Matrix metalloproteinase-9 regulates the blood brain barrier via the hedgehog pathway in a rat model of traumatic brain injury

Mu-Yao Wu, Fan Gao, Xiao-Mei Yang, Xia Qin, Guo-Zhao Chen, Di Li, Bao- Qi Dang, Gang Chen
PII: S0006-8993(19)30607-9
DOI: https://doi.org/10.1016/j.brainres.2019.146553
Reference: BRES 146553

To appear in: Brain Research

Received Date: 5 August 2019
Revised Date: 31 October 2019
Accepted Date: 11 November 2019

Please cite this article as: M-Y. Wu, F. Gao, X-M. Yang, X. Qin, G-Z. Chen, D. Li, B-Q. Dang, G. Chen, Matrix metalloproteinase-9 regulates the blood brain barrier via the hedgehog pathway in a rat model of traumatic brain injury, Brain Research (2019), doi: https://doi.org/10.1016/j.brainres.2019.146553

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Matrix metalloproteinase-9 regulates the blood brain barrier via the
hedgehog pathway in a rat model of traumatic brain injury Mu-Yao Wu 1#, Fan Gao 1#, Xiao-Mei Yang 2, Xia Qin 3, Guo-Zhao Chen 4, Di Li 5*, Bao-Qi Dang 1*, Gang Chen 6 1Department of Rehabilitation, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, China
2Department of Emergency, The First People’s Hospital of Zhangjiagang, Suzhou, China 3Department of Intensive care unit, The First People’s Hospital of Zhangjiagang, Suzhou, China 4Department of orthopedics, The First People’s Hospital of Zhangjiagang, Suzhou, China 5Department of Neurosurgery and Translational Medicine Center, The First People’s Hospital of Zhangjiagang, Suzhou, China
6Department of Neurosurgery and Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, China
#: Authors contributed equally to this work*

*: Corresponding auther:
Bao-Qi Dang, Department of Rehabilitation, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, No.77 Changan Southern Road, Suzhou 215600, China; E-mail address: [email protected] Di Li, Department of Neurosurgery and Translational Medicine Center, The First People’s Hospital of Zhangjiagang, No.68 Jiyang Western Road, Suzhou 215600, China; E-mail address: [email protected]

Abstract
The mechanisms of secondary brain injury after traumatic brain injury (TBI) are complex and are the result of multiple factors. Protecting the blood-brain barrier (BBB) and ameliorating cerebral edema are two key factors for improving the prognosis of TBI patients. The BBB is regulated by the hedgehog pathway through Scube2 and Shh protein. Matrix metalloproteinase-9 (MMP-9) influences the transport system and enzyme system of vascular endothelial cells, possibly via the hedgehog pathway. The present study aimed to investigate the role and mechanism of MMP-9 in TBI via the hedgehog pathway. Eighty male Sprague-Dawley rats were used to establish a murine model of TBI. Subsequently, the effect of SB-3CT—a specific inhibitor of MMP-9—was assessed via Western blotting, real-time PCR, immunofluorescence, apoptotic assays, and neurological scoring. The results showed that, compared with those of the sham-operation group, the mRNA and protein levels of MMP-9 were significantly increased after TBI, while the expressions of Scube2 and Shh were decreased. Application of SB-3CT at 24 hours after TBI significantly reduced neuronal apoptosis and BBB permeability, while increasing expressions of Scube2 and Shh. In conclusion, these findings demonstrate an influence of TBI-induced MMP-9 upregulation in the induction of post-traumatic nerve and BBB injury, which may be partially mediated by Scube2 and Shh via the hedgehog pathway.
Keywords: MMP-9, Scube2, Shh, traumatic brain injury, blood brain barrier

