Neurotox Res DOI 10.1007/s12640-016-9693-6
ORIGINAL ARTICLE
Coenzyme Q10 Prevents Mitochondrial Dysfunction and Facilitates Pharmacological Activity of Atorvastatin in 6-OHDA Induced Dopaminergic Toxicity in Rats Santosh Kumar Prajapati 1 & Debapriya Garabadu 1 & Sairam Krishnamurthy 1
Received: 9 August 2016 / Revised: 20 December 2016 / Accepted: 22 December 2016 # Springer Science+Business Media New York 2017
Abstract Atorvastatin (ATV) generally used to treat dyslipidemia is also reported to have effect against 6hydroxydopamine (6-OHDA) induced neurotoxicity. Additionally, atorvastatin can interfere with mitochondrial function by reducing the level of Q10. Therefore, the therapeutic effect of atorvastatin (20 mg/kg) could be compromised. In this context, the present study evaluated the effect of ATV supplemented with Q10. 6-OHDA was unilaterally injected into the right striatum of male rats. On day 8 of 6OHDA infusion, ATV (20 mg/kg), Q10 (200 mg/kg), and their combination were administered per oral for 14 days. On day 21, there was significant loss of striatal dopamine indicating neurotoxicity. The combination of ATV+Q10 showed significant amelioration of dopamine (DA) toxicity compared to individual treatments. Similarly, ATV+Q10 compared to individual treatment significantly decreased the motor deficits induced by 6-OHDA. Further, 6-OHDA induced mitochondrial dysfunction in the substantia nigra pars compacta (SNpc). There was significant decrease in mitochondrial complex enzyme activities and mitochondrial membrane potential (MMP). Treatment with ATV and ATV+Q10 ameliorated mitochondrial dysfunction by increasing complex enzyme activities; however, only ATV+Q10 were able to stabilize MMP and maintained mitochondrial integrity. Moreover, there was significant induction of oxidative stress as observed from increase in lipid peroxidases (LPO) and nitrite (NO), and decrease in super oxide dismutase (SOD). Treatment with ATV+Q10 significantly altered the above * Sairam Krishnamurthy
[email protected];
[email protected] 1
Neurotherapeutics Laboratory, Department of Pharmaceutics, Indian Institute of Technology (Banaras Hindu University), Varanasi, U.P 221 005, India
effects indicating antioxidant activity. Furthermore, only combination of ATV and Q10 decreased the 6-OHDA induced expression of cytochrome-C, caspase-9 and caspase-3. Therefore, current results provide evidence that supplementation of Q10 with ATV shows synergistic effect in reducing dopamine toxicity. Keywords Mitochondrial dysfunction . Oxidative stress . Apoptosis . Dopaminergic toxicity . Atorvastatin . Q10
Abbreviation PD Parkinson disease 6-OHDA 6-Hydroxyl dopamine SNpc Substantia nigra pars compacta cyt-C Cytochrome-C ATV Atorvastatin Q10 Coenzyme Q10 L-DOPA Levodopa MMP Mitochondrial membrane potential LPO Lipid peroxydation NO Nitric oxide SOD Superoxide dismutase
Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is primarily characterized with degenerated dopaminergic neurons in the nigrostriatal pathway (Soliman et al. 2002). 6-OHDA causes PD-like symptoms in rats after 1 week of unilateral injection (Kumar et al. 2012). 6-OHDA reduces 85% of nigral neurons and the number of tyrosine hydroxylase (TH) immunoreactive cells on the lesioned side (Bowenkamp et al. 1996; Capitelli et al. 2008; Delattre et al.
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2010). There is 50% deduction in striatal dopamine concentration in 6-OHDA infused rats (Kearns et al. 1997; Vercammen et al. 2006; Delattre et al. 2010; Silva-Adaya et al. 2011). Although the exact pathophysiology is still unknown, major processes such as neuroinflammation, oxidative stress, and mitochondrial function are considered to be important for development of PD (Sairam et al. 2003; Lee et al. 2011; Klusa et al. 2013; Wang et al. 2016). Apart from the above manifestation, 6OHDA also activates 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and produces hypercholesterolemia and an increase toxic metabolite to the neurons in substantia nigra (SN) (Lelorier et al. 1976; Kumar et al. 2012), that may be the one cause of PD, unilateral injection of 6-OHDA resulted in the behavioral alterations, oxidative damage, and mitochondrial enzyme complex dysfunction (Chen et al. 2015). Atorvastatin (ATV) may be a suitable drug for the treatment of neurodegenerative conditions due to its higher BBB penetration capacity, cholesterol lowering effect on neurons with a satisfactory safety profile, and in vitro protection against cell death in neurodegenerative condition (Sierra et al. 2011; Griffiths et al. 2014). ATV decreased TH immunoreactivity and rescued the neuronal loss in SN region (Sabogal et al. 2014). It has been reported that ATV prevents 6-OHDA induced neurotoxicity through inhibiting mitochondrial complex-I enzyme (Kumar et al. 2012). However, ATV can cause mitochondrial dysfunction by interfering cholesterol-dependent pathway and reduction of Q10 (Eghbal et al. 2014). Q10 significantly reduces mitochondrial toxicity in several studies (Bonuccelli and Del Dotto 2006; Eghbal et al. 2014). Mitochondrial dysfunction is a major contributor to neurodegeneration and causes vulnerability to oxidative stress and the activation of downstream cell death pathways. Further, it has been demonstrated that mitochondrial dysfunction is one of the predisposing factor in the pathogenesis of PD (Mizuno et al. 1998; Von Stockum et al. 2016). Previous studies have reported that ATV exhibits anti-PD-like activity in several animal models (Youssef et al. 2002). However, ATV inhibits the biological pathway involved in synthesis of Q10 that causes alteration in mitochondria integrity followed by apoptotic activity through increasing the expression of caspase-9 and caspase-3 (Caner et al. 2007; Eghbal et al. 2014). This indicates the fact that ATV may aggravate mitochondrial dysfunction. Based on these observations, we assume that drugs which can improve mitochondrial dysfunction may improve the pharmacological effect of ATV. Several agents such as selegiline, Q10, and creatine that modulate mitochondrial bioenergetics show anti-PD effect on parkinsonian rats (Bonuccelli and Del Dotto 2006). Mitochondrial dysfunction due to ATV could lead to loss of Q10. Therefore, supplementation of Q10 could improve the potency of ATV to reduced dopaminergic toxicity. Therefore, the present study evaluates the effect of ATV and its combination with Q10 in 6-OHDA model of dopaminergic toxicity in experimental rats. 6-hydroxydopamine (6-
OHDA) induced unilateral lesion is a well-known experimental model of dopaminergic toxicity (Ungerstedt 1968).
