Neuroprotective effect of cocaine- and amphetamine-regulated transcript peptide in spinal cord injury in mice

. S rsity SER a r t i c l e i n f o Article history: Received 12 May 2012 potential neuroprotective agents like progesterone, erythropoietin, alpha-melanocyte stimulating hormone, brain derived neurotropic factor (BDNF) are being investigated in preclinical trials (Labombarda et al., 2011; Lankhorst et al., 1999; Vitellaro- Zuccarello et al., 2008). Cocaine- and amphetamine-regulated transcript peptide (CART) is abundantly expressed in the brain and spinal cord (Koylu et al., 1998; Kozsurek et al., 2007). A role for the peptide in energy metabolism, regulation of food intake and reward behavior is well Abbreviations: ANOVA, Analysis of variance; BDNF, Brain derived neurotropic factor; CART, Cocaine- and amphetamine-regulated transcript peptide; GFAP, Glial fibrillary acidic protein; i.v., Intravenous; i.p., Intraperitoneal; MP, Methylprednis- olone; MFS, Motor function score; PBS, Phosphate-buffered saline; ROS, Reactive oxygen species; SCI, Spinal cord injury; SEM, Standard error of mean. * Corresponding author. Tel.: þ91 9850318502; fax: þ91 712 2500355. E-mail address: [email protected] (D.M. Kokare). 1 Contents lists available at Neuropharm journal homepage: www.elsev Neuropharmacology 67 (2013) 126e135 First two authors contributed equally to the work. � 2012 Elsevier Ltd. All rights reserved. 1. Introduction Spinal cord injury (SCI) inflicts severe health costs associated with life-long disabilities. It occurs primarily due to mechanical trauma leading to secondary injury and eventual loss of tissue and functions (Oyinbo, 2011). At cellular level SCI causes astrogliosis, a characteristic cellular response that form barrier to axonal regeneration. Marked upregulation of glial fibrillary acidic protein (GFAP) is a major index of astrogliosis (Eng and Ghirnikar, 1994; Sofroniew and Vinters, 2010; Wang and Bordey, 2008). Therapeutic strategies aim at attenuation of astrogliosis during the initial phase after SCI (Labombarda et al., 2011; Liu et al., 2008; Vitellaro- Zuccarello et al., 2008). Methylprednisolone (MP) is extensively used as a model neuroprotective reference standard (Ates et al., 2006) and an important drug in clinical practice for the treat- ment of SCI (Tohda and Kuboyama, 2011). However, at higher doses, the steroid produces side effects like sepsis and pneumonia (Gerndt et al., 1997). To circumvent these complications, a range of other Received in revised form 13 October 2012 Accepted 20 October 2012 Keywords: Cocaine- and amphetamine-regulated transcript peptide Glial fibrillary acidic protein Methylprednisolone Spinal cord injury Locomotor recovery 0028-3908/$ e see front matter � 2012 Elsevier Ltd. http://dx.doi.org/10.1016/j.neuropharm.2012.10.028 a b s t r a c t We explored the effect of cocaine- and amphetamine-regulated transcript peptide (CART), alone and in combination with methylprednisolone (MP), on the cellular pathology and locomotor recovery of mice following spinal cord injury (SCI). While cellular pathology was evaluated in terms of spinal cord histology and profile of astrocytes following immunolabeling with antibodies against glial fibrillary acidic protein (GFAP), locomotor recovery was monitored using hindlimb motor function scoring system. At 24 h post-SCI, there was a massive loss of motor function and cysts formation in the spinal cord. The SCI mice, following 3 days and onwards, showed a significant (P < 0.001) increase in the population and hypertrophy of GFAP þ astrocytes, suggesting the occurrence of reactive astrogliosis. Intra-fourth ventricular administration of CART (54e102) or intravenous treatment with MP, dose dependently improved motor function score, while CART-antibody (intra-fourth ventricular) was ineffective. This neuroprotective effect of MP was potentiated by the subeffective dose of CART and antagonized by CART- antibody. CART or MP treatment not only prevented the cysts formation, but also significantly attenuated the population of GFAP þ astrocytes at days 3, 7, 14, 21 and 28 post-SCI and the hypertrophy of astrocytes at day 14 and 28. The histological consequence of SCI, like cysts formation in the spinal cord, was rapidly improved by CART and/or MP. Taken together, the data suggest that CART may exert its neuroprotective effect via inhibition of post-SCI astrogliosis and participate in the MP mediated neuroprotection. Bhubaneswar 751 005, India c Indian Institute of Science Education and Research, First Floor, Central Tower, Sai Trinity Building, Garware Circle, Sutarwadi, Pashan, Pune 411 021, India Neuroprotective effect of cocaine- and am in spinal cord injury in mice Ashish P. Bharne a,1, Manoj A. Upadhya a,1, Gajanan P Dadasaheb M. Kokare a,* aDepartment of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj, Nagpur Unive b School of Biological Sciences, National Institute of Science Education and Research (NI All rights reserved. phetamine-regulated transcript peptide helkar a, Praful S. Singru b, Nishikant K. Subhedar c, Campus, Nagpur 440 033, India ), Institute of Physics Campus, Sachivalaya Marg, PO Sainik School, SciVerse ScienceDirect acology ier .com/locate/neuropharm arma established (Kristensen et al., 1998; Kuhar et al., 2005; Rogge et al., 2008). Using ischemic brain injurymodel, Xu et al. (2006) proposed neuroprotective role of CART. Neurotrophic effects of CART may be mediated via upregulation of BDNF (Wu et al., 2006), activation of extracellular signal-regulated kinase signaling pathways (Jia et al., 2008) and antioxidant activity (Mao et al., 2007, 2012). Interest- ingly, a direct correlation between glucocorticoids and CART systems is well established (Balkan et al., 2001; Vicentic et al., 2004; Vrang et al., 2000). We hypothesized that CART may play a role in preventing harmful effects triggered by neural damage, and promote axonal regeneration. In mouse model of SCI following contusion, we monitored the locomotor recovery using motor function scoring (MFS) scale (Farooque, 2000). Following SCI, while MP, a reference standard neuroprotective agent widely used in preclinical studies, was administered by intravenous (i.v.) route, CART or its antibody was administered into fourth ventricle. With a view to finding out the interactions, if any, CART or CART-antibody was administered prior to MP immediately following SCI. Sections of the spinal cord were stained with Luxol fast blue-cresyl violet solutions to evaluate the tissue damage following SCI, and eventual recovery, with or without various treatments. Further, to probe the underlying mechanism, we monitored the astrogliosis by GFAP immunolab- eling of astrocytes in ventral gray horn of spinal cord. 2. Materials and methods 2.1. Subjects Adult male Swiss-albino mice (25e30 g) were group housed and given free access to food (Trimurti Feeds, Nagpur, India) and drinking water. Following fourth ventricular cannulation and SCI surgery, the mice were housed one animal per cage and were maintained on a 12-12 h lightedark cycle, in controlled temperature (25� 2 �C) and relative humidity (50e70%). All surgical procedures were performed under aseptic conditions. The experimental procedures were approved by the Institutional Animal Ethics Committee. 2.2. Cannulation in the fourth ventricle The stereotaxic placement of cannula targeted at the fourth ventricle was per- formed as previously described by Bharne et al. (2011). Briefly, mice were anes- thetized with a mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg) injected subcutaneously. A permanent stainless steel guide cannula prepared in house (Kokare et al., 2011) was placed aseptically into the fourth ventricle, using stereo- taxic coordinates, �5.88 mm posterior and 3.80 mm ventral with respect to bregma (Paxinos and Franklin, 2001). The guide cannula was secured using dental cement and mounting screws anchored to the skull. A stainless steel dummy cannula was used to occlude the guide cannula when not in use (Kokare et al., 2011). Animals were allowed a recovery period of 7 days. 2.3. Surgical procedure for SCI We employed the weight-drop contusion method to induce and evaluate SCI in mice. In brief, animals were anesthetized and thoraco-lumbar vertebral region was demarcated. Using the intrascapular space as a reference point, the skin and subcutaneous tissues in the thoracic T10-T12 regionwere incised. The paravertebral muscle fascia was penetrated, and muscles were peeled laterally using blunt dissection forceps. The spinal cord segment at T10-T12 level was exposed by total laminectomy. The animals were subjected to an impact of 5 g weight (stainless steel rod, 3 mm diameter tip, Impactor device for rat and mice, VJ Instruments, Karanja, India) dropped vertically on the center of the exposed spinal cord from the height of 2 cm and allowed to remain for 30 s. In sham-operated mice, the similar procedure was carried out, except that the spinal cord contusion was not performed. The incision was sutured layer to layer using chromic catgut sutures. Necessary post-operative care of the animals was taken as previously described (Bharne et al., 2011). Briefly, the mice were treated with gentamicin (40 mg/kg, intramuscular) twice daily during the first 3 days as prophylaxis against urinary tract infection. The mice were also injected subcutaneously with 1 ml lactated Ringer’s, daily for a period of 10 days. Drinking water, softened chow and regular pellets were provided ad libitum in the cages. Bladders were emptied manually twice a day until bladder function returned to normal. A.P. Bharne et al. / Neuroph Separate sets of sham- and SCI-operated animals were used for behavioral and immunocytochemical/histological studies detailed below. 