Evaluation of Peripheral Nerve Injury According to the Severity of Damage Using 18F-FDG PET/MRI in a Rat Model of Sciatic Nerve Injury

Abstract

Reports suggest a high incidence of nerve injuries, and diverse methods, such as two-point discrimination and the pin prick test, have been attempted to evaluate the severity of nerve damage. However, these tests rely on subjective sensations and may not accurately represent the damaged area. A previous experiment revealed that ¹⁸F‑FDG positron emission tomography/magnetic resonance imaging (PET/MRI) detect peripheral nerve damage. This study aimed to assess peripheral nerve damage according to severities of damage using ¹⁸F-FDG PET/MRI in a rat sciatic nerve. Eighteen rats were divided into three groups: 30-second (G1), 2-minute (G2), and 5-minute (G3) crushing injury groups. The severity of nerve damage was measured in the third week after the crushing injury using three methods: revised withdrawal threshold (RevWT)), standardized uptake values ratio on PET/MRI (SUVR), and immunohistochemistry (intensity ratio (IntR)). There were significant differences between G1 and G3 in both SUVR and IntR. There were no significant differences in both SUVR and IntR between G2 and G3 and no significant differences in RevWT among the three groups. There was a significant difference in SUVR but no significant difference in IntR between G1 and G2. Although PET/MRI did not show results consistent with the immunohistochemistry in all respects, this study demonstrated that the severity of nerve damage as assessed by PET/MRI increased with a longer crushing time. PET/MRI showed potential as an objective diagnostic tool in this peripheral nerve injury model. If research is supplemented through further experiments, PET/MRI can be used as an effective diagnostic modality.

Introduction

Peripheral nerve injury can result from clinical accidents and trauma and accounts for 2.8% of all trauma cases [1]. Nerve tissue and repair are associated with various changes such as the development of edema, free oxygen release, and inflammatory changes. Recently, nerve injuries have been reported following dental procedures, such as tooth extraction or implant surgery [2]. When a nerve is damaged, a diagnosis must be made to determine the severity of damage, to predict the recovery period, and to assess whether surgery should be performed or not. The need for research to evaluate these points has been noted. Studies are needed to evaluate the degree of damage and monitor the recovery process. Various methods, such as two-point discrimination and the pin prick test, have been attempted to evaluate the severity of nerve damage. However, these techniques rely on subjective sensations and may not accurately represent the damaged area. Historical assessment is the most definitive method for evaluating nerve injury. However, it has limited clinical application as it is invasive and requires tissue sections. Several studies have attempted to develop accurate and objective methods to determine the severity and location of nerve damage.

Materials And Methods

Animal Model 

Eighteen male Sprague-Dawley rats (7 weeks old; weight: 200–250 g) were used. Rats were provided daily food and water, weighed once a week, and allowed to acclimatize to a specially made customized modular holder cage before weight measurement. All animal protocols were approved by the Institutional Animal Use and Care committee of the Department of Laboratory Animal Resources, Yonsei Bio-Medical Research Institute, Yonsei University College of Medicine, Korea.

Experimental Design 

The subjects were divided into three groups (n=6, respectively). All surgical procedures were performed on the left sciatic nerve. In the crushing injury model, the first group (G1) received 30 seconds of crushing time, the second group (G2) received two minutes, and the last group (G3) received five minutes. A curved hemostat (12.5 cm HB0515; HEBU, Germany) was used to apply the crushing injury. The starting point of curved portion was used as the crushing reference point of the hemostat (Fig. 1a). The clamping force between the tips of the hemostat was approximately 40 N, and the width was 3 mm. The groups were divided as shown below (Table 1).

Table 1 Experimental groups

Surgical Procedure           

Under inhalation anesthesia with isoflurane (Forane; JW, Korea), the left thigh area was shaved and cleaned with 10% povidone iodine. A 2-cm skin incision was made 0.5 cm posterior to the femur. The fascia of the biceps femoris and gluteus superficialis was exposed to access the sciatic nerve by blunt dissection through the intermuscular space. Approximately 15 mm of the nerve was carefully freed from the surrounding tissue using micro-pincettes (Fig 1a). With a curved hemostat, crushing injuries were applied in the experimental groups using the three different durations described previously. The surgical sites were closed layer-by-layer with 4-0 absorbable synthetic braided suture (Vicryl; Polygalactin 910; Ethicon, Inc., a Johnson and Johnson company, Sommerville, NJ, USA) for the muscle and fascia and 5-0 non-absorbable synthetic monofilament suture (Nylon; AILEE Co., Busan, Korea) for the skin. After performing the PWT test preoperatively and one, two, and three weeks postoperatively and PET/MRI three weeks postoperatively, all animals were sacrificed by CO₂ inhalation. The left sciatic nerves were harvested for histological analysis.

