Morphological and Functional Changes of Locus Coeruleus in Patients with Primary Open-Angle Glaucoma and Animal Models

Accepted Manuscript Morphological and Functional Changes of Locus Coeruleus in Patients with Primary Open-Angle Glaucoma and Animal Models Yuxiang Yuan, Ying Zhu, Yinwei Song, Yin Zhao, Xiaoqin Yan, Wei Chen, Mu Li, Jieling Gong, Ketao Mu, Junming Wang, Hong Zhang, Zhiqi Chen PII: DOI: Reference:

S0306-4522(17)30934-X https://doi.org/10.1016/j.neuroscience.2017.12.044 NSC 18214

To appear in:

Neuroscience

Received Date: Accepted Date:

1 June 2017 23 December 2017

Please cite this article as: Y. Yuan, Y. Zhu, Y. Song, Y. Zhao, X. Yan, W. Chen, M. Li, J. Gong, K. Mu, J. Wang, H. Zhang, Z. Chen, Morphological and Functional Changes of Locus Coeruleus in Patients with Primary OpenAngle Glaucoma and Animal Models, Neuroscience (2017), doi: https://doi.org/10.1016/j.neuroscience. 2017.12.044

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Morphological and Functional Changes of Locus Coeruleus in Patients with Primary Open-Angle Glaucoma and Animal Models Yuxiang Yuan1, Ying Zhu1, Yinwei Song1, Yin Zhao1, Xiaoqin Yan1, Wei Chen1, Mu Li1, Jieling Gong1, Ketao Mu2, Junming Wang1, Hong Zhang1, Zhiqi Chen1* 1

Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan 430030, China 2

Department of Radiology, Tongji Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan 430030, China *

Corresponding author:

Zhiqi Chen Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095# Jiefang Ave, Wuhan 430030, People’s Republic of China. E-mail:[email protected]

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Abstract Primary open angle glaucoma (POAG) is a leading cause of irreversible blindness. Magnetic resonance imaging (MRI) studies report an association between POAG and non-visual pathway alterations. Locus coeruleus(LC) is a major source of norepinephrine released in the brain, and norepinephrine can reduce intraocular pressure via increasing aqueous outflow. This study aimed to investigate the relationship between glaucoma and the LC in patients with POAG and animal models. Resting-state functional MRI was performed using a 3-Tesla MR scanner with an eight-channel phased-array head coil, and MRI data were analyzed. A rat model of chronic glaucoma was generated by episcleral vein ligation and cauterization. DBA/2J mice that develop glaucoma with age were also acquired. Immunohistochemistry and immunofluorescence staining were used to investigate LC tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DβH) expression, as well as cell apoptosis by terminal deoxynucleotidyl transferase dUTP Nick-End Labeling staining. Patients with POAG showed significantly increased amplitude of low-frequency fluctuation (ALFF) in the LC compared with controls. LC ALFF values showed significant correlations with cup-to-disc ratio, retinal nerve fiber layer thickness, and visual function (P < 0.05). Functional connectivity (FC) between the LC and frontal and insular lobes was reduced, but elevated between the LC and parahippocampal gyrus, compared with controls. Glaucoma animal models revealed reduced expression of TH and DβH in the LC, and increased cell apoptosis. In this study, we provide novel evidence for the

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relationship between the LC and glaucoma. The LC may act as a network in POAG pathogenesis and intraocular pressure regulation. Keywords: Primary Open Angle Glaucoma; Locus coeruleus; Resting-state functional MRI; Norepinephrine; Tyrosine hydroxylase; Dopamine β-hydroxylase

Introduction Primary open angle glaucoma (POAG) is one of the leading causes of irreversible blindness worldwide. It is characterized by a progressive loss of retinal ganglion cells (RGCs) and visual field damage (Quigley, 2011). Many studies have verified high intraocular pressure (IOP) as the most important risk factor for retinal ganglion cell apoptosis in glaucoma (Davis et al., 2016). Clinical evidence has already shown that neuronal damage in glaucoma involves central stations of the visual pathway (Nucci et al., 2013). Recent magnetic resonance imaging (MRI) studies have also suggested that POAG abnormalities are not limited to RGCs but extend to the entire visual pathway as well as some nonvisual pathways (Frezzotti et al., 2014; Frezzotti et al., 2016). Our previous MRI studies also confirmed this point; voxel-based morphometry revealed a significant volume reduction in patients with POAG in the intracranial part of the optic nerves and the chiasma in the left lateral geniculate nucleus (LGN) and left visual cortex (VC) (Chen et al., 2013). Furthermore, resting state functional MRI revealed abnormal brain spontaneous activity in many brain regions in patients with

