1. Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease. It is characterized pathologically by the selective and progressive loss of dopaminergic neurons in the substantia nigra (SN). As a major part of cortical-basal ganglia motor loop, the SN plays a vital role in movement control. The loss of dopaminergic neurons in the SN leads to debilitating motor dysfunction, such as resting tremor, bradykinesia, rigidity, and postural instability, which seriously affects the quality of life of PD patients [1]. It is estimated that 50% of the nigral neurons needs to be lost before PD symptoms appear [2]. Due to the progressive nature of PD, early evaluation of nigral lesions therefore is important for early diagnosis, disease progression monitoring and long–term drug impact evaluation. Diffusion tensor imaging (DTI) is an advanced non-invasive magnetic resonance imaging (MRI) technique that can assess microstructural changes in the living brain by measuring the quantity and direction of water molecule diffusion [3]. Although it has typically been used to study the integrity of white matter tracts, it also shows promise for studying the subtle changes of gray matter areas, because the loss of neuronal cells leads to a reduction in the water diffusivity restriction. Fractional anisotropy (FA), as one of the most commonly used parameters in DTI analysis, has been successfully applied to investigate brain modifications in PD [4]. Decreased FA values have been reported in the SN region of some PD patients [5-10]. However, others didn’t observe the significant change [11, 12]. In addition, a study on C57BL/6J mouse models of PD also suggested a decreased FA value after treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), while an increased FA value was detected in a rat model of PD induced by 6-hydroxydopamine(6-OHDA) injection [13]. The reason for these inconsistent results may be that these studies have evaluated the FA values at different time points, while the FA values in the SN may change over time. The purpose of this study was to use DTI to demonstrate the time course of FA values in the SN and to elucidate the relationship between FA values and motor dysfunction in a rat model of PD induced by 6-OHDA. DTI images were acquired at different stages before and after lesions in the same rats. Motor function was evaluated by rotational behavior and open-field tests. The 2

extent of nigrostriatal damage and gliosis was quantified by endpoint immunohistochemical staining.

2. Materials and methods

2.1. Animals Male Sprague-Dawley rats weighing 240-250 g were supplied by the laboratory animal center at Capital Medical University. Rats were housed three per cage with food and water available ad libitum in a laboratory equipped with a standard 12 h light/12 h dark cycle. Room temperature and humidity were maintained at 23 ± 0.5℃ and 60%, respectively. All animal procedures were performed according to the Ethics Committee on Animal Care and Use of Capital Medical University and were consistent with U.S. National Institutes of Health guidelines for the Care and Use of Laboratory Animals.

2.2. Unilateral 6-OHDA lesion Rats were anesthetized with 350 mg/kg chloral hydrate intraperitoneally and positioned in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) with the tooth bar set at -3.3 mm. A total of 8 μg 6-OHDA (Sigma, 2 μg/μl in 0.2% ascorbate saline) was injected into the right medial forebrain bundle (MFB) at a constant rate of 1 μl/min. The stereotaxic coordinates of the injection site were as follows: AP: -4.3 mm; ML: -1.5 mm; and DV: -7.6 to -7.8 mm, defined according to the rat brain Atlas [14]. After each injection, the needle was left in situ for 5 min and then withdrawn slowly to prevent backtracking of the drug.

2.3. DTI imaging and FA calculation All rats were scanned with a dedicated small animal MR scanner at 7.0T (ClinScan, Bruker, Ettlingen, Germany) before and 3, 7, 14, and 28 days after 6-OHDA lesions. An echo-planar imaging sequence was used to obtain the DTI data with parameters of TR = 4500 ms, TE = 3.7 ms, acquisition matrix size = 108 × 128, FOV = 33 × 40, voxel size is 200 × 200 μm, section thickness = 1 mm, slice number = 20, b value = 600 s/mm2, and NEX = 20. The region of SN was defined according to the rat brain atlas and analyzed by the same researcher using a custom standardized placement procedure as outlined in Fig. 1A. FA was calculated by average of a minimum cluster size of 10 voxels in the SN based on FA maps data with syngoMRB15 (Simens) software. FA value ratio was expressed as a ratio of the lesioned (right) to the unlesioned side (left).