2.4. Drug treatments and experimental outline CART (54e102), and its monoclonal antibody developed in mouse (Thim et al., 1998), were reconstituted in double distilled water and stored as stock solutions at �20 �C. The stock solution was diluted in aCSF containing 0.1% bovine serum albumin and administered via fourth ventricle. Sodium succinate salt of MP (Solu- Medrol; Pharmacia and Upjohn Company, USA) was dissolved in normal saline and administered via tail vein. Mice were divided into different groups as described below and all the treat- ments were given between 0900 and 1200 h. Group I: sham-operated (n¼ 5), Group II: mice subjected to SCI (n¼ 5), Group III: SCI mice injected with aCSF (n ¼ 8)/saline (n ¼ 6), Group IV: treated with single bolus injection of CART (0.1, 0.3 and 0.5 nM/ mouse, intra-fourth ventricle, n ¼ 8 in each group) 30 min following SCI, Group V: treated with CART-antibody (Dilution 1:500, 5 ml/mouse, intra-fourth ventricle) 30min following SCI (n¼ 7), Group VI: single bolus injection of MP (20, 30 or 40mg/ kg, i.v., n ¼ 6 in each group) 30 min following SCI, Group VII: treated with single bolus injection of CART (0.1 nM/mouse) or CART-antibody (Dilution 1:500, 5 ml/ mouse) 30 min after injury and followed byMP treatment (30 or 40mg/kg, i.v., n¼ 8 in each group) 15 min later. While CART, or its antibody, was administered into the fourth ventricle, MP was injected via tail vein in all protocols. The doses used in the present study were selected on the basis of dose dependent effect of CART, CART- antibody and MP. Hindlimb motor function was observed and scored to evaluate the locomotor recovery of each SCI mouse one day prior to injury and on days 1, 4, 7, 10, 14, 21 and 28 post-injury. 2.5. Assessment of locomotor recovery While different assays have been employed to screen the hindlimb motor functions of mice following SCI (Basso et al., 2006; Pajoohesh-Ganji et al., 2010; Ung et al., 2007), we adopted the hindlimbMFS system proposed by Farooque (2000) and standardized in our laboratory (Bharne et al., 2011). This test is based on detection of movement of ankle, knee and hip joints in freely exploring animal in the open field. We also monitored the ability of mice to walk on bars of different widths. The test detects minor deficits that may be otherwise missed in open field and other assessment methods (Farooque, 2000). Individual animal was allowed to freely explore the open andwell-illuminated arena (0.7 � 0.9 m), and observed for 1 min. Parameters like the movements in the hip, knee, and ankle joints, plantar placement, coordination between forelimbs and hindlimb as well as weight bearing capacity were carefully observed and the performance of the mouse was scored accordingly. Briefly, the score 0 was given to the animals if they showed no movement. If barely visible movement at any hin- dlimb joint (hip, knee or ankle) was observed, then animals were scored 1. Similarly, if movement of one or more hindlimb joints in one or both limbs, but no coordi- nation, alternate stepping movements or weight bearing were observed, then animals were scored 2. Score 3 was given to the animals showing alternate stepping and forward propulsive movements of the hindlimb, but no capability of weight bearing. In these animals, the hindlimbs were externally rotated and they used hindlimbs for forward propulsion. The animals showing ability to bear weight on their hindlimbs and could walk with some deficit (slight external rotation of one or both limbs and/or hip instability) were scored 4. Herein, the plantar placement of the hindlimbwas observed. However, if this deficit in animals was not observed, and the animals showed reduced mobility, then they were scored 5. Those mice with score 5 were then placed on the bars of different widths (2, 1.5, 1, 0.7 and 0.5 cm) and the walking of the animal without any slips, in at least two trials, was recorded. In addition, even if mice showed inability to walk on the bars and tried to climb under the bar, it was considered as a failure. Herein, the animals were scored 6, if theywere able towalk on bars of width 2 cm. Similarly, scores 7, 8, 9 and 10 were given to animals walking on the bars of 1.5, 1, 0.7 and 0.5 cm widths, respectively. During the study, some mortality was observed ( locomotion scores. CART treatment (0.3 and 0.5 nM/mouse) immediately following injury, via fourth ventricle, significantly improved the locomotor function inmice as compared to that of the aCSF treated animals (Fig. 1A). Application of two-way ANOVA showed significant interaction between variables like CART treat- ment (0.3 and 0.5 nM/mouse) and days [F(7,112)¼ 9.106, P< 0.0001 and F(7,112) ¼ 10.28, P < 0.0001]. Application of post-hoc Bonfer- roni’s multiple comparisons test revealed significant improvement in motor function following CART treatment at 0.3 nM/mouse on post-injury days 7 (P< 0.05), 10 (P< 0.01), 14, 21 and 28 (P< 0.001) and 0.5 nM/mouse on post-injury days 1 (P < 0.05), 4 (P < 0.01), 7, 10, 14, 21 and 28 (P < 0.001). Furthermore, application of two-way ANOVA revealed a main effect of CART treatment [F(1,112) ¼ 113.4, P < 0.0001 and F(1,112) ¼ 224.1, P < 0.0001] and days [F(7,112)¼ 279.1, P< 0.0001 and F(7,112)¼ 204, P< 0.0001] at dose of 0.3 nM/mouse and 0.5 nM/mouse, respectively. On the other hand, CART (0.1 nM/mouse) did not improve the locomotor func- tion in mice (P > 0.05) and was considered as subeffective. 