Mol Neurobiol DOI 10.1007/s12035-014-8857-8

Protective Effect of a cAMP Analogue on Behavioral Deficits and Neuropathological Changes in Cuprizone Model of Demyelination Gelareh Vakilzadeh & Fariba Khodagholi & Tahereh Ghadiri & Marzieh Darvishi & Amir Ghaemi & Farshid Noorbakhsh & Ali Gorji & Mohammad Sharifzadeh

Received: 10 June 2014 / Accepted: 6 August 2014 # Springer Science+Business Media New York 2014

Abstract Multiple sclerosis (MS) is an inflammatory demyelinating disease that leads to neuronal cell loss. Cyclic AMP and its analogs are well known to decrease inflammation and apoptosis. In the present study, we examined the effects of bucladesine, a cell-permeable analogue of cyclic adenosine monophosphate (cAMP), on myelin proteins (PLP, PMP-22), inflammation, and apoptotic, as well as anti-apoptotic factors in cuprizone model of demyelination. C57BL/6J mice were fed with chow containing 0.2 % copper chelator cuprizone or vehicle by daily oral gavage for 5 weeks to induce reversible demyelination predominantly of the corpus callosum. Bucladesine was administered intraperitoneally at different doses (0.24, 0.48, or 0.7 μg/kg body weight) during the last 7 days of 5-week cuprizone treatment. Bucladesine exhibited a protective effect on myelination. Furthermore, bucladesine significantly decreased the production of interleukin-6 proinflammatory mediator as well as nuclear factor-κB activation and reduced the mean number of apoptotic cells compared to cuprizone-treated mice. Bucladesine also decreased G. Vakilzadeh : T. Ghadiri : M. Sharifzadeh School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran G. Vakilzadeh : T. Ghadiri : M. Darvishi : A. Gorji Shefa Neuroscience Research Center, Tehran, Iran F. Khodagholi Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran A. Ghaemi Infectious Diseases Research Center, Department of Microbiology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran F. Noorbakhsh Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran

production of caspase-3 as well as Bax and increased Bcl-2 levels. Our data revealed that enhancement of intracellular cAMP prevents demyelination and plays anti-inflammatory and anti-apoptotic properties in mice cuprizone model of demyelination. This suggests the modulation of intracellular cAMP as a potential target for treatment of MS. Keywords Demyelination . Neuroinflammation . Apoptosis . Cuprizone . Cell death . Pathogenesis Abbreviations AC Adenylyl cyclase cAMP Cyclic adenosine monophosphate CNS Central nervous system ECL Electrochemiluminescence DMSO Dimethyl sulfoxide EAE Experimental autoimmune encephalomyelitis ERK Extracellular signal-regulated kinases A. Gorji Institut für Physiologie I, Klinik und Poliklinik für Neurochirurgie, Department of Neurology, Epilepsy Research Center, Westfälische Wilhelms-Universität Münster, Münster, Germany

M. Sharifzadeh (*) Department of Pharmacology and Toxicology, Pharmaceutical Sciences research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran, Iran e-mail: [email protected]

A. Gorji (*) Epilepsy Research Center, Universität Münster, Robert-Koch-Strasse 27a, D-48149 Münster, Germany e-mail: [email protected]

Mol Neurobiol

H&E HO IL-6 LFB MS NF-κB OPCs PFA PKA PNS PLP PMP-22 TUNEL

Hematoxylin and eosine Heme oxygenase Interleukin-6 Luxol fast blue Multiple sclerosis Nuclear factor-κB Oligodendrocyte precursor cell Paraformaldehyde Protein kinase A Peripheral nervous system Proteolipid protein Peripheral myelin protein 22 Terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling

responses [9], and inhibits caspase-3 activity [10]. Previous studies showed that elevation of intracellular cAMP via bucladesine, a membrane-permeable cAMP analogue, improves spatial memory retention in male rats [11], enhances endogenous neural stem cell recruitment, and attenuates demyelination in experimental model of autoimmune encephalomyelitis [12]. Bucladesine is a phosphodiesterase-3 inhibitor [13] and raises the intracellular level of cAMP [14]. Phosphodiesterase inhibitors prevent the cAMP breakdown and thereby enhancing its intracellular concentration [15]. In the present study, we evaluated the effect of increasing intracellular cAMP levels by application of bucladesine on locomotor activity, myelin protein synthesis, including proteolipid protein (PLP) and peripheral myelin protein 22 (PMP-22), as well as on apoptosis and inflammation in cuprizone-treated animals.

