Adenosine A2A receptor agonist polydeoxyribonucleotide ameliorates short-term memory impairment by suppressing cerebral ischemia-induced inflammation via MAPK pathway

Il-Gyu Ko; Jun-Jang Jin; Lakkyong Hwang; Sang-Hoon Kim; Chang-Ju Kim; Jung Won Jeon; Jun-Young Chung; Jin Hee Han

doi:10.1371/journal.pone.0248689

. 2021 Mar 18;16(3):e0248689. doi: 10.1371/journal.pone.0248689

Adenosine A_2A receptor agonist polydeoxyribonucleotide ameliorates short-term memory impairment by suppressing cerebral ischemia-induced inflammation via MAPK pathway

Il-Gyu Ko ¹, Jun-Jang Jin ¹, Lakkyong Hwang ¹, Sang-Hoon Kim ¹, Chang-Ju Kim ¹, Jung Won Jeon ², Jun-Young Chung ³, Jin Hee Han ^4,^*

Editor: Giuseppe Pignataro⁵

PMCID: PMC7971468 PMID: 33735236

Abstract

Cerebral ischemia causes tissue death owing to occlusion of the cerebral blood vessels, and cerebral ischemia activates mitogen-activated protein kinase (MAPK) and induces secretion of pro-inflammatory cytokines. Adenosine A_2A receptor agonist, polydeoxyribonucleotide (PDRN), suppresses the secretion of pro-inflammatory cytokines and exhibits anti-inflammatory effect. In the current study, the therapeutic effect of PDRN on cerebral ischemia was evaluated using gerbils. For the induction of cerebral ischemia, the common carotid arteries were exposed, and then aneurysm clips were used to occlude the common carotid arteries bilaterally for 7 minutes. In the PDRN-treated groups, the gerbils were injected intraperitoneally with 0.3 mL of saline containing 8 mg/kg PDRN, per a day for 7 days following cerebral ischemia induction. In order to confirm the participation of the adenosine A_2A receptor in the effects mediated by PDRN, 8 mg/kg 7-dimethyl-1-propargylxanthine (DMPX), adenosine A_2A receptor antagonist, was treated with PDRN. In the current study, induction of ischemia enhanced the levels of pro-inflammatory cytokines and increased phosphorylation of MAPK signaling factors in the hippocampus and basolateral amygdala. However, treatment with PDRN ameliorated short-term memory impairment by suppressing the production of pro-inflammatory cytokines and inactivation of MAPK signaling factors in cerebral ischemia. Furthermore, PDRN treatment enhanced the concentration of cyclic adenosine-3,5’-monophosphate (cAMP) as well as phosphorylation of cAMP response element-binding protein (p-CREB). Co-treatment of DMPX and PDRN attenuated the therapeutic effect of PDRN on cerebral ischemia. Based on these findings, PDRN may be developed as the primary treatment in cerebral ischemia.

1. Introduction

Cerebral ischemia, also known as stroke, is an acute disease induced by insufficient blood supply to the brain, which can result in severe complications and a high mortality rate [1, 2]. Currently, there is no effective drug therapy for acute ischemic stroke other than intravenous or intraarterial thrombolysis. However, as the time scope for treatment with a thrombolytic agent is narrow, the utility of thrombolytic agents is very limited [3, 4].

After an ischemic stroke, the development of lesions is generally associated with severity of inflammatory reaction [5]. During stroke, inflammation-inducing mediators are created, and inflammation serves to exacerbate the symptoms and progress ischemic stroke [6]. Inflammation exacerbates ischemic damage through the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 [7].

Cerebral ischemia activates mitogen-activated protein kinase (MAPK), and MAPK activation plays a selective role in determining neuronal survival or death [8]. Activation of MAPK functions primarily as a mediator for cell survival or apoptosis through phosphorylation of intracellular enzymes, transcription factors and cytoplasmic proteins [9, 10]. Extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK) and p38 kinase are included in the MAPK system, and the MAPK signaling pathway regulates expressions of inflammatory cytokines and apoptosis factors during stroke. Therefore, this MAPK signaling pathway can be a therapeutic target when developing appropriate therapeutic agents [11, 12]. Thus, studies aimed at regulating inflammation through the MAPK system in ischemic brain tissue may provide a basis for discovering new therapeutic agents targeting stroke patients.

Adenosine and its receptors are essential neuromodulators playing important roles in various pathophysiological conditions. Adenosine A₁, A_2A, A_2B and A_3A are included in the adenosine receptors, and the adenosine receptors are expressed in inflammatory cells and immune cells. Among them, stimulation of A_2A has been reported to reduce the secretion of pro-inflammatory cytokines in various neurological disease conditions [13, 14]. The physiological role of adenosine A_2A receptor-induced BDNF production was demonstrated through synapse formation from immature and mature neurons, as well as protecting neurons from excitatory toxicity and increasing neurite expansion [15].

The adenosine A_2A receptor agonist, polydeoxyribonucleotide (PDRN), exhibits an anti-inflammatory effect by inhibiting pro-inflammatory cytokine production. PDRN is also known to inhibit apoptosis in several disease states such as gastric ulcers, acute lung injury, and osteoarthritis [16–18]. Despite the excellent pharmacological effects of PDRN, few studies have evaluated the efficacy of PDRN in cerebral ischemia.

In the current study, the effect of PDRN on short-term memory and inflammation in the hippocampus after induction of transient global ischemia was evaluated using gerbils. For the experiment, the step-down avoidance task was conducted for short-term memory, and the concentrations of TNF-α, IL-1β and cyclic adenosine-3’,5’-monophosphate (cAMP) were analyzed by enzyme-linked immunoassay (ELISA). In addition, the expression of neuronal nuclei (NeuN) was determined by immunohistochemical analysis, and the levels of adenosine A_2A receptor, TNF-α, IL-1β, ERK, JNK, p38, cAMP response element-binding protein (CREB) and protein kinases A (PKA) were analyzed by western blotting.

2. Materials and methods

2.1. Animals and classification

The male adult Mongolian gerbils, weighing 50 ± 2 g (15 weeks old), were bred in the controlled temperature (23 ± 2°C) and lighting (08:00 to 20:00 h) conditions, and food and water were provided freely. The gerbils were randomly classified into the four groups such as sham-operation group, cerebral ischemia-induced group, cerebral ischemia-induced and PDRN-treated group, cerebral ischemia-induced and PDRN with 7-dimethyl-1-propargylxanthine (DMPX)-treated group (n = 10 in each group).

