The role of nitric oxide on visual-evoked potentials in MPTP-induced Parkinsonism in mice

Neurochemistry International 72 (2014) 48–57

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The role of nitric oxide on visual-evoked potentials in MPTP-induced Parkinsonism in mice Sinem Aras a, Gamze Tanriover b, Mutay Aslan c, Piraye Yargicoglu d, Aysel Agar a,⇑ a

Akdeniz University, Medical School, Department of Physiology, Antalya, Turkey Akdeniz University, Medical School, Department of Histology and Embryology, Antalya, Turkey c Akdeniz University, Medical School, Department of Biochemistry, Antalya, Turkey d Akdeniz University, Medical School, Department of Biophysics, Antalya, Turkey b

a r t i c l e

i n f o

Article history: Received 16 December 2013 Received in revised form 10 April 2014 Accepted 21 April 2014 Available online 29 April 2014 Keywords: Parkinson MPTP iNOS nNOS VEPs

a b s t r a c t The present study aimed to elucidate visual evoked potentials (VEP) changes in MPTP induced Parkinson’s disease (PD) and investigate the possible benefical effects of neuronal (n) and inducible (i) nitric oxide synthase (NOS) inhibitors on altered VEPs, lipid peroxidation and apoptosis. 3 months old C57BL/6 mice were randomly divided into 6 groups which included control (C), 7-nitra indazole treated (7-NI), S-methylisothiourea (SMT) treated, 1,2,3,6-tetrahydropyridine (MPTP) treated, 7-NI + MPTP treated and SMT + MPTP treated. Motor activity of mice was evaluated via the pole test. At the end of the experimental period VEPs were recorded, brain and retina tissues were removed for biochemical analysis. Dopaminergic neuron death at substantia nigra (SN) was determined by immunohistochemical analysis of tyrosine hydroxylase (TH). Immunohistochemical staining was also performed to determine iNOS and nNOS in all tissue sections. Mice with experimental PD exhibited decreased motor activity. Dopaminergic cell death at pars compacta of SN (SNpc) was significantly increased in MPTP treated group compared to control. Diminished Parkinsonism symptoms were observed in 7-NI + MPTP and SMT + MPTP groups. Treatment with 7-NI and SMT decreased dopaminergic cell death in MPTP treated mice. Caspase-3 activity, nitrite/nitrate and 4-hydroxynonenal (4-HNE) levels were significantly increased in SN of MPTP treated mice compared to control. Treatment with 7-NI and SMT significantly decreased elevated caspase-3 activity, nitrite/nitrate and 4-HNE levels in SN of MPTP treated mice. No significant difference in above parameters were observed in the retina. VEP latencies were significantly prolonged in MPTP group compared to control group. 7-NI and SMT treatment caused a significant decrease in VEP latencies in MPTP treated mice compared to none treated MPTP group. This data shows that 7-NI and SMT improves prolonged VEP latencies. The protective effects of 7-NI and SMT on VEP alterations can be related to decreased dopaminergic cell death and reduced lipid peroxidation. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Parkinson’s disease (PD), first described by Dr. James Parkinson in 1817, is a progressive neurodegenerative disorder of the nervous system (Kidd, 2000). The motor symptoms of PD result from the death of dopaminergic cells in the pars compacta of the substantia nigra (SNpc) Kidd, 2000; Baraboi, 1989; Kishimoto et al., 1996. The subsequent decrease in dopamine (DA) in this area results in striatal dopamine deficiency. The cardinal symptoms of PD are muscle rigidity, tremor at rest, and akinesia. The symptoms first appear ⇑ Corresponding author. Address: Department of Physiology, Faculty of Medicine Akdeniz University, Arapsuyu, 07070 Antalya, Turkey. Tel.: +90 242 249 6958; fax: +90 242 227 4483. E-mail address: [email protected] (A. Agar). http://dx.doi.org/10.1016/j.neuint.2014.04.014 0197-0186/Ó 2014 Elsevier Ltd. All rights reserved.

following 80% loss of dopaminergic cells (Kidd, 2000). Normally DA secreted in SN plays a role in the stimulation and coordination of motor functions through its effect on nucleus caudatus and nucleus putamene (Kidd, 2000). As a result of the loss of dopaminergic neurons in SN and subsequent dopamine deficiency, the affected motor functions produce motor symptoms (Kidd, 2000). The cause of this cell death is unknown, but it is within the scope of our knowledge that free radicals and lipid peroxidation increase in PD (Baraboi, 1989). Among various factors that increase lipid peroxidation, nitric oxide (NO) is of critical importance (Kishimoto et al., 1996; Krukoff, 1999). Nitric oxide is a free radical that produces various nitrogenous substances such as nitrite and nitrate in the presence of oxygen (Gutteridge, 1994; O’Donnell et al., 1997). Formation of peroxynitrite (ONOO-) in vivo has been ascribed to the reaction of