Materials and Methods Animals Inbred adult albino male rats of Charles-Foster strain (260 ± 20 g) were procured from the Central Animal House; Institute of Medical Sciences, Banaras Hindu University. The animals were housed in polypropylene cages under controlled environmental conditions of temperature of 25 ± 1 °C and 45– 55% RH and a 12:12 h light/dark cycle. The experimental animals had free access to commercial rat feed (Doodhdhara Pashu Ahar, India) and water ad libitum during the experiment. All experiments were conducted in accordance with the Principles of laboratory animal care (NIH publication number 85–23, revised 1985) guidelines. The experimental procedures were approved by the Institutional animal ethical committee, BHU (Dean/11-12/188). All animals were acclimatized for at least 1 week before using them for experiments. Materials The 6-OHDA (Sigma, St. Louis, MO, USA), atorvastatin (Ranbaxy Research Laboratories, Gurgaon, India), L-DOPA (Sigma, St. Louis, MO, USA), and Q10 (Sanofi India Ltd., India) were procured. All other chemicals for high performance liquid chromatography (HPLC) and analytical grade were procured from local supplier (HiMedia Pvt. Ltd., India). Surgery and Microinjection The scalp of an anesthetized rat (sodium pentobarbital; 35 mg/ kg; i.p.) was incised and retracted, and the head was positioned to place bregma and lambda in the same horizontal plane in a stereotaxic frame (Stoelting, USA). A small hole of 1.5 mm depth was drilled on the right sides of striatum and stainlesssteel guide cannulas were implanted (0.8 mm lateral, 2.9 mm anteroposterior, and 5.8 mm dorsoventral from bregma point; Paxinos and Watson 1986). 6-OHDA dissolved in 0.9% normal saline (100 nmol; pH 7.2; 4 μl/animal; 0.2 μl/min) was injected through a Hamilton syringe via polyethylene tube (Sairam et al. 2003, Kumar et al. 2012) into the right striatum. Experimental Design Atorvastatin and L-DOPA were suspended in 0.25% w/v sodium carboxymethyl cellulose (CMC). 6-OHDA was dissolved in saline containing 0.2 mg/ml ascorbic acid. Atorvastatin (20 mg/kg; Kumar et al. 2012) were administered in a constant volume of 5 ml/1000 g body weight.
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Q10 was also administered to rats at 200 mg/kg/day orally (Beal et al. 1998) and L-DOPA in a dose of 10 mg/kg/day orally (Alam and Schmidt 2004). The animals were randomly selected and divided in to six groups which are the control, 6-OHDA, 6OHDA+ATV, 6-OHDA+Q10, 6-OHDA+ATV+Q10, and 6OHDA+L-DOPA sequentially. All the abovementioned drugs were given orally through oral gavages needle, after the seventh day of injection of 6-OHDA. The treatment was continued up to 14 days excluding the seventh day taken to induce dopaminergic neurotoxicity. Animal were subjected to behavioral tests on D-0, 7, 14, and 21 to understand the parkinsonism-like behavioral deficits. The animals were then immediately killed by cervical dislocation. The striatum and SN from both hemispheres was microdissected according to the coordinates of Paxinos and Watson (1986). The striatum and SN were stored at −80 °C until further mechanistic studies. Estimation of Striatal Monoamines and Their Metabolites The level of dopamine (DA), 3,4-dihydroxy phenyl acetic acid (DOPAC), and homovanillic acid (HVA) were estimated in the striatal fraction of all the animals in each group following standard protocol using HPLC system with electrochemical detector (Garabadu et al. 2011). The protein content was estimated using the method of Lowry et al. (1951). Behavioral Parameters Actophotometer The locomotor activity was measured by using actophotometer (IKON Instrument). The movement of animal cut off a beam of light falling on photocell and count was recorded and displaced digitally. Subjective animals were individually placed in the instrument and the total activity count was registered for 5 min on D-7, D-14, and D-21. The locomotor activity was expressed in terms of total photobeam counts/5 min per animal (Reddy and Kulkarni 1998). Evaluation in Narrow Beam Walk Test Narrow beam test was used to measure hind-limb impairment (Henderson et al. 2005; Geed et al. 2014). The protocol described here measures foot-slips and latency to traverse the beam. The rats were preliminarily trained twice on D-1 to traverse an elevated wooden beam. On D-7, D-14, and D-21, the animals were placed horizontally 60 cm above the floor, 3 cm diameter, and 120 cm long to escape a beam end and open area. On other beam end, there was a darkened goal box measuring 25 × 20 × 18 cm. The rats were tested on the 7th day, 14th day, and 21st day by keeping each rat on the beam end, and number of hind-paw slips and the time taken to
traverse the beam was recorded. The maximum time allowed for the task was 120 s. Footprint Analysis Footprint patterns (walking tracks) measures the gait of the animal (Carter et al. 1999). In brief, rats were acclimatized to walk along a narrow track (100 cm length, 10 cm wide, and with 20 cm high walls), white paper-covered corridor (leading to a darkened enclosure), leaving a track of footprints. The fore- and hind-paws of the animals were painted with two different non-toxic water colors so as to get differential footprints (fore-limbs in red color and hind-limbs in green color). Rats were made to walk on the platform lined with white paper to obtain the footprint pattern. Once the footprints have dried, measurements of the prints was done manually (Fouad et al. 2000). The footprints were analyzed for five parameters, viz., fore- and hind-paw base width, fore- and hind-paw stride length, and overlap between fore- and hind-paw. The mean of each set of three values was used in the statistical analysis. Bar Catalepsy Test Catalepsy is defined as the acceptance and retention of abnormal posture. It was measured by means of the bar test (Sanberg et al. 1996; Geed et al. 2014). Catalepsy was measured as the sum of the latencies spent by the rats to remove one of its forelimb from a 10 cm high bar after being placed in the standing position in three consecutive trials (cutoff time = 60 s). Grip Strength Test Neuromuscular strength was determined on the D-1, D-7, D-14, and D-21 in grip strength test as per method of Khuwaja et al. (2011). The apparatus consisted of a metal wire (length, 90 cm; diameter, 1 mm) which was fixed horizontally between two vertical supports and elevated 50 cm from a flat surface. The animal was hung with its forepaws to the central position of the wire and evaluated according to the following scale: 0, fall off; 1, hangs onto string by two forepaws; 2, as for 1 but attempts to climb on string; 3, hangs onto string by two forepaws plus one or both hind-paws; 4, hangs onto string by all fore paws plus tail wrapped around string; and 5, escape from apparatus and fall down on flat surface. Rotarod Assay The rotarod test was performed the same as previously described (Lundblad et al. 2003; Rylander et al. 2009). To assess the acquisition of skilled behavior in rats with unilateral partial lesions of the nigrostriatal DA system, we performed a rotarod test (Jeljeli et al. 2000). Briefly, rats were pre-trained on the rotarod (orchid scientific) with the rod rotating at an