Pain Behavior Assessment                              

Pain behavior assessments were performed four times (preoperative, 1, 2 and 3 weeks postoperatively) in the right hind paw (control) and the left hind paw (experimental site). Both hind paws were measured thrice, and the mean value was used. Manual von Frey filament (BIO-VF-M; Bioseb Inc., Vitrolles, France), which the gold standard for determining mechanical thresholds in rats and shows force according to its size, was used for the PWT test (Fig. 2a). The rats were positioned in the customized modular holder cage for 15 minutes for acclimatization [7]. A monofilament was applied perpendicular to the plantar surface of the hind paw until it bent, delivering a constant predetermined force for two to five seconds (Fig. 2b, 2c). The response was considered positive if the rats exhibited paw withdrawal during filament application or immediately after filament removal. The plantar surface of the hind paw was the most commonly used area for testing, and the response was observed via a wire-gated floor. If a response was shown for any particular force, the mouse was re-examined with a value one step below the value, and if there was no response to the value, the particular value was recorded as the result. 

PET/MRI Imaging

All rats were fasted for 12 hours the day before the 3-week postoperative PET scan (becoming violent and dangerous after 16 hours of fasting). Animals underwent sequential small-animal PET (Inveon PET; Siemens, Germany) and small-animal MRI scans (Bruker 9.4 T 20-cm bore MRI system; Biospec 94/20 USR; Bruker, Ettlingen, Germany). A customized PET table similar to an MRI table was prepared and used to facilitate image superimposition. A dose between 1.000–1.070 uCi was injected intravenously. One-hour dynamic scans of the thigh were obtained, and T2-weighted rapid acquisition with relaxation enhancement (RARE) images of the thighs (repetition time [TR] = 2300 ms; echo time [TE] = 11 ms; slice thickness = 1 mm, acquisition matrix = 192×192; acquisition FOV 55×55 mm²) were also obtained. Co-registration of PET and MRI was performed using AMIDE image analysis software (amide.exe1.0.4; https://amide.sourceforge.net). The MRI images indicated the anatomic location of the sciatic nerves and the placement of the regions of interest (ROIs). Spherical-shaped ROIs, measuring approximately 6 mm in diameter, were placed around the nerve injury site and on the opposite side. For quantitative analysis of PET signals, the maximum standardized uptake values (SUVmax) of the ROIs were calculated using OsiriX image analysis software (Pixmeo, Geneva, Switzerland). The standardized uptake value ratio (SUVR) from two different regions within the same PET image (from a target and a reference region) was also calculated to eliminate differences between the animals as follows: 

                               SUVtarget                      SUVleft ROI

SUVR       =      _____________         =    ________________

                           SUVreference                     SUVright ROI

After the PET and MRI scans were performed, the rats were sacrificed the next day (after 24 hours, considering the drug’s half-life) to prepare tissue sections. 

Histology and Immunohistochemistry

                All harvested left sciatic nerves were fixed in 4% paraformaldehyde (PFA) for 24 hours immediately after sacrifice, and they were embedded in paraffin with threads indicating their orientation. For each specimen, three cross-sections (4 μm thickness) taken from the middle of the injury site, 1 mm proximal to the injury site, and 1 mm distal to the injury site were mounted on slides and stained with hematoxylin and eosin (H&E) for histological and IHC analysis (Fig. 1b).      For immunostaining, the ROI and proximal and distal areas were fixed with 4% PFA, permeabilized with 0.5% Triton-100 (Sigma-Aldrich), and blocked with 1% bovine serum albumin (Sigma-Aldrich). To stain cell markers, samples were incubated with specific primary antibodies: anti-S-100 beta antibody (ab52642, Abcam) for two hours at room temperature, washed with PBS thrice, and then goat anti-secondary staining was performed with goat anti-rabbit IgG H & L (horseradish peroxidase) (ab205718, Abcam) for IHC. Tissue slides obtained by staining were photographed using a Zeiss LSM780 confocal microscope system (Zeiss, Korean basic science institute in Seoul center). This analysis was performed on three randomly selected non-overlapping microscope fields. The evaluated area covered two-thirds of the whole section. All slide pictures were selected by an expert blinded to each rat’s group. The intensity of staining obtained from the microscopy images varied according to the degree of staining. The intensity was analyzed using ImageJ software [8]. The analytical protocol was implemented according to the procedures described by Crowe and Yue [9] (Fig. 3). 

