Neurochem Res DOI 10.1007/s11064-015-1604-3

ORIGINAL PAPER

Mitochondrial Division Inhibitor 1 Ameliorates Mitochondrial Injury, Apoptosis, and Motor Dysfunction After Acute Spinal Cord Injury in Rats Gang Li1 • Zhiqiang Jia1 • Yang Cao1 • Yansong Wang1 • Haotian Li1 Zhenyu Zhang2 • Jing Bi3 • Gang Lv1 • Zhongkai Fan1

•

Received: 1 December 2014 / Revised: 30 April 2015 / Accepted: 4 May 2015 Ó Springer Science+Business Media New York 2015

Abstract Mitochondrial division inhibitor 1 (Mdivi-1) is the most effective pharmacological inhibitor of mitochondrial fission. Spinal cord injury (SCI) is a common and serious trauma, which lacks efficient treatment. This study aimed to detect the role of Mdivi-1 in neuronal injury and its underlying mechanism after acute SCI (ASCI) in rats. Western blot analysis showed that Bax levels on the mitochondrial outer membrane, and release of cytochrome C (cytC) and apoptosis-inducing factor (AIF) from the mitochondria began to increase significantly at 4 h after ASCI, then peaked at 16 h, and declined significantly from 16 to 24 h. However, the mitochondrial levels of Bcl-2 increased significantly at 2 h, peaked at 4 h, and subsequently significantly decreased from 4 to 24 h after ASCI. In addition, Mdivi-1(1.2 mg/kg) significantly suppressed the translocation of dynamin-related protein 1 (Drp1) and Bax to the mitochondria, mitochondrial depolarization, decrease of ATP and reduced Glutathione, increase of the Malondialdehyde, cytC release, and AIF translocation at & Gang Lv [email protected] & Zhongkai Fan [email protected] 1

Department of Orthopaedics, The First Affiliated Hospital, Liaoning Medical University, 5-2 Renmin Street, Guta District, Jinzhou 121000, Liaoning Province, People’s Republic of China

2

Department of Orthopaedics, The First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, People’s Republic of China

3

Key Laboratory of Neurodegenerative Diseases, Liaoning Medical University, Jinzhou 121000, Liaoning Province, People’s Republic of China

16 h and 3 days after ASCI, and also inhibited the caspase3 activation and decrease of the percentage of apoptotic cells at 16 h, 3 and 10 days, further, ameliorated the motor dysfunction greatly from 3 to 10 days after ASCI in rats. This neuroprotective effect was dose-dependent. However, Mdivi-1(1.2 mg/kg) had no effects on the translocation of Bcl-2 and fission protein 1 on the mitochondria, and did not affect the expression of total Drp1 at 16 h after ASCI. Our experimental findings indicated that Mdivi-1 can protect rats against ASCI, and that its underlying mechanism may be associated with inhibition of Drp1 translocation to the mitochondria, alleviation of mitochondrial dysfunction and oxidative stress, and suppression of caspase-dependent and -independent apoptosis. Keywords Acute spinal cord injury  Dynamin-related protein 1  Mitochondrial division inhibitor-1  Mitochondrial function  Oxidative stress  Apoptosis

Introduction Spinal cord injury (SCI) often results in complete loss of voluntary motor and sensory functions below the site of injury [1]. According to a report on the National Spinal Cord Injury Center website (www.uab.edu/nscisc), it was estimated that approximately 276,000 people were living with SCI in the United States in 2014. SCI leads to highly dynamic and complex patterns of secondary destructive biochemical and pathophysiological processes that result in devastating neurological defects [2]. A prospect for improved therapies comes from an improved understanding of the secondary injury mechanisms after SCI. However, after decades of research and clinical trials, despite numerous studies reporting some measures of efficacy in animal