This experimental procedure was approved by the Institutional Animal Care and Use Committee of Kyung Hee University and received the following approval number (KHUASP[SE]-17-071). The experimental procedures were conducted in good faith in accordance with the guidelines for animal care from the National Institutes of Health and the Korean Institute of Medical Sciences. The gerbils underwent sufficient anesthesia with Zoletil 50^®(Vibac Laboratories, Carros, France) to performed surgery or sacrifice. No gerbils died or were euthanized before the end of the experiment.

2.2. Transient global ischemia induction

Transient global ischemia was surgically made in the same manner as described above [19, 20]. A bilateral neck incision was made after anesthetizing the gerbils by Zoletil 50^® (10 mg/kg; Vibac Laboratories). Then two common carotid arteries were exposed and closed using surgical clips for 7 minutes. By a Homeothermic Blanket Control Unit (Harvard Apparatus, Massachusetts, MA, USA) that wrapped around the head and body, the temperature of the head and body was maintained at 36 ± 0.5°C during the course of operation. After 7 minutes of closing, the surgical clips were removed and cerebral blood flow was allowed to reflow. Local brain blood flow on either side of the forebrain was determined using a BLF21D laser Doppler flowmeter (Transonic Systems Inc., New York, NY, USA). To prevent hypothermia, the gerbils were observed for an additional 4 hours after recovery. The animals from the sham-operation group were managed in a similar manner, but both common carotid arteries were not closed during the neck operation.

2.3. Treatment

The gerbils from the PDRN-treated groups were injected intraperitoneally with 0.3 mL of normal saline (0.9%) containing 8 mg/kg of PDRN (Rejuvenex^®, PharmaResearch Products, Pangyo, Korea), one time per a day continued for 7 days, started a day after operation. Based on preliminary data and previous studies, the PDRN concentration considered to be the most effective was used in this experiment [17, 21]. Additionally, in order to confirm that the adenosine A_2A receptor is involved in the effect mediated by PDRN, 8 mg/kg DMPX (Sigma Chemical Co., St. Louis, MO, USA), an adenosine A_2A receptor antagonist, was simultaneously administered with PDRN. In the sham-operation group and in the cerebral ischemia-induced group, the gerbils received 0.3 mL of normal saline without drugs according to the same timetable. The experiment schedule is shown in Fig 1.

2.4. Step-down avoidance task

In the same manner as described above [22, 23], the step-down avoidance task was performed to determine the short-term memory. Eight days after induction of cerebral ischemia, the gerbils implemented a step-down avoidance task. Each gerbil was placed on a 7 × 25 cm platform at height of 2.5 cm, and the platform faced a grid of parallel steel bars (45 × 25 cm) with a diameter of 0.1 cm and a spacing of 1 cm. Going down the grid during training, the animal immediately took the out of the box after receiving a 0.3 mA foot shock for 2 seconds. After 2 hours of training period, the latency of each gerbil was determined. During the test time, the gerbils were placed back on the platform, and the latency time was defined as the time until the animal descended and placed all four feet on the grid. A delay of latency time more than 180 seconds was also calculated as 180 seconds.

2.5. Tissues preparation

On the 8th day after ischemia induction, immediately after measuring the latency of the step-down avoidance task, the animals were sacrificed. After anesthetizing the gerbils using the Zoletil 50^® (10 mg/kg, ip; Vibac Laboratories), heart puncture was used to collect blood, left at room temperature for 1 hour and centrifugation was performed at 3,000 rpm for 20 minutes to obtain serum. After blood sampling, 50 mM phosphate-buffered saline (PBS) was transcardially perfused, and then fixed with a solution consisting of 4% paraformaldehyde in 100 mM phosphate buffer (PB, pH 7.4). After removing the brains, the brains were with the same fixative solution and then transferred to 30% sucrose solution to prevent freezing. Thereafter, 40-μm thick coronal sections were made using a freezing microtome (Leica, Nussloch, Germany), and an average of 10 sections was obtained in the CA1 region of each gerbil.

2.6. Pro-inflammatory cytokines and cAMP concentrations

Serum and hippocampus levels of pro-inflammatory cytokines (TNF-α and IL-1β) and cAMP concentrations were determined using enzyme-linked immunosorbent assay (ELISA). Enzyme immunoassay kits were used to detect the concentrations of TNF-α, IL-1β and cAMP in accordance with the manufacturer’s instructions (Abcam, Cambridge, UK), in the same manner as described above (n = 3 in each group) [17].

2.7. Western blot analysis

According to the same manner as described above [24, 25], analysis of western blotting was conducted (n = 4 in each group). Priority, approximately 30 mg of hippocampal tissues were extracted using 100 mg/mL of lysis buffer. The tissues were homogenized using a lysis buffer consisting of 1 mM PMSF, 1 mM EGTA, 1 mM Na2VO₄, 1.5 mM MgCl₂·6H₂O, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 150 mM NaCl, 1% Triton X-100 and 10% glycerol, and then this homogeneous mixture was centrifuged at 14,000 rpm for 30 minutes. Concentration of protein was detected by a colorimetric protein analysis kit (Bio-Rad, Hercules, CA, USA). Protein 30 μg was separated from each sample on SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The antibodies for rabbit CREB antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated (p)-CREB antibody (1:1,000; Santa Cruz Biotechnology), rabbit PKA antibody (1:1,000; Santa Cruz Biotechnology), p-PKA antibody (1:1,000; Santa Cruz Biotechnology), rabbit ERK antibody (1:2,000; Cell Signaling Technology, Danvers, USA), rabbit p-ERK antibody (1:2,000; Cell Signaling Technology), rabbit JNK antibody (1:2,000; Cell Signaling Technology), rabbit p-JNK antibody (1:2,000; Cell Signaling Technology), rabbit p38 antibody (1:2,000; Cell Signaling Technology), rabbit p-p38 antibody, (1:2,000; Cell Signaling Technology), mouse TNF-α antibody (1:1,000; Santa Cruz Biotechnology), mouse IL-1β antibody (1:1,000; Santa Cruz Biotechnology), mouse adenosine A_2A receptor antibody (1:1,000; Santa Cruz Biotechnology), and β-actin antibody (1:1,000; Santa Cruz Biotechnology) used were as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibodies (1:2,000; Vector Laboratories, Burlingame, CA, USA) for β-actin, TNF-α, IL-1β, adenosine A_2A receptor and horseradish peroxidase-conjugated anti-rabbit antibodies (1:3,000; Vector Laboratories) for p-CREP, CREB, p-PKA, PKA, p-ERK, ERK, p-JNK, JNK, p-p38, p38 were used as the secondary antibodies.