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POAG (Song et al., 2014). These findings indicate that POAG is not only an ocular disease, but also a neurodegenerative disease with rather widespread effects. The locus coeruleus (LC) is a major nucleus composed of noradrenergic fibers in the mammalian brain (Robertson et al., 2013). Rostral and dorsal LC neurons project to forebrain and cortical structures, while the caudal and ventral neurons innervate the cerebellum and spinal cord (Loughlin et al., 1986; Waterhouse et al., 1983). The LC is involved in modulating numerous behaviors, including sleep/wake states, attention and memory during cognitive tasks, and stress response (Berridge and Waterhouse, 2003). Most of the norepinephrine (NE) released in the brain is supplied by LC (Schwarz and Luo, 2015). Both animal and clinical experiments have verified NE-reduced IOP via increasing aqueous outflow (Pollack, 1973; Wang et al., 2015), and adrenergic alpha-agonists such as brimonidine have already been used as antiglaucoma drugs for several years (Fudemberg et al., 2008). Moreover, recent studies have reported a high prevalence of depression and anxiety in patients with glaucoma (Lim et al., 2016; Mabuchi et al., 2008;). These findings, which are similar to LC function, led us to consider the relationship between POAG and the LC. Moreover, Myagkov and Bryndina found that stimulation of the LC eliminated ocular hypertension of hypothalamic origin, but isolated electrical stimulation produced no significant changes in IOP ( Myagkov and Bryndina, 2004; Myagkov et al., 2004). These direct evidences indicated us that LC may play an important role in IOP regulation. Therefore, the aim of this study was to determine the role of the LC-NE

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system in POAG pathology via both a clinical MRI study and an experimental animal study.

Experimental procedures Subjects Twenty-two patients with bilateral POAG, 16 men and six women, 22–56 years of age (mean + SD, 34.15 ± 9.11 years), and 22 age-matched and sex-matched disease-free control subjects were enrolled in the study. This research adhered to the tenets of the Declaration of Helsinki, and was approved by the Institutional Human Experimentation Committee of Tongji Medical College, Huazhong University of Science and Technology (HUST). Informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study. The diagnostic and exclusion criteria were same as those in our previous study (Chen et al., 2013). All patients and control subjects underwent complete ophthalmologic examination including visual acuity (standard logarithmic visual acuity chart), IOP measurements (Goldmann tonometer), gonioscopy, fundoscopy with evaluation of the C/D by two experienced ophthalmologists, assessment of visual fields (Humphrey, 30–2: mean deviation, visual field index), and quantification of retinal nerve fiber layer thickness (RNFLT) using stratus optical coherence tomography (Carl Zeiss Meditec, Dublin, CA; scan type: fast RNFLT 3.4, scan length: 10.87 mm). All POAG patients have received therapy before rfMRI, 6 patients received trabeculectomy, 4 with travoprost,

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5 with travoprost and carteolol hvdrochloride, 4 with travoprost and carteolol hydrochloride and brinzolamide, 3 with trabeculectomy and carteolol hydrochloride, 1with carteolol hydrochloride, 1 with trabeculectomy and travoprost. The information of all patients were demonstrated in Table 1. Table 1 Information of the patients Group Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 POAG 1 2 3 4 5 6 7 8

Gender

Age

C/D

Female Female Male Male Male Male Female Female Male Male Male Female Female Male Female Female Male Male Male Male Male Male

46 34 35 47 35 26 59 30 32 40 31 32 45 34 30 28 27 27 27 47 48 25

OD 0.40 0.30 0.30 0.30 0.30 0.30 0.30 0.20 0.30 0.30 0.30 0.30 0.20 0.30 0.20 0.20 0.30 0.30 0.20 0.30 0.30 0.30

Male Female Male Female Male Male Male Female

24 40 22 22 26 33 36 42

0.90 0.90 1.00 0.60 0.70 0.90 0.90 0.80

MD(dB)

RNFLT（μm）

IOP (mmHg)

OS 0.40 0.30 0.30 0.30 0.30 0.30 0.40 0.20 0.30 0.30 0.30 0.30 0.20 0.30 0.20 0.20 0.30 0.40 0.30 0.30 0.30 0.30

OD -1.16 -1.20 -1.47 -0.87 -2.99 -0.72 -0.50 -1.40 -1.90 -2.35 -1.08 -1.96 -0.41 0.04 0.16 -0.81 -1.37 -1.04 -3.53 -0.80 -3.20 -1.37

OS -2.73 -0.30 -0.94 -1.65 -2.00 -2.11 -1.10 -2.20 -2.40 -3.68 -0.73 -2.00 -1.77 -0.52 -0.85 0.68 -1.92 -0.64 -3.19 -1.50 -0.70 -1.92

OD 120.00 109.67 101.00 112.29 95.43 109.70 105.23 107.91 124.25 119.69 121.25 109.43 103.73 106.17 118.64 114.74 100.90 119.01 118.44 113.90 130.83 100.90

OS 113.51 115.21 101.96 117.40 94.85 108.74 103.25 106.11 126.35 120.46 114.56 113.90 100.62 103.97 125.94 126.65 112.75 111.00 112.68 112.33 117.93 112.75