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accelerating speed (from 4 to 40 rpm) over 10 min, until they reached a stable baseline performance (five consecutive days of training). The training sessions were performed over five consecutive days. For each training trial, rats were gently placed on the rod in the orientation opposite to that of the already rotating rod so that they could acquire the necessary skilled behavior on the rotating rod to prevent a fall. Rats were allowed to stay on the rod for a maximum of 60 s, which was established as the cutoff period (this was deemed to be the maximum period of time that rats could stay on the rod without losing their motivation to do so). The time spent on the rotating rod was measured. Because motivation to stay on the rod was maintained during the 60-s test period, the increase of the time spent walking on the rotating rod reflects an improvement in the acquisition of skilled behavior consisting of a new combination of posture and forward locomotive steps. Assessment of Mitochondrial Integrity, Function, and Oxidative Stress Isolation of Mitochondria from Rat Brain Substantia Niagra (SN)
Evaluation of Mitochondrial Membrane Potential (MMP) in Discrete Brain Regions The rhodamine dye taken up by mitochondria was measured in spectrofluorometer (Hitachi, F-2500) at an excitation λ of 535 ± 10 nm and emission λ of 580 ± 10 nm (Huang 2002). The results were expressed as fluorescence intensity per milligram protein. Estimation of Lipid Peroxidation (LPO) and Nitric Oxide (NO) Level Mitochondrial malondialdehyde (MDA) content was measured as a marker of LPO at 532 nm (Ohkawa et al. 1979). The extent of LPO was expressed as micromoles of MDA per milligram protein. The NO level was estimated as a marker for nitrosative stress (Green et al. 1982; Samaiya and Krishnamurthy 2015) and expressed as nanomoles of NO per milligram protein. Assessment of Superoxide Dismutase (SOD) Activity
Mitochondria were isolated from SN by differential centrifugation method as described by (Pedersen et al. 1978). The mitochondrial protein content was estimated using standard method (Lowry et al. 1951).
Superoxide dismutase (SOD) activity was determined by the reduction of NBT in presence of phenazine methosulfate and NADH at 560 nm using n-butanol as blank (Kakkar et al. 1984). A single unit of the enzyme was expressed as 50% inhibition of NBT reduction per minute per milligram protein.
Estimation of Mitochondrial Respiratory Complex-I, II, IV, and V Activity
Western Blot Analysis for Cytoplasmic Cytochrome-C, Caspase-9, and Caspase-3
The activity of NADH dehydrogenase (complex-I) was measured by catalytic oxidation of NADH with potassium ferricyanide as an artificial electron acceptor at excitation, and emission wavelength for NADH were 350 and 470 nm, respectively (Shapiro et al. 1979). Activity of NADH dehydrogenase was expressed as nanomole NADH oxidized per minute per milligram protein. The mitochondrial succinate dehydrogenase (SDH; complex-II) was determined by the progressive reduction of nitro blue tetrazolium (NBT) to an insoluble colored compound, diformazan at 570 nm (Sally and Margaret 1989). The SDH activity was expressed as micromole formazan produced per minute per milligram protein. The activity of cytochrome oxidase (complex-IV) was measured in mitochondrial fraction in presence of reduced cytochromeC at 550 nm for 3 min (Storrie and Amadden 1990). Results were expressed as nanomole cytochrome-C oxidized per minute per milligram protein (ε550 = 19.6 mmol−1 cm−1). The F1F0 synthase (complex-V) was measured by incubating mitochondrial suspension in ATPase buffer (Griffiths et al. 1974), and the phosphate content was measured (Fiske and Subbarow 1925). Results were expressed as nanomole ATP hydrolyzed per minute per milligram protein.
For western blot analysis, substantia nigral region was collected and was lysed in buffer containing complete protease inhibitor cocktail. Protein concentrations were determined according to Bradford (1976). A standard plot was generated using bovine serum albumin. An aliquot of each sample were electrophoresed in 10% SDS-PAGE gels for cytochrome-C, caspase-9, and caspase-3 proteins, transferred to polyvinylidene fluoride membranes and probed with specific antibodies. The membrane was incubated overnight with rabbit anti-cytochrome-C (Abcam Plc., Cambridge, USA), anticaspase-9 (Abcam Plc., Cambridge, USA), and anti-caspase-3 (Abcam Plc., Cambridge, USA) polyclonal primary antibody at a dilution of 1:1000, 1:500, and 1:500, respectively. After detection with the desired antibodies against the proteins of interest, the membrane was stripped with stripping buffer (25 mM Glycine pH 2.0, 2% SDS for 30 min at room temperature) and re-probed overnight with rabbit anti β-actin (Santa Cruz Biotechnology Inc.; Santa Cruz, California, USA) polyclonal primary antibody at a dilution of 1:500 to confirm equal loading of protein. Further, membrane was probed with corresponding secondary antibodies. Immunoreactive band of proteins were detected by chemiluminescence using enhanced
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chemiluminescence (ECL) reagents (Amersham Bioscience, USA). Quantification of the results was performed by densitometric scan of films. The immunoreactive area was determined by densitometric analysis using Biovis gel documentation software.