2.4. Behavioral test The open-field and rotational behavior tests were performed the next day after DTI scan, i.e., day 8, 15, and 29 after 6-OHDA injection. All tests were conducted during the dark-phase (the active phase) by the same researcher. In rotational behavior tests, rats were first placed into bowls of 30 cm in diameter attached to a rotameter (Columbus Instruments, Columbus, OH, USA) and allowed to rest for 5 min to adapt to the testing environment. Then, apomorphine (Sigma, St. Louis, MO, USA) at 0.5 mg/kg was injected subcutaneously. Measurement of rotations began 5 min after injection and lasted for 30 min. The net number of turns was determined by counter-clockwise turns (contralateral to the lesion) minus clockwise turns (ipsilateral to the lesion) in 30 min [15]. The open-field test was used to evaluate spontaneous 3

locomotor activity of animals. In this test, each rat was placed in the center of the automated open-field apparatus (Columbus Instruments, Columbus, OH, USA) and the spontaneous activity was recorded for 10 min. Data were acquired and analyzed with the equipment manufacturing software (Truscan 2.0). The total distance travelled and mean velocity were used in the final analysis.

2.5. Histology and Immunohistochemical analysis Immunohistochemistry of tyrosine hydroxylase (TH) was performed as described previously [16]. Briefly, the next day after the last behavioral tests, rats were deeply anesthetized with 350 mg/kg chloral hydrate and then transcardially perfused with 0.9% saline followed by 4% paraformaldehyde. The brains were removed, postfixed overnight and kept in sucrose. Coronal sections (30 μm) were obtained from the striatum (AP: -0.3 to -1.3 mm) and SN (AP: -4.8 to -6.0 mm) by using a Leica freezing microtome (Solms, Germany). These sections were incubated overnight with a mouse antibody against tyrosine hydroxylase (TH) (1:2000; Sigma). After thoroughly washing, the sections were incubated in the biotinylated horse anti-mouse secondary antibody (1:200, Vector) for 2 h and then incubated with avidin–biotin peroxidase complex for 30 min. The number of TH positive neurons was calculated by using a method of stereotactic count with Stereo Investigator Software (Micro Bright Field, Williston, VT, USA). The optical density (OD) of TH positive neuronal fibers in the striatum was measured by using the Image-Pro Plus 6.0 software. Immunofluorescent analysis of microglia was performed almost the same way as described above, but used different antibodies. Primary antibody was against ionized calcium-binding adaptor molecule-1 (IBA-1) (1:1000, Wako Chemicals USA, Richmond, VA). The secondary Alexa 488- or Alex 594-conjugated antibodies (1:1000, Invitrogen) were used to visualize the staining. Immunofluorescent images were captured using a laser scanning confocal microscope (Leica TCS SP8, Germany).

2.6. Statistical analysis Statistical analysis was performed with Graphpad Prism 6.0 software. FA values and behavioral data were subjected to one-way analysis of variance (ANOVA), followed by post-hoc Dunnett multiple comparison tests. Whenever distributions failed the normality test, non-parametric tests such as Mann-Whitney (t-test) were used. Spearman Rank Correlation test was conducted to investigate the correlation between behavioral data and FA values. All results were presented as means ± S.E.M., and p < 0.05 was accepted as statistically significant.

3. Results

3.1. Time course of changes in FA values in the SN As shown in Fig.1, before the 6-OHDA lesion, there was no significant difference in FA values between the left and right SN. The average ratio of right to left side is 101 ± 1.61% (p > 0.05). At 3, 7, and 14 days after lesions, significantly decreased FA values were observed. The average ratios were 96.20 ± 1.40% (p < 0.05), 86.82 ± 2.81% (p < 0.01) and 96.03 ± 2.45% (p < 0.05) at 3, 7, and 14 days, respectively. However, at 28 days after lesions, a significantly less reduction of FA values was observed as compared to the values observed at 3, 7, and 14 days. 4

In fact, the FA values observed at 28 days were insignificantly different from the prelesion baseline. The absolute values of FA were presented in table 1.

3.2. Time course of changes in locomotor behavior Unilaterally lesioning the MFB resulted in a significant loss of spontaneous locomotor activity. Lesioned rats exhibited a significant decrease in total traveled distance and mean velocity during 10-min open field session at 8, 15, and 29 days after lesions. Percentages of the decrease in the total distance were 24.8 ± 4.4% (p < 0.001), 38.1 ± 5.1% (p < 0.001), and 38.9 ± 5.3% (p < 0.001) at 8, 15, and 29 days, respectively (Fig. 2A).In terms of the mean velocity, decreased percentages were 12.7 ± 3.9% (p < 0.01), 18.6 ± 3.8% (p < 0.001), and 19.1 ± 4.8% (p < 0.001) at 8, 15, and 29 days, respectively (Fig. 2B).