S. Aras et al. / Neurochemistry International 72 (2014) 48–57

the free oxygen radicals formed by auto-oxidation with the free radical nitric oxide (Hesslinger et al., 2009; Tieu et al., 2003). Peroxynitrite is a powerful oxidant; because of its oxidizing properties, peroxynitrite can damage a wide array of molecules in the body’s cells (Hesslinger et al., 2009; Tieu et al., 2003). The production of nitric oxide in mammals is catalyzed by three enzyme isoforms (Sener et al., 2001). These three enzymes are neuronal nitric oxide synthase (nNOS-NOS I) in the neurons, inducible nitric oxide synthase (iNOS-NOS II), and endothelial inducible nitric oxide synthase (eNOS-NOS III) in the endotelium (Tieu et al., 2003). Experimental animal models of PD induced by neurotoxic substances 1-methyl 4-phenyl 1,2,3,6-tetrahydropridine (MPTP) and 6-hydroxydopamine (6-OHDA) have revealed an increase in the expression and activity of iNOS and nNOS enzymes (Langheinrich et al., 2000). The mechanisms of these neurotoxins have been related to the pathogenesis of nigral cell death in PD (Langheinrich et al., 2000). In studies where inhibitors of iNOS ve nNOS are used, neurodegeneration has been found to be less (Langheinrich et al., 2000). The neurotransmitters DA and NO are amply present in the visual pathway and central nervous system (Sener et al., 2001; Langheinrich et al., 2000). PD also affects the visual system. Nitric oxide, found in many areas of the body, is also produced in the visual system. The enzymes nNOS and iNOS are also localized in the retina. The NO in the retina contributes to the regulation of basal retinal blood flow. Dopamine is affected by NO secretion. Nitric oxide directly reduces DA through oxidation (Djamgoz et al., 1995; Neal et al., 1999). Parallel to these findings, the inhibition of NOS increases the basal DA level in the retina, independent of the cGMP pathway (Bugnon et al., 1994). Considering that the measurement of visual evoked potentials (VEP) is a reliable method of functional evaluation of the visual system (Bodis-Wollner and Yahr, 1978; Celesia, 1984), the change in VEP latencies noted in our former experimental Parkinson models is the indispensable proof of the effect of PD on the visual system. Moreover, our former investigations have elucidated the effect of lipid peroxidation on VEP latencies (Kucukatay et al., 2006). Studies have proved that NO, iNOS and nNOS together play important roles in neuronal death in SN. In view of these data, this study was planned to assess, with VEP recordings, the roles of iNOS and nNOS in the mechanism of changes in the visual system of experimental Parkinson’s disease models where specific enzyme inhibitors were used. 2. Materials and methods Animal maintenance and treatment were carried out in accordance with the the Institutional Animal Care and Use Committee at Akdeniz University Medical School. Male C57BL/6 mice (3 months old, 25–30 g) were obtained from Akdeniz University Animal Care Unit. Animals were housed at an ambient temperature of 22 ± 1 °C, a 12 h light/dark cycle and were fed standart mice chow and tap water ad libitum up to day of the experiments. 2.1. Drug administration Animals were assigned to six groups: (1) control group (n = 30; intraperitoneally peanut oil injected group), (2) MPTP group (n = 30; intraperitoneally MPTP (Sigma-M 0896) injected group), (3) MPTP + 7-NI (Sigma-N778) group (n = 30; intraperitoneally specific nNOS inhibitor (50 mg/kg) plus after 1 h MPTP received group), (4) MPTP + SMT group (n = 30; intraperitoneally specific iNOS inhibitor plus after 15 min MPTP received group), (5) 7-NI group (n = 30; intraperitoneally specific nNOS inhibitor treated

49

group) and (6) SMT group (n = 30; intraperitoneally specific iNOS inhibitor treated group). SMT dissolved in normal saline was administered 12 h intervals for 2 days consecutively (Iravani et al., 2002). 7-NI dissolved in peanut oil (Di Matteo et al., 2009) was administered 12 h intervals for 2 days consecutively. MPTP at 20 mg/kg dissolved in 3 mg/ml saline was injected 12 h intervals for 2 days (Yokoyama et al., 2011). 3. Measurement of motor activity 3.1. Pole test The pole test has been utilized to measure motor coordination and balance in mouse models of PD (Ogawa, 1997). We performed the pole test on the 7th day after the last MPTP injection. In this test, animals were placed on top of a rough-surfaced iron pole (50 cm in length and 0.8 cm in diameter) and allowed to climb down to the base of the pole. Sticking plaster was wrapped around the iron pole to increase traction. Mice were placed at the top of the pole facing downward and latency to climb down the pole was measured. Trials were eliminated if the mouse jumped or slid down the pole rather than climbed down. On testing day, animals were placed head-up on the top of the pole. The time it took for the animal to turn its head downwards (movement initiation) and climb down the entire length of the pole was taken. Average of the best three measurements were taken as results. 3.2. VEPs recordings On the 7th day of MPTP and drug treatment, VEPs were recorded in a darkened room. The mice were anesthetized with a combination of ketamine (80 mg/kg, ip) and xylazine (16 mg/kg, ip) diluted in saline. For longer recording session, supplemental anesthesia (20% of initial dose) was given when required (Peachey and Ball, 2003). Recordings were collected with stainless steel sub-dermal electrodes (Nihon Kohden NE-223S, Tokyo Japan) shortened to 7 mm in length, resharpened and inserted along the longitudinal axis of the mouse. Electrode placements were Fpz (midline, just distal to the interorbital line; negative electrode) and Oz (midline, nuchal crest; positive electrode), with a ground electrode placed on the tail of the mouse (Strain and Tedford, 1993). After 5 min of dark adaptation, a photic stimulator (Biopac System; Nova-Strobe AB, Santa Barbara, California, USA) at the lowest intensity setting was used to provide the flash stimulus at a distance of 10 cm, which allowed the lighting of the entire pupilla from the temporal visual field. The repetition rate of the flash stimulus was 0.1 J. Throughout the experiments, the eye not under investigation was occluded by black carbon paper and cotton, allowing VEP recordings for both right and left eyes to be obtained. Meanwhile, the body temperature was maintained at 37.5–38 °C by a heating pad. The averaging of 100 responses was accomplished with the averager in the Biopac MP100 data acquisition equipment. Analysis time was 300 ms. The frequency bandwidth of the amplifier was 1–100 Hz. The gain was selected as 20– 50 lV/div. The microprocessor was programmed to reject any sweeps contaminated with larger artifacts, and at least two averages were obtained to ensure response reproducibility. Peak latencies of the components were measured from the stimulus artifact to the peak in millisecond. Amplitudes were measured as the voltage between successive peaks. 3.3. Tissue collection After motor performance test, at the end of the treatment period, mice were anesthetized with a combination of ketamine