Statistical Analysis All data were mean ± SD. Repeated measures of two-way ANOVA was performed for the data analysis of behavioral observations followed by Bonferroni’s post-hoc test. For all biochemical and molecular data analysis, one-way ANOVA was performed followed by Student–Newman–Keuls posthoc test. A level of p < 0.05 was considered as significant in all the data analysis.
Effect of ATV+Q10 on the 6-OHDA Induced Changes in the Footprint Analysis Table 2 shows the effect of drug treatment on 6-OHDA induced changes in the footprint analysis. One-way ANOVA showed that there were significant differences in the left fore-paw (F (5, 30) = 14.1, p < 0.05) and hind-paw (F (5, 30) = 11.1, p < 0.05) stride length and their overlay (F (5, 30) = 5.9, p < 0.05) among groups. However, there were no significant differences in either fore-paw (F (5, 30) = 0.4, p > 0.05) or hind-paw (F (5, 30) = 0.8, p > 0.05) base width among groups. Post-hoc analysis revealed that the 6-OHDA decreased significantly the stride length of both fore-paw and hind-paw of left foot and their overlap on D-21 compared to control animals. The combination of drug and standard significantly reversed the 6-OHDA induced decrease in the stride length of both fore-paw and hind-paw of left foot and their overlap in the footprint analysis.
Results Effect of ATV, Q10, and ATV+Q10 on 6-OHDA Induced Changes in Retention Time, Grip Strength Test, Locomotor Activity, Catalepsy Behavior, and Narrow Beam Walk Test The effect of ATV, Q10, and ATV+Q10 on 6-OHDA induced changes in retention time, grip strength test, locomotor activity, catalepsy behavior, and narrow beam walk test and depicted in Table 1. Repeated measure two-way ANOVA revealed significant differences in retention time, narrow beam walk test, locomotor activity, catalepsy behavior, and grip strength test among groups ((F (5, 120) = 27, p < 0.05), (F (5, 120) = 31.8, p < 0.05), (F (5, 120) = 52.0, p < 0.05), (F (5, 120) = 24.9, p < 0.05), (F (5, 120) = 44.9, p < 0.05), respectively), time ((F (3, 120) = 67, p < 0.05), (F (3, 120) = 85.3, p < 0.05), (F (3, 120) = 101.6, p < 0.05), (F (3, 120) = 60.4, p < 0.05), (F (3, 120) = 125.1, p < 0.05), respectively), and significant interaction between group and time ((F (15, 120) = 4.4, p < 0.05), (F (15, 120) = 4.8, p < 0.05), (F (15, 120) = 8.1, p < 0.05), (F (15, 120) = 4.8, p < 0.05), (F (15, 120) = 7.8, p < 0.05), respectively). Post-hoc analysis showed that 6-OHDA injection caused significant decrease in retention time, grip strength, and locomotor activity and increase in catalyptic behavior and latency to transfer into goal box on D-7. These effects were maintained up to D-21. ATV and Q10 ameliorated 6-OHDA induced alteration on grip strength capacity (on D-21), catalyptic behavior, and latency to transfer in box on D-14 and maintained up to D-21. The above treatment did not affect 6-OHDA induced alteration on retention time and locomotor activity. Whereas ATV+Q10 combination and L-DOPA was found to be effective to ameliorate the 6OHDA induced alteration on all above behavioral activities on D-14 and D-21.
Effect of ATV+Q10 on the 6-OHDA Induced Changes in the Striatal Dopamineregic System Figure 1 represents the effect of drug treatment on 6-OHDA induced changes in the striatal levels of DA (A), DOPAC (B), and HVA (D), and the ratio of DOPAC/DA (C) and HVA/DA (E). One-way ANOVA showed that there were significant differences in the levels of DA (F (5, 30) = 61.6, p < 0.05), DOPAC (F (5, 30) = 17.1, p < 0.05), and HVA (F (5, 30) = 10.8, p < 0.05) and the ratio of DOPAC/DA (F (5, 30) = 10.1, p < 0.05) and HVA/DA (F (5, 30) = 12.9, p < 0.05) among groups. Post-hoc test showed that 6-OHDA significantly decreased the levels of DA (about 50% of the control), DOPAC, and