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literature, there is currently no effective pharmacological treatment for acute or chronic SCI in humans [3]. Currently, extensive evidence indicates that therapeutic strategies targeting a specific biochemical cascade may not provide the best approach for promoting functional recovery, while a ‘‘systems approach’’ at the subcellular level-mitochondria may provide a better strategy for promoting cell survival and function and consequently improve functional outcomes following SCI [2–5]. Mitochondria are highly dynamic organelles that undergo frequent fission and fusion [6]. They are critical organelles involved in adenosine triphosphate (ATP) synthesis, calcium homeostasis, oxidative stress, and apoptosis [7]. Mitochondrial dysfunction and biogenesis, as well as apoptosis following SCI may be responsible for secondary pathophysiology [2, 4, 8–10]. Under physiological conditions, a small amount of reactive oxygen species (ROS) are generated as byproducts of ATP synthesis in mitochondria, but these ROS are rapidly removed by antioxidants in mitochondria to maintain homeostasis between production and scavenging of free radicals, and thus produce no pathological effects [11]. However, SCI has been shown to result in the release of glutamate and activation of glutamate receptors, which increases the accumulation of intracellular Ca2? and results in the formation of ROS [9]. ROS overproduction exceeding its scavenging capacity in vivo can lead to an imbalance between oxidants and antioxidants [12]. ROS increase the catabolism of membrane phospholipids, promote the increase of lipid peroxides [13], and further damage mitochondrial membranes, leading to the depolarization of the mitochondrial membrane potential and inhibition of ATP synthesis. Moreover, mitochondrial dysfunction can also lead to an increase in ROS, thus creating a vicious cycle which further increases mitochondrial membrane permeability and destroys the integrity of mitochondria, resulting in increased mitochondrial injury and apoptosis [2, 14, 15]. Mitochondrial biogenesis is a complex process involving the coordinated expression of mitochondrial and nuclear genes, the import of the products of the latter into the organelle and turnover. Disruption of any of these processes can lead to mitochondrial dysfunction and therefore to a disease state [16, 17]. The balance of mitochondrial fission and fusion is a important part of mitochondrial biogenesis [18]. Dynamin-related protein 1 (Drp1) and mitochondrial fission 1 (Fis1) are the main mitochondrial fission proteins in mammalian cells. Drp1 is a large GTPase that translocates to puncta on the mitochondrial outer membrane, where it couples GTP hydrolysis with membrane constriction and fission. Fis1 is a receptor of Drp1 that is anchored to the mitochondrial outer membrane [3, 19, 20]. Notably, recent studies have shown that mitochondrial fusion and fission are affected after ASCI [3, 21].

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Mitochondrial division inhibitor-1 (Mdivi-1) is a highly efficient small molecule that selectively inhibits mitochondrial fission by inhibiting Drp1 self-assembly and GTPase activity [22–27]. Mdivi-1 can cross the blood– brain barrier, and its half-life is estimated at 12 h [28]. Mdivi-1 is considered to be a representative drug for the treatment of amyotrophic lateral sclerosis [29], seizures [25, 27], acute kidney injury [26], acute myocardial infarction [30, 31], acute retinal ischemic injury [32], and acute cerebral ischemic injury [24]. Mdivi-1 may have therapeutic potential as a Drp1 inhibitor in tissue damage caused by different insults. However, it is unclear whether Mdivi-1 is neuroprotective after ASCI in rats. In this study, we first examined the time-dependent changes in key proteins of mitochondrial apoptotic pathway at the mitochondrial level, and further investigated the effect of Mdivi-1 pretreatment on mitochondrial dysfunction as well as oxidative stress, apoptosis and motor function after ASCI. This study contributes more insight into spinal cord secondary injury, and provides a new way of preventing and treating ASCI.

Materials and Methods Animals The adult female Sprague–Dawley rats (250–300 g) were purchased from the Experimental Animals Center of Liao Ning Medical University. All animal experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Establishment of an Acute SCI Model Using the Modified Allen’s Method Adult rats were anesthetized using pentobarbital Sodium (40 mg/kg, i.p.). The skin and muscle overlying the spinal column were incised and a laminectomy was performed at T9–11, leaving the dura intact. Rats’ spinal cords were hit extradurally with a 20 g weight-drop impactor dropped 25.0 mm onto the T10 region of the cord exposed by laminectomy as described previously [33]. Postoperatively all animals received an injection of 0.9 % normal saline (30 ml/kg) to prevent postoperative dehydration. Each animal was housed alone in a cage after surgery and exposed to 12 h light/dark cycles, with free access to food and water. Experimental Groups and Mdivi-1 Administration Rats were randomly divided into four groups: (1) Shamoperated group (Sham), (2) Sham-operation and Mdivi-1