OD 12 14 15 14 14 14 10 11 13 13 13 14 16 15 13 17 18 15 15 13 18 13

OS 13 14 14 14 15 14 10 10 16 14 13 12 16 16 14 18 18 15 13 12 19 12

1.00 0.80 0.90 0.50 1.00 0.80 0.90 0.70

-7.89 -12.10 -22.90 -9.82 -16.74 -14.60 -18.90 -4.42

-33.53 -8.50 -15.40 -1.34 -30.17 -16.90 -17.40 -1.75

73.39 58.60 50.21 58.91 78.96 58.76 35.52 67.41

50.84 82.25 56.45 88.21 60.14 55.92 36.61 74.30

17 17 18 16 18 16 15 20

17 18 19 15 17 17 14 20

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9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Female Female Male Male Male Male Male Male Male Male Male Male Male Male Male Male

43 39 56 30 32 42 27 26 47 32 46 25 35 35 29 47

0.90 0.50 0.60 0.70 1.00 0.90 0.40 0.80 0.70 0.90 1.00 0.80 1.00 0.80 0.80 0.90

0.30 0.30 1.00 0.70 0.90 0.40 0.60 1.00 0.40 0.90 0.80 0.70 1.00 0.70 0.30 0.70

-26.72 -4.67 -10.08 -4.59 -26.67 -27.37 -1.17 -5.81 -14.27 -20.33 -12.08 -15.80 -15.66 -9.32 -17.24 -25.50

-0.15 0.21 -23.18 -5.70 -14.38 -3.32 -5.30 -24.35 -0.89 -18.56 -5.95 -10.18 -39.91 -1.93 -2.57 -1.95

40.25 60.15 71.44 82.26 33.58 42.04 93.66 61.38 73.62 36.84 62.26 50.81 46.03 83.00 56.11 40.00

105.58 88.63 41.99 107.30 43.19 102.67 63.91 43.06 101.79 36.07 107.40 52.51 41.31 95.00 100.56 96.00

15 20 14 20 12 13 16 14 14 16 18 14 18 11 20 19

C/D cup to disc ratio, MD mean defect, RNFLT retinal nerve fiber layer thickness, IOP intraocular pressure before rfMRI.

Data acquisition and processing For resting-state functional MRI (rfMRI) imaging, all subjects were examined using a 3-Tesla MR scanner (Signa HDxt, GE Healthcare, Milwaukee, WI) with an eight-channel phased-array head coil. Data acquisition and processing protocols are described in our previous study (Song et al., 2014). Data analysis Based on a previous study, MRIcroN (http://www.nitrc.org/projects/mricron) was employed to create the bilateral LC (highlighted volume) adjacent to the fourth ventricle on the group-mean high-resolution T1 TSE structural slice. Red symbols indicated the position of the LC nuclei (the binary LC atlas was overlaid onto the canonical brain images, Fig. 1a). Amplitude of low-frequency fluctuation (ALFF) was used to evaluate spontaneous brain activity in patients with POAG. ALFF analysis 7

15 19 14 20 14 13 17 20 16 13 17 13 18 12 20 20

was performed using the Resting-State fMRI Data Analysis Toolkit (http://restfmri.net/forum/REST). The square root of the power spectrum of blood oxygenation level-dependent (BOLD) signals from each voxel and the sum of frequencies in the low frequency band (0.01–0.08 Hz) were computed. Region of interests (ROIs) were automatically drawn on the structural image by a spherical radial of nine voxels, including the LC. According to the results, the ALFF of the LC was extracted by a ROI, which was manually drawn by structural images. Functional voxel-based analysis was performed to determine the functional connectivity (FC) between the LC and the other voxels in the whole brain. Using REST software (http://restfmri.net/forum/REST), a one-sample t-test was used to analyze the FC maps of POAG and control groups, and then a two-sample t-test analysis was used to detect the FC differences between the two groups. Pearson’s correlation was used to assess the correlation between ALFF and cup-to-disc ratio, retinal nerve fiber layer thickness, and visual function, respectively. Animals We obtained 8-month-old male DBA/2J (J000671) and C57BL/6 mice from the Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China. In addition, male Sprague-Dawley rats weighing 200 ± 20 g (mean + SD)were obtained from the Experiment Animal Center of the HUST, Wuhan, China. Animals were maintained in a room with controlled temperature (25°C) on a 12-h light-dark cycle with ad libitum access to food and water. All animal experiments were approved by

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the Institutional Animal Care and Use Committee of the HUST in strict accordance with the guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Animal models Rats were anesthetized by intraperitoneal injection with 10% chloral hydrate (0.4 mg/kg). Three episcleral veins were exposed by making an incision through the conjunctiva and Tenon's capsule at the limbal periphery of the right eye. The selected veins were carefully ligated and then cauterized (Yu et al., 2006). Rats for control was sham-operated by isolating three episcleral veins of the right eye in a similar manner without performing the cauterization. Both eyes were treated with a topical antibiotic (Tobrex; Alcon-Couvreur, Belgium) during recovery. All rats were sacrificed 2 or 4 weeks after the procedure. DBA/2J mice develop elevated IOP and glaucoma with age (John et al., 1998) Age-matched normal C57 mice served as the control. All mice were euthanized at 8 months of age. IOP measurements A Tono-Pen (Tono-Pen XL; Medtronic Inc., Jacksonville, FL) was used to measure the IOP of awake rats and mice. To allow accurate measurements of IOP in awake rats and mice, we used a training procedure described previously (Sappington et al., 2010). For IOP measurements of awake rats and mice, one drop of 0.5% proparacaine hydrochloride was applied to each eye before measurement. All IOP measurements were obtained in accordance with the manufacturer’s recommended procedures with