1. Introduction

Traumatic brain injury (TBI) refers to the craniocerebral injury caused by blunt, penetrating, or accelerating/decelerating force, which is manifested as a decreased level of consciousness, memory loss or forgetting, other neurological/neuropsychological abnormalities, and/or even death (Dang et al., 2017). TBI occurs mostly in younger individuals and is mainly caused by traffic accidents, falls, and blows to the head (Thurman, 2016). The global incidence of TBI exceeds 294 per 100,000 people every year (Nguyen et al., 2016). After TBI, the blood-brain barrier (BBB) is often compromised, thus causing secondary pathophysiological reactions, such as brain edema, neuroinflammation, loss of nerve function, and death of neurons and glial cells (Hay et al., 2015). At present, the specific mechanisms of early brain and BBB damage after TBI remain poorly understood. Therefore, many recent studies have focused on elucidating the mechanisms of TBI-induced BBB damage and in discovering novel drugs for its effective prevention and treatment for reducing TBI-induced disability and mortality rates.

The BBB is a structural barrier between plasma and the brain and is mainly composed of brain microvascular endothelial cells, astrocytes, peripheral microglial cells, pericytes, and the basement membrane. TBI can lead to damage of tight-junction proteins (TJPs) of the BBB and degrade cell migration and astrocytic-foot processes. TBI can also induce dysfunction of BBB processes, such as from the following: exchange of molecules being perturbed due to peripheral inflammatory cells, such as neutrophils, monocytes, and macrophages, moving into the brain parenchyma; blood-borne substances—such as albumin, fibrinogen, thrombin, immunoglobulin, and glutamic acid—penetrating into the brain parenchyma. In addition, some regulatory factors—such as aquaporin and matrix metalloproteinase (MMP)—can also participate in the destruction of BBB function and mediate the formation of brain edema by affecting the transport system and enzyme system of vascular endothelial cells (Blixt et al., 2015).

(TIMP-1) is neuroprotective against traumatic and ischemic brain injury in mice (Tejima et al., 2009). Moreover, MMP-9 has been shown to be closely associated with BBB permeability (Jin et al., 2015). The increased expression of MMPs after brain injury has been shown to lead to increased BBB permeability and to mediate the occurrence of cerebral edema (Rempe et al., 2016). And in TBI, MMP-9 can cleave extracellular matrix including TJPs, induce alterations of BBB integrity (Tetsuhiro et al., 2011). MMP inhibitor can significantly reduce tPAassociated cerebral hemorrhage (Toshihisa and Lo, 2002). Intrathecal injection of SB-3CT can decrease oxidative stress and further attenuate MMP-9–mediated BBB breakdown (Fengshan et al., 2008).
As a member of the signal-peptide CUB-like domain-containing protein (Scube) gene family, Scube2 participates in sonic hedgehog (Shh) signal transduction (Johnson et al., 2012). Shh is a secretory glycoprotein and, as a bilipid protein, is released extracellularly through the release of Scube2, which interacts with Shh through the cholesterol part of Shh and specifically activates the hedgehog pathway (Creanga et al., 2012; Tukachinsky et al., 2012) to influence cell survival, growth, and differentiation of a variety of cells, including neurons (Hooper and Scott, 2005). Activation of the hedgehog pathway involves multiple aspects of brain remodeling, including nerve regeneration and axonal remodeling (Machold et al., 2003).
It has been reported that the BBB can be destroyed by down-regulation of the hedgehog pathway and destruction of TJPs secondary to increased activity of MMP-9 (Brilha et al., 2017). However, the relation of MMP-9 to the hedgehog pathway has not been reported previously. Due to the above findings, we speculated that TBI could induce MMP-9 to regulate Scube2 and Shh through the hedgehog pathway, thereby damaging TJPs of the BBB and, thus, destroying the BBB.