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(Sigma-Aldrich, St. Louis, MO; 1.20 mg/kg, i.v; Sham ? Mdivi-1) pretreatment group, (3) spinal cord injury group (0.1 ml 0.1 % dimethyl sulfoxide, SCI), and (4) SCI and Mdivi-1 pretreatment group (1.20 mg/kg, SCI ? Mdivi-1) [27, 34]. We also examined the effect of Mdivi-1 (0.24 mg/kg) on the expression of Drp1 on the mitochondrial outer membrane at 16 h after ASCI. The rats in the SCI and SCI ? Mdivi-1 groups were underwent a T9–11 laminectomy and injury at thoracic level 10 (T10), using an established Allen’s model. The animals in the Sham and Sham ? Mdivi-1 groups underwent a T9–11 laminectomy without injury. Animals in all the experiments were randomly assigned before the experiment. In the Sham ? Mdivi-1 and SCI ? Mdivi-1 group, the rats were given Mdivi-1 15 min before laminectomy or ASCI through an intravenous bolus, and the rats in the Sham and SCI group received 0.1 ml 0.1 % dimethyl sulfoxide through an intravenous bolus [24, 25]. The rats in SCI group were sacrificed at 2, 4, 8, 16, and 24 h, 3 and 10 days after ASCI. The rats in the Sham and Sham ? Mdivi-1 groups were sacrificed at 16 h after laminectomy. The rats in the SCI ? Mdivi-1 group were sacrificed at 16 h, and 3 and 10 days after ASCI. Extraction of Whole-Cell, Cytoplasm, Mitochondrial, and Nuclear Fractions In each group, rats were sacrificed under anesthesia at predetermined times. Under sterile conditions, a 1 cm segment of the spinal cord containing the compression injury site was rinsed with phosphate-buffered saline (PBS) and stored at -80 °C. However, the spinal cord tissues were freshly prepared for detecting mitochondrial membrane potential, and ATP, Malondialdehyde (MDA), reduced Glutathione (GSH) levels. The 1 cm segment of spinal cord containing the compression injury site which was used for the detection of total caspase-3 and Drp1 was sonicated in ice-cold lysis buffer (2 mM EDTA, 10 mM EGTA, 0.4 %NaF, 20 mM Tris–HCl and protease inhibitors, pH 7.5), then centrifuged at 12,0009g for 20 min at 4 °C, and the supernatants were collected. The 1 cm segment of the spinal cord tissue containing the compression injury site which are used for the isolation of mitochondrial, cytoplasmic, and nuclear fractions, were performed according to the manufacturer’s instructions of the mitochondria isolation kit and the nuclear-cytosol extraction kit (Applygen Technologies, Beijing, China). The freshly prepared mitochondria were used to detect the mitochondrial membrane potential, ATP, MDA, reduced GSH, and Bax, Bcl-2, cytochrome C (cytC), apoptosis-inducing factor (AIF), Drp1, and Fis1 on the mitochondrial outer membrane.

Measurement of Mitochondrial Membrane Potential 5,50 ,6,60 -tetrachloro-1,1 0 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide (JC-1) is a membrane potential-sensitive probe that accumulates in energized mitochondria and subsequently forms J-aggregates from monomers. The depolarization of the mitochondrial membrane is associated with a decrease in the red fluorescence, as well as an increase in green fluorescence. Briefly, at 16 h and 3 days after surgery, mitochondria were prepared as described earlier. The freshly prepared purified mitochondria were resuspended in a solution from a mitochondria isolation kit at a concentration of 0.5 lg protein/ll, and then immediately used for detecting the mitochondrial membrane potential according to the manufacturer’s instructions. JC-1 (Beyotime, Shanghai, China) was visualized [35, 36] by a fluorescence microscope (2009, Leica DMI4000B, Germany) connected to an Olympus Magnafire digital camera (Olympus Corp., Melville, NY, USA) and fluorescence spectrophotometer (Shanghai Analytical Instrument Factory, 970CRT, China) [21, 37, 38]. This process does not add any other mitochondrial substrate. A 0.3 ml suspension of purified mitochondria with a total protein content of 150 lg was incubated in 2.7 ml working solution. After mixing, 2.5 ml mitochondrial mixture was used for fluorescence spectrophotometry, and the remaining 0.5 ml mitochondrial mixture was used for fluorescence microscopy. The fluorescence intensity of both mitochondrial JC-1 monomers (k ex 490 nm, k em 530 nm) and aggregates (k ex 525 nm, k em 590 nm) were detected using a fluorescence microscope or fluorespectrophotometer. The ratio between red and green fluorescence values can be used as an indicator of the mitochondrial inner membrane potential. The experiments were repeated six times independently. Mitochondrial ATP, Malondialdehyde (MDA), and Reduced Glutathione (GSH) Measurement At 16 h and 3 days after surgery, the freshly prepared mitochondria were used for detecting the ATP, MDA, and reduced GSH in the mitochondria, according to the manufacturer’s instructions of the ATP, MDA, and reduced GSH assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) respectively and normalized to protein concentration [39, 40]. Mitochondrial ATP was measured by biochemical method with a VIS Spectrophotometer (Shanghai Precision Scientific Instrument Limited Company, 721, China) at 636 nm. Mitochondrial ATP was expressed as lmol/g protein. Mitochondrial MDA content was measured by method of thiobarbituric acid (TBA) with a VIS spectrophotometer at 532 nm. Results were