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identical animal handling and were performed in both eyes by the same blinded investigator. IOP for each eye was determined as the mean of seven consecutive, acceptable measurements. To control for the diurnal variation in IOP, all measurements were obtained between 10 A.M. and noon. Measurements of rats were taken before surgery and once a week after surgery, measurements of mice were taken at 8 months of age. Brn3a-labeled immunofluorescent staining Animals were euthanized and eyes were enucleated and fixed in 4% paraformaldehyde for 24 hours, and cut into 5-μm-thick sections. Sections were washed with PBS and blocked with bovine serum albumin for 60 min at 25 ℃. Sections were then incubated with primary monoclonal antibody Brn-3a (1:50, Table 2) overnight. After washed with PBS, the slices were incubated with fluorescein-isothiocyanate antibody (1:200; Table 2) for 2 h at 25 ℃. Sections were then observed under a fluorescence microscope (Zeiss 510 Meta, Zeiss, Jena, Germany) with an omnichrome air-cooled helium/neon laser set to produce emissions at 488 nm.

Immunohistochemistry and immunofluorescence staining For immunohistochemical studies, rats and mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (4 ml/kg for rats, 1 ml/kg for mice) and 0.9% NaCl (500 ml for rats, 20 ml for mice) perfused through the aorta, followed by administration of phosphate buffered saline (PBS) containing 4% paraformaldehyde 10

(500 ml for rats, 20 ml for mice). The brains were removed, post-fixed in perfusate for 24 h, and cut into 20-μm-thick sections. Sections were consecutively collected in PBS for immunohistochemical staining. Free-floating sections were blocked with 0.3% H2O2 in absolute methanol for 30 min, and nonspecific sites were blocked with bovine serum albumin for 60 min at 25 ℃. Sections were then incubated with primary antibodies against tyrosine hydroxylase (TH; 1:50) and dopamine β-hydroxylase (DβH; 1:50) (Table 2) overnight at 4°C. After washing with PBS, sections were incubated with biotin-labelled secondary antibodies for 1 h at 37°C. Immunoreactions were detected by incubation with horseradish peroxidase-labeled antibodies for 1 h at 37°C and visualized with the diaminobenzidine tetrachloride system (as a brownish-yellow color). For each primary antibody, two to four consecutive sections from each brain were used. For immunofluorescence staining, the slices were incubated with mouse monoclonal anti-TH receptor antibody and mouse monoclonal anti-DβH receptor antibody separately, at 4°C overnight. DAPI was used to stain the nucleus and an in situ terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay kit was used to detect cell apoptosis. Following three 10-min gentle washes with PBS, the slices were incubated with fluorescein-isothiocyanate antibody (1:200; Table 2) for 2 h at 25 ℃. Sections were then observed under a fluorescence microscope (Zeiss 510 Meta, Zeiss, Jena, Germany) with an omnichrome air-cooled helium/neon laser set to produce emissions at 488 nm.

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Table 2 Antibodies employed in the study Antibody Type IH IF WB Source Tyrosine Hydroxylase (TH) mAb 1:50 1:50 1:1000 Sigma Dopamine β-hydroxylase (DβH) mAb 1:50 1:50 1:1000 Sigma Donkey Anti-mouse IgG(Alexa Fluor® 488) IgG 1:200 Abcam Donkey Anti-rabbit IgG(Alexa Fluor® 594) IgG 1:200 Abcam In Situ TUNEL Assay Kit FITC-dUTP 1:100 Roche β-actin mAb 1:1000 Sigma Brn-3a mAb 1:50 Abcam IH: Immunohistochemistry staining, IF: Immunofluorescene staining, WB: Western blotting.

