Mitochondrial Division Inhibitor 1 Prevents Early-Stage Induction of Mitophagy and Accelerated Cell Death in a Rat Model of Moderate Controlled Cortical Impact Brain Injury

Original Article

Mitochondrial Division Inhibitor 1 Prevents Early-Stage Induction of Mitophagy and Accelerated Cell Death in a Rat Model of Moderate Controlled Cortical Impact Brain Injury Fei Niu1, Jinqian Dong2, Xiaojian Xu1, Bin Zhang2, Baiyun Liu1-5

BACKGROUND: Increasing evidence has implicated dysfunctional mitochondria in the pathophysiology of neurodegenerative disorders. Selective degradation of dysfunctional mitochondria has been termed mitophagy and constitutes a pivotal component of mitochondrial quality control to maintain cellular homeostasis. Mitochondrial fission plays a prominent role in controlling mitochondrial shape and function. However, it is unclear whether mitochondrial fission in the context of eliminating damaged mitochondria is involved in traumatic brain injury (TBI). We examined the role of mitochondrial division inhibitor 1 (Mdivi1), a small-molecule inhibitor of dynaminrelated protein (Drp1), in general autophagy and mitophagy after controlled cortical impact (CCI).

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METHODS: Mitophagy and the role of Drp1 in this process after CCI were examined using Western blotting, electron microscopy, double immunofluorescence staining, neurological severity scores, and hematoxylin and eosin staining. Statistical analysis was performed using 1-way analysis of variance, followed by the least significant difference test or the Games-Howell test.

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RESULTS: The rats exposed to CCI exhibited induction of mitophagy and fragmentation of mitochondria. When fission was blocked with Mdivi1, the mitochondria became excessively long and interconnected. Inhibition of Drp1 blocked the induction of mitophagy specifically, which aggravated neurological manifestations and neuronal

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Key words - Drp1 - Mitochondrial fission - Mitophagy - TBI Abbreviations and Acronyms CCI: Controlled cortical impact Drp1: Dynamin-related protein 1 EM: Electron microcopy Mdivi1: Mitochondrial division inhibitor 1 mtTFA: Mitochondrial transcription factor A NSS: Neurological severity score PBS: Phosphate-buffered saline PBST: Phosphate-buffered saline with Tween 20 TBI: Traumatic brain injury

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apoptosis. Mdivi1 activated caspase-3 and caspase-9, implying that selective degradation of damaged mitochondria by autophagy markedly decreased cell apoptosis induced by TBI and, thus, promoted cell survival. CONCLUSIONS: The findings from the present study support the hypothesis that Drp1-dependent mitochondrial fission contributes to mitophagy in TBI, and further understanding of the regulatory mechanisms of Drp1 will provide opportunities to develop novel strategies against TBI.

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INTRODUCTION

T

raumatic brain injury (TBI) is an increasingly common event, with high mortality and disability rates. Studies have demonstrated that mitochondrial damage, inflammation, cell functional failure, neurodegeneration, and other adverse events can result from TBI.1,2 Mitochondria are the main sites of oxidative phosphorylation and the main cellular energy source. Dysfunctional mitochondria appear to cause a destructive cycle of mitochondrial damage, fuel deficiency, and apoptosis in TBI. Also, this cycle plays a major role in neural damage.3-7 Thus, mitochondria provide vital support and protection for the function and survival of neurons. Autophagy, the intracellular process of “self-eating,” can degrade intracellular proteins and organelles via the formation of double-membraneebound autophagosomes. Increasing evidence

From the 1Neurotrauma Laboratory, Beijing Neurosurgical Institute, and 2Department of Neurosurgery, Beijing Tian Tan Hospital, Capital Medical University, Beijing; 3Nerve Injury and Repair Center, Beijing Institute for Brain Disorders, Beijing; 4China National Clinical Research Center for Neurological Diseases, Beijing; and 5Beijing Key Laboratory of Central Nervous System Injury, Beijing, China To whom correspondence should be addressed: Baiyun Liu, M.D. [E-mail: [email protected]] Citation: World Neurosurg. (2018). https://doi.org/10.1016/j.wneu.2018.10.236 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2018 Elsevier Inc. All rights reserved.