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expressed as nmol/mg protein. Mitochondrial reduced GSH was assessed by determining the conversion of 5-5dithiobis (2-nitrobenzoic acid) into the colored product 2-nitro-5-thiobenzoic acid, which is measured at 412 nm using a VIS spectrophotometer. Mitochondrial reduced GSH concentration was expressed as mg/g protein. The experiments were repeated six times independently. TUNEL Staining At 16 h, 3 and 10 days after surgery the injured spinal cords from rats perfused with 4 % paraformaldehyde were immersed in 30 % sucrose at 4 °C for 24 h. Tissue blocks were embedded in Tissue-Tek O.C.T. Compound 4583 (Sakura Finetechnical, Tokyo, Japan), frozen, and then stored at -80 °C. Serial 10-lm-thick transverse frozen sections from the epicenter of SCI site (five sections separated 100 lm apart/per animal) were prepared with a freezing microtome for TUNEL labeling with an In Situ Cell Death Detection Kit, TMR red (Roche, Basel, Switzerland, USA). Following TUNEL staining, all the sections were washed and then layered with a coverslip with 1 lg/ml 40 ,6-diamidino-2phenylindole (DAPI) for 5 min to counterstain the nuclei. The digital imaging was carried out in a double-blind manner using a fluorescent microscope (2009). Four fields of spinal cord (two horizons for each ventral horn) that do not overlap were randomly selected in the sections. The number of TUNEL-positive cells (red cells) and the total number of cells (blue cells) were counted. The percentage of apoptotic cells was defined as follows: percentage of apoptotic cells (%) = 100 9 (apoptotic cells/total cells). The experiments were independently repeated six times. Immunofluorescence Staining Ten days after surgery, serial 10-lm-thick transverse frozen sections from the epicenter of SCI site (three sections separated 100 lm apart/per animal) were prepared. All tissue sections were incubated in PBS containing 0.25 % Triton X-100 for 10 min to permeabilize the cells. Sections were incubated with 5 % normal donkey serum with 0.3 M glycine for 1 h at room temperature to block nonspecific binding of the antibodies. The tissue sections were incubated with anti-active caspase-3 antibody (ab2302) (1:50; Abcam, USA), diluted in 5 % normal donkey serum and incubated in a humidified chamber overnight at 4 °C. Tissue sections were washed three times with PBS with 0.1 % Tween 20 (PBST), and incubated with IFKine Red AffiniPure donkey anti-rabbit IgG(H ? L) (1:400; Abbkine, USA) for 1 h at room temperature in the dark. Following immunostaining, all the sections were washed and then layered with a coverslip with 1 lg/ml DAPI for 5 min to counterstain the nuclei. Negative controls included sections in which the primary

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antibody was omitted. The sections were visualized under a fluorescent microscope. To avoid counting the same cell in more than one section, we counted every eleventh Section (100 lm apart). The number of Caspase-3-positive cells in the cortex 1 mm from the wound center was counted at 4009 magnification. For each section, four separate cortex regions (two horizons for each ventral horn) were examined. Numbers of Caspase-3-positive cells (red cells) and the total number of cells (blue cells) were counted using the counting function of Photoshop CS3. The percentage of Caspase3-positive cells was defined as follows: percentage of Caspase-3-positive cells (%) = 100 9 (Caspase-3-positive cells/total cells). The experiments were independently repeated six times. Western Blot Analysis After determining the protein concentration by use of a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). The protein lysates (30 lg per lane for each sample) were fractioned on 12 or 15 % SDS–polyacrylamide gels, followed by transferring to 0.2 or 0.45 lm Polyvinylidene fluoride membranes. The membranes were blocked with 1 % Bovine serum albumin Tris-buffered saline with 1 %Tween 20 (TBST) for 1 h to block non-specific binding sites, and then incubated with DRP1 (D6C7) Rabbit mAb (1:1000; CST, USA), Fis1 Polyclonal Ab BioVision 3491-100(1:200; Bio Vision, USA), Bcl-2 (50E3) Rabbit mAb (1:1000; CST, USA), Bax Antibody (1:1000; CST, USA), Anti-Cytochrome C antibody (ab53056) (1:1000; Abcam, USA), AIF (D39D2) XPÒ Rabbit mAb (1:1000; CST, USA), Anti-active Caspase-3 antibody (ab2302) (1:500; Abcam, USA), VDAC (D73D12) Rabbit mAb (1:1000; CST, USA), Lamin A Antibody (H-102) (1:500; Santa Cruz Biotechnology, Inc. USA), b-actin (C4) (1:1000; Santa Cruz Biotechnology, Inc. USA) overnight at 4 °C and followed by each corresponding second antibody goat antirabbit IgG-HRP or goat anti-mouse IgG-HRP(1:2000; Santa Cruz Biotechnology, Inc. USA) at room temperature for 1 h at 37 °C. Reaction products were visualized using an enhanced chemiluminescence kit (Millipore, USA). The protein bands were then analyzed using the Image J software. The grayscale value of Drp1, Fis1, Bcl-2, Bax, CytC, AIF and active caspase-3 was normalized to the values of the corresponding voltage-dependent anion channel (VDAC), b-actin or Lamin A band to determine the expression level of the protein. The experiments were repeated six times independently. Motor Functional Test The motor function of rats subjected to laminectomy or compression injury was evaluated once a day until 10 days