HVA, and increased the ratios of DOPAC/DA and HVA/DA. ATV significantly reversed the 6-OHDA induced decrease in the level of DA. ATV+Q10 and L-DOPA significantly increased the level of DA compared to ATV and Q10-treated rats. ATV+Q10 ameliorated the 6-OHDA induced decrease in the level of DOPAC only. ATV+Q10 and standard drug reversed the 6-OHDA induced increase in the ratios of DOPAC/DA and HVA/DA. Effect of ATV+Q10 Treatment on 6-OHDA Induced Changes in SNpc Mitochondrial Complex-I, II, IV, and V Activity Figure 2 illustrates the effect of ATV+Q10 treatment on 6OHDA induce alteration in the mitochondrial complex-I (A), II (B), IV (C), and V (D) activities of SNpc tissue. One-way ANOVA showed that there were significant difference in complex-I (F (5, 30) = 91.9, p < 0.05), complex-II (F (5, 30) = 18.7, p < 0.05), complex-IV (F (5, 30) = 6.3, p < 0.05), and complex-V (F (5, 30) = 9.8, p < 0.05) activity among the groups. The 6-OHDA injection significantly
Neurotox Res Table 1 Effect of ATV, Q10, and ATV+Q10 on 6-OHDA induced changes in retention time, grip strength test, locomotor activity, catalepsy behavior, and narrow beam walk test Day retention time
Control
6-OHDA
ATV
Q10
ATV+Q10
L-DOPA
D-0
39.3 ± 3.0
38.5 ± 4.5
D-7 D-14 D-21
39.8 ± 2.3 40.2 ± 2.6 39.8 ± 3.4
7.3 ± 2.4a,f 9.5 ± 2.3a,f 10.3 ± 2.8a,f
39.0 ± 4.5 8.5 ± 3.0 a,f 14.7 ± 2.7a,f 17.0 ± 3.0a,f
40.6 ± 2.7 8.7 ± 2.8 a,f 13.5 ± 4.2a,f 15.7 ± 3.9a,f
39.3 ± 3.1 9.3 ± 3.4 a,f 20.8 ± 2.3a,b,f,g 28.7 ± 4.6b,c,d,f,g,h
39.7 ± 3.5 9.2 ± 2.9 a,f 26.0 ± 3.3a,b,c,d,f,g 32.7 ± 2.7b,c,d,f,g,h
D-0 D-7 D-14 D-21 Locomotor activity
11.7 ± 0.8 11.3 ± 1.0 10.9 ± 0.8 11.0 ± 0.7
11.3 ± 1.0 2.5 ± 0.5a,f 2.3 ± 0.6a,f 2.6 ± 0.5a,f
11.2 ± 0.7 2.8 ± 0.7 a,f 4.5 ± 1.2 a,f 6.0 ± 0.6a,b,f,g
11.0 ± 0.5 2.8 ± 1.1 a,f 4.0 ± 1.2 a,f 5.9 ± 0.5a,b,f,g
11.9 ± 0.6 3.0 ± 0.9 a,f 5.3 ± 0.8a,b,f,g 8.2 ± 0.7b,c,d,f,g,h
12.3 ± 0.6 3.3 ± 0.5 a,f 6.7 ± 1.0a,b,f,g 9.5 ± 1.0b,c,d,f,g,h
D-0 D-7 D-14 D-21 Catalepsy test D-0 D-7 D-14 D-21 Narrow beam test D-0 D-7
192.5 ± 6.0 190.5 ± 3.7 192.5 ± 11.1 194.2 ± 9.8
183.2 ± 4.7 89.7 ± 5.0a,f 91.2 ± 7.7a,f 87.5 ± 7.5a,f
181.9 ± 4.9 95.3 ± 5.8a,f 96.5 ± 11.1a,f 111.3 ± 11.9a,f
178.8 ± 3.4 91.5 ± 5.3a,f 93.7 ± 7.5a,f 110.8 ± 8.7a,f
176.5 ± 3.9 95.7 ± 5.5a,f 102.0 ± 7.6a,f 149.2 ± 12.0a,b,c,d,f,g,h
176.3 ± 4.9 87.2 ± 4.6a,f 108.3 ± 6.5a,f 181.2 ± 17.7b,c,d,e,f,g,h
3.3 ± 1.0 4.0 ± 0.9 4.5 ± 1.0 4.5 ± 1.0
3.7 ± 1.2 17.0 ± 1.7a,f 17.8 ± 1.7a,f 18.0 ± 1.9a,f
3.8 ± 1.5 15.5 ± 1.9a,f 10.8 ± 0.7a,b,f,g 11.3 ± 1.2a,b,f,g
4.0 ± 0.9 15.2 ± 1.2a,f 10.8 ± 0.9a,b,f,g 11.2 ± 1.1a,b,f,g
4.5 ± 1.6 15.5 ± 1.8a,f 10.3 ± 0.5a,b,f,g 6.2 ± 1.0b,c,d,f,g,h
4.0 ± 1.3 15.5 ± 2.1a,f 9.8 ± 0.6a,b,f,g 6.2 ± 0.7b,c,d,f,g,h
4.8 ± 0.7 6.2 ± 1.2
5.8 ± 1.2 42.3 ± 3.7a,f
4.8 ± 1.2 41.3 ± 3.7a,f
5.2 ± 1.5 39.3 ± 4.2a,f
5.2 ± 1.0 37.8 ± 4.2a,f
4.3 ± 1.4 39.2 ± 3.3a,f
D-14 D-21
6.0 ± 0.9 6.7 ± 1.2
43.9 ± 2.6a,f 43.2 ± 3.2a,f
30.3 ± 2.6a,b,f,g 30.0 ± 3.2a,b,f,g
30.7 ± 4.3a,b,f,g 31.3 ± 4.5a,b,f,g
31.2 ± 5.2a,b,f,g 16.3 ± 2.9a,b,c,d,f,g,h
30.4 ± 2.5a,b,f,g 13.5 ± 1.6a,b,c,d,f,g,h
Grip strength
Data is represented as the mean ± SD; n = 6; repeated measures of two-way ANOVA followed by Bonferroni post-hoc test a
p < 0.05 compared to control
b
p < 0.05 compared to 6-OHDA
c
p < 0.05 compared to ATV
d
p < 0.05 compared to Q10
e
p< 0.05 compared to ATV+Q10
f
p < 0.05 compared to day-0
g
p < 0.05 compared to day-7
h
p < 0.05 compared to day-14
reduced the respiratory enzyme activities in SNpc tissues compare to control animals. ATV+Q10 attenuated 6-OHDA induced decrease in the activity of complex-I, II, IV, and complex-V in SNpc tissues as compared to control group rats.
that 6-OHDA decreased the mitochondrial membrane potential. ATV+Q10 reversed significantly the 6-OHDA induced decrease in the mitochondrial membrane potential.