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(80 mg/kg, ip) and xylazine (16 mg/kg, ip) diluted in saline. Mice were perfused transcardially with heparinized saline and their brain and retina tissues were removed immediately and stored at 80 °C for later biochemical analysis (Smeyne and Smeyne, 2002). For immunohistochemical studies, brain tissues containing SN were fixed in 10% formaldehyde for 24 h and washed with tap water for approximately 6 h.

3.4. Immunohistochemistry For tyrosine hydroxylase (TH), iNOS and nNOS immunohistochemistry, paraffin sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. Sections were immersed in 3% hydrogen peroxide in methanol for 20 min to block endogenous peroxidase activity. Slides were then incubated with universal blocking reagent (TA-125-UB, LabVision Ultra V BlockTHERMO) for 7 min at room temperature. Afterwards, excess serum was drained and sections were incubated with primary antibodies rabbit polyclonal anti-TH (1/400; Abcam, #ab-112), rabbit monoclonal iNOS (1/500; Cell signaling; #4236), rabbit monoclonal nNOS 1/200; Cell signaling; #4231) in a humidified chamber overnight at +4°. For negative controls, the primary antibodies were replaced by PBS. After several washes in PBS, sections were incubated with biotinylated goat anti-rabbit IgG antibody (1/400; Vector, #BA-1000) for 30 min followed by LSAB streptavidin-peroxidase complex (Vector ABC kit, VECTOR #PK400) incubation for 45 min and were rinsed with PBS. Antibody complexes were visualized by incubation with diaminobenzidine (DAB) chromogen (TA-125-HD, DAB chromogen Substrate System-THERMO). Sections were counterstained with Mayer’s hematoxylin (Dako), dehydrated, mounted and examined by an Axioplan microscope (Zeiss, Oberkochen, Germany). The images were taken using a 5MP Canon A95 camera integrated to the microscope. We used semi-quantitative evaluations to determine the amount of immunohistochemical staining. Three randomly selected slides, each of ten different fields of SN were evaluated at 40 magnification. The distinct labeled cells with observed immunostaining were counted in every lm2.

3.5. Nitrite/nitrate assay Nitrite and nitrate, oxidized forms of NO, were determined by the use of the nitrite/nitrate colorimetric assay kit (CAYMAN780001). Nitrate cannot be measured directly. In this method, the nitrate in the SN and retina samples were converted into nitrite by nitrate reductase, and the total nitrite levels were determined as the total nitrate/nitrite (NOx).

3.6. Caspase 3 activity assay Activity of caspase 3 was measured by the colorimetric assay _ following manufacturer’s protocol. with (Invitrogen-KHZ220) Whole SN and retina tissues were homogenized in 200 lL lysis buffer supplied with the kit and incubated on ice for 10 min. After centrifugation for 15 min at 10,000g, supernatants were transferred to clean tubes and assayed for caspase activity based on spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrates. Samples of SN and retina tissues were mixed with 50 lL reaction buffer and 50 lL 10 mM DTT, subsequently DEVDpNA was added and samples were incubated at 37 °C for 2 h in the dark. The absorbance was then measured at 405 nm in a microplate reader.