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after SCI. Recovery from motor disturbance was graded using the modified murine Basso, Beattie, and Bresnahan (BBB) hindlimb locomotor rating scale [41, 42]. The experiments were repeated six times independently. After finishing the detection of the motor function, these animals are euthanized and the spinal cord tissue are used for immunofluorescence staning of active Caspase-3 and TUNEL staining at 10 days after ASCI.

the mitochondrial outer membrane (Fig. 2a). Mdivi-1 (1.2 mg/kg) significantly reduced Drp1 levels on the mitochondrial outer membrane (P \ 0.05) (Fig. 2b), but did not alter total cellular levels of Drp1 and Fis1 on the mitochondrial outer membrane (Fig. 2c, d). Effects of Mdivi-1 Pretreatment on Mitochondrial Membrane Potential, ATP, MDA, and Reduced GSH Levels After ASCI

Statistical Analysis Two trained observers, blinded to experiment condition, worked in conjunction with the experimenters who had conducted the experimental session to weigh and code the experimental data. However, if there was a disagreement in individual scores, both observers would reevaluate the data together by reaching a consensus agreement before combining the individual scores. SPSS version 19.0 was used for all analysis. All Data’s were expressed as mean ± SD. Differences were studied by one-way analysis of variance (ANOVA) and the LSD-t test. P \ 0.05 was considered statistically significant.

Results Changes of Key Proteins in the Mitochondrial Apoptotic Pathway After ASCI Western blots revealed that in comparison with the Sham group, the mitochondrial levels of Bax increased significantly at 4 h, and peaked at 16 h in the SCI group (P \ 0.05). In contrast, Bcl-2 levels on mitochondria increased significantly at 2 h and reached a maximum level at 4 h in the SCI group (P \ 0.05; Fig. 1a). The protein levels of cytC and AIF on mitochondria decreased significantly at 4 h and reached a minimum level at 16 h in the SCI group (P \ 0.05). Consistent with mitochondrial release, the protein levels of cytC in the cytoplasm and AIF in the nuclei increased significantly at 4 h and reached a peak at 16 h in the SCI group (P \ 0.05; Fig. 1b–d). Effects of Mdivi-1 Pretreatment on Drp1 and Fis1 After ASCI Western blot analysis showed that compared with the Sham group, Drp1 and Fis1 levels on the mitochondrial outer membrane, and the total cellular levels of Drp1 increased significantly in the SCI group (P \ 0.05), but there were no differences between the Sham and Sham ? Mdivi-1 groups (Fig. 2). Compared with the SCI group, Mdivi-1 (0.24 mg/kg) had no effects on the expression of Drp1 on

Compared with the Sham group, the mitochondrial membrane potential, ATP, and reduced GSH levels decreased significantly, while MDA level increased significantly in the SCI group (P \ 0.05); However, there is no difference between Sham and Sham ? Mdivi-1 group; compared with the SCI group at 16 h and 3 days, the mitochondrial membrane potential and ATP levels were maintained significantly, MDA levels decreased significantly, but reduced GSH levels increased significantly in the SCI ? Mdivi-1 group (P \ 0.05; Fig. 3). Effects of Mdivi-1 Pretreatment on Apoptosis After ASCI Western blot results revealed that in comparison with the Sham group, the expression of Bax and Bcl-2 on mitochondria, cytC in the cytoplasm, AIF in the nucleus, and total active caspase-3 were significantly increased in the SCI group at 16 h and 3 days (P \ 0.05). The expression of cytC and AIF in the mitochondria were significantly decreased in the SCI group at 16 h and 3 days (P \ 0.05). Compared with the SCI group at 16 h and 3 days, the expression of Bax on mitochondria and cytC in the cytoplasm, AIF in the nucleus, and total active caspase-3 were significantly decreased in the SCI ? Mdivi-1 group (P \ 0.05). The expression of cytC and AIF in the mitochondria were significantly increased in the SCI ? Mdivi1 group (P \ 0.05), but the level of Bcl-2 on the mitochondrial outer membrane did not change (Fig. 4a–e). Immunofluorescence staining results showed that compared with the Sham group, total active caspase-3 was significantly increased in the SCI group at 10 days (P \ 0.05); compared with the SCI group at 10 days, total active caspase-3 was significantly decreased in the SCI ? Mdivi-1 group (P \ 0.05, Fig. 4f, g). Fluorescence TUNEL assay showed that compared with the Sham group, the percentage of apoptotic cells increased significantly in the SCI group at 16 h, and 3 and 10 days(P \ 0.05); compared with the SCI group at 16 h, and 3 and 10 days, the percentage of apoptotic cells decreased significantly in the SCI ? Mdivi-1 group (P \ 0.05, Fig. 4h, i). The basal levels of the above indicators did not differ in the Sham group and in the Sham ? Mdivi-1 group (Fig. 4).