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Table 1. Modified Neurological Severity Score Points Test

Score

Motor Raising rat by tail

3

Flexion of forelimb

1

Flexion of hindlimb

1




Head moved 10 to vertical axis within 30 seconds

1

Placing rat on floor (normal 0; maximum 3)

3

Normal walk

0

Inability to walk straight

1

Circling toward paretic side

2

Falls down to paretic side

3

Sensory

2

Placing test (visual and tactile test)

1

Proprioceptive test (deep sensation, pushing paw against table edge to stimulate limb muscles)

1

Beam balance tests (normal 0; maximum 6)

6

Balances with steady posture

0

Grasps side of beam

1

Hugs beam and 1 limb falls down from beam

2

Hugs beam and 2 limbs fall down from beam or spins on beam (60 seconds)

3

Attempts to balance on beam but falls off (40 seconds)

4

Attempts to balance on beam but falls off (20 seconds)

5

Falls off; no attempt to balance or hang on to beam (20 seconds)

6

Reflex absence and abnormal movements

4

Pinna reflex (head shake when auditory meatus is touched)

1

Corneal reflex (eye blink when cornea is lightly touched with cotton)

1

Startle reflex (motor response to a brief noise from snapping a clipboard paper)

1

Seizures, myoclonus, myodystony

1

Maximum

18

One point is awarded for inability to perform the task or for the absence of the tested reflex; scores of 13e18 indicate severe injury; 7e12, moderate injury; and 1e6, mild injury.

from clinical and preclinical studies has implicated autophagy in the pathophysiology of TBI. Evidence from a mouse model of closed head injury showed that the protective effect of rapamycin in TBI resulted partly from the induction of autophagy.8 The results from another study have confirmed that activated autophagy might protect neurons from degeneration at an early stage after TBI and play a continuous role thereafter in eliminating aberrant cell components.9 In contrast, another study demonstrated that inhibition of autophagy is able to reduce neurological deficits and cell death caused by

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weight-drop trauma.10 Thus, the role of autophagy in TBI remains controversial. Mitochondrial autophagy, termed mitophagy, plays an especially prominent role in removing damaged mitochondria, thereby maintaining mitochondrial morphology and homeostasis.11-14 Dysfunction of mitochondrial autophagy has been shown to affect a wide variety of human brain diseases, including ischemic stroke, Alzheimer disease, and Parkinson disease.15-17 Also, many laboratories have observed that mitochondrial autophagy exists in brain trauma.18-20 Mitophagy might alleviate injury and cellular dysfunction and maintain mitochondrial dynamics by eliminating dysfunctional or damaged mitochondria after TBI.19 Therefore, mitochondrial autophagy seems to be involved in the pathological process of brain trauma. However, it is still unclear how damaged mitochondria are selectively recognized by mitophagy after TBI. Mitochondrial dynamics regulate balance through fission and fusion to maintain normal function. Mitochondrial fission seems to be required to maintain mitochondrial function and help to isolate damaged segments of mitochondria and thus promote their autophagy. Dynamin-related protein 1 (Drp1) is a predominantly controlled protein that is recruited to mitochondria from the cytoplasm during fission.21,22 It has been demonstrated in vivo that during transient hypoxia/reperfusion conditions, when mitochondrial fission is disrupted, blockage of mitophagy ensues.23 In addition, enhancement of mitochondrial fission has been observed in remotely axotomized neurons with axonal injury, further confirming the protective role of fission against neuronal injury.24 However, it is still unclear whether mitochondrial fission is changed in moderate TBI induced by CCI. We hypothesized that mitochondrial fission will result in induced mitophagy in the traumatized cortex and contribute to neuroprotection in TBI. To test this hypothesis, we first investigated mitophagy in a rat model of controlled cortical impact (CCI), a well-documented animal model of TBI. We then characterized the effects of Mdivi1, an inhibitor of Drp1, on ultrastructure and mitophagy function. Finally, we examined the influence of Mdivi1 on neurological test results of TBI and cell apoptosis. METHODS Rat CCI Model and Drug Administration Male adult Sprague-Dawley rats (weight, 290e320 g) were purchased from Beijing Vital River Laboratory (Beijing, China). All animal handling and experimental use followed protocols approved by the institutional animal care and use committee and were in accordance with the principles outlined in the National Institutes of Health “Guide for the Care and Use of Laboratory Animals.” The rats were housed in cages with food and water available and were maintained at 24
C on a normal 12-hour/12hour light-dark schedule (lights on at 7 AM) for 1 week before experimental surgery. All efforts were made to minimize suffering. Experimental modified CCI was induced to cause TBI in the rats.25 In brief, the rats were anesthetized with inhaled isoflurane and maintained at 37
C  0.5
C with a thermal mat throughout the surgical procedure. The rats were placed on a stereotaxic frame and secured using 2 ear bars and an incisor bar. A craniotomy with a 5-mm diameter was made over the right