2. Results

2.1 Post-TBI brain expression of mRNA and protein levels of MMP-9, Scube2, and Shh

The expressions of MMP-9, Scube2, and Shh at 3, 6, 12, 24, 48, 72 h and 7 d after TBI were assessed via real-time PCR and Western blotting (Figure 1). The amplification plots and melting-temperature curves showed the cycle thresholds for these genes and indicated that only one product was generated per gene (Figure 1A). The mRNA level of MMP-9 was increased beginning at 3 h after TBI and reached a peak at 24 h (Figure 1B). Conversely, the mRNA level of Scube2 was decreased at 12 h after TBI and returned back to its baseline level by 72 h post-TBI (Figure 1C). Similar to that of Scube2, the Shh mRNA level was decreased at 6 h after TBI and returned back to its baseline level by 72 h post-TBI (Figure 1D). The results of Western blotting for protein levels of MMP-9 (Figure 1E), Scube2 (Figure 1F), and Shh (Figure 1G) were consistent with their corresponding mRNA levels obtained via real-time PCR.

2.2 Post-TBI expression of MMP-9, Scube2, and Shh in peri-injury cortical cells

MMP-9, Scube2, and Shh expression were assessed by immunofluorescent staining with the neuronal marker, Neun, or the astrocytes marker, GFAP (Figure 2). Consistent with the Western blot results, immunofluorescent analyses revealed that the numbers of MMP-9-positive neurons (Figure 2A) and astrocytes (Figure 2B) in the 24 h post-TBI group were increased compared with those of the Sham group, whereas the numbers of Scube2-positive neurons and astrocytes (Figure 2C, 2D), Shh-positive neurons and astrocytes (Figure 2E, 2F) in the 72 h post-TBI group were reduced compared with those of the Sham group.

2.3 The effect of SB-3CT intervention on the protein expression of MMP-9, Scube2, and Shh after TBI
After SB-3CT intervention, MMP-9 expression was significantly altered in the TBI group compared to that of the Sham group or the TBI+DMSO group, as demonstrated by Western blotting (Figure 3A). Additionally, Scube2 (Figures 3B, 3D) and Shh (Figures 3B, 3E) were significantly higher in the Sham group, compared to those in the TBI or TBI+DMSO groups. After SB-3CT intervention, Scube2 and Shh were significantly increased compared with those of the TBI group.

2.4 The integrity of the BBB in TBI Rats after SB-3CT intervention

Collagen-IV and occludin expression was significantly different between the TBI group and the sham group, whereas there was no difference in this expression between the TBI group and the TBI+ DMSO group. Collagen-IV and occludin expression was significantly different in the TBI+SB-3CT group compared to that of the TBI group (Figure 4A). Additionally, brain edema in the injured hemispheres was significantly reduced by the SB-3CT intervention after TBI. However, the brain edema did not change significantly in the contra-lateral (Figure 4E). These results confirmed that the BBB had been significantly damaged after TBI and that the integrity of the BBB was significantly improved after SB-3CT intervention.

2.5 The effect of SB-3CT intervention on neuronal degeneration and neuronal death after TBI
The degree of necrosis of neurons in the TBI group was significantly greater than that in the sham group (Figure 4); there was no difference in the degree of necrosis between the TBI group the TBI+ DMSO group, while the degree of necrosis in the TBI+ SB-3CT group was significantly lower than that of the TBI group (Figures 4B, 4C).

2.6 Neurological behavioral scores in TBI rats after SB-3CT intervention
Neurological behavioral scores from the modified Garcia test were significantly lower in the TBI group compared to that of the sham group, while there was no difference in these scores between the TBI group and TBI+ DMSO group; the TBI+ SB-3CT group exhibited significantly improved neurological behavioral scores compared to those of the TBI group (Figure 4D). These results demonstrate that neurological behavior scores are significantly reduced after TBI and are subsequently improved after SB-3CT intervention.
In conclusion, MMP-9 may contribute to secondary brain injury, which may partially inhibit the Hedgehog pathway by reducing the secretion of Scube2 and Shh after TBI (Figure 5). This study provides new mechanistic information regarding the pathological process of TBI and suggests that targeting MMP-9 may pave the way for a new therapeutic strategy for TBI patients.