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Fig. 1 Western blot analysis (a, b) and quantitative analysis (a, c, d) of Bax, Bcl-2, cytC, and AIF at various times after ASCI. VDAC and Lamin A were used as internal controls for the mitochondrial and nuclear subfractions respectively. b-actin was used as an internal

control for total or cytoplasm protein. Data were mean ± S.D. (n = 6/group). * P \ 0.05 compared with the Sham group. Mito mitochondria, Cyto cytoplasm, Nu nucleus

Effects of Mdivi-1 Pretreatment on Hindlimb Motor Function After ASCI

found that Mdivi-1-mediated inhibition of mitochondrial fission maintained mitochondrial function, suppressed mitochondrial oxidative stress, inhibited caspase-dependent and -independent apoptosis, and promoted the recovery of motor function in rats in a dose-dependent manner. We suggest that this is a novel strategy for protecting against neural injury after ASCI. More and more studies have found that whether in animal models or human tissue, the death of spinal cord neurons and glial cells are caused primarily by apoptosis, and not due to direct damage after ASCI [4, 43]. The mitochondrial pathway is involved in the pathological process after SCI [44]. The ratio between Bax and Bcl-2 which are on the mitochondrial membrane plays an important role in maintaining the mitochondrial permeability and mitochondrial membrane potential [45, 46]. Regulators of apoptosis, such as cytochrome c (cytC) and apoptosis-inducing factor (AIF) are all localized in the mitochondria of healthy neurons, and

While motor function was only slightly impaired in Sham group rat,the rats subjected to ASCI had significant deficits in hindlimb movement (P \ 0.05). Mdivi-1 pretreatment significantly ameliorated the hindlimb motor disturbances from 3 to 10 days after ASCI (P \ 0.05), while Mdivi-1 pretreatment did not affect the hindlimb motor in Sham group (Fig. 5).

Discussion SCI is a common, devastating disease in the spinal surgery field. Although great progress has been made in elucidating the neuronal injury after ASCI, the strategies to protect the spinal cord after ASCI remain limited. In this study, we

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Fig. 2 a Effects of Mdivi-1 (0.24 mg/kg) pretreatment on Drp1 on the mitochondrial outer membrane at 16 h after ASCI. Effects of Mdivi-1 (1.2 mg/kg) pretreatment on b Drp1 and c Fis1 on the mitochondrial outer membrane, and d total cellular levels of Drp1 at 16 h after ASCI. VDAC was used as an internal control for the

mitochondrial subfractions. b-actin was used as an internal control for total cellular subfractions. Mito mitochondria. Data were mean ± S.D. (n = 6/group). * P \ 0.05 compared with the Sham group, # P \ 0.05 compared with the SCI group

the regulated release of these factors by death signals is a key event in the execution of the mitochondrial apoptotic pathway [46]. CytC release finally results in caspase-dependent apoptosis [47], while AIF release results in non-caspasedependent apoptosis [48]. In this study we examined timedependent changes in key proteins of the mitochondrial apoptotic pathway at the organelle level. The release of cytC and AIF from mitochondria and the protein levels of Bax on the mitochondrial outer membrane significantly increased at 4 h, reached a peak at 16 h, and subsequently significantly decreased from 16 to 24 h after ASCI, whereas Bcl-2 on the mitochondrial outer membrane significantly increased at 2 h

and peaked at 4 h after ASCI, which suggests a possible therapeutic window of opportunity. Our findings further add to previously published studies [49, 50]. The above results indicate that the anti-apoptotic mechanism started rapidly and reached a peak in the early stages of ASCI in vivo, which may afford some level of protection against cell death. However, with increasing time the pro-apoptotic factors dominated and led to the massive release of pro-apoptotic factors from mitochondria, ultimately activating apoptosis. The results of this study provides further insight into apoptotic mechanisms after ASCI, but the detailed mechanisms need to be further studied.