3.2. Effect of single bolus i.v. administration of MP on motor recovery following SCI Treatment with single bolus dose of MP in tail vein (30 and 40 mg/kg) following SCI, produced locomotor recovery in a dose dependent manner (Fig. 1B). Application of two-way ANOVA showed significant interaction between variables like MP treat- ment (30 and 40 mg/kg) and days [F(7,80) ¼ 3.609, P < 0.01 and armacology 67 (2013) 126e135 thiopental sodium (70 mg/kg, i.p.), perfused transcardially with heparinized phosphate-buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde fixative in 0.1 M phosphate buffer (pH 7.4). Spinal cord segment at the level of L1-L2 were left in the same fixative for overnight, cryoprotected in 30% sucrose solution in PBS, embedded, and serially sectioned using a cryostat (Leica, CM1850; Germany) at 30 mm thickness in the coronal plane and collected in PBS. GFAP-immunolabeling in the spinal cord sections was performed using streptavidin-biotin-peroxidase method. Sections were incubated in monoclonal anti-mouse GFAP-antibody (1:2000; Chemicon, USA) diluted in PBS containing 2% normal horse serum, 0.3% Triton X-100, 0.2% Kodak PhotoFlo solution and 0.08% sodium azide for 24 h at 4 �C. After rinsing in PBS, the sections were incubated in biotinylated anti-mouse IgG (Vector Laboratories, CA, USA; 1:100) for 3 h, and then in Extravidin-peroxidase conjugate (Sigma-Aldrich, St. Louis, USA; 1:100) for 2 h at room temperature. Immunoreaction was visualized using a solution containing 0.03% hydrogen peroxide and 3-amino-9-ethyl-carbazole (Sigma-Aldrich). Reddish- brown precipitate indicated the presence of antigen in the sections. The sections were washed with double distilled water and mounted in glycerol-gelatin. Spinal cord sections of different groups were processed concurrently under identical conditions to ensure reliable comparisons among different groups and maintain stringency in tissue preparation and staining conditions. Omission of the primary antibody and replacement with bovine serum albumin produced no immunoreaction. 2.7. Morphometric analysis Spinal cord sections of SCI mice treated with vehicle, CART, MP and CART þMP were subjected to morphometric analysis. The GFAP-immunoreactivity was assessed in the ventral gray horn of the spinal cord sections of mice on days 3, 7, 14, 21 and 28 post-SCI. The number of GFAP þ astrocytes was counted per unit area in the ventral horn spinal cord. Only those cells that showed clearly discernible nucleus were counted. Five sections from each spinal cordwere analyzed and the data drawn from all the mice of each control and treated group were separately pooled and the mean � standard error of mean (SEM) was calculated. To assess the hypertrophy of astrocytes, digitized images of the ventral horn gray matter of the spinal cord of the control and various treatment groups of mice at 14 and 28 days post-SCI time point were used for analysis. Background was considered as threshold, and area occupied by individual immunostained astrocyte was measured using Leica-QWin Standard software. Area of such randomly selected 100 astrocytes in different sections from each group was pooled and the mean � SEM were calculated. The skilled observer, blind to the treatments given, recorded all the behavioral as well as morphometric measurements. 2.8. Histology The segment of spinal cord at the level T12-L1 (immediately caudal to injury) was isolated from the mice of various treatment and sham-operated groups. The tissue pieces were fixed in Bouin’s fixative, cut in transverse plane at 16 mm thick- ness on a cryostat and stained with Luxol fast blue-cresyl violet method according to the protocol described earlier (Bharne et al., 2011; Santiago et al., 2009). Area covered by cysts, and that of the entire section, was measured from 5 sections of each spinal cord. The data from various control and experimental groups, drawn at different time points were respectively collated, averaged and expressed as percent of total cross sectional area. 2.9. Statistical analysis The data obtained from MFS are expressed as the mean � SEM. Locomotor recovery at each time point between different groups was analyzed using two-way analysis of variance (ANOVA) (Basso et al., 2006; Bharne et al., 2011). The morphometric data drawn from the GFAP immunostained sections and the histo- logical preparations were analyzed using one-way ANOVA. In both the test, ANOVA was followed by post-hoc Bonferroni’s multiple comparisons test. Differences were considered significant at P < 0.05. 3. Results In normal uninjured mice treated with CART, locomotor activity as monitored in the actophotometer (n ¼ 6 per group), was not different from that displayed by the aCSF treated mice (P > 0.05). The treated mice did not show any motor defects. 3.1. Effect of intra-fourth ventricular CART treatment on locomotor recovery following SCI While SCI resulted in immediate paraplegia (loss of hindlimb A.P. Bharne et al. / Neuroph128 movement), sham-operated mice showed no significant changes in Fig. 1. Effect of CART (A) and methylprednisolone (MP) (B) on hindlimb motor function score. Data are represented as mean of MFS � SEM (n ¼ 6�8) and analyzed using two-way ANOVA followed by post-hoc Bonferroni’s multiple comparisons test. *P < 0.05, **P < 0.01 and ***P < 0.001 versus vehicle (aCSF or Saline). [F(7,80) ¼ 4.98, P < 0.001] for 30 and 40 mg/kg doses of MP, 3.6. Effect of SCI, and MP and/or CART treatment(s) on GFAP- immunoreactivity in spinal cord sections GFAP-immunopositive astrocytes were evaluated in the ventral gray horn of spinal cord of control and SCI mice at each time points extending up to 28 days (Figs. 4 and 5). While isolated GFAP þ astrocytes were seen in the sham-operated mice (Fig. 4), highly significant