3. Discussion

MMPs are collectively called matrixins since they primarily participate in the degradation of the extracellular matrix (ECM). MMPs are implicated to have a pivotal role in normal growth, development, wound healing, angiogenesis, neurogenesis, bone remodeling, ovulation, and implantation (Abdulmuneer et al., 2016). MMPs are known to be secreted by a variety of different cell types and in response to inflammatory cytokines and growth factors. In healthy individuals, MMP-9 is expressed at a low level and its regulation is stable. Several studies have shown that MMPs are upregulated during TBI and are involved in its pathophysiology, including neuroinflammation and cell death (Jia et al., 2010; Muradashvili et al., 2015). Attenuation of MMP-9
levels can reduce blood–brain barrier damage, and attenuate edema after trauma induced by controlled cortical impact in mouse brain (Tatsuro et al., 2002). After mice embolus-induced focal cerebral ischemiathis, MMP-9 inhibitor SB-3CT attenuated degradation of neuronal laminin and protected neurons from ischemic cell death, without cytotoxicity after acute and repeated administration (Boguszewska-Czubara et al., 2018). Our present study showed that mRNA and protein levels of MMP-9 began to increase at 3 h after TBI and reached a peak by 24 h, after which they began to decline to the same levels as those in the sham group by 7 d. This result is consistent with findings from previous studies. Increased MMP-9 levels also cause BBB disruption, neuroinflammation, and cell death in various neurological diseases (Yong et al., 2001). Our present study assayed the expressions of collagen-IV and occludin proteins to assess whether the function of the BBB was damaged in our murine model of TBI. Collagen-IV is one of the many TJPs. Occludin is a part of the basal plate, the loss of which increases vascular permeability and contributes to the aberrant opening of the BBB (Sehba et al., 2004). A previous study found that the expression of collagen-IV and occludin was reduced after TBI, and that the hydrolyses of these proteins lead to destruction of the BBB (Rempe et al., 2016).

Scube2 was first identified in mouse as a novel gene encoding an epidermal growth factor (EGF)-related protein containing a CUB domain (Jakobs et al., 2017). Scube proteins are known to be secreted and are cell-surface associated (Wu et al., 2004; Yang et al., 2002). The hedgehog pathway includes several hedgehog proteins, including desert hedgehog, Indian hedgehog, and Shh. Among them, Shh is the most widely expressed and is the most potent protein within this pathway (Hao et al., 2013). As a secretory glycoprotein, Shh plays significant roles in normal embryonic development. Scube2 has previously been implicated in the hedgehog pathway during zebrafish myotome development. Scube2 may act upstream or adjacent to hedgehog signaling, which is supported by studies that have shown that components of the hedgehog pathway downstream of Shh are functional modulators of Scube2 (Hollway et al., 2006). Scube2 has been implicated in active Shh release from the plasma membrane of Shh-producing cells, which mobilizes Shh for signaling (Creanga et al., 2012; Jakobs et al., 2016). Scube2 may directly extract and transport Shh to promote activity of the hedgehog pathway (Machold et al., 2003; Tukachinsky et al., 2012) Our study showed that the mRNA and protein levels of Scube2 and Shh were decreased at 6 h and 12 h post-TBI and achieved the lowest levels at 72 h post-TBI, after which these levels recovered. This trend wasdifferent from that of MMP-9. This result may be due to the fact that Scube2 and Shh are secretory proteins and may have a certain delay in their expressions.

In the present study, we found that a rat model of TBI had increased levels of MMP-9 several hours after TBI, along with depressed expression of Scube2 and Shh, which induced the levels of collagen-IV and occludin to also be decreased. Indeed, this possible mechanism of MMP-9 up-regulation and the loss of basal-lamina components leading to the breakdown of the BBB has been observed in cerebral ischemia (Wagner et al., 1997). In the present study, we found that the same mechanism applies in TBI. The present study showed that, aside from directly causing the breakdown of the basement membrane and TJPs, MMP-9 affected TJP gene expression by suppressing the hedgehog pathway in brain-endothelial cells. MMP-9 may decrease Scube2 protein levels at the surface of neurons, impairing Shh processing and its delivery to endothelial cells. This process would then downregulate the hedgehog pathway and decrease TJPs expression, and this damage of tight junctions would then cause damage to the BBB.

