Neuroimmune consequences of postnatal ethanol exposure and the potential anti-inflammatory and pro-cognitive benefits of ibuprofen treatment

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Fetal alcohol spectrum disorders (FASD), despite being entirely preventable, afflict up to 5% of American school age children (May et al., 2009). Many of these individuals experience pervasive deficits in cognitive function, including impaired hippocampus-dependent long-term memory (Jacobson et al., 2011; Rangmar et al., 2015). In humans, maternal consumption of alcohol has also been linked to increased circulation of pro-inflammatory cytokines in both the mother and fetus (Ahluwalia et al., 2000). Early exposure to elevated cytokine expression can have a profound impact on cognition in later life (Bilbo and Schwarz, 2012; Rana et al., 2012), suggesting that acute alcohol-induced neuroinflammation may be an important contributor to cognitive dysfunction in FASD. The present study utilizes a FASD rat model in which pups are administered ethanol in a binge-like manner (5 g/kg/day; 5E) via intragastric intubation across postnatal days (PD) 4-9, a period of brain development that is comparable to the human 3rd trimester. Ethanol exposure during this period increases the expression of pro-inflammatory cytokines in the hippocampus (Drew et al., 2015; Tiwari and Chopra, 2011) and impairs hippocampus-dependent tasks in adolescent and adult rats such as trace fear conditioning (TFC), a cognitively-challenging form of Pavlovian conditioning (Schreiber and Hunt, 2013). In TFC, subjects must encode and store an association between a neutral conditioned stimulus (CS; tone) with an aversive unconditioned stimulus (US; footshock) when a stimulus-free trace interval is interposed between the CS offset and US onset. The trace interval length determines the necessity of the hippocampus—trace intervals exceeding 5-10 sec are hippocampus-dependent whereas trace intervals shorter than 5 sec are hippocampus-independent (Chowdhury et al., 2005; Misane et al., 2005). Ethanol has been shown to act upon toll-like receptor 4 (TLR4), a pathogen recognition protein that is critical for the activation of the innate immune system. Downstream of TLR4, nuclear factor kappa β (NFKβ) and activator protein 1 (AP1) induce the activity of the cyclooxygenase (COX)-2 enzyme (Crews et al., 2006) which, in turn, increases the expression of pro-inflammatory cytokines (Alfonso-Loeches et al., 2010). Ibuprofen, an over-the-counter non-steroidal anti-inflammatory drug (NSAID), inhibits the interaction between arachidonic acid and COX-2, the catalyzation of which is a rate-limiting step in pro-inflammatory signaling (Orlando et al., 2015). Research in rodents has demonstrated that ibuprofen, which easily crosses the blood-brain barrier (Parepally et al., 2006), is effective in attenuating increases in the cytokines IL-1β and TNF-α in postnatal rat brains following ischemia (Carty et al., 2011). However, it is currently unknown if the anti-inflammatory effects of ibuprofen may also rescue cognitive function. This study measures the expression of ethanol-induced pro-inflammatory gene expression in the hippocampus immediately after the ethanol exposure period and the efficacy of ibuprofen to reduce acute neuroinflammation and improve performance of 5E rats in TFC retention testing during adolescence. Rats (5E and sham-intubated (SI) controls) were subcutaneously injected with ibuprofen (100 mg/kg on PD4 followed every 24 hours by 50 mg/kg through PD9 (Carty et al., 2011)) or phosphate buffered saline vehicle and sacrificed on PD10 – hippocampal tissue was assessed for pro-inflammatory mRNA expression (IL-1β, TNF-α, IL-6, caspase 3) via quantitative polymerase chain reaction (qPCR). A separate group of rats were submitted to TFC on PD31, receiving 10 presentations of a 15 sec tone CS (2.8 kHz, 80 dB) followed 15 sec later by a 1 sec footshock US (0.8 mA). On PD32, rats were returned to the conditioning context and observed for context-specific freezing behavior (a metric of conditioned fear in rodents defined as the cessation of all movement except respiration) for 10 min. On PD33, rats were placed into a novel context and given 10 presentations of the 15 sec tone CS without footshocks; CS-evoked freezing was assessed during each tone CS as well as during the 15 sec following tone offset, mimicking the trace interval. Preliminary results indicate that 3rd trimester-equivalent ethanol exposure increases pro-inflammatory cytokine mRNA expression in the hippocampus on PD10 and induces impairments in freezing to the tone CS and trace interval during TFC retention testing. Ibuprofen co-treatment reduces cytokine mRNA and rescues conditioned freezing to the trace interval, suggesting that blockade of neuroinflammatory signaling during ethanol exposure ameliorates hippocampus-dependent learning and memory deficits. While the potential benefits of ibuprofen to the fetus may be significant, it is currently classified as a pregnancy category C-D drug and contraindicated for use in pregnant women and newborns. With this in mind, positive results with ibuprofen would provide valuable information regarding the etiology of cognitive deficits in FASD. It will also demonstrate the potential of anti-inflammatory therapy for the restoration of cognitive function and promote the investigation of other agents (e.g. infliximab) that are safer for use during pregnancy and after birth (Khan et al., 2014). Future studies will examine the efficacy of alternative windows (e.g. PD10-15 or during adolescence) for anti-inflammatory treatment and their potential to rescue cognitive function after ethanol exposure during early brain development.


Poster Division: Arts, Humanities, and Social Sciences: 2nd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)


neuroinflammation, fetal alcohol spectrum disorders, learning and memory, fear conditioning