Counting The retina from 8 rats and 10 mice each group were used for histochemical analysis, we chose five interval sections for counting, and the retinal ganglion cell numbers of each slice was counted using Image J software (National Institutes of Health, Bethesda, Maryland). Cell numbers for each group were determined as the mean of five counts. Quantitative image analysis DβH and TH were visualized in sagittal brain sections from 8 rats and 10 mice each group by IHC staining. Using the identical microscope and camera settings, at least four digital images per sample were taken to reflect the overall staining in the pons region of brain. For the locus coeruleus, images at 20X were used. Only one image for locus coeruleus were taken. All images were analyzed using the Image-Pro Plus 4.0 (Media Cybernetics, Silver Spring, MD). The total immunoreactivity of the selected immuno-positive area was divided by the area size, and the values relative to that of the control group are presented in the graphs. The proposed method allowed

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numerical analysis of the immunoreactivity. Western blotting analysis Western blotting was performed as described previously. Briefly, equal amounts of protein extracted from rat/mouse LC were separated by 12% or 15% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were blocked with 5% BSA at room temperature for 60–90 min and then incubated overnight at 4°C with antigen-specific primary antibodies (1:1000, Table 2). Blots were subsequently incubated with species-specific horseradish peroxidase-conjugated secondary antibodies for 60 min at room temperature. Target protein bands were visualized using a chemiluminescence substrate kit (ECL Plus; PerkinElmer Inc., Covina, CA, USA), normalized to the expression level of β-actin, and quantified using Image J software (National Institutes of Health, Bethesda, MD, USA). Statistical analysis The data were analyzed using SPSS version 19.0 software (IBM Corporation, Armonk, NY). The data were analyzed using an independent sample t-test. To determine the relationship between LC ALFF values and the clinical severity of POAG, we used Pearson’s correlation to analyze ALFF and ophthalmological measurements. A P-value < 0.05 was considered statistically significant in all cases. Statistical graphs were generated using GraphPad Prism 5 software (GraphPad Software, Inc. La Jolla, CA).

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Results Patients with POAG showed significantly increased LC ALFF values ALFF represents the intensity of low-frequency oscillations (Zuo et al., 2010). It has been reported as a valuable parameter to reflect the intensity of spontaneous neural activity (Logothetis et al., 2001). In the present study, we measured ALFF values to assess spontaneous neural activity in the LC of patients with POAG. The illustration showed the anatomic location of the LC under MRI (Fig. 1A, red symbol). As a result, ALFF values in the LC were significantly increased in patients with POAG compared with controls (Fig. 1B, p = 0.018). LC ALFF variations correlated with ophthalmological measurements There was a significant positive correlation between ALFF and cup-to-disc ratio (CDR, Fig. 2A, r = 0.505, p = 0.017), as well as mean defect (MD, Fig. 2C, r = 0.428, p = 0.047). A negative correlation was found between ALFF and RNFLT (Fig. 2B, r = -0.489, p = 0.017). The results indicated that ALFF in the LC may reflect the clinical severity of POAG. Functional connectivity of the LC in patients with POAG rfMRI examines low frequency BOLD oscillations across the brain in the absence of paradigm-driven stimulation (Biswal et al., 1995). Several regions of the brain with correlated or coherent BOLD time courses are functional connected, thus forming resting-state networks (Mallela et al., 2016). In this study, we measured FC between

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the LC and other regions (Fig. 3A, 3B). Figure 3C showed peak intensity and corresponding voxels and peak coordinate. FC between the LC and right insular and frontal lobes was significantly reduced compared with controls (p = 0.006, p < 0.001), while FC between the LC and parahippocampal gyrus yielded the opposite result (p < 0.001) (Fig. 3D). Episcleral vein ligation and cauterization elevated IOP and precipitated the loss of RGCs In order to evaluate the effectiveness of our rat model, we euthanized rats 4 weeks after episcleral vein ligation and cauterization surgery, and obtained retinal tissues for. Brn3a-labeled immunofluorescent staining to assess the number of RGCs. In addition, we measured IOP before surgery and once a week after surgery. As expected, there were fewer RGCs in the surgery group relative to the control group (Fig. 4C). Persistent elevations in IOP (Fig. 4A, p = 0.124 before surgery, p = 0.134 at 1 w, p = 0.006 at 2 w, p < 0.001 at 3 w, p < 0.001 at 4 w) were also noted in the surgery group. DBA/2J mice also showed significant elevations in IOP (Fig. 4B, p < 0.001, OD, p = 0.001, OS) and fewer cells in RGC layer (Fig. 4D) compared with wild-type C57 mice. Cell counting showed significantly difference (E, p < 0.001; F, p = 0.004). These data confirmed the effectiveness of our models. Glaucoma model decreased the expression of TH and DβH in rat LC We then performed immunohistochemistry to determine the effects of our glaucoma animal model on the expression of TH and DβH in rat LC. Decreased DβH staining

