**Ischemia-Induced Cognitive Impairment Is Improved via Remyelination and Restoration of Synaptic Density in the Hippocampus after Treatment with COG-Up® in a Gerbil Model of Ischemic Stroke**

	- sungsu.kim@famenity.com (S.-S.K.)

**Abstract:** Cerebrovascular disease such as ischemic stroke develops cognitive impairment due to brain tissue damage including neural loss, demyelination and decrease in synaptic density. In the present study, we developed transient ischemia in the forebrain of the gerbil and found cognitive impairment using the Barnes maze test and passive avoidance test for spatial memory and learning memory, respectively. In addition, neuronal loss/death was detected in the Cornu Ammonis 1 (CA1) region of the gerbil hippocampus after the ischemia by cresyl violet histochemistry, immunohistochemistry for neuronal nuclei and histofluorescence with Fluoro-Jade B. Furthermore, in the CA1 region following ischemia, myelin and vesicular synaptic density were significantly decreased using immunohistochemistry for myelin basic protein and vesicular glutamate transporter 1. In the gerbils, treatment with COG-up® (a combined extract of *Erigeron annuus* (L.) Pers. and *Brassica oleracea* Var.), which was rich in scutellarin and sinapic acid, after the ischemia, significantly improved ischemia-induced decline in memory function when compared with that shown in gerbils treated with vehicle after the ischemia. In the CA1 region of these gerbils, COG-up® treatment significantly promoted the remyelination visualized using immunohistochemistry myelin basic protein, increased oligodendrocytes visualized using a receptor-interacting protein, and restored the density of glutamatergic synapses visualized using double immunofluorescence for vesicular glutamate transporter 1 and microtubule-associated protein, although COG-up® treatment did not protect pyramidal cells (principal neurons) located in the CA1 region form the ischemic insult. Considering the current findings, a gerbil model of ischemic stroke apparently showed cognitive impairment accompanied by ischemic injury in the hippocampus; also, COG-up® can be employed for improving cognitive decline following ischemia-reperfusion injury in brains.

**Keywords:** Cornu Ammonis 1; glutaminergic synapse; ischemia-reperfusion injury; memory function; oligodendrocyte; pyramidal cells

**Citation:** Lee, T.-K.; Hong, J.; Lee, J.-W.; Kim, S.-S.; Sim, H.; Lee, J.-C.; Kim, D.W.; Lim, S.S.; Kang, I.J.; Won, M.-H. Ischemia-Induced Cognitive Impairment Is Improved via Remyelination and Restoration of Synaptic Density in the Hippocampus after Treatment with COG-Up® in a Gerbil Model of Ischemic Stroke. *Vet. Sci.* **2021**, *8*, 321. https://doi.org/10.3390/vetsci8120321

Academic Editors: Ana Faustino and Paula A. Oliveira

Received: 18 November 2021 Accepted: 9 December 2021 Published: 10 December 2021

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**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/).

#### **1. Introduction**

Ischemia/reperfusion injury in the brain is a common property of ischemic stroke, which involves a period of impaired blood circulation in the brain, followed by restoration of perfusion through medical intervention [1,2]. It was acknowledged that cognitive impairment is developed by brain ischemia/reperfusion injury which involves various neurological and behavioral changes such as paralysis in limbs, vertigo, confusion, cognitive dysfunction [3–5].

It is well accepted that the hippocampus plays a pivotal role in memory and cognitive function [6,7]. Therefore, ischemia/reperfusion in the hippocampus induces neuronal damage or death, which is accompanied by decreases in synaptic density, demyelination and axonal damage which can lead to memory and cognitive deficits [3,8–10]. It is well established that, in the hippocampus of the gerbil, delayed neuronal death is induced in the CA 1 region four to five days after in five-minute transient ischemia (TI) in the forebrain [8,11,12]. For this property, many studies have utilized this model to develop cognitive impairment followed by delayed neuronal death [3,8,13].

Many precedent studies show that the enhancement of remyelination after ischemic insults is important for the functional recovery of memory and cognition [3,8,14]. In particular, oligodendrocytes, a type of glial cells, form myelin sheath enveloping axons in the brain and spinal cord in order to accelerate neural transmission by saltatory conduction, and newly generated oligodendrocytes play an important role in remyelination [15–17]. In addition, accumulated clinical and preclinical data demonstrate that change in glutamate neurotransmission in the brain may be linked to cognitive impairment [18,19].

Numerous studies have reported the beneficial effects of medicinal herbs. *Erigeron annuus* (L.) Pers. (EALP) belongs to the Asteraceae family, and its extract shows antiinflammatory effects in lipopolysaccharide-induced activated macrophage and carrageeninduced acute inflammation in rat paws [20]. In addition, the genus *Brassica* belonging to the Brassicaceae family has beneficial attributes. For instance, the extract of *Brassica oleracea* Var. *Italica* attenuates amyloid β1–42-induced learning and memory impairment in mice [21]. However, to the best of our knowledge, experiments on the effects of the extracts from *Erigeron annuus* (L.) Pers. and *Brassica oleracea* Var. on the improvement of cognitive impairment following TI in rodent brains have rarely been conducted. Therefore, herewith we developed transient ischemia in the forebrain using gerbils, analyzed COG-up® (a combined extract of *Erigeron annuus* (L.) Pers. and *Brassica oleracea* Var.) and investigated whether COG-up® improve cognitive impairment induced by TI in the hippocampus. In addition, we examined the effects of COG-up® on remyelination and restoration of synaptic density in the damaged hippocampus.

#### **2. Materials and Methods**

#### *2.1. Experimental Animals*

Eighty-four male gerbils at the age of six months (85 ± 5 g of body weight) were provided by Experimental Animal Center of Kangwon National University (Chuncheon, Gangwon, Korea). The gerbils were housed in conventional room with optimum conditions (24 ± 1 ◦C of room temperature; 50 ± 5% of relative humidity). A steady cycle of light and dark was controlled every 12 h, and pellet feed (DBL Co. Ltd., Chungbuk, Korea) and water were freely accessible.

All experimental processes were according to the guidelines described in the "Current International Laws and Policies", a part of the "Guide for the Care and Use of Laboratory Animals". Approval for the experimental protocol was sanctioned by Institutional Animal Care and Use Committee of Kangwon National University (Chuncheon, Korea) on 18 February 2020 (approval no., KW-200113-1).

#### *2.2. Experimental Groups*

In this study, four groups were used: (1) sham+vehicle group (*n* = 21) which was given sham surgery and treated with vehicle (saline); (2) TI+vehicle group (*n* = 21) which was given TI surgery and treated with vehicle; (3) sham+COG-up® group (*n* = 21) which was undergone sham operation and treated with 100 mg/kg COG-up®; and (4) TI+COG-up® group (*n* = 21) which was undergone TI operation and treated with 100 mg/kg COG-up®.