3.3. Time course of changes in apomorphine-induced rotational behavior Fig. 2C shows the net number of rotations induced by apomorphine in rats before and 8, 15 and 29 days after 6-OHDA lesions. Before 6-OHDA lesions, slight contralateral and ipsilateral rotations could be observed and the net number of turns was 5.43 ± 4.55 per 30 min. After lesions, apomorphine caused a dramatic increase in contralateral rotations as compared to those observed before lesions. The net numbers of turns over a 30-min period were 115.07 ± 11.77, 183.29 ± 14.48, and 236.21 ± 19.52 at 8, 15, and 29 days after lesions, respectively.

3.4. Correlation between FA values ratio and motor disorder At 7 days after lesions, a maximal decrease in the FA ratio was observed in the SN. At this time point, we analyzed the potential relation between the FA ratio and movement disorder. The results revealed a positive linear correlation between the FA ratio and total distance of movement (R2 = 0.6974, p < 0.01, Fig. 3A) or mean velocity of movement (R2 = 0.6413, p < 0.05, Fig. 3B) at 10 min. In contrast, a significant negative linear correlation was detected between the FA ratio and the net number of rotations induced by apomorphine (R2 = 0.6048, p < 0.05, Fig. 3C).

3.5. Striatal and nigral immunoreactivity In order to evaluate the extent of nigrostriatal lesions in all rats, TH immunohistochemistry was carried out after the last behavioral tests. As shown in Fig. 4, 6-OHDA lesions resulted in loss of TH positive neurons in the SN (85.1 ± 3.3%, p<0.001, Fig. 4A and C) and TH positive fibers in the striatum (74 ± 6.2%, p< 0.001, Fig. 4B and D) as compared to the intact left side. After the last behavioral tests, increased number of microglia was also observed in the SN on the lesioned side, as evidenced by an increased IBA-1 immunoreactivity (p < 0.001, Fig. 5), when compared to intact left side.

4. Discussion The present study establishes the time course of changes in FA values in the SN and the relationship between FA values and motor dysfunction in a unilaterally lesioned rat model of 5

PD induced by microinfusion of 6-OHDA into the MFB. The FA values showed a V-shape change over time. A maximal decrease in FA values was observed at 7 days after lesions. At that time, a significant linear correlation between FA values and spontaneous locomotor activity or apomorphine-induced rotations was observed. PD is characterized by the selective loss of dopaminergic neurons in the SN. Animal models have contributed a large part to our understanding of the pathogenesis and to developing innovative therapeutic approaches. While none of the currently available models can completely phenocopy this disorder. The 6-OHDA model is still the most widely used tool to study the pathogenesis of PD, even though it cannot mimic the gradual evolution of the neurodegenerative process of human PD. It can produce regional nigrostriatal degeneration and exhibit some measurable motor asymmetries very similar to idiopathic PD [17-20]. Understanding the time course of FA values using DTI in this model is helpful to develop more effective treatment and means of diagnosis of PD patients. Changes in FA values in the SN after 6-OHDA injection have been reported in rats [13, 21]. The FA value is one of the most commonly used measures in DTI analysis. Completely isotropic diffusion shows an FA value of 0, whereas in highly anisotropic diffusion the FA value is close to 1 [4, 22, 23]. It may be a sensitive predicator of brain tissue damage. Soria et al. demonstrated that FA values were significantly decreased in the ipsilateral SN at 3 and 14 days after lesion in 6-OHDA-lesioned rats [21]. Decreased FA values in the SN were also observed at 3 and 5 days in an MPTP-induced mouse model of PD by Boska et al. [24]. In addition, clinical studies show that a neuronal loss in early PD patients was accompanied by a decrease in FA values in the SN [5, 8, 25-30]. Vaillancourt et al. can even distinguish early PD patients from healthy controls by using FA values in all participants [8]. These results were in agreement with our present findings. We found that FA values in 6-OHDA-lesioned rats were significantly lower in the early phase (i.e., 3, 7, and 14 days after lesion) than the prelesion level. Decreased FA values mean the abnormal water molecule diffusion which reflects the neuronal damage after 6-OHDA injury. Indeed, Boska et al. have shown that decreased FA values were correlated with the number of dopaminergic neurons lost in an MPTP mouse model of PD [24]. In the present experiment, the FA values showed a time-dependent change. They were decreased at 7 days after lesions and then gradually recovered at 14 and 28 days. At 28 days, the reduction of FA values was completely reversed. In fact, the FA value at 28 days was slightly higher than the prelesion level, although it did not reach a significant level. Of note, while a later time point was not tested in this study, an early study found that an increase in FA values in the SN was seen at 6 weeks after 6-OHDA injection into the striatum [13]. Endpoint immunohistochemical staining showed that more than 85% of nigral dopaminergic neurons were lost at 4 weeks after lesions. At the same time point, however, FA values displayed an only slightly higher level than the baseline. Thus, FA values may not be closely correlated with the loss of dopaminergic neurons in the SN at this stage of the disease. Alternatively, FA values may be corrected to ongoing neuroinflammatory responses. Such a correlation has been observed in patients with Huntington disease [31]. Similarly, in the animal model of PD, marked gliosis and neuroinflammatory responses occur, which plays an important role in the neurodegenerative process of PD [18, 32, 33]. Silva et al. even detected an increased density of microglia cell in the SN at 1 week after 6-OHDA lesion [33]. In 6