3.7. 4-HNE assay Lipid peroxide levels were measured in SN and retina tissues using a 4-hydroxynonenal (4-HNE) assay kit (Cell Bio Labs, STA-334) following manufacturer’s protocol. The utilized protocol is an enzyme immunoassay developed for detection and quantitaion of HNE-His protein adducts. Whole SN and retina tissues were homogenized in 200 lL PBS buffer and centrifuged for 15 min at 10,000g, supernatants were transferred to clean tubes and assayed for quantitation of HNE-His protein adduct. The quantitiy of HNE-His adduct in tissue samples were determined by comparing the absorbance measured at 450 nm with that of known HNE-BSA standards via a standard curve. 3.8. Protein measurements Protein concentrations were measured at 595 nm by a modified Bradford assay using Coomassie Plus reagent with bovine serum albumin as a standard (Pierce Chemical Company, Rockford, IL, USA). 3.9. Statistical analysis Analysis of variance (ANOVA) was performed on all parameters of VEPs for the factors of side (right and left) and groups. Differences of other data were also analyzed by ANOVA followed up with Tukey’s Post Hoc Test. Significance levels were set at P < 0.05. 4. Results 4.1. Animal health and survival All animals appeared generally healthy during experimental period. There were no difference in body weight gain and survivals were similar for all experimental groups. 4.2. Motor activity Motor activity are shown in Fig. 1A and B. Bradykinesia determined turn time and total time. Turn time and total time prolonged MPTP group compared with control group. Turn time decrease in MPTP + 7-NI and MPTP + SMT group compared with MPTP group. 4.3. Visual evoked potentials The means and standart errors of peak latencies, peak to peak amplitudes of VEP components and the results of statistical analysis in all experimental groups shown in Tables 1A and 1B. Differences in VEP parameters were analyzed by ANOVA. Measurements were made on five positive and four negative potentials which were seen all of the groups we did not find significant differences in latencies and amplitudes between right and left eyes. Therefore the data from stimulation of both eyes were averaged.iNOS and nNOS inhibitor caused a significant delay in P1, N1, P2,N2, P3, N3, P4, N4, P5 components of VEPs in 7 NI and SMT group compared with control group. P1, N1, P2, N2, P3, P5 latencies of VEP component prolonged in MPTP group compared with control group. P1, N1,N2, P3, N3 and P5 latencies of VEP component were significantly prolonged in 7-NI + MPTP group compared with control groups. P1 latency is decrease in 7-NI + MPTP group compared with MPTP group. The means and standart errors of peak to peak amplitudes of VEP components of all groups are given in Table 1B. Component P1N, N2P3 were significantly increased in SMT group compared

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Time required to turn (T-Turn) (S)

A

Total Turn Time (S)

B

MPTP group. Caspase 3 activity did not show any significant change in the retina of all groups (Fig. 2B).

T-TURN (S) 8

*

6

4.5. Nitrite/nitrate levels

4

Nitrite/nitrate levels of SN shown Fig. 3A. Nitrite/nitrate levels increased in MPTP group compared with control group. Nitrite/ nitrate level was not found to be different in 7- NI and SMT group compared with control. Nitrite/nitrate levels of SN decreased in 7-NI + MPTP and SMT + MPTP group compared with MPTP group. Nitrite/nitrate levels did not show any significant change in the retina of all groups (Fig. 3B).

2 0 Control

7-NI

SMT

MPTP

7-NI+MPTP SMT+MPTP

TOTAL TURN TIME (S)

4.6. 4-HNE levels

*

20 15

4-HNE is an indicator of lipid peroxidation. The level of 4-HNE increased in MPTP group compared control group. Specific NOS inhibitors decreased the level of 4-HNE in SN of 7-NI + MPTP and SMT + MPTP groups compared with MPTP (Fig. 4A). 4-HNE levels did not show any significant change in the retina of all groups (Fig. 4B).

10 5 0 Control

7-NI

SMT

MPTP

7-NI+MPTP

SMT+MPTP

Fig. 1. Determination of bradykinesia by pole test. (A) Time required to turn (s). (B) Total turn time. At day 7 post-intoxication, each mouse was placed head upward at the top of a rough-surfaced pole double wrapped with gauze to prevent slipping. Data are means ± SEM. P < 0.05.  vs. Control.

with control. P2N2, P4N4, N4P5 amplitude were significantly elevated in 7-NI + MPTP group compared with MPTP (Table 1B). 4.4. Substantia nigra and retina caspase-3 activity Fig. 2A shown SN caspase 3 activity levels of study groups at the end of experimental period. Caspase 3 activity increased in the MPTP group compared with control group. SMT and 7-NI had no effect in caspase 3 activity of SN. However 7-NI, SMT decreased caspase 3 activity in 7-NI + MPTP and SMT + MPTP compared with

4.7. Expression of TH in the SN The compact, reticular and lateral parts of mice SN were easily distinguished by TH immunochemistry. The immunoreactivity for TH was observed in neuron bodies and processes. NO immunoreactivity was observed in glial cells and endothelium. The TH immunoreactive neuron number in all groups were counted independently by two observers blinded to the source and type of tissue according to these counts the dopaminergic neuron numbers in MPTP groups (%63) were significantly lower compared to all the other groups. The quantitative number of immunostained cells are presented in Fig. 5C given below. Dopaminergic neuron numbers significantly increased in 7-NI + MPTP and SMT + MPTP group compared with MPTP group. There were no significant differences in neuron number of SN in 7-NI and SMT groups were compared control (Fig. 5C).