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Figure 1. Changes in LC3, Beclin-1, and p62 over time in the traumatized cortex of controlled cortical impact rats. (A) Representative images and (BeD) quantitative analysis of Western blot bands labeled with antibodies

parietal cortex (3.8 mm posteriorly and 2.5 mm laterally to the bregma) using a dental drill. All efforts were made to avoid disrupting the dura and associated vasculature during the craniotomy. A PCI 3000 PinPoint Precision Cortical Impactor (Hatteras Instruments, Cary, North Carolina, USA) was used to deliver an impact at a velocity of 2.0 m/second with 2.5-mm deformation and a dwell time of 85 ms using an impactor tip 4 mm in diameter. After CCI, the removed skull section was immediately replaced and sealed with bone wax, and the incision was closed with interrupted 4-0 silk sutures. The sham group underwent the same procedure as the injured rats, except for the impact. For the rats treated with Mdivi1 (1 mg/kg body weight; Sigma-Aldrich, St. Louis, Missouri, USA), Mdivi1 was administered by intracerebroventricular injection after the onset of CCI. The stereotaxic coordinates for injection were 1 mm posterior and 1.5 mm lateral with respect to the bregma and 4.0 mm ventrally to the dura, with the tooth bar set at 0 mm. Mdivi1 was dissolved in dimethyl sulfoxide, and 3 mL of this solution was injected into the ventricle. Neurobehavioral Evaluation For all rats, the neurobehavioral tests were scored in a blinded manner 24 hours after CCI. Neurological function was measured in terms of the neurological severity score (NSS), with 0 points representing normal function and 18 representing the most severe deficit. The scores were used to assess the functional neurological

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against LC3, Beclin-1, p62, and b-actin after a sham operation and 1, 3, 6, 12, and 24 hours after controlled cortical impact. Data presented as the mean  standard error of the mean (n ¼ 5).

status according to the presence of certain reflexes and the rats’ ability to perform motor and behavioral tasks (Table 1).26 Histological Findings After behavioral testing, the rats were sacrificed and perfused through the left cardiac ventricle with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. The excised brain tissue was fixed in 4% buffered paraformaldehyde for 24 hours, embedded in paraffin, and sectioned. The sections were cut 4-mm thick and stained with hematoxylin and eosin. To evaluate the histopathological changes, we examined the stained tissue samples with a light microscope. Western Blot Analysis At the indicated time after CCI, the rats were transcardially perfused with ice-cold PBS, and the brain tissues of the traumatized right cortex and the corresponding cortex of the sham-operated rats were dissected and homogenized in lysis buffer. Total cell protein was acquired by lysing cells in phenylmethane sulfonyl fluoride-treated cell lysis buffer for Western blotting. Isolation of mitochondrial and cytosolic proteins was performed using the Mitochondria/Cytosol Fractionation Kit (Applygen Technologies, Beijing, China). Next, the homogenates were centrifuged at 12,000g for 30 minutes at 4
C. The protein concentration was determined using a bicinchoninic acid protein