increase in the astrocyte population was noticed as early as day 3 (P < 0.001) which is peaked at day 7 (P < 0.001) in vehicle treated SCI mice (Fig. 5). Although significantly more than that in the control, the population of GFAP þ astrocytes gradually decreased at days 14, 21 and 28 time point in SCI mice. CART treatment immediately following SCI decreased the population of GFAP þ astrocytes at day 3 (P < 0.05), 7 (P < 0.05), 14 (P < 0.01), 21 (P < 0.05) and 28 (P < 0.01) as compared with vehicle treated time matched SCI groups. Similarly, treatment with MP decreased the population of GFAP þ astrocytes at day 3 (P < 0.01), 7 (P < 0.01), 14 (P < 0.001), 21 (P < 0.05) and (P < 0.05) as compared with the time matched vehicle treated SCI group. SCI mice treated with the combination of CART and MP showed increased population of GFAP þ astrocytes at day 3, which sustained at all time points till day 21. In addition, in the SCI þ vehicle groups, the astrocytes appeared hypertrophic with large and clearly recognizable somata at days 14 and 28 post-SCI (Fig. 6). However, we noticed significant Fig. 2. Effect of pretreatment with subeffective dose of CART (0.1 nM/mouse) in combination with subeffective [20 mg/kg, (A)] and effective [30 mg/kg, (B)] doses of methylprednisolone (MP) on motor function score in mice subjected to spinal cord injury. Data are represented as mean of MFS � SEM (n ¼ 6e7) and analyzed by two- way ANOVA followed by post-hoc Bonferroni’s multiple comparisons test. *P < 0.05, **P < 0.01 and ***P < 0.001 versus respective dose of MP. #P < 0.05, ##P < 0.01 and ###P < 0.001 versus control. arma respectively (Fig. 3A, B). Post-hoc Bonferroni’s multiple compari- sons test revealed that pretreatment of CART-antibody significantly blocked the effect of MP (30 mg/kg) on locomotor recovery at days 1 (P < 0.05), 4, 7 (P < 0.01), 10, 14, 21 and 28 (P < 0.001). Furthermore, CART-antibody also significantly attenuated the effect of higher dose of MP (40 mg/kg) at days 7 (P < 0.05) and 10 (P < 0.01), 14, 21 and 28 (P < 0.001). Statistical analysis revealed a main effect of combination treatment [30 mg/kg MP; F(1,96) ¼ 117.1, P < 0.001 and 40 mg/kg MP; F(1,80) ¼ 96.46, P < 0.001] and days [30 mg/kg MP; F(7,96) ¼ 126.3, P < 0.001 and 40 mg/kg MP; F(7,80) ¼ 95.35, P < 0.001]. However, CART-antibody per se did not produce any effect on the locomotor recovery 3.5. Effect of CART-antibody on MP induced locomotor recovery following SCI Co-treatment with CART-antibody significantly blocked the MP- induced locomotor recovery in SCI mice. Application of two-way ANOVA confirmed the significant interaction between combina- tion treatment and days [F(7,96) ¼ 2.919, P < 0.001] and 3.4. Effect of intra-fourth ventricular CART treatment on MP induced locomotor recovery following SCI Administration of subeffective doses of CART (0.1 nM/mouse) and MP (20 mg/kg) in combination, immediately following SCI, produced significant locomotor recovery on days 7 (P < 0.05), 10 (P < 0.01), 14, 21 and 28 (P < 0.001) as compared to that following MP (20 mg/kg) at corresponding time points (Fig. 2A). Application of two-way ANOVA confirmed significant interaction between combination treatment and days [F(7,96) ¼ 6.764, P < 0.001]. In addition, statistical analysis also revealed amain effect of combined treatment [F(1,96) ¼ 93.41, P < 0.001] and days [F(7,96) ¼ 291.2, P < 0.001]. Furthermore, pretreatment with CART (0.1 nM/mouse) potentiated the locomotor recovery observed following effective dose of MP (30 mg/kg) per se (Fig. 2B). Statistical analysis showed significant interaction between variables like combined treatment and days [F(7,80) ¼ 3.657, P < 0.001]. Application of post-hoc Bonferroni’s multiple comparisons test revealed significant loco- motor recovery from day 1 (P < 0.05). Application of two-way ANOVA showed a main effect of combined treatment [F(1,80) ¼ 91.45, P < 0.001] and days [F(7,80) ¼ 84.66, P < 0.001]. F(7,80) ¼ 5.784, P < 0.001]. Application of post-hoc Bonferroni’s multiple comparisons test revealed progressive locomotor recovery following 30 mg/kg dose from day 1 (P < 0.05). Signif- icant locomotor recovery was observed on days 4 (P < 0.05), 7 (P < 0.01), 10, 14, 21 and 28 (P < 0.001). In addition, MP, at higher dose (40 mg/kg), produced more significant locomotor recovery starting from day 1 and 4 (P < 0.01), which was sustained at all the time points till day 28 (P < 0.001). While MP at 30 and 40 mg/ kg doses significantly prevented the locomotor dysfunction, lower dose (20 mg/kg) did not influence the locomotor recovery following SCI (P > 0.05) and was considered as subeffective. 3.3. Comparison between CART and MP induced locomotor recovery following SCI Treatment with single dose of CART (0.5 nM/mouse) or MP (30 mg/kg) to SCI mice produced comparable locomotor recovery (Fig. 1). No significant differences were encountered across the two groups (P > 0.05). A.P. Bharne et al. / Neuroph (P > 0.05). cology 67 (2013) 126e135 129 reduction in the size of the GFAP þ cells in the CART (P < 0.01) and A.P. Bharne et al. / Neuropharma130 MP (P < 0.01) treated mice at days 14 and 28 as compared to the vehicle treated SCI mice. Hypertrophic GFAP þ cells were seen in the SCI mice treatedwith CARTþMP at days 14 (P> 0.05), although some reduction in size was noticed by day 28 (P < 0.05). 