The entire experimental step was carried out under normal laboratory conditions, except for membrane transfer performed at 4°C using pre-cooled buffer with cold pack. An enhanced chemiluminescence (ECL) detection kit (Santa Cruz Biotechnology) was used for band measurements. Each sample was loaded twice, and the number of samples was 4 per group.

2.8. Immunohistochemistry for NeuN

Immunohistochemistry for NeuN was conducted in the same manner as described above [26, 27]. The sections were treated with mouse anti-NeuN antibody (1:500; Abcam, Cambridge, MA, USA) overnight, and then treated with biotinylated mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) for an additional 1 hour. Secondary antibody was amplified using the Vector Elite ABC kit^® (1:100; Vector Laboratories), and then 0.03% 3,3′-diaminobenzidine was used to show antibody-biotin-avidin-peroxidase complexes. The sections were then mounted on gelatin-coated slides, air-dried at room temperature overnight, and finally Permount^® (Fisher Scientific, New Jersey, NJ, USA) was used to mount the coverslips.

2.9. Data analysis

The area of the hippocampal CA1 region on each slide was observed by optical microscope (Olympus, Tokyo, Japan), and the number of NeuN-positive cells was calculated using the Image-Pro^® plus computer-assisted image analysis system (Media Cybernetics Inc., Silver Spring, MD, USA). The number of NeuN-positive cells was calculated using the following equation: N = N_v × V_ref. N is the total number of NeuN-positive cells of the CA1 region, which is counted by multiplying the NeuN-positive cell numerical density N_v by the reference volume (mm³) V_ref. N_v is the average numerical density of NeuN-positive cells and it is calculated based on the sum of counts within the CA1 region of each section and the volume of the CA1 region contained in each section. V_ref is calculated according to Cavalieri’s method as follows [28, 29]: V_ref = a × t × s. In this equation, a is the average area of the CA1 cell layer, t represents the average thickness (40 μm) of the microtome section, and s indicates the total number of sections through the reference volume.

In order to compare the relative levels of protein expressions, the bands were detected densitometrically by Molecular Analyst^TM version 1.4.1 (Bio-Rad, Hercules). For relative quantification, a random value of 1.00 was given to the results of the sham-operation group (western blotting). One-way ANOVA with Duncan’s post-hoc test was used for data analysis, and the results are presented as mean ± standard error of the mean. For statistical analysis, the value of P < 0.05 was determined to be statistically significant.

3. Results

3.1. Changes of cerebral blood flow

The flow of brain blood during carotid artery occlusion and reperfusion is shown in Fig 2. Occlusion of the carotid artery reduced brain blood flow and increased brain blood flow during reperfusion.

3.2. Changes of short-term memory latency and NeuN-positive cell number in the CA 1 region

Fig 3A shows the results of the latency in the step-down avoidance task. Following the induction of cerebral ischemic damage, short-term memory was impaired (P < 0.05), and treatment with PDRN ameliorated this ischemia-induced memory impairment (P < 0.05). The co-administration of PDRN and DMPX failed to increase the latency observed with PDRN in cerebral ischemia.

Fig 3B and 3C are the photomicrographs of NeuN-positive cells in the hippocampal CA1 region. Cerebral ischemic damage decreased the number of NeuN-positive cells, indicating reduced neuronal survival in the hippocampal CA1 region (P < 0.05), and treatment with PDRN improved neuronal survival in cerebral ischemia (P < 0.05). The co-administration of PDRN and DMPX failed to enhance the neuronal survival observed with PDRN in cerebral ischemia.

3.3. Changes of pro-inflammatory cytokine expression

To determine whether PDRN improves cerebral ischemia, ELISA and western blot analysis were performed by examining the effect on PDRN on production of pro-inflammatory cytokines, TNF-α (Fig 4A) and IL-1β (Fig 4B). Following cerebral ischemic damage, TNF-α and IL-1β expressions were enhanced in the serum and hippocampus (P < 0.05), and treatment with PDRN suppressed the expressions of TNF-α and IL-1β (P < 0.05). The co-administration of PDRN and DMPX failed to decrease TNF-α and IL-1β expressions observed with PDRN in cerebral ischemia.

Fig 4 — A-upper. Concentration of tumor necrosis factor-α (TNF-α) in the serum. A-lower. The relative level of TNF-α in the hippocampus. B-upper. Concentration of interleukin (IL)-1β in the serum. B-lower. The relative level of IL-1β in the hippocampus. Sham, sham-operation group; CI, cerebral ischemia-induced group; CI-PDRN, cerebral ischemia-induced and polydeoxyribonucleotide (PDRN)-treated group; CI-PDRN+DMPX, cerebral ischemia-induced and PDRN with 7-dimethyl-1-propargylxanthine (DMPX)-treated group. * indicates P < 0.05 compared with the sham-operation group. # indicates P < 0.05 compared with the cerebral ischemia-induced group.

3.4. Changes of phosphorylation of MAPK cascade

To determine whether cerebral ischemia is improved by PDRN, the effect of PDRN on MAPK phosphorylation was investigated using western blotting (Fig 5). Induction of cerebral ischemia promoted MAPK cascade phosphorylation, such as ERK, JNK and p38 (P < 0.05). Interestingly, treatment with PDRN more enhanced the phosphorylation of ERK, JNK and p38 (P < 0.05). The co-administration of PDRN and DMPX failed to further enhance ERK, JN and p38 phosphorylation observed with PDRN in cerebral ischemia.

Fig 5 — Left. Ratio of phosphorylated extracellular signal-regulated kinases (p-ERK) to ERK. Middle. Ratio of phosphorylated c-Jun NH₂-terminal kinases (p-JNK) to JNK. Right. Ratio of phosphorylated p38 kinase (p-p38) to p38 in the hippocampus. Sham, sham-operation group; CI, cerebral ischemia-induced group; CI-PDRN, cerebral ischemia-induced and polydeoxyribonucleotide (PDRN)-treated group; CI-PDRN+DMPX, cerebral ischemia-induced and PDRN with 7-dimethyl-1-propargylxanthine (DMPX)-treated group. * indicates P < 0.05 compared with the sham-operation group. # indicates P < 0.05 compared with the cerebral ischemia-induced group.

3.5. Changes of cAMP concentration and adenosine A_2A receptor expression

The cAMP concentration in the serum and hippocampus and adenosine A_2A receptor expression in the hippocampus are shown in Fig 6. Induction of cerebral ischemia decreased the levels of cAMP concentration and adenosine A_2A receptor expression (P < 0.05), and treatment with PDRN improved the levels of cAMP concentration and adenosine A_2A receptor expression (P < 0.05). The co-administration of PDRN and DMPX failed to increase the levels of cAMP concentration and adenosine A_2A receptor expression observed with PDRN in cerebral ischemia.