Table 1A Peak latencies of VEPs. Groups

P1 (ms)

N1 (ms)

P2 (ms)

N2 (ms)

P3 (ms)

N3 (ms)

P4 (ms)

N4 (ms)

P5 (ms)

Control 7-NI SMT MPTP 7-NI + MPTP SMT + MPTP

22.71 ± 0.73 30.21 ± 1.34# 28.50 ± 1.27# 33.96 ± 1.24# 26.08 ± 1.00# 27.21 ± 0.93#,

30.58 ± 1.27 45.00 ± 2.47# 42.42 ± 1.89# 40.38 ± 1.32# 35.33 ± 1.73# 37.71 ± 1.78#

34.67 ± 1.41 53.42 ± 2.78# 48.21 ± 1.95# 45.88 ± 1.57# 40.25 ± 1.94# 43.54 ± 2.19#

43.21 ± 1.29 62.92 ± 2.72# 57.92 ± 2.31# 54.67 ± 1.86# 52.38 ± 2.04# 54.33 ± 2.76#

50.04 ± 1.92 78.08 ± 3.80# 72.88 ± 4.00# 60.58 ± 2.20# 56.92 ± 4.23# 64.58 ± 3.76#

58.79 ± 2.27 87.38 ± 4.38# 80.87 ± 4.07# 67.67 ± 2.03# 65.58 ± 5.05# 73.67 ± 3.87#

82.21 ± 4.50 103.88 ± 3.40# 97.33 ± 4.08# 88.96 ± 2.65# 77.08 ± 6.03* 90.88 ± 6.59#

90.92 ± 4.23 120.04 ± 3.95# 110.04 ± 3.94# 95.54 ± 2.90# 84.38 ± 8.39* 101.63 ± 7.49#

111.58 ± 2.95 157.08 ± 5.53# 146.71 ± 3.93# 130.50 ± 2.12# 124.67 ± 6.30#, * 146.25 ± 5.75#, *

*

The means and standart errors of peak latencies for each VEP component. The mean value of each component was determined by average the data both eye. # Compared with control P < 0.05. * Compared with MPTP P < 0.05.

Table 1B VEPs amplitudes. Groups

P1N1 (lV)

N1P2 (lV)

P2N2 (lV)

N2P3 (lV)

P3N3 (lV)

N3P4 (lV)

P4N4 (lV)

N4P5 (lV)

Control 7-NI SMT MPTP 7-NI + MPTP SMT + MPTP

8.80 ± 2.88 15.21 ± 3.49 19.26 ± 3.96# 7.97 ± 1.90 13.99 ± 3.54 13.17 ± 2.82

5.71 ± 1.34 6.13 ± 1.26 5.31 ± 1.41 5.12 ± 1.39 3.63 ± 0.89 4.41 ± 1.12

9.34 ± 2.06 8.45 ± 2.07 10.74 ± 3.03 7.69 ± 1.68 15.79 ± 3.30* 14.20 ± 2.85*

5.90 ± 1.65 12.17 ± 3.10 15.23 ± 3.79# 3.82 ± 0.68 6.29 ± 1.80 10.22 ± 2.73

8.41 ± 3.18 4.70 ± 1.70 6.69 ± 1.47 3.65 ± 0.88 5.65 ± 1.36 6.77 ± 1.86

18.14 ± 4.72 8.93 ± 2.06 11.57 ± 3.66 14.34 ± 3.08 9.57 ± 2.97 17.33 ± 4.04

3.79 ± 1.03 5.79 ± 1.10 5.44 ± 1.73 2.85 ± 0.50 6.15 ± 1.58* 2.64 ± 0.66*

11.38 ± 2.70 13.65 ± 2.15 18.64 ± 4.59 8.92 ± 1.84 17.97 ± 4.24* 14.30 ± 2.18*

The means and standart errors of peak to peak amplitudes of each groups. The mean value of each component was determined by averaging the data both eyes. Values are expressed as means ± SEM for 10 mice each group. # Compared with control P < 0.05. * Compared with MPTP P < 0.05.

S. Aras et al. / Neurochemistry International 72 (2014) 48–57

SN Caspase-3 activity (fold increase)

A

1,6 1,4

*

1,2

**

1

**

0,8 0,6 0,4

A

5

SN 4-HNE (ng/ug tissue protein)

52

4

0,2 0 Control

SMT

1,2

3 2 1 0

MPTP 7-NI+MPTP SMT+MPTP

B

Control

7-NI

SMT

MPTP 7-NI+MPTP SMT+MPTP

Control

7-NI

SMT

MPTP 7-NI+MPTP SMT+MPTP

40

1

Retina 4-HNE (ng/ug tissue protein)

Retina Caspase -3 activity (fold increase)

B

7-NI

*

0,8 0,6 0,4 0,2

32

24

16

8

0 Control

7-NI

SMT

MPTP 7-NI+MPTP SMT+MPTP

0 Fig. 2. The effect of 7-NI and SMT on caspase-3 activity in the control and MPTP treated groups in SN and retina tissues. (A) Substantia nigra Caspase-3 activity. (B) Retina Caspase-3 activity. Caspase-3 activities in all groups were measured by a caspase colorimetric activity assay kit. Data are means ± SEM.  Compared with control group P < 0.05,  compared with MPTP group P < 0.001.

SN Nitrate/Nitrite (nmol/mg protein)

A

MPTP group. The reactions were clearly increased in MPTP groups (Fig. 6A and B).

*

10 8

5. Discussion

6 **

4

**

**

2 0

Retina Nitrate/Nitrite (nmol/mg protein)

B

Fig. 4. 4-Hydroxynonenal levels. (A) Substantia nigra and (B) retina. Data are means ± SEM.  Compared with control group P < 0.05.