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Figure 3. Time course of Drp1 in controlled cortical impact rats. (A) Representative images of Drp1 in the cytoplasm or mitochondria of the sham-operated and vehicle groups at different times after controlled cortical impact. (B) The results were quantified and are presented as the mean  standard error of the mean; the ratio of the gray level of the Drp1 band to that of the

assay kit (Applygen Technologies, Beijing, China). The samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany) for immunoblotting. The membranes were incubated in blocking buffer (5% bovine serum albumin and 0.1% TWEEN 20 in Tris-buffered saline) for 2 hours at room temperature, followed by incubation with primary antibody in TWEEN 20 in Tris-buffered saline overnight at 4
C. The next day, the membranes were washed 3 times in PBS with Tween 20 (PBST) and incubated with secondary antibody for 2 hours at room temperature. The primary antibodies used were as follows: anti-LC3 (1:1000; Cell Signaling Technology, Danvers, Massachusetts, USA), anti-Beclin-1 (1:1000; Novus Biologicals, Centennial, Colorado, USA), anti-p62 (1:1000; Novus Biologicals, Centennial, Colorado, USA), anti-Drp1 (1:1000; Abcam, Cambridge, United Kingdom), anti-TIM23 (1:1000; Santa Cruz

b-actin or COX IV band in the sham-operated group is

shown. (C, D) Representative images and quantitative analysis of Western blots labeled with Drp1, b-actin, and COX IV antibodies in cytoplasm or mitochondria with or without mitochondrial division inhibitor 1. Data presented as the mean  standard error of the mean from 3 rats.

Biotechnology, Santa Cruz, California, USA), anti-TOM20 (1:1000; Cell Signaling Technology, Danvers, Massachusetts, USA), anti-mitochondrial transcription factor A (mtTFA; 1:500; Abcam, Cambridge, United Kingdom), anti-active caspase-3 (1:1000; Cell Signaling Technology, Danvers, Massachusetts, USA), anti-COX IV (1:1000; Abcam, Cambridge, United Kingdom), anti-active caspase-8 (1:1000; Cell Signaling Technology, Danvers, Massachusetts, USA), anti-active caspase-9 (1:1000; Cell Signaling Technology, Danvers, Massachusetts, USA), and anti-b-actin (1:1000; Sigma-Aldrich, St. Louis, Missouri, USA). The horseradish peroxidase-conjugated secondary antibodies used were goat anti-rat IgG and goat anti-rabbit IgG (1:3000; Millipore, Darmstadt, Germany). The level of b-actin or COX IV in the same membrane was simultaneously assessed as the internal reference. The predicted bands were detected using chemiluminescence (Bio-Rad Laboratories, Hercules, California, USA). The relative

Figure 2. Ultrastructural investigation of mitochondrial autophagy in controlled cortical impact rats by electron microscopy. (A) Sham-operated group at 3 hours; (B) controlled cortical impact group at 3 hours; (C) sham-operated group at 6 hours; (D) controlled cortical impact group at 6 hours (scale bar ¼ 5.0 mm). (E, H, I) Magnification of A or C showing mitochondrial integrity (white stars), lysosomes (white arrows), and Golgi (white arrowhead). (F, G) Magnification of B showing autophagosomes containing mitochondria. (J, K) Magnification of D showing hyperdense mitochondrial membranes (scale bar ¼ 500 nm). (L) Quantification of hyperdense mitochondria. n ¼ 3 rats per group. Data presented as the mean  standard error of the mean. Stars denote damaged mitochondria characterized by hyperdense membranous material. Red arrows indicate autophagosomes. Green arrows indicate magnification of mitochondria with hyperdense membranous material.