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Fig. 3 Fluorescence microscopy and a fluorescence spectrophotometric analysis b of the spinal cord mitochondrial membrane potential. The addition of CCCP depolarized the mitochondria and red fluorescence was markedly reduced. Scale bar 100 lm. c Measurement of mitochondrial ATP. d Measurement of MDA in the

mitochondria. e Measurement of reduced GSH in the mitochondria. Data were mean ± S.D. (n = 6/group). @ P \ 0.05 compared with the CCCP group, * P \ 0.05 compared with the Sham group, # P \ 0.05 compared with the SCI group (Color figure online)

Mitochondrial fission and fusion play an important role in maintaining mitochondrial morphology, number, motility, distribution, membrane potential, ATP synthesis, mtDNA, quality control, mitophagy, and apoptosis [51, 52]. The critical importance of mitochondrial fusion and fission in the nervous system has been firmly established [53, 54]. After transient cerebral ischemia there was

continuous mitochondrial fission and fusion in the ischemic penumbra [55]. Similarly, mitochondrial fission was increased after seizures [27]. Meanwhile, Owens et al. [56] reported extensive fusion of mitochondria in cultured spinal cord motor neurons. Notably, Cao et al. [3] found that mitochondria tended to elongate and fuse in the injured spinal cord at 3 and 6 h after ASCI. However,

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Fig. 4 Western blot analysis and quantitative analysis of Bax (a, b), Bcl-2 (a, b), CytC (a, c), AIF (a, d) and active caspase-3 (e). VDAC and Lamin A were used as internal controls for the mitochondrial and nuclear subfractions respectively. b-actin was used as an internal control for total or cytoplasm protein. Mito: mitochondria; Cyto: cytoplasm; Nu nucleus. f Fluorescence staining and g quantitative analysis of caspase-3 expression. The blue particles represent total

number of cells and the red particles represent caspase-3-positive cells. Scale bar 50 lm. h Photographs of fluorescence TUNEL staining. i Quantitative analysis of apoptosis. The blue particles represent total number of cells and the red particles represent apoptotic cells. Scale bars are 100 lm. Data were mean ± S.D. (n = 6/group). * P \ 0.05 compared with the Sham group, # P \ 0.05 compared with the SCI group (Color figure online)

mitochondrial fission occurred at 12 and 24 h after ASCI in ASCI rat model. In the present study, western blot analysis showed Drp1 and Fis1 increased significantly at 16 h after ASCI, which is in agreement with previous studies. However, we found that Mdivi-1 (1.2 mg/kg) had already inhibited Drp1 translocation to the mitochondria, but there were no changes in total Drp1 expression in cells and no changes in the levels of Fis1 on the mitochondrial outer membrane at 16 h after ASCI. In addition, Mdivi-1 (0.24 mg/kg) had no effects on Drp1 translocation to the mitochondria. These results indicate that Mdivi-1 markedly inhibits Drp1 translocation to the mitochondria selectively

in a dose-dependent manner after ASCI, which is consistent with studies by Park et al. [32] and Qiu et al. [27]. Measurement of mitochondrial membrane potential can be used to evaluate mitochondrial function; it can indirectly reflect the overall functional status of mitochondria, and is also the first event in an apoptotic cascade [29]. We found that there was significant decrease in the mitochondrial membrane potential at 16 h and 3 days after ASCI, which is consistent with previously described findings [21]. Notably, Mdivi-1 (1.2 mg/kg) significantly maintained mitochondrial membrane potential at 16 h and 3 days after ASCI. These findings indicate that there are abnormalities

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Fig. 4 continued

in mitochondrial function after ASCI, and inhibition of mitochondrial fission can have a protective effect on mitochondrial membrane potential after ASCI, which is in agreement with studies by Zungu et al. [57], Ong et al. [31], and Xie et al. [34]. Based on previous reports, we speculate that Mdivi-1 may protect mitochondrial membrane potential after ASCI by inhibiting excessive mitochondrial fission, leading to a tendency for mitochondrial fusion, thereby compensating for defective mitochondria by sharing their contents, consequently inhibiting decreases in mitochondrial membrane potential [31, 57, 58]. However, the exact mechanism needs further study. Normal mitochondrial membrane potential is necessary to ensure mitochondrial oxidative phosphorylation and

