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Resveratrol Protects Purkinje Neurons and Muscle Activity in Rat Model of Cerebellar Ataxia

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Resveratrol Protects Purkinje Neurons and Restores Muscle Activity in Rat Model of Cerebellar Ataxia

 

Abstract

Cerebellar ataxia (CA) is a miscellaneous cluster of brain disorders with ataxia as the leading symptom. Resveratrol is a naturally-occurring polyphenolic compound. Piece of evidence indicates that resveratrol confers neuroprotection in various animal models of brain disorder. We actually considered it invaluable to investigate whether a treatment with resveratrol has a therapeutic role against CA induced by 3-acetylpyridine (3-AP) in rats. In addition, no investigation has examined neuroprotective effect of resveratrol in rat model of CA. Initially, 3-AP administration generated CA rat models followed by intraperitoneal injection with resveratrol. Then, motor performance and muscle EMG activity were assessed. Moreover, the anti-apoptotic role of resveratrol in CA and its relationship to protection of purkinje cells were explored. According to what we have found, resveratrol administration improved the muscle activity and movement coordination in 3-AP-lesioned rats. Also under resveratrol treatment, the total number of the purkinje neurons increased whereas a reduction in apoptotic bodies was observed. In conclusion, post-treatment with resveratrol evidently ameliorated motor performance as well as muscle activity accompanied by protection of purkinje cells in ataxic rats.

Introduction

Cerebellar ataxia (CA) is a wide spectrum of brain disorders associated with dysfunction of the cerebellum (Manto et al., 2009). As different phenotypes have specific pathogenesis, it is very challenging to envisage a remedy to exclusively target CA based on etiological factors until the pathogenic mechanism for each type is well defined (Teive et al., 2009). Furthermore, with a high prevalence of ataxia, these series of disorders have several clinical and pathologic manifestations in common, including ataxic gait, cerebellar atrophy and Purkinje cell damage (Tatsuoka et al., 1985).

Resveratrol is an antioxidant-like compound produced in at least 72 plant species such as grapes, raspberries, mulberries, pistachios and peanuts (Jang et al., 1997; Vastano et al., 2000). Research findings have disclosed that several precious properties of this compound, including cardioprotection, cancer prevention and prolongation of lifespan in several species (Das et al., 2007; Hung et al., 2004; Jang et al., 1997; Howitz et al., 2003). Large body of evidence suggests that resveratrol confers neuroprotection in various animal models of brain disorder. For instance, in rat traumatic brain injury, a single high dose of resveratrol given immediately after trauma reduced brain edema and oxidative stress, and attenuated brain pathology (Ates et al., 2007). Similarly, resveratrol decreased the frequency of seizures and enhanced pathological damage to the hippocampus CA1 and CA3a neurons after kainic acid-induced temporal lobe seizures (Wu et al., 2009). Two recent studies exhibited the positive effects of resveratrol in the treatment of Parkinson’s disease model in rats (Jin et al., 2008) and mice (Blanchet et al., 2008). In the rat model, 6-hydroxydopamine was used to induce dopaminergic neurotoxicity. Upon resveratrol administration, a significant protection on dopaminergic neurons in substantial nigra was achieved, accompanied by the decreased expression of cyclooxygenase-2 (COX-2) and tumor necrosis factor α (TNFα) mRNA levels (Jin et al., 2008). Moreover, chronic application of resveratrol was beneficial in colchicine-induced rat Alzheimer’s disease model (Kumar et al., 2007) and in 3-nitropropionic acid (3-NP)-induced Huntington’s disease model in rats (Kumar et al., 2006). In colchicine-induced Alzheimer’s disease model, after treatment by  resveratrol, the cognitive impairment was greatly improved and this neuroprotection was associated with reduced malondrialdehyde (MDA) and nitrite content and increased glutathione and acetylcholinesterase levels (Kumar et al., 2007). The really important point to choose a drug is that successful treatment should fight pathogenic elements of the disease and also promote cellular neuroprotective pathways. Accordingly we found that resveratrol could be valuable because of its therapeutic role against CA induced by 3-acetylpyridine (3-AP) in rats. Also, no research has been done so far about its treating role in rat model of CA. The goal of this study is to assess motor performance and muscle activity in resveratrol-treated ataxic rats. Moreover, the anti-apoptotic role of resveratrol in CA and its relationship to protection of purkinje cells were explored.

