Introduction

Parkinson’s disease (PD) is a neurodegenerative disorder in which the primary motor symptoms are associated with a progressive loss of dopamine (DA) in the nigrostriatal pathway. Clinically, the typical motor symptoms of PD do not appear until approximately 50–60% of the nigral DA neurons have been destroyed (Liu et al., 2017) and striatal DA has been depleted by 70–80% (Bezard et al., 2001; Stoessl,2011; Willard et al., 2015; Barber et al., 2017; Segura-Aguilar,2017). Although several molecular imaging techniques have recently been used to evaluate neuronal loss in clinical PD,including positron emission tomography and single-photon emission computerized tomography (SPECT) (Stoessl,2011; Bor-Seng-Shu et al., 2014; Joutsa et al., 2015; Niñerola-Baizán et al., 2015; Suwijn et al., 2015), it is difficult to differentiate between tremor-dominant PD and other forms of tremor. Moreover, because there are relatively limited structural changes in the early stages of PD (Stoessl, 2011;Pagano et al., 2016), the identi fi cation of neurodegeneration as early as possible, particularly during the premotor phase, is essential for the prevention and treatment of the disease.

绿色矿业和绿色矿山两个概念有着相互联系但不完全相同。对于绿色矿业，21世纪初时任国土资源部副部长的寿嘉华，针对西部大开发中环境问题的严重性，较早提出了“绿色矿业”的概念，即在矿山环境扰动量小于区域环境容量的前提下，实现矿产资源开发最优化和生态环境影响最小化[14]，形成了“绿色矿业”概念的雏形。朱训认为绿色矿业是既能为当代人提供物质资源，又不影响当代人和后代人的生存环境与可持续发展的矿业[15]。

Several researchers have noted that dopamine transporters(DAT) in the striatum regulate synaptic DA levels and affect locomotor activity in PD (Huang et al., 2003; Chotibut et al.,2012; Niñerola-Baizán et al., 2015; Suwijn et al., 2015). In addition, dopaminergic (DAergic) neuron degeneration in the substantia nigra pars compacta (SNc) results in a decrease in the density of DAT in the striatum (Bor-Seng-Shu et al.,2014; Kawaguchi et al., 2016). DAT binding can be used to assess DA function (Stoessl, 2011; Ba and Martin. 2015; Georgiopoulos et al., 2015; Suwijn et al., 2015; Badoud et al., 2016;Caminiti et al., 2017), and SPECT scans using 99mTc-TRODAT-1 (a 99mTc-labeled tropane derivative) can be used to image DAT (Wu et al., 2011; Shinto et al., 2014; Huang et al., 2015). Therefore, a measurement of DAT density in the DA nerve terminal can indicate the severity of DA neuronal loss. Although the decrease in striatal DAT density has been described in clinical PD patients (Cummings et al., 2011; Bor-Seng-Shu et al., 2014; Saari et al., 2017) and 99mTc-TRODAT-1 SPECT imaging has been demonstrated as a useful method for diagnosing PD in its early stages (Wu et al., 2011), few reports have been published regarding the association between the degree of 6-hydroxydopamine (6-OHDA)-induced DAergic degeneration and in vivo 99mTc-TRODAT-1 SPECT/computed tomography (CT) imaging in rats.

In the present study, DAT imaging was used with 99mTc-TRODAT-1 SPECT/CT to evaluate DAT density in the striatum of unilaterally 6-OHDA-lesioned rats. To mimic the progressive neuronal loss in PD patients, different degrees of nigrostriatal DA depletion were generated by injecting different doses of 6-OHDA. The severity of the lesions was tested by monitoring animal motor behavior and using tyrosine hydroxylase (TH) immunohistochemistry.

Materials and Methods

Animals

A total of 32 male adult Sprague-Dawley rats aged 8—10 weeks and weighing 295–310 g were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China;license no. SCXK [Hu] 2012-0001). All rats were housed in a controlled environment at 23 ± 2°C with humidity at 55 ± 5%in a 12-hour light/dark cycle, and were supplied with standard rat chow and drinking water ad libitum. All procedures were approved by the Laboratory Animal Welfare & Ethics of Fujian Medical University of China (approval No. 2015-26).

Of the 32 rats used in this study, 2 died during the experiment. Thirty rats were divided into fi ve groups (n = 6 per group): control, vehicle, and 2, 4, or 8 μg 6-OHDA groups.