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was observed in the LC of model rats compared with control rats at both 2 and 4 weeks post-surgery (Fig. 5A, B). TH staining yielded similar results (Fig. 5C, D). The difference between model rats and control rats was significant (Fig. 5E, p = 0.021, p = 0.005, p = 0.048, p =0.007, separately), suggesting that chronic high IOP decreased TH and DβH expression in rat LC. Expressions of TH and DβH were reduced in DBA/2J mice LC We then performed the same experiment on DBA/2J mice and wild-type C57 mice. Reductions in TH and DβH staining were observed in the LC of DBA/2J mice compared with C57 mice at 8 months of age (Fig. 6A, B), quantitative image analysis showed significantly difference (Fig. 6C, p = 0.034, p = 0.045, separately). The immunofluorescence staining showed similar results (Fig 6D). Cell apoptosis was elevated in DBA/2J mice LC and glaucoma model rats LC To determine reductions in TH and DβH expression in DBA/2J mice LC and glaucoma model rats LC, we used the TUNEL assay to detect cell apoptosis in the LC. There was an increase in cell apoptosis in DBA/2J mice compared with C57 mice, along with reduced expression of TH and DβH (Fig. 7A, B). Quantitative analysis showed significant difference. (Fig. 7C, p = 0.007, p = 0.023, p = 0.003, p = 0.012, separately). We repeated the same experiments on glaucoma model rats and found the results consistent with that on DBA/2J mice. Cell apoptosis also increased in rats after sugery, compared with control, along with reduced TH and DβH expression. (Fig. 8A, B). Quantitative analysis showed significant difference. (Fig. 8C, p = 0.006, p = 0.012,

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p = 0.002, p = 0.009, separately). These data suggest that chronic high IOP activated cell apoptosis in the LC, which resulted in decreased expression of TH and DβH. Western blotting analysis showed decreased expressions of TH and DβH in DBA/2J mice LC and glaucoma model rats LC To verify our findings, we used another methodology, western blotting, to assess the TH and DβH levels in DBA/2J mice LC and glaucoma model rats LC. We found reduced level of TH and DβH in DBA/2J mice compared with C57 mice (Fig. 9C). Quantitative analysis showed significant difference. (Fig. 9D, p = 0.041, p = 0.030, separately). The results were in accordance with that in our immunohistochemistry and immunofluorescence experiments. As expected, experiments on rats showed the same results (Fig. 9A, B, p = 0.034, p =0.021, separately).

Discussion This study investigated the relationship between glaucoma and the LC in both patients with POAG and in animal models. In our clinical MRI study, we used rfMRI to measure LC ALFF and FC between the LC and other regions in patients with POAG. Patients with POAG showed significantly increased ALFF values in the LC compared with controls, and the variations in ALFF values correlated with ophthalmological measurements. FC between the LC and insular and frontal lobes was significantly reduced compared with controls, while FC between the LC and parahippocampal gyrus yielded the opposite result.

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The increased LC ALFF in patients with POAG indicated increased spontaneous neural activities in the LC. Since ALFF bears similarities to fluctuations in neurophysiological, dynamic, and metabolic parameters, brain regions with decreased ALFF could experience dynamic and metabolic problems (Shmuel and Leopold, 2008). In our previous study, we already observed decreased spontaneous neuronal activity in the primary and secondary visual cortex in patients with POAG, mainly caused by apoptosis of retinal ganglion cells (Song et al., 2014). The results in the present study showed the increased spontaneous neural activities in the LC, indicating that the LC may also play an important role in POAG pathogenesis. However, it was unclear whether the LC acts as a protective or pathogenic factor. Moreover, we found a significant correlation between ALFF and ophthalmological measurements (CDR, MD, and RNFLT), which reflect the severity of POAG. This discovery may lead us to consider the possibility that ALFF in LC be used as a marker in evaluating POAG clinical severity. Reduction of elevated IOP is currently the only evidencebased treatment strategy for glaucoma (Casson et al., 2012). IOP-lowering medications include prostaglandin analogues (e.g. travoprost), b-adrenergic receptor antagonists (e.g. carteolol), carbonic anhydrase (CA) inhibitors (CAIs) (e.g. brinzolamide) and a-adrenergic receptor agonists (e.g. brimonidine) (Higginbotham, 2010). In our clinical study, all POAG patients received anti-glaucoma therapy before rfMRI and their IOPs were controlled within normal range. However, the sample size was too small and the therapy varied

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between POAG patients. Thus we were unable to group patients by therapy. Larger sample studies are needed to elaborate medication effect on LC changes in POAG patients. Studies from nonhuman primates showed that the parahippocampal gyrus plays a key role in the information flow between cortical association areas and hippocampal information (Insausti and Amaral, 2008). Echávarri et al. suggested that the parahippocampal gyrus is a highly sensitive marker that can detect AD at a very early stage (Echávarri et al., 2011). In our study, elevations in FC between LC and parahippocampal gyrus were detected in patients with POAG compared with controls, which means that parahippocampal gyrus may cooperate with the LC in POAG pathogenesis and IOP regulation. This suggests a potential new target, the parahippocampal gyrus, to study the relationship between AD and POAG. The insular lobe is a triangular area of neocortex that lies underneath the lateral (Sylvian) fissure. Recent studies found that this region is quite complex. Shura et al. concluded that this region is important in various higher-level and multimodal integrative functions, especially in relation to dysfunctions involving higher-order cognitive, emotional, and social networks (Shura et al., 2014). The frontal lobe is a large region comprising several frontal-subcortical circuits, and damage to each circuit is associated with different syndromes. Welmoed and Yolande concluded that the frontal cortical and subcortical structures, forming functional networks, are essential for maintaining a homeostatic affective and behavioral status, and for