3.7. Histology Spinal cord segments from sham-operated mice showed intact white and gray matter (Fig. 7A). SCI resulted in massive cysts Fig. 4. Transverse section of spinal cord viewing the ventral horn gray matter from spinal cord at the level of L1-L2 of sham-operated mice sacrificed at day 3. Arrow shows the glial fibrillary acidic protein (GFAP)þ astrocytes. Fig. 3. Effect of CART-antibody (CART-Ab) and/or methylprednisolone (MP) [30 mg/kg (A) or 40 mg/kg (B)] on the motor function score in mice subjected to spinal cord injury. Data are represented as mean of MFS � SEM (n ¼ 6e7) and analyzed by two- way ANOVA followed by post-hoc Bonferroni’s multiple comparisons test. *P < 0.05, **P < 0.01 and ***P < 0.001 versus respective doses of MP and #P < 0.05, ##P < 0.01 and ###P < 0.001 versus control. formation in the white matter and the percentage area covered by cysts was significantly increased at days 1 (Fig. 7B, P < 0.001) and 7 (Fig. 7C, P < 0.001). Percentage of area covered by cysts was grad- ually reduced in vehicle treated group and significant reduction was noticed at day 28 (Fig. 7G, P< 0.05) as compared to day 1 post- SCI. CART (0.5 nM/mouse) treatment resulted in significant reduction in the percentage cyst area in the white matter at days 7 (Fig. 7D, P< 0.05) and 28 (Fig. 7H, P< 0.05) post-SCI comparedwith timematched vehicle treatment group. Similar improvement in the tissue profile was observed following MP (40 mg/kg) treatment at days 7 (Fig. 7E, P < 0.05) and 28 (Fig. 7I, P < 0.05). Combination treatment with CART and MP, immediately following SCI, resulted in significant reduction in percentage of area covered by cysts at days 7 (Fig. 7F, P < 0.01) and 28 (Fig. 7J, P < 0.01). 4. Discussion In the present study, weight-drop contusion method was used to induce SCI at T10-T12 level of spinal cord. The method is reproducible and resembles typical SCI condition in humans in terms of gliosis, connective tissue deposition, sensory fiber growth, demyelination, and cysts formation (Kuhn and Wrathal, 1998; Stokes and Jakeman, 2002). We observed tissue deformation and appearance of cysts in the white matter as also reported in previous studies (Bharne et al., 2011; Isaksson et al., 2005). We also noticed that, within 24 h following SCI, the mice were paraplegic and unable to walk on the hindlimb. The disability was reflected in the reduced locomotor function score. A single injection of CART, immediately following ischemic brain injury was found to offer neuroprotection (Xu et al., 2006). Simi- larly, in the present study, CART bolus administration via fourth ventricular route immediately following SCI, resulted in positive effect on locomotor recovery at all the time points. While the present results suggest a causal relationship between CART and neuroprotection, the involvement of other endogenous factors cannot be ruled out. We observed some motor recovery in the SCI mice treated with aCSF, which may be attributed to the response of the surviving neural elements in the spinal cord. CART (55e102 or 42e89), at the dose of 1 mg or more, admin- istered intracerebroventricularly influenced locomotion and produced motor defects, while the lower doses were found to be ineffective (Aja et al., 2001; Kristensen et al., 1998; Vrang et al., 1999). Dandekar et al. (2008, 2009) reported no influence of CART (54e102) on the locomotor activity at the dose 200 ng/rat. In the present study also, we did not observe any motor defects or influence on spontaneous locomotor activity following intra-fourth ventricular administration of CART (54e102) at dose 0.5 nM/mouse (260 ng/5 ml/mouse). Furthermore, we administered single dose of CART immediately following SCI and the assessment of locomotor recovery was performed at time points from day 1 through day 28 post-SCI. Thus, it is rather unlikely that, CART, given by intra-fourth ventricular route, will influence locomotor activity at these delayed time points. MP treatment showed the significant locomotor recovery by inhibiting the early lipid peroxidation and then producing favorable environment for remyelination (Christie et al., 2008). In the present study, i.v. bolus administration of MP dose dependently showed significant attenuation of the locomotor dysfunction at day 1 and further facilitated the locomotor recovery as compared to that of the saline treated group. CART potentiated the effect of MP with refer- ence to the locomotor function. Physiological interaction between glucocorticoids and CART system has already been studied. Vrang et al. (2000) reported that central injection of CART increased plasma corticosterone level. Balkan et al. (2001) mentioned that cology 67 (2013) 126e135 CART expression in the hypothalamus is under the control of arma A.P. Bharne et al. / Neuroph glucocorticoids. Since the neuroprotective effect of MP was blocked by prior administration of CART-antibody, there is a possibility that MPmight upregulate the endogenous CART system. Xu et al. (2006) reported that CART-antibody blocked the ability of estradiol to reduce cell death inmodel of brain ischemia. In this background,we Fig. 5. Transverse sections through