第三，旅游业发展水平各指标中与机场关联度最大的是旅游外汇收入。首先，旅游外汇收入代表旅游经济水平，旅游经济水平的提高是带动机场建设的最大原因；其次，旅游外汇收入相对于国内旅游收入往往更能体现某一地区或景区的旅游业发展程度，旅游外汇收入越高代表该旅游目的地具有更加广泛的知名度，具备吸引更远游客到来的能力。在单一指标关联中，与旅游外汇收入关联度最高的是机场密度，旅游目的地的发展水平直接影响该区域内的机场数量。

All data are expressed as the mean ± SD. All statistical analyses were performed using SPSS 17.0 software (IBM,Armonk, NY, USA). Data between groups were analyzed using two-way analysis of variance. Significant differences were determined by Tukey’s post hoc tests. The effects of the 6-OHDA-induced lesion between the ipsilateral and contralateral sides were determined by a paired t-test. The correlations between DAT density and TH-positive neurons or apomorphine-induced rotations were analyzed using the Pearson’s correlation test. A value of P ＜ 0.05 was considered statistically signi fi cant.

Nigrostriatal 6-OHDA lesion

TH-positive neurons were counted under a light microscope (Olympus, Tokyo, Japan) at 20× magnification. For quanti fi cation of DA depletion, three adjacent sections were counted per animal, which were selected from the substantia nigra at the level of the optic nerve. Imaging was performed on a Motic microscope system (Motic BA600-4) equipped with a Moticam Pro 285A camera (Olympus), and TH-positive neurons in the SNc of both hemispheres were counted in a blinded fashion.

Apomorphine-induced rotational behavior

个性化的教育还应该具有动态性。大数据形态和速度的优势，使得它对于教育的反馈不是一劳永逸的，能够持续观察、持续分析、持续反馈、持续调整。

Preparation of 99mTc-TRODAT-1 and SPECT/CT imaging

The TRODAT-1 kit was produced by the Institute of Jiangsu Atomic Medicine (Wuxi, China). A dried sample of TRODAT-1 was reconstituted with 2 mL freshly eluted sodium pertechnetate 99mTcO4-, and heated at 100°C for 30 minutes.After cooling to room temperature, the radiochemical quality was tested by thin layer chromatography. Methylbenzene and acetonitrile at a ratio of 80:20 were used as the mobile phase, and the distribution of radioactivity was determined by a thin layer chromatography scanner (Mini-Scan). The radiochemical purity was over 90%.

Eight weeks after 6-OHDA injection, rats under iso fl urane(2%) anesthesia were injected with 99mTc-TRODAT-1 (148—185 MBq/300 μL) via the tail vein. The changes in DAT density in the striatum were detected by a nanoScan SPECT/CT preclinical imager (Mediso, Budapest, Hungary). Rats maintained spontaneous breathing during the scan. CT data were acquired using an x-ray voltage biased to 50 kVp with a 670 μA anode current, and the projections were 720°. At 30 minutes post injection, SPECT images were acquired with low-energy, high-resolution collimators. Emission data were acquired in a 256 × 256 matrix size through 360° rotation at 15° intervals for 30 seconds per angle step.

Distribution of 99mTc-TRODAT-1 in the striatum

6-OHDA is widely used to induce degeneration of midbrain DA neurons to create animal models of PD (Duty and Jenner, 2011; Blesa et al., 2012; Bäck et al., 2013; Le et al.,2014). Our behavioral studies showed that although both the 4 and 8 μg 6-OHDA groups responded to apomorphine with significantly increased numbers of rotations, the number of rotations in the 4 μg 6-OHDA group was ＜ 86 turns/30 minutes at 1—8 weeks post-lesion. However, the rotational response in the 8 μg 6-OHDA group was ＞ 264 turns/30 minutes at 2 weeks post-lesion, and the rotational frequency increased a further 15% during 4—8 weeks post-lesion. These fi ndings are similar to those from previous research, which showed that injecting increasing concentrations of 6-OHDA into the medial forebrain bundle can produce behavioral changes in a dose-dependent manner (Truong et al., 2006).This result also supports the idea that apomorphine-induced rotational behavior is not sensitive enough to detect partial lesions (Boix et al., 2015).