Effect of ATV+Q10 on the 6-OHDA Induced Changes in the Mitochondrial Membrane Potential in SNpc
Effect of ATV+Q10 Treatment on 6-OHDA Induced Alterations in Mitochondrial LPO Activity, NO Level, and SOD Activity in SNpc
Figure 3 depicts the effect of drug treatment on 6-OHDA induced alterations in the mitochondrial membrane potential. One-way ANOVA revealed that there were significant differences in the mitochondrial membrane potential (F (5, 30) = 13.2, p < 0.05) among groups. Post-hoc test showed
Figure 4a–c sequentially illustrates the effect of ATV+Q10 treatment on mitochondrial LPO activity, NO level, and SOD activity in 6-OHDA infused rats. Mitochondrial MDA level was estimated as an index of activity of LPO. One-way ANOVA showed that there were significant differences in
Neurotox Res Table 2
Effect of drug treatment on 6-OHDA induced changes in the footprint analysis
Groups
Fore-paw base width (cm)
Hind-paw base width (cm)
Left fore-paw stride length (cm)
Left hind-paw stride length (cm)
Left overlap (cm)
Control
2.7 ± 0.5
3.3 ± 0.6
11.0 ± 0.7
6-OHDA ATV Q10
3.3 ± 0.2 3.1 ± 0.2 3.1 ± 0.2
4.2 ± 0.3 3.7 ± 0.4 3.3 ± 0.3
8.8 ± 0.5a 8.8 ± 0.8a 9.0 ± 0.6a
10.7 ± 0.8 8.4 ± .51a 9.1 ± 0.6a 8.9 ± 0.6a
1.5 ± 0.3 2.2 ± 0.1a 2.1 ± 0.2a 2.1 ± 0.1a
ATV+Q10 L-DOPA
2.8 ± 0.3 3.1 ± 0.2
3.3 ± 0.5 3.6 ± 0.5
10.4 ± 0.5b,c,d 10.4 ± 0.7b,c,d
10.4 ± 0.7b,c,d 10.4 ± 0.6b,c,d
1.9 ± 0.3b,c,d 1.7 ± 0.4b,c,d
Effect of ATV, Q10, ATV+Q10, and L-DOPA on 6-OHDA induced changes on footprint analysis done in terms of fore-paw base width, hind-paw base width, left fore-paw stride length, and left overlap expressed in centimeters. Data is represented as the mean ± SD; n = 6; one-way ANOVA followed by Student–Newman–Keuls test a
p < 0.05 compared to control
b
p < 0.05 compared to 6-OHDA
c
p < 0.05 compared to ATV
d
p < 0.05 compared to Q10
MDA (F (5, 30) = 16.7, p < 0.05), NO level (F (5, 30) = 40.6, p < 0.05), and SOD activity (F (5, 30) = 22.1, p < 0.05) among groups. Post-hoc test showed that 6-OHDA increased MDA
Fig. 1 Effect of ATV, Q10, ATV+Q10, and L-DOPA on 6OHDA induced alterations in DA (a), DOPAC (b), and HVA (c) levels and ratios of DOPAC/DA (d) and HVA/DA (e) in ipsilateral striatum. All values are mean ± SD; n = 6; ap 0.05 compared to control, bp 0.05 compared to 6-OHDA, cp 0.05 compared to ATV, dp 0.05 compared to Q10, and ep 0.05 compared to ATV+Q10 (one-way ANOVA followed by Student– Newman–Keuls test)
activity as compared to control. ATV+Q10 reversed significantly the 6-OHDA induced increase MDA activity. Post-hoc analysis showed that 6-OHDA increased NO level as
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Fig. 2 Effect of ATV, Q10, ATV+Q10, and L-DOPA on 6-OHDA induced alterations in mitochondrial complex-I (a), complex-II (b), complex-IV (c), and complex-V (d) activity in ipsilateral striatum. All values are mean ± SD; n = 6; ap 0.05 compared to control, bp 0.05
compared to 6-OHDA, cp 0.05 compared to ATV, dp 0.05 compared to Q10, and ep 0.05 compared to ATV+Q10 (one-way ANOVA followed by Student–Newman–Keuls test)
compared to control. ATV+Q10 significantly ameliorated the 6-OHDA induced increase in NO level. SOD activity was significantly reduced in 6-OHDA infused rats compared to control rats and that effect significantly attenuated by ATV+ Q10 combination treatment.
Effect of ATV+Q10 on the 6-OHDA Induced Changes in the Cytochrome-C, Caspase-9, and Caspase-3 in SNpc Figure 5 depicts the effect of drug treatment on 6-OHDA induced alterations in the level of cytochrome-C, caspase-9, and caspase-3 in nigral tissues. One-way ANOVA revealed that there were significant differences in the mitochondrial cytochrome-C (F (5, 12) = 12.85, p < 0.05), caspase-9 (F (5, 12) = 32.80, p < 0.05), and caspase-3 (F (5, 12) = 43.58, p < 0.05) among groups. Post-hoc test showed that 6-OHDA caused significant increase in all the protein levels compared to control rats. The combination decreased the 6-OHDA induced increase in all the protein levels.
Discussion
Fig. 3 Effect of ATV, Q10, ATV+Q10, and L-DOPA on 6-OHDA induced alterations in mitochondrial membrane potential in ipsilateral striatum. All values are mean ± SD; n = 6; ap 0.05 compared to control, bp 0.05 compared to 6-OHDA, cp 0.05 compared to ATV, d p 0.05 compared to Q10, and ep 0.05 compared to ATV+Q10 (oneway ANOVA followed by Student–Newman–Keuls test)
One of the salient findings of the study is that behavioral and neurochemical deficits induced by 6-OHDA induced dopaminergic toxicity was a significantly attenuated addition of Q10 to ATV treatment in rats. The combination significantly increased the 6-OHDA induced loss of DA. Although ATV and Q10 mitigated the 6-OHDA induced behavioral deficits
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Fig. 4 Effect of ATV, Q10, ATV+Q10, and L-DOPA on 6-OHDA induced alterations in mitochondrial LPO (a), NO (b), and SOD (c) activity in ipsilateral striatum. All values are mean ± SD; n = 6;
a
p 0.05 compared to control, bp 0.05 compared to 6-OHDA, cp 0.05 compared to ATV, dp 0.05 compared to Q10, and ep 0.05 compared to ATV+Q10 (one-way ANOVA followed by Student–Newman–Keuls test)
in the rats, the combination of ATV and Q10 was more effective than either of them administered alone. Further, the combination protected against 6-OHDA induced mitochondrial dysfunction, loss of mitochondrial integrity, and oxidative stress in substantia nigra. At the molecular level, the combination improved mitochondrial function and anti-apoptotic effects by markedly decreasing 6-OHDA induced release in cytochrome-C, and caspase-9 and capsase-3 protein expression in nigral tissues. 6-OHDA model is used to evaluate pharmacological effect of drugs in PD (Silva-Adaya et al. 2011). DA is the primary neurotransmitter which is involved in motor coordination and its loss directly affects physical movements (Ungerstedt 1968). 6-OHDA contributes to the clinical symptoms of PD in humans and animal models of the disease by depleting striatal DA content (Kumar et al. 2012). In the present study, intrastriatal injection of 6-OHDA causes marked reduction in DA level as reported by earlier studies (Blandini et al. 2008). We also found marked decrease in DOPAC and HVA levels and increase in their turnover at ipsilateral striatum in 6OHDA injected animals (Geed et al. 2014). Increased DA turnover in terms of DOPAC/DA and HVA/DA ratios in the ipsilateral striatum site was consistent with other findings in models of DA neurodegeneration (Thiffault et al. 2000; Van