the spinal cord (AeT) at L1-L2 showing GFAP þ neuron indicated as columns and durations in rows. Note highly significant increase in the populati days 3 (A), 7 (E), 14 (I), 21 (M) and 28 (Q) compared to that in sham-operated mice (Fig. 4). S with CART (B,F,J,N,R) as well as MP (C,G,K,O,S) at day 3, 7, 14, 21 and 28 respectively. H GFAP þ astrocytes. Morphometric analysis of the GFAP-immunoreactivity in the ventral ho scale) shown in the spinal cord schematic demarcates the area from which GFAP þ cells we horn; VH, ventral horn; VWM, ventral white matter. One-way ANOVA followed by post-ho CART and/or MP treatment following SCI and inter-day effect of SCI on GFAP-immunoreactiv ***P < 0.001 versus vehicle treated SCI group at respective time points. Scale bar ¼ 50 mm cology 67 (2013) 126e135 131 suggest that the endogenous CART may enhance locomotor improvement and facilitate the effects of MP in SCI. Reactive astrogliosis is commonly associated with injury of nervous system including SCI (Sofroniew, 2009). Post-SCI astro- gliosis is characterized by activation of astrocytes and their s in the ventral gray horn of spinal cord injured (SCI) mice. The various treatments are on of glial fibrillary acidic protein (GFAP)þ astrocytes in the vehicle treated SCI mice at ignificant reduction in the GFAP þ immunoreactive cells was seen following treatment owever, co-treatment of CART and MP (D,H,L,P,T) did not result in the reduction of rn gray matter following various treatments is also represented (U). Rectangle (not to re counted. cc, central canal; DWM, dorsal white matter; GM, gray matter; LH, lateral c Bonferroni’s multiple comparisons test was employed for the intra-day effect of SCI, ity. $P < 0.01 and $$P < 0.001 versus sham-operated mice, and *P < 0.05, **P < 0.01 and . Fig. 6. Transverse sections through the spinal cord (AeH) at L1-L2 showing GFAP þ cells in the ventral gray horn of spinal cord injured mice. While the astrocytes in the vehicle treated mice showed considerable hypertrophy (A,E), the cell size was significantly reduced in the mice treated with CART (B,F) or MP (C,G). The astrocytes in the mice treated with CART þ MP showed hypertrophy at day 14 (D) but their size was reduced by day 28 (H). Bar graph (I) represents average immunoreactive area of astrocytes in different groups of treated and control mice. One-way ANOVA followed by post-hoc Bonferroni’s multiple comparisons test was employed for analysis. #P < 0.05, *P < 0.01 versus vehicle treated SCI group. Scale bar ¼ 20 mm. A.P. Bharne et al. / Neuropharmacology 67 (2013) 126e135132 arma A.P. Bharne et al. / Neuroph precursors that migrate and show hypertrophic and proliferative changes (Eng and Ghirnikar, 1994; Wang and Bordey, 2008). In the acute phase of SCI, reactive astrocytes play pro-inflammatory role in the center or immediate vicinity of lesions contributing to the Fig. 7. Transverse sections through the spinal cord, at the level of T12-L1, of various control a method.While broken line demarcates the grayandwhitematter, rectangle demarcates the are white and graymatter (A). Significant increase in thepercentageof cysts areawas noticed in the (A). However, at day7, treatmentwithCART (D),MP (E) or CARTþMP(F), showed reducedperc group (C). Percentage cysts area invehicle treated groupwas found tobe significantly reduced a or CARTþMP (J) also showed significantly decreased cysts area compared to that in the SCImic the total cross sectional area of spinal cord. One-way ANOVA followed by post-hoc Bonferron different time points. $P< 0.01, $$P< 0.001, compared to sham-operated; *P< 0.05, **P< 0.01 vehicle treatment group at day 1 post-SCI group. Scale bar ¼ 500 mm for section of the spina cology 67 (2013) 126e135 133 inhibition of axonal regeneration (Liuzzi and Lasek, 1987; Rudge and Silver, 1990). Thus, inhibition of astroglial activation might facilitate axonal regeneration after SCI, and the phenomenon has been demonstrated using therapeutic interventions in rodents nd treated groups, at 1, 7 and 28 days post-SCI, stained with Luxol fast blue-cresyl violet a shownasmagnified view. Spinal cord of sham-operatedmice showednormal profile of whitematter at days 1 (B) and7 (C) post-SCI, as compared to that in sham-operatedmice entage of cysts area in thewhitematter as compared to that in the SCIþ vehicle treatment t day28 (G) as compared to that in the day1post-SCI (B). TreatmentwithCART (H),MP (I) e treatedwith vehicle (G). Bar graph (K) represents the percentageof cysts area relative to i’s multiple comparisons test was employed for the comparison of treatment groups at compared to vehicle treatment group at respective time points; #P< 0.05, compared to l cord, while 100 mm for the magnified view. arma (Labombarda et al., 2011; Tian et al., 2007; Vitellaro-Zuccarello et al., 2008). Immediate release of pro-inflammatory cytokines and generation of reactive oxygen species (ROS) are key mediators of reactive astrogliosis in SCI (Gwak et al., 2012). Recently, neuro- protective effect of CART in brain injury was correlated with the neuroimmunomodulation (Chang et al., 2011) and its antioxidant property (Mao et al., 2012). Marked upregulation of GFAP- immunoreactivity and hypertrophy of astrocytes are hallmark of