We have demonstrated that MMP-9 can affect the integrity of the BBB by inhibiting Scube2 and Shh, but this mechanism is extremely complex. Studies have shown that, in TBI, the hedgehog pathway plays a role in the BBB through a specific mechanism. Namely, Shh binds to patched (Ptch), allowing activation of Smoothened (smo), and Gli translocation to the nucleus, which then leads to the expression of TJPs (Brilha et al., 2017). Whether or not this mechanism is applicable to TBI needs to be confirmed by further studies. In addition, the specific mechanism of MMP-9 influencing the hedgehog pathway during TBI requires further elucidation. Our present study has several limitations. The sample size of our study was small and only male rats were used. As such, we were unable to investigate any sex differences in the TBI-induced expression of MMP-9 and Scube2. Hence, our results should be interpreted with this caveat taken into consideration. Additionally, we did not investigate whether hedgehog pathway agonists could directly improve nerve damage and protect the BBB. We also did not investigate the specific mechanism of interaction between the hedgehog pathway and vascular endothelial cells. Future studies should investigate these important phenomena.

4. Experimental Procedure

4.1 Study Design and Experimental Groups

Two separate experiments (Figure 6).Experiment 1: There was no obvious difference in weight, feed intake, and motor ability of all rats. To determine the time course of MMP-9 after TBI,48 rats(48 surviving out of 50) were randomly divided into eight groups according to a computer-based randomization (EXCEL randbetween function), specifically sham, TBI 3 h, TBI 6 h, TBI 12 h, TBI 24 h, TBI 48 h, TBI 72 h and TBI 7 d (Dash et al., 2016). Brain tissue surrounding the damaged area was sampled. Tissue in the front was used to perform Western blot analysis (WB) to assess the expression of MMP9, Scube2 and Shh in TBI rat brains. Tissue from the sides was used to perform Real-time PCR analysis. Tissue from the rear part close to the cerebellum was used to perform double immunofluorescence (Figure 6B).Experiment 2: To establish the role of MMP-9 through Scube2 and Shh on Hedgehog Pathway in TBI, 48 rats (48 surviving out of a group of 55) were randomly divided into four groups according to EXCEL and between function, specifically sham, TBI, TBI+DMSO, and TBI+ SB-3CT. At 24h after TBI, which was based on the results of experiment 1, the rats were killed and damaged brain tissue was collected. Neurological testing was examined in all groups before decollation. 32 rats (8 rats of each group) were studied using Western blot analysis to measure the expression of MMP-9, Scube2 and Shh, fluoro-jade B (FJB) staining to measure the neuronal apoptosis and necrosis. Brain tissue from the front area near the damage was used for WB, and tissue from the back was prepared into frozen sections. 16 rats (4 rats of each group) were studied to brain edema (Figure 6C). The experiment abides by the blind method strictly. All the samples were encoded by an independent investigator. All the experimenters were blinded to all the sample types during the analysis.

4.2 Animals

Here, 105 male Sprague-Dawley rats weighing 280-300 g were purchased from the Animal Center of Chinese Academy of Sciences, Shanghai, China. Of which eighty were used for statistical analysis. Animals were housed in 12 h light/dark cycles at a controlled temperature and humidity with free access to food and water. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Soochow University and were performed in accordance with guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

4.3 Traumatic Brain Injury Model

The TBI model was generated based on a previously described protocol (Hang et al., 2004). Briefly, animals were fixed in the stereotactic instrument after intraperitoneal anesthetization with isoflurane gas. Behind the cranial coronal suture and beside the midline, we made a right parietal bone window of 5 mm in diameter with a bone drill under aseptic conditions, and the skull disk was removed without disturbing the dura. Only animals with intact dura were used for inducing contusion model. A copper block (4 mm in diameter and 5 mm in height) was placed on the dura mater and a 40 g flat-headed steel rod was dropped from a height of 25 cm onto the block to introduce trauma. The pillar was allowed to compress the brain tissue to a maximum depth of 3 mm.The blood was wiped away with gauze after the weight was removed, and the scalp was sutured with a silk thread. The animals were allowed to recover in a warmed chamber before send back to their home cages. The Sham group animals underwent the exact same procedure but were not impacted with the steel rod.