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deciding what to do (or not to do) next (Krudop and Pijnenburg, 2015). In our study, FC between the LC and frontal and insular lobes significantly decreased in patients with POAG compared with controls. This result revealed dysfunction in the frontal and insular lobes in patients with POAG, which may be correlated with the behavior changes in patients POAG mentioned above (Lim et al., 2016; Mabuchi et al., 2008). However, owing to limitations with the imaging technique, we were unable to differentiate the subcortical structures. Future studies focused on detailed structures are needed to verify our discovery. Our rfMRI study suggested that POAG is a neurodegenerative disease affecting multiple brain regions, which is in line with the findings from recent studies (Chen et al., 2013; Frezzotti et al., 2014; Frezzotti et al., 2016; Nucci et al., 2013; Song et al., 2014). Moreover, we found that the LC and some related regions may act as a network in POAG pathogenesis and IOP regulation, but the specific effect still remains unknown. To verify our discovery, we then performed an experimental animal study. We tested TH and DβH expression in the LC in DBA/2J mice and glaucoma rats generated by episcleral vein ligation and cauterization. The levels of TH and DβH were significantly reduced compared with wild-type C57 mice and control rats. Moreover, TUNEL staining showed increased cell apoptosis in the LC in DBA/2J mice compared with C57 mice, this phenomenon also occurred in glaucoma model rats compared with control rats.

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TH is a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines, such as dopamine, NE, and adrenaline (AD) (Nagatsu T and Nagatsu I, 2016). DβH is a glycoprotein that is specifically localized in NE-containing synaptic vesicles in NE and AD neurons in the human brain (Kitahama et al., 1996). In our study, both TH and DβH expression were significantly decreased in model rats and DBA/2J mice compared with the control, indicating the dysfunction of LC and decreasing levels of NE in the brain. Moreover, TUNEL staining in DBA/2J mice showed increasing cell apoptosis in the LC, this phenomenon accounts for the LC dysfunction. However, the mechanism of cell apoptosis in the LC still remains unknown. There were studies already reported that in DBA 2/J mice, alterations in the norepinephrine uptake in the LC may associated with susceptibility to stress-induced behavioral depression (Hwang et al., 1999; Shanks et al., 1991). Here, we offered another possibility that LC alterations in DBA 2/J might also be associated with elevated IOP. Moreover, recent studies also found a high prevalence of depression and anxiety in patients with glaucoma (Mabuchi et al., 2008; Lim et al., 2016). Thus elevated IOP might also be associated with the genetic alteration of the strain, or even secondary to LC alterations caused by genetic alteration. Obviously, our studies at present are unable to explain this phenomenon. Since rare studies focused on the relationship between LC and glaucoma, further investigations are required to extend our findings and we will continue our studies on LC and glaucoma.

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In fact, our MRI study showed increased ALFF in the LC in patients with POAG, which means increased activity in the LC. Our previous study and other studies already showed degeneration in the LGN and VC in patients with POAG (Chen et al., 2013; Frezzotti et al., 2014; Frezzotti et al., 2016). Takuya et al. found increased NA terminals in the LGN in VC-ablation rats, which were supplied by LC neurons (Sakaguchi et al., 1984). Nakamura et al. also reported increased projection from the LC to LGN and VC in unilateral eye-enucleated rats (Nakamura et al., 1984). These findings suggest that the increased ALFF in the LC in patients with POAG may act as compensatory effect secondary to LGN and VC abnormalities. In our experimental animal study, decreased NA neurons and increased cell apoptosis in the LC were recorded in high IOP rats and DBA/2J mice, the results seemed inconsistent with that in our clinical study. Several differences may partially explain this phenomenon. First, in clinical study, all patients were diagnosed with POAG. In experimental animal study, however, the glaucoma animal model was secondary to high IOP, the etiology difference may account for this phenomenon. Second, Experimental animals showed elevated IOP compared with controls. However, in our clinical experiments, the IOP of all patients with POAG was controlled by anti-glaucoma medicine. This difference may partially explain the phenomenon, clinical fMRI studies on OHT patients are needed to verify this prediction. Finally, the LC effects may be diverse in different phase of glaucoma. In the early stage of glaucoma, the LC may act as compensatory effect, then turn into decompensation as the disease progress. In our clinical study, the

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sample size was too small that we were unable to group patients by clinical stage of glaucoma, further study with larger sample size may help clarify this prediction. There are still limitations in our study. In our clinical study, the sample size was too small that we were unable to divide patients into groups. Larger sample rfMRI studies are needed to have better understanding on LC alterations and POAG, such as clinical stage of glaucoma, medication effects. Moreover, elevated IOP is not the only etiology of glaucoma, not all of the patients include ocular hypertension (OHT). Thus it is necessary to include a group of patients with OHT and without glaucoma in our future study. In the animal studies, since the major concern of our study was on clinical research at present, we only conducted simply preliminary animal experiments. More functional assessments and intervention measures on animal studies are needed to better understanding the detailed relationship between LC and glaucoma. In conclusion, our study revealed a strong correlation between the LC and POAG; ALFF in the LC measured by rfMRI may act as a marker in evaluating POAG clinical severity. LC may participate in POAG pathogenesis by cooperating with other brain regions. Further investigations are required in order to confirm and extend our findings.

