**1. Introduction**

Stroke is an acute life-threatening condition in which poor blood perfusion in the brain causes cell death. Life style modification and pharmacological interventions to manage risk factors have been used to lower the incidence of strokes worldwide [1]. Despite such efforts, stroke is still associated with high mortality and morbidity across the globe [2]. Ischemic reperfusion injury in the brain is detrimental and elicits major dysfunctions within the body, such as impairments in movement, cognition and other vital functions [3]. Ischemic reperfusion injury in the brain encompasses abnormal production of oxygen radicals that may exacerbate initial ischemic injuries [4]. Such injury can occur in the current clinical therapies for stroke, such as thrombectomy. These surgeries are effective to ameliorate ischemic damage in the brain by the elimination of blockages [5]. However, it is currently arduous to prevent further oxidative damage caused by reperfusion. Again, secondary damage caused by reperfusion is intractable and could render leaving the reversible areas, such as the penumbra, vulnerable to oxidative damage [6]. Meanwhile, increases in inflammatory markers have been shown to reflect ischemic injuries in clinical settings. Cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), ficolin-1 and others, have been reported to increase in relations with the incident of stroke [7–10]. Therefore, decreasing inflammatory activity may be a goal for effective therapies. In previous studies, the administration of recombinant interleukin-1 receptor antagonist in rat models of middle cerebral artery occlusion (MCAO) successfully provided protection against ischemic injury [11,12]. However, the protective effects of interleukin-1 receptor antagonist have not been able to translate to neuroprotection in clinical trials but remain as a potential therapy through continued research [7].

Previous studies have implicated the importance of the canonical Wnt signaling pathway in the case of ischemic reperfusion injury [13,14]. The activation of glycogen synthase kinase-3β (GSK-3β) and the downregulation of protein kinase B (Akt) have been reported as important factors that contribute to the death of neurons [15,16]. The modulation of these proteins has been reported to affect survival of neurons in excitotoxic conditions [16,17]. The activity of GSK-3β leads to the degradation of downstream survival markers such as β-catenin through ubiquitination. Furthermore, canonical Wnt signals, which are lipid-modified glycoproteins, have been reported to inactivate GSK-3β through phosphorylation and thereby promote cell survival [18]. A recent study reported that motor exercise stimulated the canonical Wnt/β-catenin pathway for recovering from focal cerebral ischemic reperfusion injury in juvenile rats. Motor activity regulated canonical Wnt/β-catenin pathway and promoted neurogenesis and myelin repair [19]. Another study reported that electroacupuncture in multiple acupoints of a paralyzed limb stimulated the proliferation of neural progenitor cells through the activation of the Wnt/β-catenin signaling pathway and suppression of GSK-3β [20].

Lipid emulsion (LE) was approved for clinical use as a component of parenteral nutrition in 1962 [21]. LE is composed of 20% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin and water for injection (Intralipid™ 20%, Fresenius Kabi, Uppsala, Sweden). The major fatty acids that constitute soybean oil are linoleic acid (44–62%), oleic acid (19–30%), palmitic acid (7–14%), linolenic acid (4–11%) and stearic acid (1.4–5.5%). In 1998, Weinberg et al. [22] shed light on the cardioprotective properties of LE against local anesthetic toxicity. Clinical reports have utilized the administration of LE for resuscitation from cardiac toxicity induced by lidocaine [23], ropivacaine [24] or bupivacaine [25]. The mechanism of protection of cardiac cells from excitotoxic conditions by LE depends on the phosphorylation of Akt [26] and GSK-3β [27] that contributes to cell survival. LE also has been reported to provide cardioprotection against ischemic reperfusion injury in the isolated rat heart [28]. While research into the utilization of LE against various cardiotoxic conditions is being actively conducted, investigations delineating the protective properties of LE in the central nervous system are still lacking. Neurons and cardiomyocytes share key similarities such as excitability and conductibility, which lead to their vulnerability to excitotoxic conditions. Relevant reports have claimed that distinct cardioprotective properties of LE might provide therapeutic targets or diagnostic tools for neuroprotection [29,30]. Our recent study has reported the protective effects of LE against kainic acid-induced excitotoxicity when administered directly into the brain, thereby revealing the potential neuroprotective aspects of LE [31]. However, the neuroprotective effects of LE against ischemic reperfusion injury in the brain have not been clearly elucidated.

Therefore, the present study investigated the neuroprotective effects of LE against ischemic reperfusion injury and elucidates the mechanism involved in the protection process. We examined the neuroprotective roles of LE in an in vivo rat model of the middle cerebral artery occlusion (MCAO) and reperfusion. We assessed the neurological deficits using the modified Bederson score [32] and extracted the brain to measure the severity of infarctions in experimental groups. We assessed the changes in protein and mRNA expression of distinct genes related to cell survival, Wnt/β-catenin signaling pathway and inflammation. We verified that XAV939, a Wnt/β-catenin signaling pathway inhibitor, reversed the protective effects. Based on our results, we propose that LE provides neuroprotection against ischemic reperfusion injury in the brain by regulating the Wnt/β-catenin signaling pathway.

**Figure 1.** Neuroprotective effects of lipid emulsion (LE) or vehicle (Veh) after the middle cerebral artery occlusion (MCAO) and reperfusion injury. (**a**) Triphenyltetrazolium chloride (TTC)-stained brain slices for infarction measurement. Decrease in infarction volumes were observed in MCAO+LE 10% and MCAO+LE 20% groups. Sham group did not show ischemic reperfusion injury; (**b**) Measurement of infarction volume from TTC staining. The MCAO+Veh group increased significantly in infarction volume compared to Sham+Veh group. Both MCAO+LE 10% and MCAO+LE 20% decreased in infarction volume but only the MCAO+LE 20% group had significant difference compared to the MCAO+Veh group; (**c**) Bederson scores of the experimental groups. The MCAO+Veh group increased in Bederson score significantly compared to Sham+Veh group. Both MCAO+LE 10% and MCAO+LE 20% groups decreased in Bederson scores compared to MCAO+Veh group; however, only the MCAO+LE 20% group decreased significantly. Data are presented as mean ± standard error of the mean (SEM); *n* = 16 for each group; \*\*\* *p* < 0.001 vs. Sham+Veh, # *p* < 0.05 vs. MCAO+Veh. Statistical analysis for the measurement of infarction volume was performed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Statistical analysis for Bederson scores was performed using Kruskal-Wallis non-parametric test followed by Dunn's post hoc test.