Zhao et al. observed that greater damage was correlated with more active regeneration of Schwann cells. Therefore, we expected that the intensity of S-100, a Schwann cell biomarker, would be strongly expressed [10]. Experimental data were obtained from three randomly selected sections. Herein, the development of strong intensity was considered a positive response to the S-100 antibody. The average intensity measured in these three sections was used as the result. The brown-stained part was recorded as a positive reaction for the S-100 antibody. The mean gray value was evaluated using ImageJ software particle analysis; the mean value was the average of intensity in the selection, indicating the raw integrated density/pixel number (Fig. 3). Dubovy et al. reported that Schwann cells distal to the crushed sciatic nerve showed increased immunostaining for proinflammatory and anti-inflammatory cytokines [11]. The number of Schwann cells distal to the crushed sciatic nerve was expected to increase as greater damage is correlated with higher S-100 intensity values. To compensate for the difference between individual rats, the S-100 intensity of the distal part of the ROI was divided by the S-100 intensity of the proximal part of the ROI. The corresponding value was expressed as intensity ratio (IntR).

                        Mean Gray Value _distal

IntR   =    __________________________

                      Mean Gray Value _proximal

Statistical Analysis

              Statistics were analyzed using GraphPad Prism (Version 8.0.0 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com). The Shapiro-Wilk test was used to determine whether each group had a normal distribution. All the results did not follow the normal distribution based on the normality test, and the number of rats was six in each group; therefore, non-parametric tests were conducted. The independent sample Kruskal-Wallis test was performed to examine whether there was a significant difference in the results between each group. The Kruskal-Wallis test was conducted to determine the differences between the three methods. Dunn’s multiple comparison test was used to determine which groups were significantly different. P<0.05 was considered statistically significant.

Results

Pain Assessment 

 First, the Kruskal-Wallis test was used for the three groups to determine whether there was a significant difference between each result. The Kruskal-Wallis test showed no significant difference between PWT test results the results showed that the right hind paws (intact side) had lower standard deviations than the left hind paws (injured side). Overall, G2 and G3 showed more severe hypoesthesia in the second week than in the first week; however, hypoesthesia recovered significantly in the third week, possibly due to significant nerve recovery. The level of hypoesthesia appeared to increase with an increased intensity of nerve damage. However, in the third week, all three groups showed almost complete recovery (Fig. 4a). However, the right hind paw graph showed that G1, G2, and G3 were constant without any statistically significant differences (Fig. 4b). 

 Withdrawal threshold values were revised to compensate for different conditions of individual rats (such as sensitivity, nervousness, fear, adaptation, and blunting) before comparison between variables at three weeks postoperatively in the three groups. Revised withdrawal threshold (RevWT) values were calculated as follows: 

                                    Withdrawal thresholds value of the injured side

RevWT  =  ____________________________________________________________

                   Average of withdrawal threshold values of the intact side in each group

For example, the G1 preoperative left paw (preL) value was 26.0 g, and the average of the withdrawal threshold values of the intact side for G1 was 27.7 g. Thus, the RevWT value was 26.0 g / 27.7 g = 0.983064 (preL/R).

The Kruskal-Wallis test demonstrated that G1, G2, and G3 all showed significant differences among RevWT values (p=0.0144, p=0.0237, and p=0.005 respectively). In Dunn’s multiple comparisons test, G1 only showed a significant difference between the preoperative value and the first week postoperative value (p=0.0469). However, G2 showed a significant difference between the preoperative value and the second week postoperative value (p=0.0226). G3 showed a significant difference between the preoperative value and second week postoperative value (p=0.0112). When changing from one filament to the next, the increase in force was not linear, and the gap between filaments was very large. As the force increased, the thickness of the filament also increased. Therefore, it was not possible to accurately measure the force alone. Herein, pressure, which is the force per unit area of the filament, was calculated according to Aesthesio’s precision tactile sensory evaluator data chart. However, there was no significant difference in pressure among the groups. 

