archer PG, Rumack BH (1989) Acute acetaminophen overdose during pregnancy. Obstet Gynecol 74:247\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoliman A, Michelsen KS, Karahashi H, Lu J, Meng FJ, Qu X, Crother TR, Rabizadeh S, Chen S, Caplan MS, Arditi M, Jilling T (2010) Platelet-activating factor induces TLR4 expression in intestinal epithelial cells: implication for the pathogenesis of necrotizing enterocolitis. PLoS One 5:e15044. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0015044\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0015044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSulistyo A, Rahman A, Biouss G, Antounians L, Zani A (2018) Animal models of necrotizing enterocolitis: review of the literature and state of the art. Innov Surg Sci. 3:87\u0026ndash;92. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1515/iss-2017-0050\u003c/span\u003e\u003cspan address=\"10.1515/iss-2017-0050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiest DB, Chang E, Fanning D, Garner S, Cox T, Jenkins DD (2014) Antenatal pharmacokinetics and placental transfer of N-acetylcysteine in chorioamnionitis for fetal neuroprotection. J Pediatr 165:672-7.e2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jpeds.2014.06.044\u003c/span\u003e\u003cspan address=\"10.1016/j.jpeds.2014.06.044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZmora O, Gutzeit O, Segal L, Boulos S, Millo Z, Ginsberg Y, Khatib N, Dabbah-Assad F, Fainaru O, Weiner Z, Beloosesky R (2020) Prophylactic antenatal N-Acetyl Cysteine administration combined with postnatal administration can decrease mortality and injury markers associated with necrotizing enterocolitis in a rat model. PLoS One 15:e0233612. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0233612\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0233612\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZmora O, Gutzeit O, Segal L, Boulos S, Millo Z, Ginsberg Y, Khatib N, Fainaru O, Ross MG, Weiner Z, Beloosesky R (2021) Maternal N-acetyl-cysteine prevents neonatal brain injury associated with necrotizing enterocolitis in a rat model. Acta Obstet Gynecol Scand 100:979\u0026ndash;987. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/ 10.1111/aogs.14054\u003c/span\u003e\u003cspan address=\" 10.1111/aogs.14054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"necrotizing enterocolitis, brain injury, TLR-4, N-Acetyl-Cysteine","lastPublishedDoi":"10.21203/rs.3.rs-1684513/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1684513/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eObjective: Necrotizing enterocolitis (NEC) brain injury is mediated through Toll-like receptor 4 (TLR4) on the intestinal epithelium and on brain microglia. We sought to determine whether postnatal and/or prenatal NAC can modify NEC associated intestinal and brain TLR4 expression and brain glutathione levels in a rat model of NEC.\u003c/p\u003e\u003cp\u003eStudy Design: Newborn Sprague-Dawley rats were randomized into 3 groups: Control (n=33); NEC (n=32) subjected to hypoxia and formula feeding; NEC-NAC (n=34) received NAC (300mg/kg IP) in addition to NEC conditions. Two additional groups included pups of dams who were treated once daily with NAC (300mg/kg IV) for the last 3 days of pregnancy: NAC-NEC (n=33) or NAC-NEC-NAC (n=36) with additional postnatal NAC. Pups were sacrificed on the fifth day, ileum and brains harvested for TLR-4 and glutathione protein levels. \u003c/p\u003e\u003cp\u003eResults: NEC offspring had significantly increased brain and ileum TLR-4 protein levels compared to control (brain 2.5\u003cu\u003e+\u003c/u\u003e0.6 vs 0.88\u003cu\u003e+\u003c/u\u003e0.12 U; ileum 0.24\u003cu\u003e+\u003c/u\u003e0.04 vs 0.09\u003cu\u003e+\u003c/u\u003e0.01 U, p\u0026lt;0.05). NAC administered only to dams (NAC-NEC) significantly decreased NEC offspring brain (1.53\u003cu\u003e+\u003c/u\u003e0.41 vs 2.5\u003cu\u003e+\u003c/u\u003e0.6 U; p\u0026lt;0.05) and ileum (0.12\u003cu\u003e+\u003c/u\u003e0.03 vs 0.24\u003cu\u003e+\u003c/u\u003e0.04 U; p\u0026lt;0.05) TLR-4 protein levels compared to NEC. The same pattern was demonstrated when NAC was administered only or also postnatally. The decrease in offspring brain glutathione levels observed under NEC condition was reversed with all NAC treatment groups.\u003c/p\u003e\u003cp\u003eConclusion: NAC could reverse the increase in ileum and brain TLR-4 levels and the decrease in brain glutathione levels associated with NEC in a rat model, and thus may protect from NEC associated brain injury.\u0026nbsp;\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Intestine and brain TLR-4 modulation following N-Acetyl-Cysteine treatment in NEC rodent model","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-07-08 13:34:20","doi":"10.21203/rs.3.rs-1684513/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-01-05T18:02:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-01-04T14:51:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1f29a50f-076c-45a7-821d-f1eb76161201","date":"2022-12-28T14:45:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-12-08T06:50:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c3e8fafd-618c-48ab-b8dd-dca23d136786","date":"2022-12-05T15:25:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5a4a65cc-87ad-4cf0-98db-bbd06259533c","date":"2022-12-03T08:58:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-10-25T20:04:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-10-25T17:24:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-06-23T11:32:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-06-23T11:30:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-06-15T10:12:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"45fadef2-7c0e-4607-b226-603e092d962f","owner":[],"postedDate":"July 8th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T21:05:51+00:00","versionOfRecord":{"articleIdentity":"rs-1684513","link":"https://doi.org/10.1038/s41598-023-35019-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-05-22 20:57:54","publishedOnDateReadable":"May 22nd, 2023"},"versionCreatedAt":"2022-07-08 13:34:20","video":"","vorDoi":"10.1038/s41598-023-35019-5","vorDoiUrl":"https://doi.org/10.1038/s41598-023-35019-5","workflowStages":[]},"version":"v2","identity":"rs-1684513","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1684513","identity":"rs-1684513","version":["v2"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}