3.6. Changes of ratio in p-CREB vs CREB and p-PKA vs PKA

Western blot analysis was used to measure the relative expressions of p-CREB vs CREB and p-PKA vs PKA (Fig 7). Induction of cerebral ischemia decreased the ratio of p-CREB vs CREB and the ratio of p-PKA vs PKA when compared with the sham-operation group (P < 0.05). Treatment with PDRN increased the ratio of p-CREB vs CREB and ratio of p-PKA vs PKA in the hippocampus (P < 0.05). The co-administration of PDRN and DMPX failed to enhance the phosphorylation of CREB and PKA observed with PDRN in cerebral ischemia.

4. Discussion

Damage by ischemia particularly destroys pyramidal neurons in the hippocampal CA1 region, and ischemia induces apoptotic cell death in the hippocampal CA1 neurons [19, 30]. Pyramidal neurons are crucial for learning and memory, and appearance of passive avoidance memory impairment after ischemia is associated with damage in the neurons of the CA1 region [31, 32]. In the current study, the cerebral ischemic injury resulted in cell loss in the hippocampal CA1 neurons, and this neuronal cell loss reduced short-term memory when compared with the gerbils of the sham-operation group. The present findings are similar to previous studies showing that loss of neurons in the CA1 region caused short-term memory impairment [31, 33].

The ischemic brain exhibits inflammation characterized by the accumulation of inflammatory cells and mediators. Previous studies have suggested that induction of ischemic damage enhances neuronal cell loss owing to increased levels of inflammatory exudates and pro-inflammatory cytokines [6, 7, 34]. Furthermore, an increment of inflammatory cytokines in the brain acts as a major causes of neuronal cell loss and memory impairment [35, 36]. Based on current findings, enhanced secretion of TNF-α and IL-1β, pro-inflammatory cytokines, in the serum, hippocampus, and basolateral amygdala (Supplement 1 in S1 File) exacerbated the symptoms of ischemic injury. These results indicate that symptoms were worsened by excessive production of pro-inflammatory cytokines during cerebral ischemia. Inhibiting the secretion of pro-inflammatory cytokines is one of the important treatment strategies for the brain ischemic injury.

Most of cells involved in wound healing express the adenosine A_2A receptor [17], and this adenosine A_2A receptor is locates in several brain regions and modulates the pathophysiological response to ischemic stroke [14]. Agonists on adenosine A_2A receptor have been reported to be useful for the treating of inflammatory diseases [14, 37]. In the previous studies, adenosine A_2A receptor knockout mice exhibited symptoms of chronic cerebral ischemia such as working memory impairment, increased demyelination, glial proliferation, and increased pro-inflammatory cytokines [4, 38]. Reduced functional capacity of BDNF in adenosine A_2A receptor knockout mice was associated with a decrease in hippocampal BDNF level, and pharmacological blockade of adenosine A_2A receptors significantly reduced BDNF level in the hippocampus of normal mice [39]. These results indicated that tonic activation of adenosine A_2A receptor is required for BDNF-induced potentiation of synaptic transmission and for sustaining a normal BDNF tone in the hippocampus [39]. The facilitating action of BDNF on hippocampal long-term potentiation is critically dependent on the presence of extracellular adenosine and activation of the A_2A receptor through a cAMP/PKA-dependent mechanism [40]. Activation of the adenosine A_2A receptor regulates BDNF production in rat cortical neurons, which provides neuroprotective action [15].

In the current study, PDRN treatment substantially suppressed the secretion of pro-inflammatory cytokines. Inflammation is a compensatory response to cellular and tissue damage caused by ischemia-reperfusion injury, and the inflammatory response is primarily regulated by the signaling pathway of the MAPK cascade [8, 12]. MAPK is essential for the regulation of various inflammatory mediators, and MAPK is a kind of kinases that regulate cellular response to external stress signals or inflammatory cytokines [2, 41]. It was demonstrated that the induction of ischemic damage activated phosphorylation of the MAPK cascade, and this activation of MAPK controls a wide range of cellular processes [2, 12]. Our current study found that phosphorylation of the MAPK cascade pathway was increased by ischemic damage and this phosphorylation of the MAPK cascade pathway was reduced by PDRN treatment.

The intracellular concentration of cAMP is increased by stimulation of the adenosine A_2A receptor, and this increased cAMP concentration serves as a physiological inhibitor to function of inflammatory neutrophil [42]. Furthermore, adenosine A_2A receptor activation promotes signal from the cAMP-PKA pathway and accelerates the level of CREB phosphorylation. Elevated cAMP concentration suppresses the level of phosphorylation of the MAPK cascade pathway in stimulated cells [42, 43]. Adenosine A_2A receptor agonist, PDRN, has been proposed to have therapeutic potential in inflammatory diseases [16, 17]. In the current study, PDRN treatment increased the cAMP concentration in gerbils presenting cerebral ischemia, and this increased cAMP concentration inhibited phosphorylation of the MAPK cascade pathway, thereby inactivating the MAPK cascade pathway in the hippocampus and basolateral amygdala (Supplements 2–4 in S1 File).

The current study has revealed that PDRN treatment inhibits inflammation, improves neuronal cell survival, and prevents a decline in short-term memory in a brain ischemia animal model (S1 Graphic abstract). Co-administration of PDRN and adenosine A_2A receptor antagonist DMPX attenuated the therapeutic effect of PDRN in cerebral ischemia. Based on these findings, PDRN may be developed as the primary treatment in cerebral ischemia.

Supporting information

S1 File

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S1 Graphic abstract

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S1 Raw images

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Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

• Jin Hee Han • NRF-2017R1D1A1B03032827 • National Research Foundation of Korea • https://www.nrf.re.kr/ • Research fund support.

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PLoS One. doi: 10.1371/journal.pone.0248689.r001

Decision Letter 0

Giuseppe Pignataro

18 Dec 2020

PONE-D-20-25445

Adenosine A2A receptor agonist polydeoxyribonucleotide ameliorates short-term memory impairment by suppressing cerebral ischemia-induced inflammation via MAPK pathway

PLOS ONE

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Reviewer #1: Results

In the present work Il-Gyu Ko and colleagues demonstrated that the administration of an Adenosine A2a receptor agonist, polydeoxyribonucleotide (PDRN), following the induction of transient global ischemia in gerbils results in an improvement in short-term memory tested 8 days following induction of ischemia and in the survival of NeuN-positive cells in the CA1 region of Hippocampus. PDRN induced a decrease in proinflammatory cytokine production and in the activation of MAPK signalling which were increased following ischemia. Moreover an increase in the concentration of C-AMP in serum and hippocampus and of A2a receptor expression, accompanied by an increase of CREB and PKA phosphorylation and a decrease of the phosphorylation of ERK, p38 and JNK were observed. The co-treatment with the A2a receptor antagonist DMPX attenuated the therapeutic effect of PDRN.