Keuren et al. 1998; Yazdani et al. 2006). 6-OHDA toxicity showed several motor behavioral deficits that directly linked to reduced DA level (Lancu et al. 2005; DeJesus-Cortes et al. 2016). Ascorbic acid has been used as an antioxidant to prevent oxidation of 6-OHDA. Earlier study has reported that ascorbic acid supplementation (1 mg/ml in water daily) on submucosal vasoactive intestinal polypeptideimmunoreactive (VIP-IR) neurons in the jejunum of rats played a neuroprotective role and its efficacy depends on age, dose, and interaction with other antioxidants (De Freitas et al. 2012). Even though ascorbic acid could have pharmacological activity, it is reported that the concentration used in the study is too negligible to exert any discernable activity (Zbarsky et al. 2005; Kumar et al. 2012). ATV and L-DOPA, but not Q10, reversed the 6-OHDA induced decrease in the DA level in the ipsilateral site of the striatum similar to earlier observation (Beal and Matthews 1997; Alam and Schmidt 2004; Kumar et al. 2012; Chohan et al. 2014). However, the combination of ATV+Q10 significantly increased the DA level compared to the drugs used alone. Several motor behavioral tests such as rotarod performance, narrow beam walk test, locomotor activity, catalepsy test, and grip strength test were used to characterize the motor effects of DA toxicity (Borlongan et al. 1996; Yazdani et al. 2006; Chung
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Fig. 5 Effect of treatment with 6-OHDA, individual and combination of drug on cytoplasmic cytochrome-C, caspase-9 and caspase-3 protein expression in the substantianigra tissues. The blots are representative of cytoplasmic cytochrome-C, caspase-9 and caspase-3 (a) in the substantianigra tissues. The results in the histogram are expressed as ratio of relative intensity of levels of protein expression of either
cytochrome-C (b) or caspase-9 (c) or caspase-3 (d) to β-actin. All values are mean ± SD of three separate sets of independent experiments. ap 0.05 compared to control, bp 0.05 compared to 6OHDA, cp 0.05 compared to ATV, dp 0.05 compared to Q10, and e p 0.05 compared to ATV+Q10 (one-way ANOVA followed by Student–Newman–Keuls test)
et al. 2015). Unilateral injection of 6-OHDA leads to alteration of motor behavior in Parkinsonian rats along with dopaminergic deficits in striatal regions (Khan et al. 2010; Hansen et al. 2016). In the rotarod test, it was observed that the mean time taken on rotating drum was less in 6-OHDA lesion group, indicating compromised motor performance (Haddadi et al. 2015). This was attenuated by ATV+Q10 and L-DOPA on D-21. However, ATVand Q10 alone did not show any effect on motor functions. Narrow beam walk test was performed to assess hindlimb functions in terms of latency to transfer in goal box and numbers of right and left hind-paw slip by each animal on the beam (Geed et al. 2014). In our study, right hind-limb slips (contra lateral to lesioned side) were found to be significantly
increased as compared to left hind-limb slips in 6-OHDA treated rats. All the drug treatments showed significant decrease in the 6-OHDA induced increase in the latency to goal box on D-14 of the experimental protocol. However, the combination showed better motor behavior improvement in the 6-OHDA induced alteration in behavior in narrow beam walk test. Footprint test was measured to evaluate the walking pattern in terms of stride length, overlap, and base width (Forster and Lewy 1912; FrussaFilho et al. 1997; Carter et al. 1999; Rial et al. 2014). 6-OHDA treated rats have decreased fore- and hind-paw stride length and overlap between consecutive fore-paw and hind-paw showing gait abnormality (Klein et al. 2009). ATV with Q10 showed better improvement in motor activity in the 6-OHDA induced
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behavioral deficits in footprint test compared to ATV and Q10 drug treatment alone. Preclinical studies have reported that 6OHDA significantly reduced locomotor activity as observed in open field test (Kumar et al. 2012; Machado et al. 2014). LDOPA reduced the 6-OHDA induced decrease in the locomotor activity in the rats similar to earlier reports (Khaldy et al. 2003). However, in the present study, combination of ATV+Q10 and L-DOPA showed increase of locomotor behavior in 6-OHDA induced decrease in the locomotor activity on D-21, whereas treatment with individual was found to be ineffective. In the grip strength test, 6-OHDA infused rats have been shown to have limb rigidity with longer hanging time in comparison to the control rats (Ma et al. 2014). In the present study, lesioning with 6-OHDA decreased grip strength score in grip strength test. ATV+Q10 and L-DOPA ameliorated the loss in grip strength in contrast to the individual treatment which had no effect. However, the combination showed improvement in motor activity on D-14 and it became more pronounced on D-21. Treatment with ATV+Q10 showed significantly reversed behavioral deficits on D-14 (seventh day of treatment) and D-21 compared to drug treatment with ATVor Q10. Earlier study reported that the neuroprotective efficacy of ATV and Q10 started after 3 week of treatment schedule (Dormoi et al. 2013; Hyson et al. 2010). Therefore, it can be assumed that the combination of ATV+Q10 synergistically improved the 6-OHDA induced behavioral deficits. These results show that neurochemical recovery with the combination is more robust in 6-OHDA model. Therefore, treatment with combination of ATV and Q10 improved functional recovery in a rat model of dopaminergic neurotoxicity. In contrast, the individual treatment with ATVor Q10 was found to be ineffective or had limited functional recovery. Catalepsy test is performed in rodents treated with toxin and drugs that interfere with dopaminergic neuronal system and widely used for the selection of drugs with therapeutic potential in the treatment of PD (Duvoisin 1976). Our study showed that all the drugs (individual as well as combination of ATV+Q10) were effective in reducing the 6-OHDA induced increase in the cataleptic behavior on D-14. However, the combination was more effective than other drug treatment groups on D-21 of the protocol. It is interesting to note that although ATV increased DA levels in 6-OHDA rats, this had no effect or limited effect on functional recovery. The neurochemical recovery was translated into significant function recovery by addition of Q10. Therefore, ATV+Q10 treatment was significantly better in functional and neurochemical recovery following 6-OHDA induced neurotoxicity. However, the exact role of supplementation of Q10 in presumed case of Q10 deficiency with ATV could be better validated by measuring the blood Q10 concentration during the experimental schedule. This is a limitation of the study which would otherwise have given a linear relationship between ATV and Q10. The improvement in PD-like symptom of ATV by Q10 may be related to its mitochondrial effects, as Q10