Acknowledgments

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Yuxiang Yuan and Ying Zhu contributed equally to this work and should be considered co-first authors. This study was supported by grants from the National Natural Science Foundation of China (grant numbers 81300760, 81470632)

Conflicts The authors have no conflicts of interest to disclosure.

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Figure Legends Figure 1. Amplitude of low-frequency fluctuation (ALFF) values measured in the LC. Red symbol shows the anatomic location of the LC. (A) ALFF values in patients with primary open-angle glaucoma are significantly higher than those of the control. (B, * p = 0.018, N = 22). Figure 2. Correlation between amplitude of low-frequency fluctuation (ALFF) values and ophthalmological measurements. There is a positive correlation between ALFF and cup-to-disc ratio (A, r = 0.505, p = 0.017), as well as mean defect (C, r = 0.428, p = 0.047). A negative correlation is found between ALFF and retinal nerve fiber layer thickness (B, r = -0.489, p = 0.017). Figure 3. Functional connectivity (FC) between the locus coeruleus (LC) and other brain regions. The illustrations show coronal (A) and horizontal (B) sections of the magnetic resonance (MR) image. Peak intensity and corresponding voxels and coordinate are demonstrated (C). FC between the LC and parahippocampa gyrus is significantly increased (*** p < 0.001), while it is significantly reduced between the LC and right insular lobe (** p = 0.006) and frontal lobes (*** p < 0.001) (D). 31

Figure 4. Effectiveness tests of the glaucoma animal model after episcleral vein cauterization and ligation. Intraocular pressure (IOP) is elevated at 2, 3, and 4 weeks after episcleral vein cauterization and ligation compared with controls (A, * p = 0.124, p = 0.134, p = 0.006, p < 0.001, p < 0.001, separately, N = 8). DBA/2J mice also show elevated IOP compared with C57 mice at 8 months of age (B, *** p < 0.001, N = 10). Brn3a-labeled immunofluorescent staining of the rat retina show decreased numbers of retinal ganglion cells at 4 weeks after episcleral vein cauterization and ligation (C). Mice at 8 months of age also show similar results (D). Cell counting show significantly difference (E, *** p < 0.001; F, *** p = 0.004). Figure 5. Pathological changes in the locus coeruleus (LC) after episcleral vein cauterization and ligation. Dopamine β-hydroxylase (DβH) expression decreases both at 2 and 4 weeks after surgery (A, B). Tyrosine hydroxylase (TH) expression shows the same results (C, D). There is a significant difference in the relative immunoreactivity between model rats and control rats (E). * p = 0.021, p = 0.005, p = 0.048, p =0.007, separately, N = 8. Figure 6. Pathological changes in the locus coeruleus (LC) in DBA/2J mice. Dopamine β-hydroxylase (DβH) and tyrosine hydroxylase (TH) expression decreased in DBA/2J mice compared with controls (A, B). There is a significant difference in the relative immunoreactivity between DBA/2J mice and C57 mice both at 8 months and 10 months of age (C). * p = 0.034, * p = 0.045, N = 10. Immunofluorescence staining shows the same results (D, E).

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Figure 7. Cell apoptosis examined in DBA/2J mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining shows increased cell apoptosis with decreased tyrosine hydroxylase (TH) expression in the locus coeruleus (LC) in DBA/2J mice compared with controls (A). Dopamine β-hydroxylase (DβH) yielded the same results (B). Quantitative analysis shows significant difference. (C, * p = 0.007, p = 0.023, p = 0.003, p = 0.012, separately, N = 10). Figure 8. Cell apoptosis examined in the locus coeruleus (LC) after episcleral vein cauterization and ligation. TUNEL staining shows increased cell apoptosis with tyrosine hydroxylase (TH) expression in the locus coeruleus (LC) after surgery, compared with controls (A). Dopamine β-hydroxylase (DβH) showed the same results. (B) Quantitative analysis shows significant difference. (C, * p = 0.006, p = 0.012, p = 0.002, p = 0.000, separately, N = 8). Figure 9. Western blotting analysis in the locus coeruleus (LC) in DBA/2J mice and glaucoma model rats. The levels of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DβH) in the locus coeruleus (LC) reduce in rats 4 weeks after surgery, compared with control. (Fig. 9A, B, p = 0.034, p =0.021, separately, N = 8). Tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DβH) levels also decrease in DBA/2J mice, compared with C57 mice. (Fig. 9D, p = 0.041, p = 0.030, separately, N = 10)

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LC ALFF variations correlated with clinical severity of POAG Glaucoma model decreased the expression of TH and DβH in LC Cell apoptosis was elevated in DBA/2J mice LC

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