The manuscript provides a demonstration of the efficacy of an adenosine A2a receptor agonist on cognitive decline following cerebral ischemia by inhibiting the activation of endogenous inflammation mechanisms.

However, the neuroprotective effect of adenosine neurotransmission in ischemia is not emphasized in the introduction and a greater emphasis could underline the involvement of the pathway involved in neuronal plasticity in the discussion.

English should be checked in all the text and in some places there is no correspondence between text and figures.

Regarding the treatment, the injection volume is unclear as different values are reported in the abstract and in the text.

In Figure 2C the spatial reference of the magnification at 200 μm is missing.

Regarding the cognitive task it is not clear when the training phase was carried out with respect to induction of ischemia.

Moreover, only the hippocampus was considered in the measurements. Other brain areas, for example the basolateral amygdala, are also important in the acquisition and consolidation of the memory trace relating to the association between the shock and the position of the animal.

Reviewer #2: Paper by Jin-Hee Han and collegues describes the effects of adenosine A2A receptor agonist, polydeoxyribonucleotide (PDRN), on short-term memory in gerbils subjected to global ischemia. The authors speculate that treatment with PDRN ameliorated short-term memory impairment by suppressing the production of pro-inflammatory cytokines and inactivation of MAPK signaling factors in cerebral ischemia. The work is discreet, well written and is the first to analyze the effect of polydeoxyribonucleotide (PDRN) in an ischemia model. A couple of issues need to be resolved:

1. There is no indication of the calculation of the sample size for in vivo experiments together with the number of experimental groups for western blotting evaluations etc.

2. What evidence is available regarding the ability of the experimental compound PNDR to cross the blood brain barrier when administered intraperitoneally?

**********

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Reviewer #1: No

Reviewer #2: Yes: Antonio Vinciguerra

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PLoS One. 2021 Mar 18;16(3):e0248689. doi: 10.1371/journal.pone.0248689.r002

Author response to Decision Letter 0

5 Feb 2021

Answer to Reviewers’ Comments

Manuscript ID: PONE-D-20-25445

Title: Adenosine A2A receptor agonist polydeoxyribonucleotide ameliorates short-term memory impairment by suppressing cerebral ischemia-induced inflammation via MAPK pathway

Authors: Il-Gyu Ko, Jun-Jang Jin, Lakkyong Hwang, Sang-Hoon Kim, Chang-Ju Kim, Jung Won Jeon, Jun-Young Chung, Jin Hee Han

We sincerely appreciate for your kind advice and comments to our manuscript. We revised the manuscript according to the reviewer’s comments. We added new experimental data, and modifications were expressed in red (Please check the attached file).

Reviewer #1: Results

Q1. The manuscript provides a demonstration of the efficacy of an adenosine A2a receptor agonist on cognitive decline following cerebral ischemia by inhibiting the activation of endogenous inflammation mechanisms. However, the neuroprotective effect of adenosine neurotransmission in ischemia is not emphasized in the introduction and a greater emphasis could underline the involvement of the pathway involved in neuronal plasticity in the discussion.

Answer 1. According to reviewer comment, we have added sentences to emphasize the neuroprotective effect of adenosine A2A receptor in the manuscript.

• Following sentence was added into the Introduction.

The physiological role of adenosine A2A receptor-induced BDNF production was demonstrated through synapse formation from immature and mature neurons, as well as protecting neurons from excitatory toxicity and increasing neurite expansion [15].

• Following sentences were added into the Discussion.

Reduced functional capacity of BDNF in adenosine A2A receptor knockout mice was associated with a decrease in hippocampal BDNF level, and pharmacological blockade of adenosine A2A receptors significantly reduced BDNF level in the hippocampus of normal mice [39]. These results indicated that tonic activation of adenosine A2A receptor is required for BDNF-induced potentiation of synaptic transmission and for sustaining a normal BDNF tone in the hippocampus [39]. The facilitating action of BDNF on hippocampal long-term potentiation is critically dependent on the presence of extracellular adenosine and activation of the A2A receptor through a cAMP/PKA-dependent mechanism [40]. Activation of the adenosine A2A receptor regulates BDNF production in rat cortical neurons, which provides neuroprotective action [15].

• Following references were added into the Reference list.

15. Jeon SJ, Rhee SY, Ryu JH, Cheong JH, Kwon K, Yang SI, et al. Activation of adenosine A2A receptor up-regulates BDNF expression in rat primary cortical neurons. Neurochem Res. 2011; 36(12):2259-69. http://doi.org/10.1007/s11064-011-0550-y.

39. Tebano MT, Martire A, Potenza RL, Grò C, Pepponi R, Armida M, et al. Adenosine A2A receptors are required for normal BDNF levels and BDNF-induced potentiation of synaptic transmission in the mouse hippocampus. J Neurochem. 2008; 104(1):279-86. http://doi.org/10.1111/j.1471-4159.2007.05046.x.

40. Fontinha BM, Diógenes MJ, Ribeiro JA, Sebastião AM. Enhancement of long-term potentiation by brain-derived neurotrophic factor requires adenosine A2A receptor activation by endogenous adenosine. Neuropharmacology. 2008; 54(6):924-33. http://doi.org/10.1016/j.neuropharm.2008.01.011.

Q2. English should be checked in all the text and in some places there is no correspondence between text and figures.

Answer 2. According to reviewer comment, we carefully checked spelling and grammar throughout text. Also, the text and figure have been modified to match.

• (Fig. 1) was deleted from the “Transient global ischemia induction” of the Materials and Methods.

Local brain blood flow on either side of the forebrain was determined using a BLF21D laser Doppler flowmeter (Transonic Systems Inc., New York, NY, USA) (Fig. 1).

• Fig. 1 was inserted in the “Treatment” of the Materials and Methods.

The experiment schedule is shown in Fig 1.

• Following words were modified in the Results.

• Following hyphens were added in the Fig. 4 and Fig. 6.

Fig 4. Altered expression of pro-inflammatory cytokines in the serum and hippocampus. A-upper. Concentration of tumor necrosis factor-α (TNF-α) in the serum. A-lower. The relative level of TNF-α in the hippocampus. B-upper. Concentration of interleukin (IL)-1β in the serum. B-lower. The relative level of IL-1β in the hippocampus. Sham, sham-operation group; CI, cerebral ischemia-induced group; CI-PDRN, cerebral ischemia-induced and polydeoxyribonucleotide (PDRN)-treated group; CI-PDRN+DMPX, cerebral ischemia-induced and PDRN with 7-dimethyl-1-propargylxanthine (DMPX)-treated group. * indicates P < 0.05 compared with the sham-operation group. # indicates P < 0.05 compared with the cerebral ischemia-induced group.

