Maternal N‐acetyl‐cysteine prevents neonatal brain injury associated with necrotizing enterocolitis in a rat model

Abstract

Introduction Preterm infants with necrotizing enterocolitis (NEC) are at increased risk of cerebral injury and neurodevelopmental dysfunction. N‐acetyl‐cysteine (NAC) is a known anti‐inflammatory and antioxidant agent. Currently, there is no prophylactic treatment in clinical use to prevent NEC and its neurodevelopmental sequelae. We sought to determine whether brain inflammation/apoptosis accompanies NEC systemic inflammation, and whether it can be attenuated by maternal NAC treatment during pregnancy and/or in the neonatal period in a rat model. Material and methods An established NEC newborn model (hypoxia 5% O2 for 10 min and formula feeding thrice daily, beginning on day 1 for 4 days) was used in Sprague‐Dawley rat pups (n = 32). An additional group of pups (n = 33) received NAC (300 mg/kg intraperitoneal thrice daily) in addition to NEC conditions (NEC‐NAC). Control pups (n = 33) were nursed and remained with the dam in room air. Two additional groups included pups of dams treated once daily with NAC (300 mg/kg intravenous) in the last 3 days of pregnancy. After birth, pups were randomized into NAC‐NEC (n = 33) with NEC conditions and NAC‐NEC‐NAC (n = 36) with additional postnatal NAC treatment. Pups were sacrificed on the fifth day of life. Pup serum interleukin (IL)‐6 protein levels, and brain nuclear factor kappa B (NF‐κB) p65, neuronal nitric oxide synthase (nNOS), Caspase 3, tumor necrosis factor alpha (TNF‐α), IL‐6 and IL‐1β protein levels were determined by ELISA, western blot and TUNEL staining, and the groups were compared using analysis of variance (ANOVA). Results NEC pups had significantly increased serum IL‐6 levels compared with the control group as well as increased neuronal apoptosis and brain protein levels of NF‐κB, nNOS, Caspase 3, TNF‐α, IL‐6 and IL‐1β compared with control. In all NAC treatment groups, levels of serum IL‐6, neuronal apoptosis and brain NF‐κB, nNOS, Caspase 3, TNF‐α, IL‐6 and IL‐1β protein levels were significantly reduced compared with the NEC group. The most pronounced decrease was demonstrated within the NAC‐NEC‐NAC group. Conclusions NAC treatment can attenuate newborn inflammatory response syndrome and decrease offspring brain neuroapoptosis and inflammation in a rat model of NEC by inhibition of NF‐κB, nNOS and Caspase 3 pathways.

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

“…Our novel nding that NAC administered to either dams or offspring decreased TLR-4 protein levels in offspring ileum is of great importance. These ndings are consistent with reports of decreased NEC observed in previous studiesfollowing NAC treatment (Zmora et al 2020;Zmora et al 2021).…”

“…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-in ammatory 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 neuroin ammatory response (Dean et al 2011;Durieux et al 2015).…”

Neurologic Consequences of Neonatal Necrotizing Enterocolitis

“…Neonatal inflammation is known to adversely affect the processes of maturation in the immune and nervous systems during a critical period of development [87][88][89]. Systemic and brain inflammation in various prenatal and neonatal conditions including chorioamnionitis, sepsis, meningitis, and bronchopulmonary dysplasia, have been shown to be associated with neonatal brain injury and worse developmental outcomes [90,91]. NEC is known to involve both local and systemic inflammation.…”

“…In mice with NEC, the activated microglia were found to be distributed in regions of the brain where myelination deficits were most evident, including the hippocampus, frontal cortex, and midbrain [2, 33]. Activation of microglia correlated with an accumulation of ROS and increased brain gene expression and protein levels of markers of inflammatory signaling and injury including IL-6, IL-1, TNFa , NF-kB, nNOS, caspase 3, and lipocalin-2 (Lcn2) [2, 33, 90]. In further support of the role of microglia in brain injury, attenuation of microglial activation in a mouse model of NEC resulted in significant reduction of ROS accumulation and prevented a decrease in myelin basic protein (Mbp) expression, loss of pre-OLs, and neurocognitive impairments, as evidenced by the performance on the Y-maze and novel object recognition memory tests [2].…”

Maternal N-Acetyl-Cysteine Prevents Neonatal Hypoxia-Induced Brain Injury in a Rat Model

“…NAC mechanisms of action include increased glutathione peroxidase levels, antioxidant effect by free-radical scavenging, inhibition of the activation of NF-κB, inhibition of TNF toxicity, NOS inhibition, and prevention of mitochondrial dysfunction [34]. We previously demonstrated that NAC has a protective effect on pups' brain damage in the Entero Colitis model (NEC) [35]. NEC model is based on pups' formula feeding thrice daily which induces intestinal inflammation complemented with hypoxia treatment.…”