TH immunohistochemistry

After SPECT imaging, the degree of DAergic neuron loss in the SNc was con fi rmed by immunohistochemistry. Rats were anesthetized with an overdose of pentobarbital (90 mg/kg, intraperitoneally) and intracardially perfused with 100 mL 0.9% saline solution, followed by 150 mL 4% paraformaldehyde. The entire midbrain was post-fixed for 24 hours in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) and embedded in paraffin. Coronal sections 5 μm thick were mounted on poly-L-ornithine-coated (Sigma-Aldrich) glass slides. TH immunostaining was used to identify DAergic neurons in the SNc. Brie fl y, sections were treated with 0.3% hydrogen peroxide for 20 minutes to quench endogenous peroxidase activity, and then blocked with 5% normal goat serum in PBS for 30 minutes. Sections were then incubated with mouse anti-TH monoclonal antibody (1:1000; Millipore, Bedford, MA, USA) overnight at 4°C. After washing with PBS, the sections were incubated with peroxidase-labeled anti-mouse IgG secondary antibody(1:200; Boster, Wuhan, China) at room temperature for 1 hour. After rinsing with PBS, staining was visualized using 3,3-diaminobenzidine (Boster) as the chromogen.

Lesioning with 6-OHDA (6-OHDA hydrochloride, Sigma-Aldrich, St. Louis, MO, USA) was performed as previously described (Meng et al., 2015), with minor modi fi cations. Brie fl y, rats were anesthetized with 100 mg/kg chloral hydrate through intraperitoneal injection before surgery,and were immobilized in a stereotaxic frame to target the right medial forebrain bundle at the following coordinates relative to the bregma, according to the Paxinos and Watson(2006) rat brain atlas: anterior—posterior: –4.4 mm, medial—lateral: –1.4 mm, and dorsal—ventral: +8.5 mm. To reduce brain damage and allow precise targeting into the medial forebrain bundle, a modi fi ed injection procedure was used according to a previous study (Gonzalez-Perez et al., 2010).Brie fl y, the solution was delivered by a glass capillary needle with a diameter of 100 μm, which was mounted on a microinjection pump connected to a 5 μL Hamilton syringe via polyethylene tubing with a diameter of 1 mm. The 2, 4, or 8 μg of 6-OHDA in a total of 2 μL in 0.02% ascorbic acid was infused with a glass capillary needle at a rate of 1 μL/min.The needle was left in place for an additional 10 minutes for maximal diffusion. Lesions were not induced in the control group; however, the vehicle groups were infused with 2 μL of saline containing 0.02% ascorbic acid in an identical manner to the 2, 4, or 8 μg 6-OHDA groups.

Statistical analysis

水利部发展研究中心主任杨得瑞在中欧水资源管理对话会上做主旨发言，从发生在2011年、对于中国水利改革发展具有里程碑意义的两件大事谈起，分水利改革发展的政策与实践两个方面向与会者介绍了中国的情况。他说，中国水利专家正与欧盟专家合作开展在水资源管理政策和法规方面的对比研究，并建议中欧双方在水交流平台的框架下，进一步加强水政策和水法律方面的交流合作，分享经验，共同探寻“治水良策”。

To assess the severity of 6-OHDA-induced DAergic degeneration, apomorphine-induced contralateral rotational behavior was monitored after a single injection of apomorphine (0.5 mg/kg, intraperitoneally; Sigma-Aldrich) at 1, 2, 4,6, and 8 weeks post-6-OHDA infusion (i.e., behavioral tests were repeated fi ve times). After 5 minutes of apomorphine injection, individual rats were placed in a plastic container that had a circumference and depth of 35 and 15 cm, respectively. The number of rotations was video recorded, and the rotations were counted for 30 minutes.

Results

Apomorphine-induced rotational behavior

Apomorphine-induced contralateral rotation is a reliable indicator of nigrostriatal DA depletion. As shown in Figure 1, there were no statistically significant differences among the control, vehicle, and 2 μg 6-OHDA groups. However,the rotational behavior in the 4 μg and 8 μg 6-OHDA groups was signi fi cantly higher than in the control group (P ＜ 0.01).