Materials and Methods

Experimental Model of Cerebellar Ataxia in Rats

Induction of ataxia (lack of coordinated movements) in rats (Sprague–Dawley, 200–220 g) was achieved by intraperitoneal (IP) injection of 3-acetylpyridine (3-AP, 75 mg/kg of body weight), a neurotoxin that particularly degenerates inferior olive neurons in brain stem (Wüllner et al., 1997).

Tentatively protocol and drug management

All rats were randomly arranged into three groups as follows: control group (n=10), 3-AP group (n =10) and resveratrol +3-AP group (n=10). Resveratrol (Sigma Chemicals, St. Louis, MO) was dissolved in 50% ethanol and diluted in physiological saline at the concentration of 10 mg/kg  was administered intraperitoneally. Then, three days after 3-AP injection, the rats were treated with resveratrol per day for 7 days.

Assessment of motor coordination

A behavioral test was performed one day before the induction of ataxia, and at the 1st, 2nd, 3rd, and 4th week after the injection of 3-AP. Rats were placed on the accelerating cylinder at speeds increasing from 4 to 40 rpm over a 5-min test session. The test was stopped if the animal fell off the rungs or gripped the device and spun around for two consecutive revolutions without attempting to run. The maximum time that each animal remained on the device was recorded.

Electromyography (EMG)

Animals were placed under general anesthesia via IP injection of ketamine hydrochloride (60 mg/kg) and xylazine (8 mg/k). After that the right hind limb of animal was shaved and cleaned with a betadine solution. A 3 cm skin incision was made longitudinally on the posterior aspect of each thigh, from the greater trochanter to the knee. Then dissection was performed between the gluteus maximus and biceps femoris muscles, and sciatic were exposed, along with the gastrocnemius muscle. With appearance of the sciatic nerve using forceps and cautiously in order to avoid damage to nerve, it is separated from the surrounding connective tissue which stimulation electrodes could able to pass under sciatic nerve. For electrical stimulation two monopolar subdermal teflon needle electrodes were used, arranged in parallel at a fixed distance of 7 mm from each other. The recording electrodes had an insulating coating over leaving the distal uncoated. The sciatic nerve was then stimulated (1 A, 0.2 Hz frequency, 100 s long) and the compound muscle action potential was recorded in gastrocnemius muscle on the side ipsilateral to the stimulation. The compound muscle action potential parameters which analyzed were amplitude and latency. Besides, during stimulation and recording, ringer solution was used in order to prevent drying of the nerve.

Western Blot

The animal’s cerebellums were extracted while the rats were strangled with CO2 and decapitated formerly. Then their cerebellum was homogenized in tissue lysis buffer. To determine the protein concentration in the sample, bradford’s method was performed. Sixty microgram of each protein sample was loaded on 12% SDS-PAGE gel and electrophoresis followed by transfer to poly vinylidene difluoride membranes (Millipore. Billerica, MA, USA). Blots were blocked by blocking solution and incubated with primary antibody against caspase-3, obtained from Cell Signaling Technology (Beverly, MA, USA), at 4 °C overnight. After being washed with TBST, blots were incubated with secondary antibody [Cell signaling Technology (Beverly, MA, USA], for 90 min at the room temperature. Finally by using ECL kit (Amersham Bioscience. Piscataway, NJ, USA); the immunoreactivity of polypeptides was detected. Blots were stripped in stripping buffer [100 mM 2-mercaptoethanol, 2 % (w/v) SDS, 62.5 mM Tris–HCl (pH 6.7)] and probed with anti β-actin antibody to normalize protein content. By using image J software; densitometric analysis of the protein bands was quantified.