Fig 6. Changes in cAMP concentration and adenosine A2A receptor expression. A-upper. Concentration of cAMP in serum. A-lower. Concentration of cAMP in the hippocampus. B. The relative expression of the adenosine A2A receptor in the hippocampus. Sham, sham-operation group; CI, cerebral ischemia-induced group; CI-PDRN, cerebral ischemia-induced and polydeoxyribonucleotide (PDRN)-treated group; CI-PDRN+DMPX, cerebral ischemia-induced and PDRN with 7-dimethyl-1-propargylxanthine (DMPX)-treated group. * indicates P < 0.05 compared with the sham-operation group. # indicates P < 0.05 compared with the cerebral ischemia-induced group.

Q3. Regarding the treatment, the injection volume is unclear as different values are reported in the abstract and in the text.

Answer 3. According to reviewer comment, we corrected injection volume in the Abstract.

• Following word was modified in the Abstract.

In the PDRN-treated groups, the gerbils were injected intraperitoneally with 0.3 mL of saline containing 8 mg/kg PDRN, per a day for 7 days following cerebral ischemia induction.

Q4. In Figure 2C the spatial reference of the magnification at 200 μm is missing.

Answer 4. According to reviewer comment, we have marked the reference of the enlarged figure in the form of a box.

• Following figure was modified in the Results.

Q5. Regarding the cognitive task it is not clear when the training phase was carried out with respect to induction of ischemia.

Answer 5. The step-down avoidance task evaluates short-term memory ability. In this study, training was performed 8 days after the induction of cerebral ischemia, and testing was performed 2 hours after training. This was explained in the “Step-down avoidance task” of the

Materials and Methods.

According to reviewer comment, the experiment schedule has been newly inserted to make it easier to check the experiment schedule.

• Following sentence was added in the Materials and Methods.

The experiment schedule is shown in Fig 1.

• Following figure was added in the Results.

Q6. Moreover, only the hippocampus was considered in the measurements. Other brain areas, for example the basolateral amygdala, are also important in the acquisition and consolidation of the memory trace relating to the association between the shock and the position of the animal.

Answer 6. According to reviewer comment, we further evaluated the factors identified in the hippocampal region in the basolateral amygdala region. In the isolated amygdala, the concentration of inflammatory cytokines (TNF-α and IL-1β) was evaluated using ELISA. The expression of MAPK cascade (ERK, JNK, and p38), adenosine A2A receptor, CREB, and PKA was confirmed using western blotting. The results of additional research are same as in this study, and the results are shown in supplement 1-4.

In the results of additional study, concentration of pro-inflammatory cytokines and expression of MAPK cascade were increased in the basolateral amygdala following cerebral ischemic insult. Expression of adenosine A2A receptor, p-CREB, and p-PKA was decreased in the basolateral amygdala by cerebral ischemic damage. However, PDRN treatment suppressed concentration of pro-inflammatory cytokines and expression of MAPK cascade, whereas enhanced adenosine A2A receptor, p-CREB, and p-PKA expression in the basolateral amygdala. The co-treatment of PDRN and DMPX did not show any improvement effect in basolateral amygdala, as shown in the hippocampus. Additional study results indicate that PDRN treatment is effective in improving cerebral ischemia by acting effectively on the basolateral amygdala.

• Following sentences were added in the Discussion.

Based on current findings, enhanced secretion of TNF-α and IL-1β, pro-inflammatory cytokines, in the serum, hippocampus, and basolateral amygdala (supplement 1) exacerbated the symptoms of ischemic injury.

In the current study, PDRN treatment increased the cAMP concentration in gerbils presenting cerebral ischemia, and this increased cAMP concentration inhibited phosphorylation of the MAPK cascade pathway, thereby inactivating the MAPK cascade pathway in the hippocampus and basolateral amygdala (supplement 2-4).

• Following figures and figure legends were added in the supplement results.

Reviewer #2

Paper by Jin-Hee Han and collegues describes the effects of adenosine A2A receptor agonist, polydeoxyribonucleotide (PDRN), on short-term memory in gerbils subjected to global ischemia. The authors speculate that treatment with PDRN ameliorated short-term memory impairment by suppressing the production of pro-inflammatory cytokines and inactivation of MAPK signaling factors in cerebral ischemia. The work is discreet, well written and is the first to analyze the effect of polydeoxyribonucleotide (PDRN) in an ischemia model. A couple of issues need to be resolved:

Q1. There is no indication of the calculation of the sample size for in vivo experiments together with the number of experimental groups for western blotting evaluations etc.

Answer 1. According to reviewer comment, we have inserted the sentences about the sample size for in vivo experiments together with the number of experimental groups for western blotting evaluations in the manuscript.

• Following sentences were added in the “Western blot analysis” of the Materials and Methods.

Each sample was loaded twice, and the number of samples was 4 per group.

Q2. What evidence is available regarding the ability of the experimental compound PNDR to cross the blood brain barrier when administered intraperitoneally?

Answer 2. When considering the pharmacological action of PDRN, there is reason to infer that it acts on the brain through the BBB. The rationale for the action of PDRN can be classified into two categories: 1. Adenosine receptor signaling and BBB permeability regulation and intracellular action. 2. Adenosine receptor signaling increases immune cell entry by increasing BBB permeability. On this basis, it is likely that an active carried-mediated transport enables PDRN to traverse the blood-brain barrier as already reported for nucleosides.

1. Adenosine receptor signaling and BBB permeability regulation and intracellular action.

Studies have shown that adenosine-based drugs may play a substantial modulatory role in CNS barrier permeability. Koszalka et al. (2004) reported that adenosine produced extracellularly during disease in an experimental autoimmune encephalomyelitis (EAE) animal model positively regulates lymphocyte entry into the brain and spinal cord. Mills et al. (2008) suggested that experimental recruitment of adenosine receptors either by the broad-spectrum agonist NECA or the engagement of both A1 and A2A receptors by selective agonists (CCPA and CGS21680) cumulatively and transiently augmented BBB permeability facilitating the entry of intravenously infused macromolecules into the CNS. Furthermore, the analysis of engineered mice lacking adenosine receptors reveals a limited entry of macromolecules into the brain upon exposure to adenosine receptor agonists.