DAT binding of 99mTc-TRODAT-1 in the striatum measured by nanoScan SPECT/CT

As indicated in Figure 2, a unilateral injection of 6-OHDA resulted in a significant decrease in striatal 99mTc-TRODAT-1 binding (bq/mm3), both in the contralateral (unlesioned, P ＜ 0.05) and the ipsilateral (lesioned, P ＜ 0.01)sides, in the 4 and 8 μg 6-OHDA groups compared with the corresponding side in the control or vehicle group at 8 weeks post-lesion (P ＜ 0.05). When lesions were induced by 2 μg 6-OHDA, the amount of 99mTc-TRODAT-1 binding on the ipsilateral side was signi fi cantly reduced compared with the same side in the control group (P ＜ 0.01). There was no signi fi cant difference in 99mTc-TRODAT-1 binding between each side of the striatum in the control or vehicle groups.Representative images are shown in Figure 3.

TH-immunoreactive neurons in the SNc

At 8 weeks post-lesion, the remaining numbers of TH-immunoreactive neurons in the ipsilateral SNc were significantly lower than those on the contralateral side(P ＜ 0.01).Injections of 6-OHDA thus resulted in the loss of TH-immunoreactive neurons in the ipsilateral SNc compared with the contralateral side (Figure 4A–D). A signi fi cant decrease in the numbers of TH-immunoreactive neurons in both the ipsilateral and contralateral (P ＜ 0.05) SNc was found in the 8 μg 6-OHDA group compared with the control group. There was no signi fi cant difference in the numbers of TH-immunoreactive neurons between the control and vehicle groups (Figure 4E).

Correlation between DAT radioactivity and numbers of TH-immunoreactive neurons

As shown in Figure 5, there was a positive correlation between DAT radioactivity in the ipsilateral striatum and the numbers of TH-immunoreactive neurons in the ipsilateral SNc at 8 weeks post-lesion. The Pearson’s correlation coeffi-cient was 0.899 at the 0.01 level (n = 30).

Correlation between DAT radioactivity and apomorphine-induced rotations

Because there were no significant differences in apomorphine-induced rotations among the control, vehicle, and 2 μg 6-OHDA groups, correlation analysis was only performed between the 4 and 8 μg 6-OHDA groups. As indicated in Figure 6, there was a negative correlation between DAT radioactivity in the ipsilateral striatum and apomorphine-induced rotations at 8 weeks post-lesion. The Pearson’s correlation coefficient was −0.887 at the 0.01 level (n = 12).

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Figure 1 Apomorphine (APO)-induced rotational behavior in rats.

The numbers of rotations towards the contralateral side to the 6-hydroxydopamine (6-OHDA) injection within 30 minutes were recorded at 1, 2, 4,6, and 8 weeks post-lesion. **P ＜ 0.01, vs. control group (mean ± SD, n = 6;two-way analysis of variance followed by Tukey’s post hoc test).

Figure 2 6-Hydroxydopamine (6-OHDA)-induced changes in striatal dopamine transporter (DAT) radioactivity at 8 weeks post-lesion.

DAT radioactivity (99mTc-TRODAT-1 binding) in the striatum was measured in the 2 μg, 4 μg, and 8 μg 6-OHDA groups and in the control group.*P ＜ 0.05, **P ＜ 0.01, vs. control group (corresponding side ) (mean ± SD,n = 6; two-way analysis of variance followed by Tukey’s post hoc test).

Figure 3 Representative SPECT images of striatal 99mTc-TRODAT-1 binding at 8 weeks post-lesion.

A relatively symmetrical uptake was observed in the striatum in the control group (A). DAT with 99mTc-TRODAT-1 binding gradually decreased on the ipsilateral side as the dose of 6-OHDA increased (B—D). Arrows indicate decreased 99mTc-TRODAT-1 binding. SPECT: Single-photon emission computed tomography; 6-OHDA: 6-hydroxydopamine;DAT: dopamine transporter.

Figure 4 Changes in the numbers of TH-immunoreactive neurons in the substantia nigra pars compacta after 6-OHDA-induced injury.