Introduction Materials and Methods Multiple sclerosis (MS) has long been known as an inflammatory demyelinating disease of the central nervous system (CNS) in which myelin sheaths that surround axons are destroyed [1]. Despite the fact that pathogenesis of the disease is not fully understood, oligodendrocyte death with subsequent demyelination, neuroinflammation, and axonal damage play crucial roles in the pathophysiology of MS. Acute axonal damage results in irreversible axonal loss and enhances the disability in patients suffering from MS [2]. Several toxins have been used to induce demyelination experimentally, including cuprizone, ethidium, and bromide lysolecithin. Feeding of cuprizone, a copper-chelating agent, induces demyelination in a number of CNS regions, most frequently in the corpus callosum [3]. Partial remyelination can be detectable by 3 weeks after feeding, and therefore, this model is particularly useful for investigation of concurrent demyelination and remyelination [4]. The toxic effect of cuprizone is correlated with inhibition of copper-dependent mitochondrial enzymes in the cell respiratory chain that leads to oligodendrocyte injury and death [4–6]. Extensive apoptosis has been observed before acute demyelination in this model [5]. Astrocytes together with microglia and macrophages accumulate within lesions and play a role in cytokine proliferation and release [5]. There are several studies that have characterized specific components as anti-inflammatory and anti-apoptotic agents for treatment of neuroinflammatory disorders. Among those, the enhancement of cyclic adenosine monophosphate (cAMP) within immune cells in different experimental models has been reported to have neuroprotective and anti-inflammatory effects. cAMP works as a second messenger mediating signal transduction [7]. After ligand binding to G protein-coupled receptors, cAMP can activate protein kinase A (PKA) that allows phosphorylation of various downstream targets and promotes axonal regeneration [8], regulates inflammatory

Animals and Demyelination Male C57BL/6 mice were purchased from Pasteur Institute, Tehran, Iran, with body weight ranging between 19 and 21 g (8 to 9 weeks old). They had free access to food and water and were maintained on a 12-h light/dark cycle at room temperature (20–22 °C). Demyelination was induced by feeding a diet containing 0.2 % cuprizone mixed into ground standard rodent chow for 5 weeks. Control animals were fed with normal powdered chow. All animal manipulations were carried out according to the Ethical Committee for the use and care of laboratory animals of Tehran University of Medical Sciences and Shefa Neuroscience Research Center. Experimental Design Mice were divided randomly into four groups: (i) control group which received normal powdered chow for 5 weeks with intraperitoneal (i.p.) injection of 10 % dimethyl sulfoxide (DMSO) solution for the last 7 days (n=7); (ii) cuprizone group that were fed with powdered chow mixed with 0.2 % cuprizone for 5 weeks and received i.p. injection of 10 % DMSO solution for the last 7 days (n=7); (iii) bucladesine groups that were divided into three separate subgroups, treated with 0.24, 0.48, or 0.7 μg/kg body weight of bucladesine (i.p.) during the last 7 days of 5-week cuprizone feeding period (seven mice per subgroup); and (iv) healthy group which were divided into three subgroups and received three doses of bucladesine (0.24, 0.48, or 0.7 μg/kg body weight) separately for 7 days (seven mice per subgroup). All mice from different groups were investigated by behavioral, molecular, and histopathological tests. All measurements were performed by an observer blinded to group assignments.

Mol Neurobiol

For Western blot analysis, the caudal region of corpus callosum was dissected from the brain. Tissues were homogenized in lysis buffer containing a complete protease inhibitor cocktail and centrifuged at 12,000 rpm for 20 min. Protein levels were determined according to Bradford’s method [18]. A standard plot was generated using bovine serum albumin. Then, total proteins were electrophoretically separated in 12 % SDS-PAGE gels, transferred to polyvinylidene fluoride membranes, and probed with specific antibodies. Immunoreactive polypeptides were detected by chemiluminescence using enhanced electrochemiluminescence (ECL) reagents (Amersham Bioscience, USA) and subsequent autoradiography. Quantification of results was performed by densitometric scan of films, and data analysis was done by ImageJ, measuring integrated density of bands after background subtraction. Nuclear and cytoplasmic proteins were isolated as described earlier [19].