PET/MRI Evaluation

MRI enabled clear visualization of the anatomy, and PET/MRI fusion images showed sciatic nerve injury sites as hot spots (Fig. 5). A longer injury time was correlated with a higher SUVR (Fig. 6). For the quantitative analysis of PET images, the SUVmax and SUVR of ROIs were calculated using OsiriX image analysis software (Table 2). The SUVR demonstrated a significant difference in the Kruskal-Wallis test results for the three groups (Table 3). In Dunn's multiple comparisons test, G1 showed a significant difference from G2 (p=0.0135) and G3 (p=0.0080). However, there was no significant difference between G2 and G3 (p≥0.9999). Although G2 and G3 had different crushing injury times, there was no statistically significant difference between these two groups (Fig.7b).

Table 2 Imaging analysis of PET/MRI

Table 3 PET/MRI results (Mean, (SD))

Histology and Immunohistochemistry

Hemorrhage was observed on the tissue slides. The diameter and thickness of the epineurium were relatively increased. Dense myelinated axons surrounded by perineural epithelium were visible in all sections. Some slides showed intact vital cellular features. In contrast, some other slides showed folded tissues, and the tissue condition was not well organized due to errors in tissue processing. Some slides showed fragmentation, atrophy of myelin and axons, swollen neuronal bodies, and interstitial fibrosis. Here, S-100 IHC was used to determine the severity of damage by calculating the S-100 intensity (Fig. 8a). With S-100-stained slides, the intensity was analyzed using ImageJ software [8]. The analytical protocol was implemented according to a study by Crowe and Yue (Fig. 8b) [9]. The Kruskal-Wallis test showed a significant difference between groups (p=0.038). Dunn’s multiple comparison test was conducted among the groups, and the p-values were 0.8385, 0.0386, and 0.4792 for G1 vs G2, G1 vs G3, and G2 vs G3, respectively (Table 4, Fig. 7c). There were no significant differences between G1 and G2 or between G2 and G3. However, there was a significant difference between G1 and G3 (Fig. 7c).

Table 4 IntR results. There was a significant difference between G1 and G3 (Dunn's multiple comparisons test p=0.0386) and no significant difference between G1 and G2 or G2 and G3)

Comparison of Assessment Methods

In terms of RevWT, there was no significant difference among the groups in the third week (Kruskal-Wallis test p=0.7668) (Fig 7a). However, there was a significant difference in the SUVR between G1 and G2 and between G1 and G3 (Dunn’s multiple comparisons test both p=0.001). However, there was no significant difference in the SUVR between G2 and G3 (p>0.99). Finally, there was a significant difference in IntR between G1 and G3 (Dunn’s multiple comparisons test p=0.0386) and no significant difference between G1 and G2 or between G2 and G3 (p=0.8385 and p=0.4792, respectively) (Table 5, Fig. 7).

Table 5 Comparison of Assessment Methods

Discussion

Peripheral nerve injury produces loss of sensory and motor function, resulting in critical economic and psychological issues and diminished quality of life. Common causes of peripheral nerve injuries are traffic accidents, firearm injuries, chemical injuries, cutting tool injuries, and crushing injuries [1]. Nerve injuries may also result from dental procedures, such as extraction, implantation, minor surgery, and root canal treatment (chemical injury), and trauma [2]. There is no doubt that an accurate diagnosis leads to accurate treatment planning. However, these diagnoses are based on the patient's subjective symptoms. An objective and accurate diagnosis can help achieve successful treatment outcomes. To our knowledge, this is the first study to evaluate peripheral nerve injury according to the severity of damage using ¹⁸F-FDG PET/MRI in a rat model of sciatic nerve injury. 

The nerve damage models were divided into three groups according to the crushing time. Alvites et al. reported that in most studies, the crushing time was different for each paper [12]. There was no standardized protocol for crushing time, which ranged from 15 seconds to two hours [12-19]. An et al. attempted to objectify the damage intensity by varying the number of notches on the forceps and the pinching part of forceps to damage nerves [20]. This study attempted to examine the differences in sciatic nerve damage according to the crushing time. 