(A–D) Representative images of tyrosine hydroxylase (TH)-immunoreactive neurons at 8 weeks post-lesion: Rats were injected with saline(A) or different doses of 6-OHDA (B—D). The remaining numbers of TH-immunoreactive neurons were imaged in the substantia nigra pars compacta at 8 weeks post-lesion. Arrows indicate a gradual decrease in TH-immunoreactive neurons. Scale bar: 500 μm. (E) Histogram representing dopaminergic cell loss determined by the quanti fi cation of TH-positive neurons in the substantia nigra pars compacta at 8 weeks post-lesion. *P ＜ 0.05, vs. control group (mean± SD, n = 6; two-way analysis of variance followed by Tukey’s post hoc test). ##P ＜ 0.01, vs.corresponding side in the same group (mean ±SD, n = 6; paired t-test). 6-OHDA: 6-Hydroxydopamine; TH: tyrosine hydroxylase.

Figure 5 Correlation between DAT 99mTc-TRODAT-1 binding and TH-immunoreactive neurons.

DAT 99mT c-TRODAT-1 binding in the ipsilateral striatum was positively correlated to TH-immunoreactive neuron number in the ipsilateral substantia nigra pars compacta at 8 weeks post-lesion. The Pearson’s correlation coefficient (R) is shown (r = 0.899, P ＜ 0.01, n = 30). DAT: Dopamine transporter; TH: tyrosine hydroxylase.

Figure 6 Correlation between DAT 99mTc-TRODAT-1 binding and APO-induced rotations.

DAT 99mTc-TRODAT-1 binding in the ipsilateral striatum showed a high correlation with APO-induced rotations at 8 weeks post-lesion (4 and 8 μg 6-OHDA groups). The Pearson’s correlation coefficient (R) is shown (r =0.887, P ＜ 0.01, n = 12). DAT: Dopamine transporter; 6-OHDA: 6-hydroxydopamine; APO: apomorphine.

Discussion

The present study investigated the relationship between 6-OHDA-induced DAergic degeneration and DAT imaging using 99mTc-TRODAT-1 SPECT/CT.

SPECT images (Mediso, Budapest, Hungary) were reconstructed with Tera-TomoTM software (Mediso, Budapest,Hungary), and processing and quantification were performed using VivoQuantTM software (Boston, MA, USA).For the analysis of striatal 99mTc-TRODAT-1 binding, the reconstructed image with the highest signal in the striatum was summed together with its two adjacent slices as a single composite image. Regions of interest were drawn in the striatum in each hemisphere. The DAT radioactivity[99mTc-TRODAT-1 binding (bq/mm3)] was counted and corrected for background activity from the cerebellum. All images were examined by two observers who were blinded to the identities of the subject and group.

Pathologically, PD is characterized by the loss of DAergic neurons in the SNc that primarily project to the striatum (Shi and Chen, 2017). Our data indicate significant changes in rotational asymmetry following administration of apomorphine (with DAergic neuron loss approximately 70% in the SNc), which is in agreement with the fi ndings of prior studies in rats with DAergic neuron loss of ＞ 50% (Hefti et al.,1980; Hudson et al., 1993). However, our results differ from fi ndings by other researchers showing that apomorphine-induced rotations resulted from DAergic neuron losses as low as 40% (Przedborski et al., 1995; Truong et al., 2006). This inconsistency could result from variations in experimental design, such as the duration of behavioral tests (Gui et al.,2011) and the concentration of apomorphine used.

Some studies have suggested that, in rat PD models,chronic injections of the DA receptor agonist R-apomorphine provide neuroprotective effects (Yuan et al., 2004; Gui et al., 2011). In the current study, apomorphine-induced behavioral tests were repeated fi ve times (at 1, 2, 4, 6, and 8 weeks after 6-OHDA injection). There were no obvious protective effects under our experimental conditions. A possible explanation is that the anti-parkinsonian effects of apomorphine are largely dependent on experimental procedures.Although rats received apomorphine fi ve times in this study,each time only 0.5 mg/kg was injected intraperitoneally.In contrast, Yuan et al. (2004) and Gui et al. (2011) used R-apomorphine (10 mg/kg per day subcutaneously) for 11 consecutive days.