For the evaluation of myelination, paraffin sections were stained with Luxol fast blue (LFB). After anhydration, brain sections were incubated with 0.1 % LFB solution at 60 °C overnight. Following a rinse with 95 %, 70 % ethanol and distilled water, the slides were differentiated in lithium carbonate solution for 30 s and then in 70 % ethyl alcohol for another 30 s. After distilled water washes for the second time, the sections were counterstained with 0.1 % Cresyl fast violet for 10 s. After washing with distilled water, sections were dehydrated in a graded series of alcohols, then cleared in xylene and mounted finally. The ventral body of the corpus callosum was observed with an Olympus light microscope (BX51, Olympus, Japan) and photographed with an Olympus digital camera (Olympus, Japan) linked to a microscope. ImageJ software was used to evaluate the extent of demyelination based on the ratio of blue to pink fibers in the corpus callosum as the percentage of volume fraction in damage tissue/total area for 16 sections in each animal (n=4). Furthermore, hematoxylin and eosin (H&E) staining was performed to study infiltration of hematogenous cells into the corpus callosum. To visualize DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) test was used for staining DNA fragmentation using an in situ Cell Death Detection Kit. Briefly, three 6-μm-thick tissue sections from each block were dewaxed and dehydrated by heating to 60 °C, followed by washing in xylene and rehydration through washing with diluted alcohol. After being washed with 10 mM Tris-HCl (pH 7.6), sections were incubated in methanol containing 0.3 % H2O2 for 10 min to inhibit endogenous peroxidase activity. They were then treated with proteinase K (Roche, 20 μg/ml in Tris buffer) at 37 °C for 30 min. The sections were incubated in TUNEL reaction mixture (450 μl of label solution and 50 μl of enzyme solution) at 37 °C for 60 min and in POD solution for 30 min. The color reaction was developed in 3-3′diaminobenzidine (DAB, Roche; 0.5 μl DAB and 1.5 μl peroxide buffer) for 5–10 min, and counterstaining was performed with Cresyl violet. The percentage of TUNEL-positive neurons was calculated by counting 500 cells in each specimen (five visual fields/specimen). Sections were observed on an Olympus microscope (CX31, Tokyo, Japan) with a ×40 objective lens, and images were captured using a digital camera (Olympus, Japan; [20, 21]).

Histopathological Studies

Drugs

After performing behavioral assessment tests, mice were anesthetized with ketamine (i.p, 50 mg/kg) and xylezine (4 mg/kg). Thereafter, mice were perfused with 4 % paraformaldehyde (PFA). Brains were dissected and post-fixed for 24 h in 4 % PFA and embedded in paraffin. Finally, coronal sections were obtained in 6-μm thickness (1.58 to 2.30 mm from the bregma).

Bucladesine (dibutyryl cyclic AMP), ketamine, and xylazine were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies directed against β-actin, astrocyte nuclear factor-κB (NF-κB), caspase-3, Bax, and Bcl-2 were obtained from Cell Signaling Technology. Lamin-B2, interleukin-6 (IL-6), PLP, and PMP-22 antibodies were purchased from Santa Cruz Biotechnology. HO-1 antibody was obtained from ABCAM.

Behavioral Experiments Open-Field Test To evaluate the effect of bucladesine on motor impairment, at the end of the fifth week of experiments, all groups (healthy, control, cuprizone, and bucladesine groups) were assessed by open-field test. Animals were placed in an open-field box, and locomotion was tracked over a 3-min period. The EthoVision tracking system (Noldus Information Technology, Wageningen, The Netherlands) was used to evaluate motor function by measuring the total distance moved (cm) and movement velocity (cm/s) [16, 17]. Tail-Flick Test A standardized tail-flick apparatus (tail-flick Unit 7360, Ugo Basile, Italy) with a radiant heat source connected to an automatic timer was used to assess acute nociception response. After the end of the fifth week of experiments, each animal was placed in a restrainer and the tail-flick latency was measured by focusing a beam of light on the distal 2 cm of the tail until the animal exhibited a flick of the tail. Cutoff time (10 s) was used to minimize tissue damage. Western Blot Analysis

Mol Neurobiol

ECL kit was obtained from Amersham Bioscience. Cresyl violet was purchased from Santa Cruz, Germany, and Cell Death Detection Kit was obtained from Roche, Germany. All other reagents, unless otherwise stated, were purchased from Sigma-Aldrich, USA. Statistical Analysis For all experiments, data were analyzed using GraphPad Prism 5. Comparison between groups was tested by a two-way analysis of variance (ANOVA), followed by a post hoc Bonferroni’s test. Statistical significances were gained when p