CNS entry of intravenously delivered macromolecules was also induced by the FDA-approved, adenosine A2A receptor agonist Lexiscan: 10 kDa dextran was detectable within the CNS of mice as soon as 5 min after drug injection (Carman et al., 2011). Actually, adenosine receptor activation by agonists was indeed associated with augmented actinomyosin stress fiber formation indicating that adenosine receptors signaling initiates changes in cytoskeletal organization and cell shape. These processes are reversed as the half-life of the adenosine receptor agonist decreases. In agreement with these findings and with the observation that human brain endothelial cells do respond to adenosine in vitro, agonist-induced A2A receptor signaling transiently permeabilized a primary human brain endothelial cell monolayer to the passage of both drugs and human T cells in vitro (Kim and Bynoe, 2014).

Hence, by regulating the expression level of factors crucially involved in tight junction integrity/function, signaling induced through receptors for adenosine acts as a potent, endogenous modulator of BBB permeability in mouse models or human brain endothelial cells.

2. Adenosine receptor signaling increases immune cell entry by increasing BBB permeability.

Adenosine contributes to restraining leukocyte recruitment and platelet aggregation and might be important to control vascular inflammation. This is notable as most studies show that leukocyte migration into the CNS or in vitro BBB models occur by both paracellular and transcellular pathways (Zimmermann, 1992; Yegutkin, 2008). Thus, in vivo paracellular T-cell transendothelial migration under physiological conditions may be mediated by adenosine receptor signaling. Previous study observed that CD73-generated adenosine promotes the entry of inflammatory lymphocytes into the CNS during EAE development (Mills et al., 2008). For adenosine to exert biological effect, CD73 and adenosine receptors must be present on the same cell or on adjacent cells, because adenosine acts locally due to its short half-life. CD73, adenosine A1 and adenosine A2A receptor are indeed expressed on BBB endothelial cells in mice and humans. While CD73 is highly and constitutively expressed on choroid plexus epithelial cells that form the blood to CSF barrier, its expression on brain endothelial barrier cells is low under steady state conditions.

In addition, selective adenosine A2A receptor agonist CGS21680 caused an increase in CX3CL1 level in the brain of treated mice. Conversely, the A2A adenosine receptor antagonist SCH58261 protected mice from CNS lymphocyte infiltration and EAE induction recapitulating the phenotype of CD73 null mutant mice (Imai et al.,1997; Mills et al., 2012). Thus, the augmented CX3CL1 expression level seen in the brain of EAE developing mice can be regulated by adenosine A2A receptor signaling. This suggests that CD73/A2A receptor signaling may preferentially regulate inflammatory immune cells entry into the CNS but confers less stringency on these suppressor T cells.

Moreover, pharmacological activation or inhibition of the adenosine A2A receptor expressed on BBB cells opens and tightens the BBB, respectively, to entry of macromolecules or cells in the mice. The observation that adenosine can modulate BBB permeability upon A2A receptor activation suggest that this pathway might represent a valuable strategy for modulating BBB permeability and promote drug delivery within the CNS (Kim and Bynoe, 2014). Especially, FDA-approved, adenosine A2A receptor agonist, Lexiscan, or a broad-spectrum agonist, NECA, increased BBB permeability and supported macromolecule delivery to the CNS (Carman et al., 2011).

In other words, inhibiting adenosine receptor signaling on BBB cells restricts the entry of macromolecules and inflammatory immune cells into the CNS with limited impact on anti-inflammatory, T regulatory cells. Conversely, activation of adenosine receptors on BBB cells promotes entry of small molecules and macromolecules in the CNS in a time-dependent manner. The duration of BBB permeabilization depends on the half-life of the adenosine receptor activating agent or agonist, suggesting that adenosine receptor modulation of the BBB is a tunable system.

These actions can eventually be seen as a predictable mechanism for the in vivo actions of PDRN in relation to BBB permeability through adenosine A2A receptor stimulation. PDRN acting on adenosine A2 subtype receptors in the cerebrovasculature (increase in E and I prostaglandin levels with consequent regional vasodilatation, inhibition of platelet aggregation, reduction of leukotriene B4 levels) may increase oxygen and substrate supply using a range of doses between 30 and 200 mg/kg in different species (Palmer and Goa, 1993). Furthermore, PDRN has been shown to directly inhibit lipid peroxidation activated during ischemic events in the rats. In addition, PDRN has been shown to inhibit the activation of neutrophils through adenosine A2 subtype receptors, so indirectly reducing the release of free oxygen radicals and other cytotoxic substances (Di Perri et al., 1991).

It is likely that an active carried-mediated transport enables PDRN to traverse the blood-brain barrier as already reported for nucleosides (Pardridge, 1983; Paschen et al., 1988). Finally, it can be hypothesized that PDRN could interact with polyamines whose synthesis can be greatly stimulated in response to ischemia.

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PLoS One. doi: 10.1371/journal.pone.0248689.r003

Decision Letter 1

Giuseppe Pignataro

12 Feb 2021

PONE-D-20-25445R1

Adenosine A2A receptor agonist polydeoxyribonucleotide ameliorates short-term memory impairment by suppressing cerebral ischemia-induced inflammation via MAPK pathway

PLOS ONE

Dear Dr. Han,

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: The manuscript is in line with the previous comments.

The abstract lacks a reference to the brain areas where the measurements were made.

Reviewer #2: The present paper by Il-Gyo Ko and collegues aims to demonstrate the therapeutic effect of PDRN (polydeoxyribonucleotide), an adenosine A2A receptor agonist, on cerebral ischemia in gerbils by suppressing the secretion of pro-inflammatory cytokines with an anti-inflammatory effect. This action is accompanied by increased phosphorylation of MAPK in hippocampus and an amelioration in short-term memory in gerbils. The model of global cerebral ischemia performed is of 2-common carotid arteries occlusion for 7 minutes.

The manuscript is not original, since there is a lot of evidence about the neuroprotective role for PDRN in cerebral ischemia in rodents. At the same time, it has been widely demonstrated the involvement of A2A adenosine receptors in cerebral ischemia reperfusion injury, in the signaling to phosphorylated extracellular signal-regulated protein kinase (pERK1/2). The survival pathway evoked is clearly linked to an amelioration in short-term memory performances in rodent models of global cerebral ischemia, where CA1 ippocampal populations are mainly impaired. Therefore the paper is not innovative, not suitable of publication on PLOS journal.