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Figure 4. Effects of mitochondrial division inhibitor 1 on selective mitophagy in controlled cortical impact rats. Representative immunostaining images of COX IV (green) and LC3 (red) in the traumatized

density of the bands was analyzed using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). Electron Microscopy Electron microcopy (EM) was performed as previously described. In brief, the rats were deeply anesthetized and perfused with precooled PBS, followed by 4% paraformaldehyde. The brains were then removed, and part of the selected ipsilateral cortex (3 mm from the core of the controlled cortical impact injury) was cut into small blocks (1 mm3). The blocks were fixed with 2.5% glutaraldehyde for 2 hours, washed with 0.1 M PBS (pH 7.4) 3 times, and then postfixed with 1% osmium tetroxide for 2 hours. After dehydration in graded alcohol solutions, the blocks were embedded in Epon. Randomly selected ultrathin sections were then poststained with lead citrate and uranyl acetate and viewed by transmission EM (Hitachi, Tokyo, Japan). The images were quantified by counting the percentage of damaged mitochondria per image (n ¼ 3). Immunofluorescence Staining The brain samples were fixed in 4% paraformaldehyde for 48 hours and then embedded in paraffin. The brain tissues were cut into 4-mm-thick coronal sections. After being deparaffinized in xylol and rehydrated with gradient alcohols, the slices were incubated in sodium citrate buffer (0.01 M; pH 6.0) and heated in a microwave for antigen retrieval. After natural cooling, the

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cortex of vehicle- or mitochondrial division inhibitor 1-treated mice. Scale bar ¼ 10 mm.

slices were washed in PBST 3 times and treated with 5% goat serum for 1 hour to block nonspecific protein binding. Next, they were incubated with rabbit anti-LC3B polyclonal antibody (1:100; Abcam, Cambridge, United Kingdom) and mouse antiCOX IV polyclonal antibody (1:100; Abcam, Cambridge, United Kingdom) overnight at 4
C. The next day, the slices were washed with PBST and incubated for 2 hours at room temperature with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody or Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:200, Jackson, West Grove, Pennsylvania, USA). After washing in PBST, the slices were imaged on the side of the CCI regions with a fluorescence microscope (Carl Zeiss, Oberkocken, Germany). Statistical Analysis The data are presented as the mean  standard error of the mean. Statistical analysis was performed using 1-way analysis of variance, followed by the least significant difference test or the GamesHowell test. Differences were considered significant when P values were <0.05. RESULTS Mitochondrial Autophagy Induced by CCI in the Model At present, the commonly used autophagy markers are LC3B and Beclin-1, whose expression levels are positively correlated with

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Figure 5. Effect of mitochondrial division inhibitor 1 (Mdivi1) on morphological changes in mitochondria in controlled cortical impact (CCI) rats. Rats in the sham and CCI groups were treated with dimethyl sulfoxide or Mdivi1 after traumatic brain injury or sham surgery and once daily after surgery. Representative images from rats 3 hours after CCI surgery (A) without and (B) with

autophagy levels,27,28 and autophagy substrate protein p62, whose accumulation correlates negatively with autophagy activity.29 We first analyzed the induction status of LC3, Beclin-1, and P62 in the traumatized cortex from sham-operated and vehicle-treated rats. Compared with the sham-operated group, the CCI group showed increased levels of LC3B/LC3A and Beclin-1 at 3 and 6 hours (Figure 1A,B; P < 0.05), although the level of p62 was reduced to its minimum level at 6 hours after CCI (Figure 1A,C; P < 0.05). These results suggest that the experimental CCI treatment induced nonselective autophagy. Liu et al.30 showed that damaged mitochondria were removed by mitochondrial autophagy in certain conditions. Thus, we focused our attention on mitophagy after CCI. We next used EM to observe the state of mitochondria after CCI. The sham-operated group showed

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Mdivi1. Scale bar ¼ 5.0 mm. (C) Magnification of A showing autophagosomes containing mitochondria. (D) Magnification of B showing injured mitochondria. Scale bar ¼ 500 nm. Stars denote damaged mitochondria characterized by hyperdense membranous material. Red arrows indicate autophagosomes. Red arrowhead indicate elongated mitochondria.