Other papers have used various crushing load tools, such as Jeweler’s forceps, Kocher's forceps, pincers, mosquito forceps, and aneurysmal clips [12]. Serrated and non-serrated forceps have also been used without distinction. In this experiment, a curved serrated hemostat was used; however, the serration was in one specific direction, and a crushing injury could be applied only to a specific part of the nerve by clamping one time. Thus, there may have been part of the nerve where a crushing injury was not applied. Thus, it was difficult to inflict more than a certain level of injury. Beer et al. used a non-serrated clamp to standardize their nerve crush model to ensure that the pressure on the injury site was uniformly transmitted [21]. Future studies should develop a consistent crushing injury model using tools, such as non-serrated hemostats, generating a crushing injury by turning the tool 90 degrees after crushing once, or causing a crushing injury by changing the angle of two hemostats by 90 degrees. 

There were several limitations in measuring the PWT with von Frey filament. One limitation of the von Frey filament test was that it did not allow for differentiation between a pain response and routine bodily grooming/itching. Mason et al. stated that it was important to note the duration of a specific behavior to differentiate between pain and grooming. Typically, a pain response is one swipe after filament application; however, a grooming motion tends to be longer and can last from a few seconds to a few minutes. If the grooming/itching behavior is indistinguishable due to irritability, it is best not to record it as a positive response [22]. When a manual filament was used, there was a possibility that the experimenter may have had a subjective interpretation. Therefore, both hind paws were measured three times, and the mean value was used for analysis. 

When changing from one filament to the next, the increase in force was not linear, and the force gap between filaments was very large. For example, after using a 60 g filament, the next filament force was 100 g, followed by 180 g and 300 g. As the force increased, the filament thickened, and it was not possible to obtain accurate measurements for force alone. Therefore, the pressure, which is the force per unit area of the filament, was calculated. The results showed no significant difference between force and pressure. However, future studies should use pressure units instead force to obtain more precise data. In light of these points, the PWT test showed subjective results similar to those of neurosensory testing in clinical practice. 

Nevertheless, the withdrawal threshold result was consistent with the results of other research. Roman et al. demonstrated hypoesthesia by time in the first, second, and third weeks; the peak was in the first and second weeks, and hypoesthesia gradually improved and recovered by the third week [17]. The histological findings were also consistent with those of other studies. In a sciatic nerve injury model by Zhang et al., the IHC findings showed Schwann cell proliferation over time, with a peak in the first and second weeks, followed by a decrease in the third week [23]. 

Histological analysis was conducted using the IHC method. Schwann cells play vital roles in peripheral nerve regeneration [24]. However, identification by conventional histological methods is difficult. Schwann cells can be identified using an antibody against the S-100 protein. Detection of Schwann cells by IHC staining is considered to be a positive indicator of nerve regeneration [25]. Furthermore, accumulation of S-100 protein indicates the proliferation of sciatic nerve Schwann cells. Proliferating Schwann cells promote the sustained regeneration and functional recovery of the sciatic nerves [26]. Zhao et al. used S-100 as a Schwann cell marker to compare the effect of decimeter wave therapy on the proliferation of Schwann cells after nerve injury [10]. S-100 may serve as a marker for the proliferation of Schwann cells in sciatic nerve regeneration research [27]. Wang et al. reported that ginsenoside Re significantly increased S-100 expression in Schwann cells to promote rat sciatic nerve regeneration [28]. In this experimental study, S-100 intensity was measured, and the values (IntR) were compared with the SUVR results.

Many studies have focused on noninvasive diagnostic tools using imaging methods such as US to evaluate nerve hypertrophy and intraneural vascularization [29, 30]. The quantity and quality of nerve damage have been studied using diffusion tensor imaging [31]. Moreover, radioactive tracers for PET scan have been used to assess the severity of nerve damage. PET alone has some advantages and disadvantages. Although it can identify nerve damage, it is difficult to determine the exact location of damage. Purohit et al. stated that researchers should be aware of several specific patterns of FDG uptake and suggested that contrast-enhanced CT, US, or MRI be used together to prevent errors in PET interpretation [3]. In particular, in this experiment, PET/MRI fusion was used to avoid the limitations of PET and utilize the advantages of MRI. This study used PET/MRI fusion to obtain metabolic ¹⁸F-FDG PET images with a more sensitive anatomical evaluation with MRI.