Measurement of GSH content

Reduced glutathione (GSH) reacts with 5-5 –dithio-bis-2(- nitrobenzoic acid) (DTNB) to form a yellow dianion of 5′-thio-2-nitrobenzoic acid (TNB) which is measured by its absorbance at 412 nm (Ellman 1959) (24). The buffers used in this experiment were provided as follow: 50 μL of the DTNB solution was mixed with 100 μL of Tris solution and 840 μL of molecular grade biology water [Sigma Aldrich (St. Louis, MO, USA]. First the UV/VIS spectrum of mix solution was quantified alone to display the maximum absorption band. Afterwards, 10 μL of tissue lysed buffer were carefully added to 990 μL of DTNB reagent and incubated at room temperature for 5 minutes. Finally, the absorbance at 412nm was recorded using UV/VIS spectrophotometry. GSH concentrations were expressed as mol/mg protein.

Immunohistochemistry

Rats were deeply anesthetized by chloral hydrate and perfused transcardially using chilled saline followed by fixative consisting of 4% paraformaldehyde in 0.1M phosphate-buffere saline (PBS).Then, cerebellum were removed and placed in formalin, prepared and placed on slides. The primary antibody was diluted with PBS containing 0.3% Triton X-100 and 1% bovine serum albumin (BSA). Sections were incubated in primary antibody against calbindin (1:300) overnight in 4 °C.  Sections were then incubated with the avidin–biotin complex substrate and treated with 0.05% 3, 3-diaminobenzidine tetra- hydrochloride and 0.03% hydrogen peroxide in 0.05 m Tris-buffer (pH 7.6). After immunohistochemical reaction, sections were mounted, counter- stained and observed under a light microscope.

Estimation of the number of purkinje cells

The total number of purkinje cells was determined using the optical disector method. The position of the microscopic fields was selected by an equal interval of moving the stage and systematic uniform random sampling. Microcator was used for measurement of Z-axis movement of the microscope stage.  An unbiased counting frame with inclusion and exclusion borders was superimposed on the images of the sections viewed on the monitor.  A nucleus was counted if it was placed completely or partially within the counting frame and did not reach the exclusion line. Numerical density (Nv) was calculated with the following formula:

Nv (Purkinje/cerebellum) = [ΣQ- / (h ×x a/f × Σp)] × (t / BA)

where “ΣQ-” is the number of the nuclei, “h” is the height of the disector, “a/f” is the frame area, “ΣP” is the total number of the unbiased counting frame in all fields, “t” is the real section thickness measured in every field using the microcator, and BA is the block advance of the microtome which was set at 10μm. The total number of the purkinje cells was estimated by multiplying the numerical density (Nv) by the total V (cerebellum).

N (purkinje cell) = NVv ×V (final)

Data analysis

All data are represented as the mean ± SEM. Comparison between groups was made by one-way analysis of variance (ANOVA) followed by multiple comparison test of Tukey to analyze of the difference. The statistical significances were achieved when P<0.05.

Results

The role of resveratrol on enhancing motor performance and muscle EMG activity in 3-AP injected rats

To determine whether the administration of resveratrol in 3-AP injected rats improved the coordination of movement, the rotarod test was performed. Significant enhancement in the motor skills was found in the resveratrol-treated animals compared with the 3-AP injected rats which did not treat by resveratrol (P < 0.05) (Fig. 1). During the treatment by resveratrol, the motor performance was increased by the time and it was statistically significant (P < 0.05).

Measurment of muscle activity in ataxic rats treated with resveratrol were conducted by EMG test (Fig. 2A, B). EMG amplitude was downgraded in 3-AP group compared with control. However, after treatment by resveratrol, amplitude was significantly rose in treatment group in comparison to 3-AP injecting animals (P < 0.05).