supplementation improves mitochondrial electron transport chain activity and reduces mitochondrial oxidative stress (Duberley et al. 2014). It is reported that 6-OHDA irreversibly inhibits complex-I activity that decreases the ATP production and subsequently causes DA depletion (Kumar et al. 2012). In the present study, 6-OHDA decreased the activity of striatal mitochondrial complex enzymes such as complex I, II, IV and V indicating mitochondrial dysfunction. ATV (20 mg/kg) treatment for 14 day significantly increased mitochondrial enzyme complex-I activities in 6OHDA treated animals. However, the effects of ATV+Q10 on mitochondrial complex-Ι and IV activities were more pronounced compared to ATV and Q10 treatment alone. On the contrary, treatment with L-DOPA did not show any statistical effects on the above complex enzyme activities. Decreased activity of complex-ΙΙ enzyme SDH was found to be associated with enhanced peroxide levels causing oxidative stress (Ishii et al. 1998). This ultimately leads to disturbance in electron transport chain (ETC) on mitochondria. This dysfunction was attenuated by the combination of ATV+Q10, while the other treatment schedules had no effect. Electrons generated by ETC are utilized by complex-V for the production of ATP that is irreversibly inhibited by 6-OHDA injection (Reed et al. 2015). Reduced ATP level reduces DA level in PD rats (Domanskyi et al. 2011; Dias et al. 2013). It is interesting that only combination of ATV+Q10 was effective in improving complex-V activity which may have led to ATP-dependent increased DA level in striatum. Therefore, combination of ATV with Q10 attenuated the 6-OHDA induced decrease in complex I, II, IV, and V, while individual treatment with ATV and Q10 was only effective to improve complex-I activity. Therefore, the combination therapy was more effective in maintaining the mitochondrial function compared to other treatments. Intrastriatal injection of 6-OHDA increased formation of free radicals may arise from activated microglial cells present in degenerating SNpc (McGeer et al. 1988), which produce nitric oxide (NO) and cytokines (Hunot et al. 1996; Beckman et al. 1990; Goeth et al. 1990). Our study shows that combination of ATV+Q10 and L-DOPA reduced the level of NO. However, ATV and Q10 treatment separately was not able to show anti-nitrosative stress effect. Decrease in the activity of mitochondrial SOD in the substantia nigra is reported in PD (Saggu et al. 1989; Li et al. 2012). This may indicate a compensatory mechanism to nullify the augmented oxidative stress. ATV+Q10 in combination attenuates the formation of superoxide radicals formed by 6-OHDA. Therefore, in the present study, there was an increase in the striatal mitochondrial oxidative damage in terms of increased LPO and NO levels and decreased SOD levels 7 days after 6-OHDA injection. ATV with Q10 significantly compensates the increased LPO in mitochondria.
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6-OHDA infusion decreased the MMP in striatal tissues as that of an earlier study suggesting loss of mitochondrial integrity and compromised mitochondrial bioenergetics in the ipsilateral striatum (McGeer et al. 1988; Parker et al. 1989; Camilleri and Vassallo 2014). ATV combination with Q10 attenuated the 6-OHDA induced decrease in MMP in ipsilateral striatum indicating maintenance of mitochondrial integrity. Hence, ATV combination with Q10 potentially maintained the striatal mitochondrial function and integrity in 6-OHDA induced dopaminergic neurotoxicity in rats. Reduced MMP disturbs mitochondrial potential as well as releases some small matrix molecules like reducing equivalents, resulting in blockade of ETC activity and necrotic or apoptotic cell death (Iijima 2006; Qu et al. 2012). Oxidative stress leads to mitochondrial stress decrease in MMP and leak of cytochrome-C (Huang 2002; Singh et al. 2013; Bracken et al. 2016). CytochromeC activates the proapoptotic factor which leads to initiation of caspase-dependent apoptosis (Gottlieb et al. 2003; Dias et al. 2013). Clinically, it has been shown that 6-OHDA administration significantly enhanced the expression of capsase-9/3 and cytochrome-C and induces mitochondrial-linked apoptosis (Haddadi et al. 2015). ATV+Q10 combination had significantly reduced the expression of these proteins and ameliorates 6-OHDA induced mitochondrial-linked apoptosis. However, individual drugs did not show any effect of apoptosis. Therefore, treatment in combination could limit apoptosislinked dopaminergic toxicity. Clinical studies show the risk of the development of PD in association with untreated hyperlipidemia and with hyperlipidemia treated with lipid-lowering drugs. Statins have been shown to have potentially beneficial effects on PD patients (Becker et al. 2008). We have shown that while Q10 by itself does not show any anti-PD-like effects, its combination with ATV significantly improved the behavioral and neurochemical deficits induced by 6-OHDA. Interestingly, addition of Q10 with ATV improved mitochondrial function and inhibited the mitochondrial-linked apoptosis. Therefore, we presume that combined treatment of Q10 with ATV may significantly improve the treatment due to the favorable effects on mitochondrial function. Additionally, Q10 concentration can be measured to determine the appropriate dose length of treatment with ATV.
Summary Hence, treatment with combination of ATVand Q10 attenuated 6OHDA induced depletion of DA level in striatum which leads to improvement of motor deficits. ATV with Q10 supplementation preserved striatal mitochondrial function and integrity. Further, maintenance of mitochondrial integrity attenuated mitochondrial-linked apoptosis. Therefore, supplementation of Q10 improved neurochemical and functional behavioral recovery by ATV due to 6-OHDA induced dopaminergic neurotoxicity.
Acknowledgments Santosh Kumar Prajapati is thankful to the University Grant Commission (UGC), New Delhi, India, for the student fellowship.
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