clear mitochondria and crista structures, lysosomes, and Golgi (Figure 2E,H,I). In the vehicle-treated group, in contrast, vague cristae and dense membranous material emerged in numerous damaged mitochondria (Figure 2B; stars).31 Autophagosomes appeared in partially degraded mitochondria at 3 hours after CCI (Figure 2F,G; arrows), suggesting that mitophagy occurred after CCI and might be independent of nonselective autophagy. Mitochondrial Morphological Abnormalities Induced by Experimental CCI The CCI model at 6 hours rarely showed mitochondrial autophagy but large mitochondria (Figure 2J; arrowheads) and a disordered arrangement of mitochondrial cristae and many hyperdense membranous mitochondria (Figure 2D; stars) were observed.31

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Figure 6. Effect of mitochondrial division inhibitor 1 on nonselective autophagy. Representative Western blot images of (A) TIM23, TOM20, mitochondrial transcription factor A, (B) LC3, P62, and b-actin for each group are shown. (C, D) The results were quantified as

For quantification, dense membranous material was used as the index. We found that the amount of hyperdense membranous material in the model group at 6 hours after CCI was significantly different from that in the model group at 3 hours after CCI group (Figure 2L; P < 0.001). These results indicate the accumulation of injured mitochondria in the absence of mitophagy. We next determined the level of the fission-related protein Drp1. The Drp1 levels in the cytoplasm were significantly reduced in the model group at 3 hours and 6 hours after CCI (Figure 3B; P < 0.05) and then increased with time (Figure 3A). In addition, the Drp1 levels in the mitochondria were significantly increased from 3 hours after CCI (Figure 3B; P < 0.05), peaking at 6 hours (Figure 3B; P < 0.01), and then steadily decreased with time (Figure 3A). Mdivi1 Prevented Induction of Mitochondrial Autophagy To determine whether the process of mitochondrial dynamics plays a vital role in CCI rats, we used Mdivi1 as a selective inhibitor of Drp1. The Western blot results showed that the levels of Drp1 were decreased in the cytoplasm and had increased significantly in the mitochondria of vehicle-treated animals. Alsl, these alterations were effectively reversed by treatment with Mdivi1 (Figure 3C,D; P < 0.05). Immunofluorescence examination of the colocalization of LC3 and COX IV in the traumatized cortex showed that LC3immunoreactive puncta were increased in number at 3 hours in

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the gray level of the TIM23, TOM20, mitochondrial transcription factor A, or P62 band normalized to the b-actin band of the control group. Data presented as the mean  standard error of the mean.

the vehicle-treated rats and colocalized with COX IV immunopositivity, suggesting mitophagy was present in the CCI model. Mdivi1 treatment induced LC3 puncta that was more concentrated in cytoplasm rather than fully colocalized with COX IV (Figure 4). As expected, the blockade of mitochondrial fission by Mdivi1 led to the interruption of mitophagy and caused an accumulation of damaged, even elongated, mitochondria (Figure 5). Mdivi1 Increased Mitochondrial Mass with No Effect on Mitochondrial Biogenesis It has previously been reported that the mitochondrial outer membrane protein Tom20 and the mitochondrial inner membrane protein Tim23 are stably expressed when the mitochondria are fully formed.32,33 In contrast, mtTFA regulates the proliferation and function of mitochondria. We, therefore, analyzed Tom20, Tim23, and mtTFA levels in the traumatized cortex of rats subjected to CCI with or without Mdivi1. In the CCI rat model at 6 hours after impact, the levels of Tom20 and Tim23 were increased compared with those in the sham-operated rats. These alterations were further strengthened by treatment with Mdivi1 (Figure 6A). However, no significant change was found in the mtTFA level of either group (Figure 6C). These results suggest that the accumulation of damaged mitochondria in the Mdivi1-treated group could result in a significant increase in mitochondrial mass without affecting mitochondrial biogenesis.