Control

7-NI

SMT

MPTP 7-NI+MPTP SMT+MPTP

Control

7-NI

SMT

MPTP 7-NI+MPTP SMT+MPTP

25 20 15 10 5 0

Fig. 3. Nitrite/nitrate levels (A) substantia nigra and (B) retina. Data are means ± SEM.  Compared control and 7-NI groups P < 0.05,  compared with MPTP group P < 0.001.

4.8. NOS immunoreactivity iNOS and nNOS immunoreactivities were slightly observed in neuron cyctoplasm in control and 7NI, SMT treatment groups. Moreover, both of the antibodies immunoreaction resulted in moderate staining intensities in 7NI-MPTP and SMT-MPTP groups. However, iNOS and nNOS showed strong immunoreactivities in

Parkinson’s disease also affects the visual system; this effect is elucidated by a reliable parameter, the elongation of VEP latencies (O’Mahony et al., 1993). In our study, the use of specific inhibitors of iNOS and nNOS resulted in improvement. This finding was supported by biochemical parameters. NO plays a role in the processing of vision from the lowest level of retinal tranduction to the control of neuronal excitability in the visual cortex (Lima et al., 2014). Thus, there is no doubt that parkinson induced lipid peroxidation and increase in NO synthesis can cause changes in brain and retinal functions. Consequently, it could be expected that iNOS and nNOS inhibitors improve visual system changes. In this regard, our study was undertaken to investigate the effect of iNOS and nNOS on visual system by means of visual evoked potentials which consist of several components arising from retina, optic pathway, subcortical and cortex. We have focused on the effect of lipid peroxidation on altered VEP latencies in this study. Other parameters reported within the manuscript validate the MPTP-induced Parkinsonism in our experimental settings. Since induction of experimental PD in man is not possible, animal models of PD have been used in the search for clues to the underlying cause of the disorder and in the discovery of novel treatments. The neurotoxic chemicals widely used in animal models of PD are 6-OHDA, MPTP, paraquat, rotenon and manneb (Baltazar et al., 2014; Qi et al., 2014). These neurotoxins cause mitochondrial dysfunction by inhibiting the Complex-I or Complex-III in the mitochondria. 6-OHDA is the hydroxyl analog of DA. It destroys almost all dopaminergic neurons and also adrenergic and serotonergic neurons. Since 6-OHDA cannot pass through the blood–brain barrier, it is injected into the Medial Forebrain

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Fig. 5. (A) Localization of tyrosine hydroxylase (TH) protein in SN. TH immunoreactivity in the control group was almost the same as in 7NI and SMT groups. The immunostaining observed in the MPTP group was lower than other Parkinson experimental groups. Also, the immunoreactivity of 7NI-MPTP and SMT-MPTP groups were almost the same as in control, 7NI and SMT groups. No immunoreactivity was observed on when TH antibody was replaced with normal rabbit IgG (negative control). Magnification is presented 2.5. (B) Photomicrographs of TH immunohistochemistry in the dopaminergic neurons. The neuronal morphological alterations, the decrease of TH immunoreactivity and loss dopaminegic fibers can be seen in the MPTP group. TH increased in 7-NI and SMT groups. Representative pictures are presented in high magnification (10). (C) TH+ cell number per lm2.

Bundle (MFB) unilaterally or bilaterally by using the stereotaxic method (Betarbet et al., 2002). The MPTP-treated primate model of PD, which closely mimics the clinical features of PD is undoubtedly the most clinicallyrelevant of all available models. This model which also mimics the first stage of PD is the most widely-used model for the study of neurochemical and neuropathologic changes in this disorder (Wichmann and DeLong, 2003). There are also MPTP-treated mouse and rat models for the study of PD (Bazzu et al., 0000. MPTP selectively shows toxic effect on dopaminergic neurons. This chemical is administered to rats by using the stereotaxic method. In mice it destroys the dopaminergic neurons in the SN (Betarbet et al., 2002). Lesion formation is dependent on the dose of MPTP administered. Rats are resistant to the degenerative effect of MPTP on dopaminergic neurons (Betarbet et al., 2002). MPTP can be administered to mice by gavage feeding, subcutaneously,

intravenously, intraperitoneally or intramuscularly (Bazzu et al., 0000). The degree of dopaminergic damage depends on the dose and application protocol of the chemical. The C57BL/6 strain of mice is more susceptible to systemic MPTP than other strains of mice, where the chemical selectively damages the dopaminergic neurons (Castagnoli et al., 2001). In view of these data in literature, in our animal model we preferred to administer MPTP intraperitoneally at a dose of 4  20 mg/kg with 12-h intervals for 2 days (Date et al., 1990). The purpose of this application was to decrease the risk of death in the animals. In experiments with higher doses there was a higher rate of death. The damage induced in animal models, particularly the damage in their dopaminergic and adrenergic neurons, is assessed with motor performance tests. In our study, peg test was used to assess the motor activity in Parkinson models. With peg test we determined the degree of bradykinesia, one of the cardinal symptoms

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Fig. 5 (continued)

of PD. Time of delay in executing the command to move shows the degree of bradykinesia (Ogawa, 1997). In our model, prolonged reaction time was noted both in starting to move as well as in return. This result showed that our animal model was successfully built. Upon administration of iNOS and nNOS with MPTP, the level of bradykinesia fell down to that of the control, a result which underlines the role of iNOS and nNOS in motor movement impairement in PD. To obtain additional proof of the success of our animal model, the number of TH immunoreactive cells was assessed with immunohistochemical methods. We found a decrease of 63% in the cells of SN in the MPTP group. This result also indicates the success of our model. We also noted the protective effect of the inhibitors of iNOS and nNOS, a finding supported by biochemical parameters.