Resveratrol effects on decreasing apoptotic marker of caspase-3 in ataxic rats

As shown in Figure 3, Western blotting analysis displayed that cleaved caspase-3 level in 3-AP injected group was 2.9-fold higher than the control group level. Although, the level of cleaved caspase-3 in the group which received resveratrol was approximately 1.6 times lower in comparison to 3-AP injected ones (P<0.001).

Resveratrol increases GSH content in ataxic rats

As shown in Figure 4, the results showed that the level of GSH significantly decreased in 3-AP-injecting rats about 1.4-fold compared to the control group, while this value in the animal’s pretreatment with resveratrol increased about 1.2-fold compared with 3-AP-injected group (P < 0.05).

Resveratrol’s protective role on Purkinje cells against 3-AP neurotoxin

Estimation of total number of purkinje cells was done by nissle staining (Fig. 5A). On the other hand, immunohistochemistry showed calbindin positive cells in the study groups (Fig. 5B). The results showed that the total number of the purkinje neurons in the cerebellum was significantly reduced in the 3-AP group in comparison to the control (P<0.001) (Fig. 6). However, the number of the purkinje cells increased in the resveratrol group in comparison to the 3-AP group (P<0.001). It revealed that treatment of the 3-AP rats with resveratrol protected the purkinje cells from toxicity of 3-AP.

Discussion

In this study, we investigated the neuroprotective effect of resveratrol on the adult male rats with CA. According to our findings, resveratrol administration improved motor performance and muscle activity in 3-AP-lesioned rats. In addition, under resveratrol treatment, total number of the purkinje neurons increased whereas a reduction in apoptotic bodies was observed.

The term ‘cerebellar ataxias’ encompasses a plethora of neurological disorders associated with dysfunction of the cerebellum (Manto et al., 2009). The intraperitoneal (IP) administration of 3-AP induces discerning obliteration of inferior olive nucleus in the rat, leading to degeneration of cerebellar climbing fibers which basically form synapses on Purkinje cells (Aoki and Sugihara, 2012). In this regard, we found a significant decline of purkinje neurons in the cerebellum by 3-AP adminsteration. Besides, this neurotoxin adversely affected motor coordination, including staggering of their hind limbs and gait disturbances. Additionally, a dramatic reduction in time spent in the rotarod as compared with intact animals was noticed as also documented by (Wecker et al., 2013; Jiang et al., 2015).

As a bioactive polyphenol, resveratrol has a profusion of positive effects including neuroprotection and anti-inflammatory activities (Zhang et al., 2010; Bastianetto et al., 2015). A dose of 10 mg/kg/day resveratrol was selected in this study since it was been shown to be beneficial in several rat models of cardiovascular diseases (Rivera et al., 2009; Toklu et al., 2010). Further, 10 mg/kg resveratrol IP injection for 48 prior cerebral ischemia induction in rat models rendered the protection of CA1 region of the hippocampus (Della-Morte et al., 2009).   However, due to the in vivo poor bioavailability of resveratrol, the physiologically effective dose still needs to be determined (Amri et al., 2012).

Next, the rat neuromuscular response was studied from the brain and/or spinal cord to the arms and legs using electromyography (EMG), recording the electrical activity. EMG results disclosed that latency was prolonged in the non-treated rats compared with the control group and compound muscle action potential amplitude was significantly decreased (Akman et al., 2015)

Our motor skill finding indicated in vivo efficacy of resveratrol and support the suitability of this treatment for CA. We found that treatment with resveratrol improved motor performance and normalized the cerebellar pathology in the lesioned group. This is in agreement with the previous report demonstrating pretreatment with resveratrol substantially ameliorated motor and cognitive impairment in a Huntington’s model (Kumar et al., 2006).