consistent with these studies, we also observed the gliosis in the SN by IBA-1 staining at 4 weeks after 6-OHDA lesion which may account for the increase of FA values over time. In the current study, motor function was evaluated by rotational behavior and open-field tests. Previous studies have shown that the unilateral damage of the nigrostriatal dopaminergic system by 6-OHDA led to a decrease in dopamine levels and functional supersensitivity of postsynaptic dopamine receptors (D1/D2, especially D2) in the denerved striatum. These changes can be evaluated by apomorphine, apomorphine is a dopamine receptor agonist capable of stimulating both D1 and D2 receptors. The administration of APO induces contralateral rotation [34, 35]. In addition, the 6-OHDA model often exhibit motor asymmetries such as short stride, forelimb akinesia and shuffling gait patterns very similar to the key features of the human parkinsonian gait, this spontaneous locomotor activity can be assessed by an open-field test. Rotational behavior and open-field tests are considered to provide reliable behavioral indicators for nigrostriatal dopamine loss. Thus, in this study, we used these two methods to evaluate the correlation of FA values with behavioral activities. We found that FA values were correlated with behavioral changes at an early time point. At 7 days after lesions, a decrease in FA values was significantly correlated with spontaneous locomotor activity and apomorphine-induced rotations. The fact that FA values reached the lowest level at 7 days is noteworthy. Consistent with this result, other studies show that a decrease in FA values in the SN was inversely correlated with clinical severity [6, 10]. However, impaired motor activity was not correlated with an insignificant change in FA values at 28 days (this study). This seems to suggest that FA values are selectively useful for evaluating the disease severity in the early phase of PD. In summary, our findings demonstrate, for the first time in vivo, a V-shape change in FA values in the SN of a PD rat model induced by microinfusion of 6-OHDA into the MFB. A maximal decrease in FA values was observed at 7 days after lesions. At the same time point, a significant correlation was detected between reduced FA values and motor symptoms. These results suggest that DTI may be a useful tool in evaluating the SN lesion in an early phase of PD and may thus facilitate the early diagnosis of PD in clinical practice. Acknowledgments We are grateful to Prof. Shaowu Li at the Department of Radiology of Beijing Tian Tan Hospital for his excellent technical assistance during the image production. This work was supported by the National Natural Science Foundation of China (31100766, 81527901), the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges Under Beijing Municipality (IDHT20140514) and Beijing Municipal Science and Technology Commission (Z161100002616007). References [1] A.J. Lees, J. Hardy, T. Revesz,;1; Parkinson's disease, Lancet. 373 (2009) 2055-2066.