DAT is located in presynaptic DAergic nerve terminals,and plays a critical role in removing DA from the synaptic cleft and in regulating extracellular DA concentrations(Shen et al., 2012; Tian et al., 2012). Therefore, the density of DAT in the striatum may represent a presynaptic DAergic function. Because DAT is selectively expressed by DA neurons, speci fi c SPECT ligands for DAT imaging (FP-CIT, beta-CIT, IPT, TRODAT-1) provide a marker for presynaptic neuronal degeneration (Goebel et al., 2011; Bor-Seng-Shu et al., 2014; Hsiao et al., 2014). 99mTc-TRODAT-1 is a recently developed radiotracer that selectively binds to DAT (Wang et al., 2012). There was only a marked reduction in the ipsilateral striatal DAT uptake with 2 μg 6-OHDA in the present study. It could be that injection with 4 and 8 μg of 6-OHDA reduces DAT uptake progressively in both the contralateral and ipsilateral striatum. However, the increase over time in apomorphine-induced rotations indicates that damage to DAergic neurons is largely unilateral. The reasons for this inconsistency between apomorphine-induced behavior and DAT uptake need to be studied further.

Our results showed agreement between the distribution of 99mTc-TRODAT-1 in the ipsilateral striatum and the remaining TH-positive neurons in the SNc, indicating that reduced DAT binding corresponds with a loss of DAergic neurons. This result is in line with previous studies from animal and clinical studies, showing that a decrease of DAergic neurons in the SNc may lead to a reduction of DAT uptake in the striatum (Bäck et al., 2013; Bor-Seng-Shu et al., 2014; Kraemmer et al., 2014). However, the opposite result has been reported recently by others, who found that postmortem SNc neuron numbers had no relationship with striatal DAT binding in PD patients (Saari et al., 2017). This inconsistency may partly be explained by the loss of DAergic neurons in the SNc in late-stage PD, which is associated with other factors such as a loss of co-transmitter release from DA neurons, inflammatory responses, and gliosis(Seutin, 2005; Nagatsu and Sawada, 2006; Whitton, 2007).Moreover, it is difficult to show a close relationship between biomarker changes over time and changes in severity in clinical PD (Stoessl et al., 2014), especially when the degeneration of SNc neurons and striatal DA is greater than 50%(Saari et al., 2017). Although DAT density, visualized using 99mTc-TRODAT-1 binding, was remarkably decreased in the bilateral striatum in both the 4 and 8 μg 6-OHDA groups after 8 weeks, increasing concentrations of 6-OHDA tended to decrease DAT uptake from the 2 μg 6-OHDA group, in which the mean DA neuron loss was approximately 40%.This supports the idea that 99mTc-TRODAT-1/SPECT imaging may detect nigrostriatal DA neuronal degeneration in the early stages of PD, and that this imaging technique may be of use to assess the severity of nigrostriatal DA neuronal degeneration.

It is also important to note that the correlation between the number of DAergic neurons and SPECT results was strong, because there were large differences between groups(control, low, and high dose of 6-OHDA). This is similar to in the clinical situation, where the DaT-SCAN works well to differentiate PD patients from controls (Stoessl, 2011; Suwijn et al., 2015). In contrast, the correlation within groups (e.g.,the 4 μg or 8 μg group) between the number of TH-positive neurons and SPECT results was poor. This is consistent with clinical data that suggests that the DaT-SCAN is not suitable for monitoring disease progression (Saari et al., 2017).

The correlations between DAT radioactivity and apomorphine-induced rotations or TH-immunoreactive neuron numbers further supports the idea that PD symptoms are associated with both a loss of DA neurons in the SNc and a reduction of DAT uptake in the striatum.

Some clinical studies have suggested that there is a more marked reduction of striatal DAT binding on the contralateral side to the more affected limbs than on the ipsilateral side (Huang et al., 2001; Booth et al., 2015); however, there was no significant difference in 99mTc-TRODAT-1 binding between the contralateral and ipsilateral sides in this study.In the present study, both DAT binding in the striatum and TH-immunoreactivity in the SNc were bilaterally impaired after a unilateral injection of 8 μg 6-OHDA to the medial forebrain bundle (there was a loss of approximately 95% of DAergic neurons, similar to what is observed in advanced PD). This fi nding indicates that severe unilateral ablation of DA neurons might impair the bilateral nigrastriatal pathway.This phenomenon may re fl ect the dysfunction in striatal DA homeostasis in the later stages of PD. It is worth noting that the approximately 75% decrease in TH-immunoreactivity in the 4 μg 6-OHDA group reported in the present study is higher than that of a previous study (Truong et al., 2006).This discrepancy may be associated with our modi fi ed injection procedure, which signi fi cantly improves accuracy.