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Reviewer #1: Yes: Mario Campanile

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PLoS One. 2021 Mar 18;16(3):e0248689. doi: 10.1371/journal.pone.0248689.r004

Author response to Decision Letter 1

1 Mar 2021

Answers to Reviewers’ Comments

Manuscript ID: PONE-D-20-25445

Title: Adenosine A2A receptor agonist polydeoxyribonucleotide ameliorates short-term memory impairment by suppressing cerebral ischemia-induced inflammation via MAPK pathway

Authors: Il-Gyu Ko, Jun-Jang Jin, Lakkyong Hwang, Sang-Hoon Kim, Chang-Ju Kim, Jung Won Jeon, Jun-Young Chung, Jin Hee Han

Reviewer #1

The manuscript is in line with the previous comments. The abstract lacks a reference to the brain areas where the measurements were made.

Answer: According to reviewer comment, we have added word in the abstract.

• Following word was added into the Abstract.

In the current study, induction of ischemia enhanced the levels of pro-inflammatory cytokines and increased phosphorylation of MAPK signaling factors in the hippocampus and basolateral amygdala.

Reviewer #2

The present paper by Il-Gyo Ko and collegues aims to demonstrate the therapeutic effect of PDRN (polydeoxyribonucleotide), an adenosine A2A receptor agonist, on cerebral ischemia in gerbils by suppressing the secretion of pro-inflammatory cytokines with an anti-inflammatory effect. This action is accompanied by increased phosphorylation of MAPK in hippocampus and an amelioration in short-term memory in gerbils. The model of global cerebral ischemia performed is of 2-common carotid arteries occlusion for 7 minutes.

The manuscript is not original, since there is a lot of evidence about the neuroprotective role for PDRN in cerebral ischemia in rodents. At the same time, it has been widely demonstrated the involvement of A2A adenosine receptors in cerebral ischemia reperfusion injury, in the signaling to phosphorylated extracellular signal-regulated protein kinase (pERK1/2). The survival pathway evoked is clearly linked to an amelioration in short-term memory performances in rodent models of global cerebral ischemia, where CA1 hippocampal populations are mainly impaired. Therefore, the paper is not innovative, not suitable of publication on PLOS journal.

Answer: A search for "PDRN" and "ischemia" in PubMed only shows 6 results: renal ischemia, skin ischemia, testicular ischemia, and peripheral arterial occlusion. There have been no studies on the effect of PDRN on the central nervous system, particularly the brain. Therefore, this study is considered to be the first to evaluate the action and mechanism of PDRN on brain ischemia. In this study, we first identified the pharmacological action of PDRN in brain ischemia, and proved that this paper has sufficient originality.

We have demonstrated that PDRN treatment promoted the signaling of the cAMP-PKA pathway by activating the adenosine A2A receptor and enhanced phosphorylation of CREB. It was also found that increased cAMP concentration reduced the expression of pro-inflammatory cytokines by inhibiting the phosphorylation level of the MAPK cascade pathway. This mechanism of action of PDRN has been proven to be effective in ameliorating short-term memory loss. In this paper, we identified the mechanism of action of PDRN during brain ischemia, and contained very innovative contents.

In addition, the PDRN used in this study is one of many adenosine A2A receptor agonists, whose origin differs from conventional drugs. PDRN (source: DNA extracted from salmon sperm cells) used in this study has very high stability and pharmacological action unlike compounds extracted from mammalian organs or PDRN (eg Defibrotide). Thus, this study evaluated the therapeutic efficacy of cerebral ischemia using PDRN, a highly stable drug. In this paper, we deal with PDRN, a very stable drug, and contain novel discoveries.

Therefore, we disagree with the reviewers' opinion that our work is not innovative. We think this paper has originality containing innovative contents and novel discoveries.

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PLoS One. doi: 10.1371/journal.pone.0248689.r005

Decision Letter 2

Giuseppe Pignataro

4 Mar 2021

Adenosine A2A receptor agonist polydeoxyribonucleotide ameliorates short-term memory impairment by suppressing cerebral ischemia-induced inflammation via MAPK pathway

PONE-D-20-25445R2

Dear Dr. Han,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Giuseppe Pignataro, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0248689.r006

Acceptance letter

Giuseppe Pignataro

9 Mar 2021

PONE-D-20-25445R2

Adenosine A_2A receptor agonist polydeoxyribonucleotide ameliorates short-term memory impairment by suppressing cerebral ischemia-induced inflammation via MAPK pathway

Dear Dr. Han:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Giuseppe Pignataro

Academic Editor

PLOS ONE

Associated Data

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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Adenosine A2A receptor agonist polydeoxyribonucleotide ameliorates short-term memory impairment by suppressing cerebral ischemia-induced inflammation via MAPK pathway

Il-Gyu Ko

Jun-Jang Jin

Lakkyong Hwang

Sang-Hoon Kim

Chang-Ju Kim

Jung Won Jeon

Jun-Young Chung

Jin Hee Han

Roles

Abstract

1. Introduction

2. Materials and methods

2.1. Animals and classification

2.2. Transient global ischemia induction

2.3. Treatment

Fig 1. Experimental schedule.

2.4. Step-down avoidance task

2.5. Tissues preparation

2.6. Pro-inflammatory cytokines and cAMP concentrations

2.7. Western blot analysis

2.8. Immunohistochemistry for NeuN

2.9. Data analysis

3. Results

3.1. Changes of cerebral blood flow

Fig 2. Brain blood flow during occlusion and reperfusion of both common carotid arteries.

3.2. Changes of short-term memory latency and NeuN-positive cell number in the CA 1 region

Fig 3. Changes in short-term memory and neuronal survival in the CA1 region.

3.3. Changes of pro-inflammatory cytokine expression

Fig 4. Altered expression of pro-inflammatory cytokines in the serum and hippocampus.

3.4. Changes of phosphorylation of MAPK cascade

Fig 5. Changes in the mitogen-activated protein kinase (MAPK) cascade in the hippocampus.

3.5. Changes of cAMP concentration and adenosine A2A receptor expression

Fig 6. Changes in cAMP concentration and adenosine A2A receptor expression.

3.6. Changes of ratio in p-CREB vs CREB and p-PKA vs PKA

Fig 7. Changes in phosphorylated cAMP response element-binding protein (p-CREB) to CREB ratio and phosphorylated protein kinases A (p-PKA) to PKA ratio.

4. Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Giuseppe Pignataro

Roles

Author response to Decision Letter 0

Decision Letter 1

Giuseppe Pignataro

Roles

Author response to Decision Letter 1

Decision Letter 2

Giuseppe Pignataro

Roles

Acceptance letter

Giuseppe Pignataro

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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Adenosine A_2A receptor agonist polydeoxyribonucleotide ameliorates short-term memory impairment by suppressing cerebral ischemia-induced inflammation via MAPK pathway

3.5. Changes of cAMP concentration and adenosine A_2A receptor expression

Fig 6. Changes in cAMP concentration and adenosine A_2A receptor expression.