**1. Introduction**

Oxidative stress is caused by reactive oxygen species (ROS) and leads to structural and functional cellular changes in neurons, including apoptosis and necrosis [1]. Neurons are especially vulnerable to oxidative stress due to their high lipid content and high metabolic rates. Under physiological conditions, ROS are removed by a cellular antioxidant defense system that includes redox homeostasis and cellular signal transduction. Even though ROS are considered as damaging agents, ROS generation is initially necessary for cell function, as ROS act as intracellular messengers and serve as a platform for the transmission of physiological cellular redox signals in mitochondria and the cellular environment [2,3]. However, over-produced ROS lead to an accumulation of excess oxidant radicals that could damage the mitochondria and neuronal cells [4]. Increased ROS in the aging process progressively reduces the maintenance of tissue homeostasis and increases the likelihood of degenerative diseases. Studies on aging over the past several decades have shown that free radicals and oxidative stress increase with age and cause age-related increases in oxidative damage to many human cellular molecules [5]. These previous studies have implicated ROS as a source of oxidative damage to DNA, proteins, and lipids [6–9]. Mitochondria-generated ROS, as byproducts of mitochondrial respiration, are primary targets for oxidative damage and play an important role in aging [10,11]. Vitamins C and E are the main dietary antioxidants that protect erythrocytes from damage caused by ROS [12].

**Citation:** Lee, K.H.; Kim, U.J.; Cha, M.; Lee, B.H. Chronic Treatment of Ascorbic Acid Leads to Age-Dependent Neuroprotection against Oxidative Injury in Hippocampal Slice Cultures. *Int. J. Mol. Sci.* **2020**, *22*, 1608. https:// doi.org/10.3390/ijms22041608


Academic Editor: Volkmar Lessmann Received: 29 December 2020 Accepted: 2 February 2021 Published: 5 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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/).

Ascorbic acid (AA), also known as vitamin C, is more highly concentrated in the brain than in other organs and plays an important role in neuronal differentiation and myelin formation [13]. For normal homeostasis of the nervous system, AA is considered to be the most important nutrient due to its crucial role in the brain's antioxidant defense system responsible for scavenging reactive oxygen and nitrogen species produced during cellular metabolism [14,15]. High concentrations of AA have been observed predominantly in neuron-rich areas of the hippocampus and neocortex in the human brain [16], and the ascorbate content in neurons compared to glia appears to be significantly different [17]. Therefore, AA is very important for brain function and maintenance. Furthermore, many degenerative central nervous system diseases, including Alzheimer's disease, multiple sclerosis, Parkinson's disease, and Huntington's disease [18], as well as psychiatric disorders, such as depression, anxiety disorders, and schizophrenia [13,14], are highly associated with AA deficiency. Brain levels of α-tocopherol, ascorbate, and glutathione have all been reported to decrease with aging, and the ascorbate level decrease was accompanied by decreased ascorbate synthesis and altered ascorbate transport characteristics [19]. These decreased ascorbate levels could either contribute to and/or result from the aging process. Interestingly, Michels et al. [20] demonstrated that declines in hepatic ascorbate in rats were associated with an age-related change in ascorbate uptake. Nevertheless, few studies have dealt with age-related functional changes in the brain to sustained ascorbate supplementation or the optical activity of neurons capable of restoring functional activity using optical imaging.

To investigate the results according to age and AA treatment effects, the present study evaluated the neuroprotective properties of ascorbate treatment for various age-related oxidation states in hippocampal slices, as well as the difference between acute and chronic administration of ascorbate during aging. In addition, optical images were used to evaluate the functional role of ascorbate-rescued neurons after oxidative damage caused by kinetic acid (KA).

#### **2. Results**

#### *2.1. Different Neuroprotection Effects of AA*

At 3 w or 9 w after hippocampal slice culture, 5 μM KA was applied for 18 h to induce oxidative injury and was included in fresh medium (vehicle group). AA with medium was replaced for 24 h after KA treatment (AA treatment). For the 9 w experiment groups, one was treated daily with AA from week 3 to week 9 (9 w-daily), and the other was treated only with medium from week 3 to week 9 (9 w). The experimental groups and the overall design are presented in Figure 1.

Figure 2 shows the different neuroprotective effects of AA in 3 w, 9 w, and 9 w-daily organotypic hippocampal slice cultures (OHSCs) before (Pre, 2A upper line) and 24 h after oxidative injury. KA treatment (5 μM) led to progressive cell death in the CA3 area of the hippocampus after 24 h (24 h, 2A bottom line). Hippocampal neurons in the 3 w KA + AA group showed reduced propidium iodide (PI) uptake compared to the 3 w KA + vehicle group. Similar to the results of 3 w group, the 9 w KA + AA and 9 w-daily KA + AA groups also showed decreased PI uptake compared to the 9 w KA + vehicle and 9 wdaily KA + vehicle groups. These results indicate the neuroprotective effects of AA after KA exposure. Statistical analysis of neuroprotective effects of AA is shown in Figure 2B. The 3 w KA + vehicle and KA + AA PI uptake values were significantly increased compared to the 3 w normal PI uptake (normal: 3.01 ± 1.58, KA + vehicle: 90.03 ± 3.02, KA + AA: 33.33 ± 4.75). In addition, 9 w of KA + vehicle and KA + AA and 9 w-daily KA + vehicle and KA + AA value were significantly increased (9 w normal: 3.80 ± 2.01, KA + vehicle: 91.83 ± 2.33, KA + AA: 74.46 ± 4.50; 9 w-daily normal: 2.73 ± 1.38, KA + vehicle: 76.54 ± 3.68, KA + AA: 59.37 ± 3.78). Moreover, 9 w KA + AA group showed significantly increased cell death compared to 3 w KA + AA group. These results indicate that the aging group (9 w) was more vulnerable compared to the young group (3 w) to cell death and less sensitive to the protective effect of AA treatment following an oxidative injury. In

addition, 9 w-daily KA + vehicle and 9 w-daily KA + AA groups showed significantly reduced PI uptake compared to 9 w KA + vehicle and 9 w KA + AA groups. These results indicate that chronic AA treatment protects hippocampal neurons from KA-induced oxidative injury and that prolonged antioxidant treatment during the aging process has a neuroprotective effect.

**Figure 1.** Experimental design and paradigm. Organotypic hippocampal slice cultures (OHSCs) were cultured for 3 w and 9 w. Slices were treated with propidium iodide (PI) 2 h before 5 μM kinetic acid (KA) treatment for all cultures (PRE). All received KA treatment except for the normal controls in each experimental group. After 18 h exposure to KA, the medium containing KA was changed to a fresh culture medium containing 500 μM ascorbic acid (AA). PI uptake, Western blot, and electrophysiological studies were conducted 24 h after AA treatment. Experimental groups were divided by culture time and treatment type, such as 3 w, 9 w, and 9 w-daily. In the 9 w-daily group, 500 μM AA was added to the culture medium for 6 w before studies began.