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[13] N. Van Camp, I. Blockx, M. Verhoye, C. Casteels, F. Coun, A. Leemans, J. Sijbers, V. Baekelandt, K. Van Laere, A. Van der Linden,;1; Diffusion tensor imaging in a rat model of Parkinson's disease after lesioning of the nigrostriatal tract, NMR Biomed. 22 (2009) 697-706. [14] G. Paxinos, C. Watson,;1; The Rat Brain in Stereotaxic Coordinates, Compact 6th ed. Academic Press. 2006. [15] X.B. Liang, Y. Luo, X.Y. Liu, J. Lu, F.Q. Li, Q. Wang, X.M. Wang, J.S. Han,;1; Electro-acupuncture improves behavior and upregulates GDNF mRNA in MFB transected rats, Neuroreport. 14 (2003) 1177-1181. [16] L. Liu, Y. Wang, B. Li, J. Jia, Z. Sun, J. Zhang, J. Tian, X. Wang,;1; Evaluation of nigrostriatal damage and its change over weeks in a rat model of Parkinson's disease: small animal positron emission tomography studies with [(11)C]beta-CFT, Nucl Med Biol. 36 (2009) 941-947. [17] F. Blandini, M.T. Armentero,;1; Animal models of Parkinson's disease, Febs j. 279 (2012) 1156-1166. [18] F. Blandini, M.T. Armentero, E. Martignoni,;1; The 6-hydroxydopamine model: news from the past, Parkinsonism Relat Disord. 14 Suppl 2 (2008) S124-129. [19] J. Bove, C. Perier,;1; Neurotoxin-based models of Parkinson's disease, Neuroscience. 211 (2012) 51-76. [20] S.A. Jagmag, N. Tripathi, S.D. Shukla, S. Maiti, S. Khurana,;1; Evaluation of Models of Parkinson's Disease, Front Neurosci. 9 (2015) 503. [21] G. Soria, E. Aguilar, R. Tudela, J. Mullol, A.M. Planas, C. Marin,;1; In vivo magnetic resonance imaging characterization of bilateral structural changes in experimental Parkinson's disease: a T2 relaxometry study combined with longitudinal diffusion tensor imaging and manganese-enhanced magnetic resonance imaging in the 6-hydroxydopamine rat model, Eur J Neurosci. 33 (2011) 1551-1560. [22] D. Le Bihan,;1; Looking into the functional architecture of the brain with diffusion MRI, Nat Rev Neurosci. 4 (2003) 469-480.

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[35] J.L. Waddington, A.J. Cross, A. Longden, F. Owen, M. Poulter,;1; Apomorphine-induced rotation in the unilateral 6-OHDA-lesioned rat: relationship to changes in striatal adenylate cyclase activity and 3H-spiperone binding, Neuropharmacology. 18 (1979) 643-645.

Figure 1 - Time course of changes in FA values in the SN of 6-OHDA rat models of PD. A, A schematic diagram and a series of five images illustrating changes in FA values in the regions of interest (red dotted line) in rat atlas and FA maps before and 3, 7, 14, and 28 days after lesions. B, Changes in FA values based on FA maps at corresponding time points. Data are expressed as means ± SEM (n = 8-10). *p < 0.05, **p < 0.01 vs. prelesion baseline; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. day 28 after lesions.

Figure 2 - Time course of motor impairment in 6-OHDA rat models of PD. A, Changes in total distance of movement measured 10 min before and 8, 15, and 29 days after lesions. B, Changes in mean velocity measured 10 min before and 8, 15, and 29 days after lesions. C, Apomorphine-induced rotations measured before and 8, 15, and 29 days after lesions. Data are expressed as means ± SEM (n = 8-10). ***p < 0.001 vs. prelesion baseline.

Figure 3 - Correlation of FA ratios and motor function at 8 days after lesions. A, A significant positive linear correlation between FA ratios and total distance of movement observed during 10 min. B, A significant positive linear correlation between FA ratios and mean velocity of movement observed during 10 min. C, A significant negative linear correlation of FA ratios and the net number of rotations observed during 30 min after the apomorphine injection. R2, coefficient of determination.

Figure 4 - Loss of TH positive neurons in the SN and TH positive fibers in the striatum. A and B, Immunostaining images illustrating a loss of TH positive neurons in the SN (A) and TH positive fibers in the striatum (B) on the lesioned side at 30 days after 6-OHDA injection. C and D, Quantifications of the loss of TH positive neurons in the SN (C) and TH

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positive fibers in the striatum (D) on the lesioned side. Data are expressed as means ± SEM (n = 8). ***p< 0.001 vs. left side.

Figure 5 - Immunofluorescent analysis of microglia in the SN. A, Representative immunofluorescent images illustrating TH (red) and IBA-1 (green) costaining in the SN at 30 days after 6-OHDA injection. B, Optical density analysis indicating an increased IBA-1 positive microglia density in the SN on the lesioned side. Data are expressed as means ± SEM (n = 5). ***p < 0.001 vs. left side. Table 1. Time course of changes in FA values on both sides of the SN Prelesion 3d 7d 14d 28d Left

0.368 ± 0.007

0.363 ± 0.005

0.365 ± 0.006

0.367 ± 0.005

0.364 ± 0.004

Right

0.372 ± 0.011

0.349 ± 0.008

0.316 ± 0.010

0.352 ± 0.012

0.381 ± 0.012

TDENDOFDOCTD

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