2.1.5 精密度试验 精密吸取混合对照品溶液10 μL，按照“2.1.1”项下条件重复进样6次，测定10种黄酮的峰面积，结果金丝桃苷、朝藿定B、朝藿定A、朝藿定C、淫羊藿苷、木犀草素、槲皮素、川陈皮素、山柰酚、宝藿苷I峰面积的RSD值分别为1.31%、0.48%、0.25%、0.63%、0.44%、1.81%、1.43%、1.11%、0.41%、0.57%，表明该条件下仪器精密度良好。

The limitations of the present study are: (1) We did not differentiate 99mTc-TRODAT-1 binding in the subregions of the striatum (such as the caudate or putamen), and (2) the small size of the animals for 99mTc-TRODAT-1 binding. It would be helpful for future studies to increase the sample size, and to observe 99mTc-TRODAT-1 binding in different subregions of the striatum in detail.

在关系数据库中，附属实体表与主实体表的联系类型为“1对多”，每一个附属实体的键对应若干个主实体表的键。而主实体表与联系表之间的联系类型也是“1对多”，每一个主实体表的键，对应多个联系表的键。对于“多”方的多个键可以通过链表的方式连起来。而对于联系类型“1对多”，可以创建一个哈希表，实现从“1”方到“多”方的映射。该哈希表的键为“1”方的键，哈希表的值为与“1”方对应的“多”方的键的链表。因此，可以采用如下方法，构建全局的元组ID传播哈希表，将多个关系实现虚拟连接。

前几日，微博上一组“紫禁城的花开了”的图片令我深有感触。照片中，宫殿前的树上开满了花朵，树还是数百年前的那棵树，而当年“一肌一容，尽态极妍，缦立远视，而望幸焉”的佳人早已化为一抔黄土。花开又一年丝毫不见苍老，偌大的宫殿悄悄地多了几道细碎的裂痕，百花齐放笑得何尝不是物是人非的荒唐？

In conclusion, our data support the use of DAT imaging with 99mTc-TRODAT-1 SPECT for the diagnosis of early PD In the future, earlier diagnosis of PD using this technique may allow for presymptomatic intervention.

Acknowledgments: We thank Dr. Wen-Xin Chen from Fujian Provincial Hospital of China for providing TRODAT-1 reagent, and staff from the Center for Molecular Imaging in Xiamen University of China for their professional technical assistance.

Author contributions: LL, JY and ZHZ conceived and designed the experiments, analyzed the data, and wrote the paper. JY, HZ and ZFH performed the experiments. ZHZ and LL contributed reagents/materials/analysis tools. All authors approved the fi nal version of the paper.

Con fl icts of interest: The authors declare no competing fi nancial interests.

绘画语言的发展从来都不是既有范式的复制，它的语言价值体现在自然生成的原创性上，也体现在与时代精神的契合上。在近百年来的现代变革中，中国工笔绘画艺术突破传统审美和表现的藩篱，以当代意识为自觉，在题材内容、表现手法以及工具材料等方面出现了大量的新的拓展，展现出了惊人的创造力和活力，新的审美境界在逐渐形成。罗春辉是中国工笔画坛上的菁英，也是创造这种新的审美境界的积极实践者，他是在工笔画艺术的传统技法与符号程式的基础上，尝试创造多种因素复合的新的表现形式，力求使作品获得崭新审美品质的成功者之一。其成绩斐然，声名远播。

Financial support: This study was supported by the National Natural Science Foundation of China, No. 81571250. The funder did not participant in the study design, in the collection, analysis and interpretation of data, in the writing of the paper, and in the decision to submit the paper for publication.

Experimental study on the removal of different C/N wastewater using sequencing batch reactor

Institutional review board statement: This study was approved by the Laboratory Animal Welfare & Ethics in Fujian Medical University(2015-26). The experimental procedure followed the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985).

Copyright license agreement: The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement: Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check: Checked twice by iThenticate.

Peer review: Externally peer reviewed.

Open access statement: This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-Non-Commercial-ShareAlike 4.0 License, which allows others to remix, tweak,and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewer: Peng Luo, Fourth Military Medical University, China.

Additional fi le: Open peer review report 1.

在全球化大背景下，企事业单位可能会面临新的形势、新的任务、新的机遇、新的挑战，要想在新环境下有出色的表现，在现代经济中占一席之地，完善单位内部控制是关键，进而制定出合理的内部控制建设方案。

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