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Figure 7. Effect of mitochondrial division inhibitor 1 on neurological severity score and apoptosis. (A) Neurological severity score and (B) hematoxylin and eosin staining at 24 hours after controlled cortical impact with or without mitochondrial division inhibitor 1. Data presented as the mean  standard error of the mean (n ¼ 5). ###P < 0.001 compared with sham-operated rats; ***P < 0.001 compared with vehicle-treated group. (C) Representative

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Western blot images of active caspase-3, active caspase-8, active caspase-9 and b-actin of each group shown. (D) The results were quantified as the ratio of the gray level of active caspase-3, active caspase-8, or active caspase-9 band to that of the b-actin band of control group. Data presented as the mean  standard error of the mean.

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We next asked whether inhibition of Drp1 by Mdivi1 would affect nonselective autophagy. The Western blot results showed that treatment with Mdivi1 reduced the levels of LC3B/LC3A (Figure 6B) to the levels found in the sham group. Thus, Mdivi1 seemed to have an effect on the regulation of autophagy. However, Mdivi1 had no effect on p62/b-actin (Figure 6D). Therefore, it appears that inhibition of mitochondrial fission would specifically block mitophagy. Mdivi1 Aggravated CCI Injury in Rats and Promoted Apoptosis We next examined the neurologic function of all the rats and evaluated the role of mitophagy in CCI rats. The rats in the vehicletreated group at 24 hours exhibited significant neurological deficits after CCI (Figure 7A; P < 0.001), illustrating that a model of moderate injury was established. Treatment with Mdivi1 significantly heightened the behavioral deficits (P < 0.001). We also determined the morphology of the traumatized cortex using hematoxylin and eosin staining. Histological observation of the cortex at the site of the false CCI in the sham group showed clearly distinguishable neurons and normal cortical structures of moderate size. The corresponding brain regions in the rats at 24 hours after CCI exhibited vacuolar degeneration of local cortical tissue, neuronal loss, and gliosis (Figure 7B), and Mdivi1 aggravated the injury of the CCI rats by causing significant pyknosis of the neuronal nuclei, implying that mitophagy might be a promising target for the early treatment of TBI. We also analyzed the effect of Mdivi1 on apoptosis-related proteins. At 24 hours after surgery, the levels of active caspase3, active caspase-9, and active caspase-8 were significantly greater in the CCI rats than in the control rats (Figure 7C). Mdivi1 treatment further upregulated the levels of active caspase-3 (P < 0.05) and active caspase-9 (P < 0.01) but decreased the level of active caspase-8 (Figure 7D; P < 0.05). Together, these results suggest that the inhibition of mitochondrial autophagy by Mdivi1 causes mitochondria-related apoptosis rather than cell death-related apoptosis. DISCUSSION The results of the present study have provided experimental evidence regarding the function of mitophagy in a rat model of TBI. These results have demonstrated that rats exposed to Mdivi1 exhibit reduced Drp1 expression, inhibition of mitochondrial autophagy with decreased colocalization of LC3 and COXIV, and accumulation of dysfunctional and elongated mitochondria. These results have also indicated that Mdivi1 aggravates the NSSs of CCI rats and promotes the expression of the mitochondrial quality control proteins TIM23 and TOM20 without affecting bulk autophagy or producing mitochondrial-like apoptosis. Together, our data support the hypothesis that mitophagy dysfunction and aggravated injury in CCI rats might contribute to the inhibition of Drp1. However, we could not rule out the possibility that CCI injury also induced bulk autophagy. We speculated that mitochondria are more sensitive to CCI injury. Previous studies have shown that secondary injuries from TBI lead to alterations in mitochondrial homeostasis, inflammation, and peroxide generation, in addition to the propagation of these injuries involved in autophagy.34,35 Our results are consistent with