4.4 Drug Injection

As to the TBI+SB-3CT group, intraperitoneal injection of SB-3CT (50 mg/kg, 10% DMSO) was injected 30 minutes after TBI, followed by the second and third injection at 6 h and 12 h (Jia et al., 2014). Weight-matched control animals were injected with 10% DMSO of equal volume. Intraperitoneal injection was selected as an effective delivery route for the inhibitor to the brain, and the same route has been previously reported in adult rats (Gu et al., 2005). In addition, similar dosages have been shown to be effective in inhibiting MMP-9 activity in adult rat models of spinal cord injury (Ranasinghe et al., 2012) and ischemia (Gu et al., 2005) without any confounding toxic effects.

4.5 Tissue Collection and Sectioning

Rats were deeply anesthetized with Isoflurane gas at an equivalent time point after injury. For the isolation of proteins and messenger RNAs (mRNAs),rats were transcardially perfused with 200 ml of 4℃ 0.9% saline, and a sample of the cortex surrounding the contusion area that was located < 3 mm from the margin of the contusion site (or the region located < 3mm from the parietal craniotomy in the sham group) was collected on ice(Figure 6A). The obtained tissue samples were rapidly frozen and stored at −80℃ until further use.
For brain sections, brains were removed, immersed overnight in 4% paraformaldehyde at 4℃, and cryoprotected in a 15% sucrose solution overnight, followed by a 1-day incubation in a 30%sucrose solution. Frozen brain sections were cut at a 15 µm thickness using a sliding microtome (Leica CM1950, Germany). All the processes used for tissue resection and selection were conducted by two pathologists who were blinded to the experimental conditions.

4.6 Real-Time PCR

Total RNA was isolated from peri-injury brain tissues using the Trizol Reagent (Invitrogen, United States) according to the manufacturer’s instructions. According to the protocol provided by the manufacturer (Thermo Fisher, United States), complementary DNA (cDNA) was synthesized using 1µg of the total RNA. Real-time PCR was then performed using the QuantStudioTM Dx Real-Time PCR Instrument (Life Technologies Corporation, United States) with a PowerUpTM SYBRTM Green Master Mix kit (Thermo Fisher, United States). The phases included the following: the template was denatured at 95℃ for 2 min, followed by 40 cycles of amplification (95℃ for 15 s, 60℃ for 15 s, and 72℃ for 1 min). Three replicate wells are provided for each sample. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as the internal reference for each sample, and the relative mRNA expression levels of the target genes were calculated using relative quantification. The sequences of the forward and reverse primers of each gene are as follows: MMP-9: F: 5’-CCACCGAGCTATCCACTCAT-3’ R: 5’-GCTCCGGTTTCAGCATGTTT-3’
Scube2: F: 5’-GCAAGTTTGCGTCAACACAG-3’ R: 5’-GACGCCCTTTACTTCCACAC-3’
Shh: F: 5’-TGGAGTGAAGCTTCGAGTGA-3’ R: 5’-ACTGCTCGACCCTCATAGTG-3’