According to our results, the expression of active caspase 3 was down regulated in ataxic rats, but following treatment with resveratrol, this event was considerably reversed. This anti-apoptotic characteristics of resveratrol shown to be associated with the activation of SIRT1, probably facilitated by p53 inhibition (Van Ginkel et al., 2007). Likewise, activation of PI3K/Akt pathway provided neuronal survival in resveratrol-treated subarachnoid hemorrhage rat models (Zhou et al., 2014). Further, it was shown resveratrol through activation of Ras-extra cellular signal-regulated kinase (ERK) cascade rendered neuroprotection in several Huntington’s  models (Maher et al., 2010). The sirtuin-dependent regulation of Akt signaling and Ras-ERK cascade might explain the resveratrol-mediated neuroprotection of purkinje cells against neurotoxin 3-AP in our study, since these pathways are involved in cellular survival and proliferation (Steelman et al., 2011; Pillai et al., 2014).

These preliminary results provide a platform for more well-tailored studies aimed exclusively at resveratrol-mediated neuroprotection at the molecular levels. Nonetheless, this study falls short of several aspects. First, only resveratrol post-treatment were performed in 3-AP-lesioned rats, and the effect of resveratrol pre-treatment or both was not inspected. Second, alternative resveratrol administration routes were not explored. Considering rapid metabolism of resveratrol, effective formulations to enhance its bioavailability in the brain are highly demanded. Third, we didn’t thoroughly study survival pathways namely PI3K/Akt and Ras-ERK cascade in resveratrol-treated ataxic rats.

In order to translate the physiological benefits of resveratrol, observed in animal models, into clinic, development of human clinical trials are of high importance. Regarding ataxia and resveratrol, there is one published clinical study to our knowledge (Smoliga et al., 2011; Yiu et al., 2015). An open trial of resveratrol in patients with inherited Friedreich’s ataxia indicated encouraging clinical values of resveratrol (5g/day) despite the fact that no changes detected in frataxin expression levels. Nonetheless, more clinical trials with specific focus on CA are required to further measure the potential therapeutic use of resveratrol for human medicine. In conclusion, our findings suggest post-treatment with resveratrol evidently ameliorated motor performance as well as muscle activity accompanied by protection of purkinje cells in ataxic rats.

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Captions:

Fig.1.The administration of resveratrol in 3-AP injected rats improved motor coordination.  # indicates the difference between the 3-AP injected group and control, whereas * shows the difference between resveratrol-treated 3-AP and 3-AP injected group (*, # P<0.05;** ,# P<0.01; ***, ### P<0.001).

Fig.2. Resveratrol treatment enhanced muscle EMG activity in 3-AP injected rats. A) The sciatic nerve was stimulated and the muscle action potential was recorded in gastrocnemius muscle. B) EMG amplitude was measured in control, and 3-AP injected and resveratrol-treated 3-AP. # indicates the difference between the 3-AP injected group and control, whereas * shows the difference between resveratrol-treated 3-AP and 3-AP injected group (*, # P<0.05)

Fig.3. Westen blot analysis to evaluate the effect of resveratrol pretreatment on the expression levels of an apoptotic marker Caspase-3 in 3-AP injected rats. Western blots for cleaved caspase-3 are shown. Equal amounts of total proteins (60 μg) were separated by SDS-PAGE and blots were probed with anti caspase-3 and anti β-actin antibody. The results showed high expression of cleaved caspase-3 in 3-AP injected rats. b The densities of cleaved caspase-3 bands were measured and their ratios to β-actin bands were calculated. # indicates the difference between the 3-AP injected group and control, whereas * shows the difference between resveratrol-treated 3-AP and 3-AP injected group (***, ### P<0.001).

Fig.4. The effect of resveratrol on changes in GSH content in rats receiving 3-AP and resveratrol (One representative GSH content is shown; n=3). Each point shows the mean±S.E.M. ** P<0.01 significantly different from resveratrol treated groups and 3-AP groups; ## P<0.01 much different from control group.

Fig.5. Nissl staining (A), immunohistochemistry against calbindin (B)

Fig.6. Resveratrol protected Purkinje cells against 3-AP neurotoxin. Total number of the purkinje cells in the cerebellum was significantly reduced in the 3-AP group in comparison to the control group. # indicates the difference between the 3-AP injected group and control, whereas * shows the difference between resveratrol-treated 3-AP and 3-AP injected group (***, ### P<0.001).



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