Although the pathogenesis of PD has not been fully clarified until present time, studies have elucidated the role of lipid peroxidation, a process implicated in the pathogenesis of various disorders (Pall et al., 1986). Nitric oxide is quite reactive and unstable gas taking part with other agents in lipid peroxidation (Jenner, 1998). Studies have shown the role of NO in forming free radicals and neurodegeneration (Jenner, 1998). Nitric oxide has been found to be localized to the central nervous system (hippocampus, striatum) as well as to the visual system (Vielma et al., 2012). Former studies have shown an increase in the expression and activity of nNOS and iNOS in PD (Levecque et al., 2003). In relevance to these findings, we assessed the role of NO in visual system changes in PD, using the inhibitors of nNOS and iNOS. The doses and routes of administration of the inhibitors were determined after considering

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Fig. 5 (continued)

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other studies on the subject in literature (Przedborski et al., 1996; Tran et al., 2001). Because nitric oxide is a molecule that is rapidly oxidized by the interaction of oxygen to nitrite and nitrate and has a very short half-life, its direct measurement in biologic tissues is difficult. Although sensors for instant measurements have been developed in recent years, measurement methods related to oxides of nitrogen such as nitrite and nitrate are still in use (Boo et al., 2007) For this reason, we measured nitrite/nitrate as NO index. A significant increase was detected in the nitrite–nitrate level in SN of the MPTP group when the control group was compared with the MPTP group. This finding shows that MPTP induces nNOS and iNOS, a result in agreement with studies demonstrating an increase in nitrite/nitrate levels in MPTP- and 6-OHDA models (Guo et al., 2007). Moreover, while the basal level did not change in groups receiving only inhibitors of nNOS and iNOS, nNOS and iNOS combined with MPTP decreased the nitrite/nitrate levels, a result

Fig. 6. Localization of iNOS and nNOS expression in substantia nigra. Representative pictures are presented from each experimental group. Magnifications are presented as 2.5 (upper panel) and 20 (lower panel). (A) SN iNOS immunoreactivity in all groups. iNOS immunoreactivity was observed in neurons. Higher immunoreactivity was seen in the MPTP group. iNOS immunolabelling was not observed in dopaminergic neurons in SN of control and SMT groups (arrowheads). SMT decreased iNOS immunoreactivity induced by MPTP. (B) SN nNOS immunoreactivity in all groups. Localization of nNOS immunoreactivity was clearly seen in MPTP group compared to the control and 7NI groups (arrows). The immunoreaction was also observed in 7NI-MPTP group (arrows).

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supporting former studies on the subject (Levecque et al., 2003). This result indicates the importance of nNOS and iNOS in neuronal apoptosis. No change was detected in the levels of nitrite/nitrate in the retina. Therefore, our result indicates that the administration of MPTP does not cause an increased expression of the neurotransmitter NO or of nNOS and iNOS in the retina as much as it does in SN, or it indicates that the neuromelanin-containing dopaminergic neurons in the retina are less than those found in SN. Another interpretation of this result is that MPTP in the retina cannot be converted to MPP+. The active form of MPTP is the free radical MPP+; conversion to MPP+ is mediated by the enzyme MAO-B in glial cells (Kalgutkar and Castagnoli, 1992). This mechanism could not be efficient in the retina, or other mechanisms could be more effective. In this study, the levels of lipid peroxidation were determined with 4-HNE measurements in order to evaluate the relationship between changes in VEP and oxidative damage. By dissociation of lipid peroxides, aldehydes such as 2-alkenes, 2,4 alkenes and 4-hydroxyalkenes form. The hexzonal Omega-6, formed by the oxidation of fatty acids and 4-HNE are the primary aldehydes. 4-HNE bears cytotoxic, mutagenic and genotoxic properties (Soulere et al., 2007). Oxygen radicals make up the secondary toxic messenger of lipid peroxidation. The role of lipid peroxidation in certain pathologies is well known. In PD, lipid peroxidation and the level of 4-HNE in SN are increased (Andersen, 2004). The increase in 4-HNE stimulates the caspase 3-8-9 activation which triggers the apoptotic process (Ji et al., 2001). Moreover, 4-HNE causes aggregation of a-synuclein (Hattoria et al., 2009) and fragmentation of DNA (Zhou and Zhu, 2009). Our study showed that 4-HNE increased quantitatively when MPTP was administered. The effect of MPTP is mediated by nNOS and iNOS (Levecque et al., 2003). This increase in 4-HNE is mediated by NO and free radicals formed by nNOS and iNOS (Levecque et al., 2003). In agreement with this fact, in groups that received iNOS and nNOS inhibitors we found a significant decrease in 4-HNE in SN, but no significant decrease in the retina. These results indicate that, beside the roles of iNOS and nNOS, the COX-2 pathway and other pathways might play a role in lipid peroxidation. In our former study we showed that MPTP caused an increase in the COX-2 activity and lipid peroxidation (Ozsoy et al., 2011). On the other hand, there was no difference between the groups in regard to the levels of MPTP in the retina. This result indicates that the retina is not affected by MPTP as much as SN. In literature, no data exists on the effect of MPTP on 4-HNE and nitrite–nitrate levels in the retina. When the caspase-3 activity as indicator of apoptosis was studied, in SN and retina the following were observed: in MPTP-groups of PD the caspase-3 activity in the retina showed no change, but was increased in the SN, and in groups receiving nNOS and iNOS inhibitors, the caspase-3 activity was less than that found in MPTP-groups. These findings are in agreement with the results of the study by Brzozowski et al. (2011). It has been reported that nNOS and iNOS have roles in apoptosis, and inhibition of iNOS leads to a decrease in caspase-3 activity (Du et al., 2001). These results support our study. The inhibition of nNOS and iNOS shows a protective effect (Brzozowski et al., 2011). The immunohistochemical results and biochemical parameters found in this study affirm that in experimental PD the inhibition of iNOS and nNOS exerts a corrective effect. The examination of TH-stained dopaminergic neurons in SN showed a decrease in the number of TH-stained neurons when MPTP was administered. This result is in agreement with literature (Kastner et al., 1994). When MPTP was given in combination with SMT and 7-NI, the consecutive specific inhibitors of iNOS and nNOS, the formerly decreased neuron count showed an increase. This result indicates the crucial roles played by iNOS and nNOS in apoptosis and

