**Astaxanthin Confers a Significant Attenuation of Hippocampal Neuronal Loss Induced by Severe Ischemia-Reperfusion Injury in Gerbils by Reducing Oxidative Stress**

**Joon Ha Park 1,†, Tae-Kyeong Lee 2,†, Dae Won Kim 3, Ji Hyeon Ahn 4, Choong-Hyun Lee 5, Jong-Dai Kim 6, Myoung Cheol Shin 7, Jun Hwi Cho 7, Jae-Chul Lee 8, Moo-Ho Won 8,\* and Soo Young Choi 9,\***


**Abstract:** Astaxanthin is a powerful biological antioxidant and is naturally generated in a great variety of living organisms. Some studies have demonstrated the neuroprotective effects of ATX against ischemic brain injury in experimental animals. However, it is still unknown whether astaxanthin displays neuroprotective effects against severe ischemic brain injury induced by longer (severe) transient ischemia in the forebrain. The purpose of this study was to evaluate the neuroprotective effects of astaxanthin and its antioxidant activity in the hippocampus of gerbils subjected to 15-min transient forebrain ischemia, which led to the massive loss (death) of pyramidal cells located in hippocampal cornu Ammonis 1-3 (CA1-3) subfields. Astaxanthin (100 mg/kg) was administered once daily for three days before the induction of transient ischemia. Treatment with astaxanthin significantly attenuated the ischemia-induced loss of pyramidal cells in CA1-3. In addition, treatment with astaxanthin significantly reduced ischemia-induced oxidative DNA damage and lipid peroxidation in CA1-3 pyramidal cells. Moreover, the expression of the antioxidant enzymes superoxide dismutase (SOD1 and SOD2) in CA1-3 pyramidal cells were gradually and significantly reduced after ischemia. However, in astaxanthin-treated gerbils, the expression of SOD1 and SOD2 was significantly high compared to in-vehicle-treated gerbils before and after ischemia induction. Collectively, these findings indicate that pretreatment with astaxanthin could attenuate severe ischemic brain injury induced by 15-min transient forebrain ischemia, which may be closely associated with the decrease in oxidative stress due to astaxanthin pretreatment.

**Keywords:** antioxidant enzymes; DNA damage; ischemia and reperfusion; lipid peroxidation; lipid-soluble carotenoid; neuroprotection

**Citation:** Park, J.H.; Lee, T.-K.; Kim, D.W.; Ahn, J.H.; Lee, C.-H.; Kim, J.-D.; Shin, M.C.; Cho, J.H.; Lee, J.-C.; Won, M.-H.; et al. Astaxanthin Confers a Significant Attenuation of Hippocampal Neuronal Loss Induced by Severe Ischemia-Reperfusion Injury in Gerbils by Reducing Oxidative Stress. *Mar. Drugs* **2022**, *20*, 267. https:// doi.org/10.3390/md20040267

Academic Editors: Donatella Degl'Innocenti and Marzia Vasarri

Received: 22 March 2022 Accepted: 13 April 2022 Published: 14 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

### **1. Introduction**

Brains are very sensitive and vulnerable to disruption of the blood supply. Transient cerebral ischemia is a medical emergency that results from the temporary cessation of the blood supply to the brain, which results in irrecoverable neuronal damage in various brain regions [1,2]. Compared to other brain regions, the hippocampus is the region most vulnerable to ischemic injury, where neuronal loss (death) in the hippocampal CA1 occurs a few days after five minutes of transient forebrain ischemia [3,4]. The extent and time of neuronal death in the hippocampus are closely correlated with the duration of transient ischemia [5–7]. The diverse mechanisms of hippocampal neuronal death following transient ischemia (ischemia and reperfusion, IR) have been addressed, and, among the mechanisms, oxidative stress caused by the overgeneration of reactive oxygen species (ROS) following IR is well understood [8,9]. Hence, antioxidant strategies to improve cellular defenses against oxidative stress in IR injury have been proposed as useful therapeutic approaches [10,11]

Astaxanthin (ATX), a lipid-soluble carotenoid, is a powerful biological antioxidant and exists widely in natural organisms, including marine organisms, green plants, and microorganisms [12]. It exerts a broad spectrum of pharmacological effects, such as anticoagulant, anti-inflammatory, and antioxidant activities with the characteristic of low toxicity [13–15]. ATX has been reported to have higher antioxidant activity than a range of carotenoids including α-carotene, β-carotene, lycopene, and lutein [16]. In addition, ATX can cross the blood-brain barrier (BBB) and accumulate in the brain, where it can potentially provide a beneficial effect [17]. Due to these advantages, ATX has been regarded as a promising therapeutic agent for neurological diseases [18].

ATX has been demonstrated to exert neuroprotective effects via antioxidant activity in experimental animal models of cerebral ischemia. Zhang et al. (2020) showed that pretreatment with ATX protected the SH-SY5Y neuronal cells from oxygen and glucose deprivation-induced oxidative damage [19], and Pan et al. (2017) showed that pretreatment with ATX significantly reduced infarction volume in rat brains after transient focal brain ischemia, via activation of the antioxidant defense pathway [20]. Pretreatment with ATX was also reported to protect hippocampal neurons from repeated IR-induced brain injury in mice, suggesting that the effect might be related to the alleviation of oxidative stress [21]. Furthermore, it has been demonstrated that treatment with ATX significantly reduces brain injury following transient focal cerebral ischemia induced by middle cerebral artery occlusion in rats via antioxidant efficacy [22,23]. In addition, treatment with ATX ameliorates neuronal loss in the spinal cord following IR in rats via activation of the PI3K/Akt/GSK-3 beta pathway [24].

However, to the best of our knowledge, the neuroprotective effects of ATX against severe IR injury in the forebrain induced by a long period of transient ischemia have not been explored yet, and it is unclear whether ATX can attenuate severe IR injury in the forebrain by antioxidative activity. Thus, the aim of this experiment was to investigate the neuroprotective effects of ATX in a gerbil model of 15-min transient forebrain ischemia which results in massive neuronal loss (death) in the hippocampal CA1-3 [7,25]. In addition, we examined whether the neuroprotective effects were closely associated with the potent antioxidant activity of ATX.