                The results confirmed that PET imaging and IHC values increased as the nerve-crushing time increased. Both the imaging and histological results showed a significant difference between 30 seconds and five minutes of crushing injury. However, there was no significant difference between two and five minutes for both results. One reason is that with sufficient strength, a curved hemostat can produce a crushing injury even in two minutes; therefore, there was no difference between two and five minutes. To elicit a difference in time through crushing injury, it is worth considering reducing the crushing time to one minute or ratcheting the clamp a different number of times. Conversely, investigator should consider methods other than clamping for crush durations of more than two minutes. Another study reported that crushing injury through clamping could not produce more severe injuries, such as neurotmesis [20]. Therefore, no significant difference was expected between two and five minutes of crush injury.

                Unlike the imaging analysis, the histological results showed no statistically significant difference between 30 seconds and two minutes. This finding could result in a subjective interpretation of histological characteristics. In addition, obtaining reliable histological data may be difficult because specialized equipment for each process is required for histological analysis [32]. Therefore, this study used computer-aided IHC analysis to minimize subjectivity in interpretation. The biggest advantage of IHC compared with immunofluorescence (IF) is that IHC staining is permanent with no change in color. Moreover, tissue morphology is clearly visible, simplifying interpretation. With IF, fluorescence eventually disappears; therefore, photographing and scans are necessary to maintain a record [33]. A disadvantage is that fluorescence can be automatically generated when using formalin-fixed paraffin-embedded tissue (FFPE) and seen in autofluorescent elements such as, collagen, elastin, neutrophils, and blood vessels. Therefore, it is very important to restore slides that are not stained with isotype controls and use frozen sections instead of FFPE tissues. An advantage of IF is that it allows immediate visualization of cell populations through various staining techniques on one slide. Recently, IF has been widely used in immune-oncology research. Fluorescence quantification can provide better visualization and more sensitive results for future experiments [34, 35].

                This experiment is very important in that crush damage was imposed differently over time. This study aimed to quantify the SUVR according to the severity of injury in a rat sciatic nerve injury model using PET/MRI. When a nerve is damaged, it will be possible to objectively quantify the severity of damage using PET imaging. As a result, it will be also possible to predict how long it will take to recover if long-term data are accumulated. 

                There are errors and difficulties in obtaining measurements with manual filaments. However, these aspects are similar to the neurologic examinations currently performed in clinical practice, and the findings may vary depending on the measurer’s subjective interpretation and the subject’s sensory diversity, which are current limitations. With advancements in computer-aided image capture systems, non-communicating and non-stimulus-evoked pain evaluations may become more accurate and useful [36]. The sciatic nerve can be evaluated by analyzing the toe angle during the gait stance duration, ankle kinematics, and gait through video recording for motion evaluation without considering the method of evaluation by applying pressure [12]. The analysis is technically complex and necessitates appropriate equipment. However, more precise results can be obtained if this method is also used for further research.

                As there were only six rats in each group, there were some differences in the values, but no statistically significant differences. Future studies should include at least 30 rats and use statistical methods with parametric tests to yield more sensitive and accurate results. Moreover, PET/MRI can be used to quantify the severity of nerve damage and predict prognosis. Thus, PET/MRI can objectively evaluate treatment efficacy when studying new drugs used for neuropathic pain. Doctors often face the challenges of defining a diagnosis and prognosis based only on the patient's subjective symptoms. Patients have difficulties describing the symptoms of their sensory numbness and may feel frustrated. However, objective tools can diagnose nerve injury, predict prognosis, and provide treatment satisfaction for doctors and patients.

Conclusion

Following our previous study, this study demonstrated that the severity of nerve damage as assessed by PET/MRI increased with an increased nerve-crushing time. Although PET/MRI did not show results consistent with the histological analysis in all respects, PET/MRI showed potential as an objective diagnostic tool for assessing this peripheral nerve injury model, even considering the errors in the experimental design and the results of the PWT test, which represents a clinical neurosensory test. If this research is supplemented through further experiments, PET/MRI may be used as an effective diagnostic modality.

Declarations

Data Availability  

The data presented in the study are included in the article materials and are available from the corresponding author on reasonable request.

Conflict of interest

The authors declare that they have no competing interest.

Funding

This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. 2019R1G1A1085187). 

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