the astrogliosis (Wang and Bordey, 2008;Wilhelmsson et al., 2006). While Rothman and Winkelstein (2007) reported proliferation of GFAP þ astrocyte from day 7, we observed the increase from day 3 onward, reaching a high at day 7, which was sustained till day 21 post-SCI time point. Increase in the population of GFAP þ cells, till day 14 following SCI, has been reported (Schnell et al., 1999; Wu et al., 2005). However, CART treatment per se immediately following SCI, attenuated the GFAP over-expression from day 3 onward post-SCI suggesting its inhibitory effect on astrocytic proliferation and possible promotion of cellular healing. This data is well correlated with the locomotor recovery of CART treated paraplegic mice from day 1 post-SCI. MP treatment also decreased the GFAP þ cell population at all days examined following SCI. Liu et al. (2008) reported the attenuation of GFAP þ astrogliosis following MP treatment and suggested its role in neuronal repair following excitotoxic insult of spinal cord. SCI is known to cause an increase in size of somata with prominent enlargement of processes, known as astrocytic hyper- trophy (Vitellaro-Zuccarello et al., 2008). We also observed hyper- trophy of astrocytes in SCI-inducedmice at days 14 and 28 post-SCI, which was attenuated by CART or MP treatment. Hypertrophy of astrocytes following SCI is mainly attributed to the extracellular ionic imbalance (Bender et al., 1998; Gwak et al., 2012). Although at this stage we do not know if there is any causal relationship between CART and the regulation of ion channels following injury, we may recall that CART inhibited voltage-dependent intracellular Ca2þ signaling in hippocampus (Yermolaieva et al., 2001). We speculate that, CART via modulation of the ion channels may attenuate the hypertrophy of the GFAP þ astrocytes. Although MP and CART individually caused reduction in the GFAP þ astrocyte population, surprisingly, an opposite effect was encounteredwhen a combination of the agents was given to the SCI mice. In these animals a high number of GFAP þ astrocytes was consistently noticed following SCI. This is in spite of the fact that the mice showed considerable improvement in locomotion score and also inhistological profile.Whilewedonot know the reasons for this discrepancy, we speculate that, the two agents, interact by some unknownmechanism, and fail to attenuate GFAPþ astrogliosis, and that this effect might be independent of the recovery process. While the exact mechanisms by which CART attenuated GFAP þ astrogliosis are not known, SCI is known to cause the release of pro-inflammatory cytokines and ROS, key mediators of reactive astrogliosis (Gwak et al., 2012). Recently, CART is reported to inhibit early cytokines expression in brain injury model (Chang et al., 2011) and scavenge ROS (Mao et al., 2012). The neuro- protective effect of CART involves upregulation of BDNF (Wu et al., 2006) that reduced astrogliosis (Schabitz et al., 2004). In this background, we suggest the following possibilities: (i) receptors for CART may be present on astrocytes, (ii) CART may upregulate BDNF that indirectly reduces astrogliosis, (iii) CART may inhibit the expression of early pro-inflammatory cytokines, and (iv) CART may scavenge the ROS generated following SCI. 5. Conclusion SCI causes hindlimb paraplegia, tissue deformity at injury site A.P. Bharne et al. / Neuroph134 with cysts formation and increased GFAP þ astrogliosis. CART treatment facilitated the locomotor recovery, reduced the struc- tural deformity in spinal cord and decreased the post-SCI GFAP þ astrogliosis. Neuroprotective effects of CART were similar to those of the reference drug MP. 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Xu, Y., Zhang, W., Klaus, J., Young, J., Koerner, I., Sheldahl, L.C., Hurn, P.D., Martinez- Murillo, F., Alkayed, N.J., 2006. Role of cocaine- and amphetamine-regulated transcript in estradiol-mediated neuroprotection. Proc. Natl. Acad. Sci. U. S. A. 103, 14489e14494. Yermolaieva, O., Chen, J., Couceyro, P.R., Hoshi, T., 2001. Cocaine- and amphetamine- regulated transcript peptide modulation of voltage-gated Ca2þ signaling in hippocampal neurons. J. Neurosci. 21, 7474e7480. Neuroprotective effect of cocaine- and amphetamine-regulated transcript peptide in spinal cord injury in mice 1. Introduction 2. Materials and methods 2.1. Subjects 2.2. Cannulation in the fourth ventricle 2.3. Surgical procedure for SCI 2.4. Drug treatments and experimental outline 2.5. Assessment of locomotor recovery 2.6. Immunohistochemistry 2.7. Morphometric analysis 2.8. Histology 2.9. Statistical analysis 3. Results 3.1. Effect of intra-fourth ventricular CART treatment on locomotor recovery following SCI 3.2. Effect of single bolus i.v. administration of MP on motor recovery following SCI 3.3. Comparison between CART and MP induced locomotor recovery following SCI 3.4. Effect of intra-fourth ventricular CART treatment on MP induced locomotor recovery following SCI 3.5. Effect of CART-antibody on MP induced locomotor recovery following SCI 3.6. Effect of SCI, and MP and/or CART treatment(s) on GFAP-immunoreactivity in spinal cord sections 3.7. Histology 4. Discussion 5. Conclusion Conflicts of interest Acknowledgment References