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ATP production [59]. Malondialdehyde (MDA) and reduced Glutathione (GSH) are a stable metabolite of the free radical-mediated lipid peroxidation cascade and an important endogenous antioxidant respectively, which are widely used as markers of oxidative stress after ASCI [60]. Previous studies showed that MDA levels were significantly increased and ATP and GSH levels were significantly increased after SCI [10, 21, 60–62]. Our study showed that MDA levels increased, and ATP and reduced GSH levels decreased significantly at 16 h and 3 days after ASCI, which is consistent with previous reports. Notably, we also found that Mdivi-1 significantly maintained ATP levels, reduced MDA levels, and increased reduced GSH levels in the mitochondria at 16 h and 3 days after ASCI,

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Fig. 4 continued

Fig. 5 Effects of Mdivi-1 on hindlimb motor disturbance within 10 days after ASCI. Data were mean ± S.D. (n = 6/group). * P \ 0.05 compared with the Sham group, # P \ 0.05 compared with the SCI group

suggesting that Drp1 inhibition by Mdivi-1 maintains mitochondrial membrane potential, maintains ATP level, thereby decreasing MDA production, increasing the reduced GSH production, and ultimately attenuating the oxidative stress after ASCI. Other similar studies also reported. For instance, Qiu et al. [27] found that Mdivi-1 decreased 8-hydroxy deoxyguanosine content and

increased superoxide dismutase (SOD) activity during seizures. In addition, Tang et al. [26] reported that Mdivi-1 inhibited the production of ROS and maintained ATP level significantly in rhabdomyolysis acute kidney injury. Above results indicate that Mdivi-1 can extenuates mitochondrial dysfunction effectively in different insults. Most scholars believe that excessive Drp1 is related to permeabilization of mitochondrial outer membrane (MOMP), and Drp1 inhibition is an effective neuroprotective strategy [24, 29, 63]. However, how Drp1 participates in MOMP remains to be deciphered. MOMP inhibition in isolated mitochondria indicates a Drp1 activity in MOMP that extends beyond mitochondrial fission [23, 64, 65]. Meanwhile, mitochondrial fission is merely an accompanying phenomenon in apoptosis, rather than a prerequisite for apoptosis [66, 67]. However, later, Montessuit et al. [68] and Brooks et al. [69] reported that mitochondrial fission may sensitize the cells to Bax insertion and oligomerization in mitochondria, promote the formation of the mitochondrial apoptosis-induced channel, thereby enhancing MOMP, facilitating the release of apoptogenic factors and consequently activating apoptosis [20]. Our study found that Mdivi-1(1.2 mg/kg)

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significantly inhibited the translocation of Bax to the mitochondrial outer membrane, and the release of cytC and AIF in mitochondria at 16 h and 3 days after ASCI, while there were no significant effects on the protein level of Bcl2 on mitochondria. Moreover, Mdivi-1 markedly decreased the level of active caspase-3 and the number of apoptotic cells at 16 h, and 3 and 10 days. The results of this study indicated that Mdivi-1 may play a protective role, mainly by inhibiting Bax insertion to the mitochondira, then inhibiting the MOMP, but not by enhancing the Bcl-2 translocation to the mitochondria after ASCI, as described in other [22, 24, 25, 69]. Our study also showed that Mdivi1(1.2 mg/kg) pretreatment significantly improved the hindlimb motor function from 3 to 10 days after ASCI. In summary, inhibition of mitochondrial fission has neuroprotective effects between the acute stage and subacute stage after SCI. However, mitochondrial fission is also essential for the survival of young neurons and preventing mitochondrial fission impairs mitochondrial function [70]. Thus, we speculate that mitochondrial fission may be a double-edged sword. In normal conditions, mitochondrial fission may be beneficial, but under stress, excessive Drp1mediated mitochondrial fission may activate a pro-apoptosis cascade or other signals and form a strong positive feedback mechanism, which contributes to the release of pro-apoptotic factors in the mitochondria, and lead to neural injury. The regulation of the mitochondrial fusionfission balance may be a novel strategy for the prevention and treatment of ASCI. In conclusion, we report for the first time that Mdivi-1 plays a neuroprotective role after ASCI. The underlying mechanism may be through inhibition of mitochondrial fission, protection of mitochondrial function and suppression of mitochondrial oxidative stress and inhibition of caspase-dependent or -independent apoptosis. The results of this study provide novel targets for the treatment of patients with SCI. However, our findings do not specify the cell types (neurons vs. glial cell types) that are affected, and whether post-treatment with Mdivi-1 after injury can also provide beneficial effects for motor function recovery. Thus, further studies are necessary to address these issues. Acknowledgments This study was supported by a Grant from National Natural Science Foundation of China (Grant No. 81272074); Doctoral Scientific Research Starting Fundation of Liao Ning Province (Grant No. 20121094); Program for Liaoning Excellent Talents in University (Grant No. 2014091).

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