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the findings from these studies, demonstrating the occurrence of nonselective autophagy after TBI. Dysfunctional mitochondria produce excessive reactive oxygen species, resulting in defects in energy generation and activation of cell death pathways.36 Therefore, mitochondrial quality control is essential for maintaining cellular homeostasis,37 which depends on the balance between mitochondrial biogenesis and degradation. Mitophagy acts as a critical quality control mechanism by clearing away damaged or malfunctioning mitochondria.38 However, whether mitochondria are randomly or selectively targeted by autophagy is not yet clear. Some studies have shown that damaged mitochondria can be selectively degraded by mitophagy. For example, mitophagy is regulated independently of nonselective autophagy in yeast cells.39 In the present study, we demonstrated that TBI can induce mitophagy in the traumatized cortex and that mitophagy appeared earlier than nonselective autophagy. Zuo et al.17 noted that permanent middle cerebral artery occlusion induced mitophagy independently of bulk autophagy. Taken together, these results suggest that the removal of damaged mitochondria relies mainly on sensitive and specific mitophagy in TBI. Drp1 is the primary protein necessary for mitochondrial fission and is important for the integrity of mitochondrial structure. Importantly, the expression of Drp1 mainly regulates the shape of mitochondria and changes in their morphology.40 One patient with a defect in Drp1 showed perinuclear tangles of elongated, large-diameter mitochondria.41 In addition, in vitro research has demonstrated that Drp1 expression is responsible for preventing changes in mitochondrial shape and attenuating cell death.42 Our data have also provided evidence that the inhibition of Drp1 causes structural and functional abnormalities in the mitochondria when the rats subjected to CCI were treated with Mdivi1. Considering that mitochondrial fission had no effect on mitochondrial biogenesis, that mitophagy does not proceed in the absence of Drp1 strongly suggests that mitophagy is responsible for the changes in mitochondrial morphology. Treatment with Mdivi1 did not affect bulk autophagy, implying that the damaged segments of mitochondria were isolated by mitochondrial fission and then selectively cleared away by promotion of autophagy. Although some insights into mitochondrial fission have emerged, the interactions between fusion and fission remain to be clarified in further investigations. To date, it has remained obscure whether the protective role of mitophagy is involved in TBI. Evidence has shown that the protective effect of melatonin in CCI rats is a result of mitophagy induction.19 Consistent with that study, our results revealed greater severity of neurological impairment and accumulated necrosis of neurons in the Drp1-absent group, implying a protective role of mitophagy against TBI. In contrast, Wu et al. reported that the prevention of cell death in mice resulted, in part, from inhibition of mitophagy activation. This seeming contradiction regarding the role of mitochondrial autophagy in TBI can be attributed to the diverse objects of study, stimulation conditions, and pathological stages investigated. Loss of mitochondrial fission might also promote neuronal death via either apoptosis or impaired autophagy. Destructive mitochondrial damage often results in apoptosis, and if mitophagy is too impaired to remove excessive mitochondria, the dynamic balance

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of mitochondria will be disrupted, allowing secondary injury after TBI.43,44 The acceleration of cell apoptosis by Mdivi1 in our study showed that mitochondrial autophagy is required for the prevention of cell death in the early stage of TBI. In the near future, we are eager to determine the mechanisms regulating the balance between cell survival and cell death after TBI.

malfunctioning mitochondria, a process that is dependent on the presence of Drp1. These results are of considerable importance because mitophagy is involved in the development and progression of TBI. An improved understanding of mitophagy can provide new insights for the treatment of TBI. ACKNOWLEDGMENTS

CONCLUSION Using Mdivi1, we have provided evidence that mitophagy in the traumatized cortex exerts a protective effect by degrading

requires autophagy for mitochondrial network maintenance. Cell Metab. 2013;18:844-859.

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Conflict of interest statement: This study was supported by the Beijing Neurosurgical Institute Youth Programme (grant 2016002) and the National Natural Science Foundation of China (grants 81471238 and 81771327). Received 2 August 2018; accepted 31 October 2018 Citation: World Neurosurg. (2018). https://doi.org/10.1016/j.wneu.2018.10.236 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2018 Elsevier Inc. All rights reserved.

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WORLD NEUROSURGERY, https://doi.org/10.1016/j.wneu.2018.10.236