neurodegeneration. Nevertheless, the inhibitors of iNOS and nNOS never caused neuron counts equal to those in the controls. This result points out the role of other mechanisms in apoptosis. With conversion of MPTP into MPP+ apoptosis is induced. As MPTP turns into MPP+, mitochondria damage 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) and increase NO production, thus by violating redox balance cause the death of dopaminergic cells. Excess production of NO causes the loss of mitochondrial membrane potential, secretion of cytochrome-c and binding with APAF-1 which together increase the caspase-3 activity (Eberhardt and Schulz, 2003). nNOS, iNOS and eNOS can also be expressed under normal circumstances. The conversion of MPTP into MPP+ causes an elevation of intracellular Ca+2 which stimulates nNOS activity and also causes increased NO production. Moreover, factors secreted by microglia and astrocytes play a role in apoptosis by increasing the expression of iNOS and leading to the production of MPP+. Our findings show that the inhibition of iNOS and nNOS prevents apoptosis, but not in the level of the control group. The intracellular accumulation of Ca+2 damages mitochondrial functions and activates phospholipase, protease and endonuclease; moreover, apoptosis has a role in the COX-2 pathway (Brown, 2010). In our study, the inhibitors of iNOS and nNOS prevented apoptosis to some degree, decreased the caspase-3 activity, and diminished the 4-HNE quantity. These decrements would not be caused only by iNOS and nNOS, but also by other pathways as well. Since no change indicative of apoptosis was observed in the retina, parameters such as caspase-3 activity, 4-HNE and nitrite/nitrate were not immunohistochemically evaluated. In our former study on PD, no immunohistochemical change had been found (Ozsoy et al., 2011). In patients with PD the visual system is also disturbed (Buttner et al., 1996). Varying with the severity and duration of PD, VEPs show prolongation of P100 latency and changes in amplitude (Okuda et al., 1995). Also ERG amplitudes are lowered and threshold values are increased (Nightingale et al., 1986). Particularly the P100 latency is prolonged, which is identical with the P3 latencies in animals. Nitric oxide and sGC are amply present in photoreceptors and the retina, within Müller, amacrine, bipolar, horizontal and ganglion cells. nNOS and iNOS are present in the retina. Nitric oxide has a role in the regulation of light-dependent gap junctions (Vielma et al., 2012; Pang et al., 2010). Nitric oxide modulates the phototransduction cascade and neurotransmitter release while inhibiting DA release. When the inhibitors of iNOS are nNOS were given, the latencies of all VEP components were prolonged in relation to the control group. MPTP was found to cause prolongation in the latencies of VEP components. This result is in agreement with the data in literature (Ozsoy et al., 2011). Following the administration of the nNOS inhibitor, correction was observed in P1, P4, and N4; while P1 was corrected, P5 was prolonged. Our study shows that in the visual system the presence MPTP increases the expression of nNOS whereas its inhibition corrects many components. In the visual system nNOS and iNOS induce the production of NO, a significant neurotransmitter and neuromodulator. The data obtained indicates that nNOS and iNOS exert their effect in the central nervous system rather than in the retina. It has been concluded that in MPTP-treated mouse models of PD the visual system is affected and the inhibitors of nNOS and iNOS provided a protective effect against damage in dopaminergic neurons. Nevertheless, the observation that the VEP values in the treated groups did not attain the VEP values of the control groups indicates the likely contribution of other pathways in the involvement of the visual system. For elucidation, further studies are required.

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