4.7 Western Blot Analysis

Western blot analysis was performed as previously described (You et al., 2016). Firstly, protein extraction from peri-injury cortex tissues was performed by gently homogenizing the samples in RIPA lysis buffer with protease Inhibitor (CWBIO, China) with further centrifugation at 13,000×g at 4℃ for 20min. The supernatant was collected, and the protein concentration was determined using the bicinchoninic acid (BCA) method with the PierceTM BCA Protein Assay Kit (Thermo Fisher, United States). Equal amounts of extracted proteins were loaded and subjected to electrophoresis on 8% SDS-polyacrylamide gels (Beyotime, China) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, United States). Blocking buffer with 5% defatted milk was used to block the membranes for 2 h at room temperature, and membranes were incubated afterward with the following primary antibodies overnight at 4℃: rabbit anti-MMP-9 (1:1000, Abcam, United States), rabbit anti-Scube2 (1:1000, Abcam, United States), rabbit anti-Shh (1:500, Proteintech, United States), rabbit anti-Collagen-Ⅳ (1:500, Abcam, United States)and rabbit anti-Occludin (1:500, Santa, United States). Rabbit anti-GAPDH (1:10,000,Sigma, United States) was used as an internal loading control. The membranes were then incubated with horseradish peroxidaseconjugated secondary antibodies, including goat anti-rabbit IgG-HRP (Invitrogen, United States) for 2 h at 4℃. Immunoblots were finally probed with the ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore, United States) and visualized with an imaging system (BioRad, United States). All data were analyzed using ImageJ software (National Institutes of Health, United States).

4.8 Immunofluorescence Staining
Double immunofluorescence staining was conducted as described previously (Wang et al., 2016). After three washes with 0.3% Triton in phosphate-buffered saline (PBS) to rupture the cell membranes, frozen brain coronal sections (15 µm) were blocked with 10% goat serum for at least 1h at room temperature and incubated at 4℃ overnight with the following primary antibodies: rabbit anti-MMP-9 (1:200, Abcam, United States), rabbit anti-Scube2(1:200, Abcam, United States), rabbit anti-Shh (1:200, Proteintech, United States), mouse anti-NeuN (1:300, Millipore, United States), mouse anti-CD11b (1:300, Bio-Rad, United States), and mouse anti-GFAP (1:300, Bio-Rad, United States). The sections were then incubated with secondary antibodies, including Alexa Fluor 488 donkey anti-rabbit IgG antibody (Invitrogen, United States) and Alexa Fluor 555 donkey anti-mouse IgG antibody (Invitrogen, United States), for 1h at room temperature at a dilution of 1:800. Finally, slides were counterstained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 10min and were observed with a laser confocal microscope Leica DMi8 (Leica Microsystems, Germany), and images were obtained using LAS X software.
For each animal, three sets of three sections were randomly selected for immunostaining and quantification. The region of interest was defined and delineated under a 10× objective on each section as the MMP-9, Scube2 or Shh-positive cells in the contusion margin along the cortex. Using a 20× objective, six randomly selected, nonoverlapping adjacent fields with an area of 690µm in width and 520 µm in height were examined that surrounded the edge of the contusion cortex. MMP-9, Scube2 and Shh-positive cells and the total number of neurons/microglia/astrocytes were counted manually and expressed as the mean numbers per field of view. The results are presented as percentages of positive cells. All the processes, including sectioning, field selection, and cell counting, were performed by an investigator who was blinded to the treatments among groups.

4.9 Neurological Score

Neurological deficiency was assessed by a blinded investigator at 24h after TBI with the modified Garcia score as previously reported (Feng et al., 2017; Yang et al., 2016). Briefly, the following seven parameters were included: spontaneous activity, body proprioception, response to vibrissae touch, symmetry of limb movement, lateral turning, forelimb walking and climbing ability. Each subtest is scored from 0 to 3, with a composite maximum score of 21 (no neurological deficits).4.10 FJB StainingFluoro-Jade B staining served as a marker of neuronal injury and was conducted per the manufacturer’s instructions (Millipore, United States). Briefly, after incubation with 1% sodium hydroxide in 80% alcohol for 5 min and 70% alcohol for 2 min, the slides were transferred to a solution containing 0.06% potassium permanganate for 10min. The slides were then immersed in 0.0004% Fluoro-Jade dye staining solution (0.1% acetic acid) for 20min followed by rinsing in deionized water. After being washed and dried in an oven (50℃) for 5–8 min, the sections were cleared by immersion in xylene for at least 1min before coverslipping with distyrene plasticizer xylene (DPX), a non-aqueous non-fluorescent plastic mounting media. The total number of FJB-positive cells was expressed as the mean number per field of a view. All the processes, including sectioning, field selection, and cell counting, were conducted by an investigator who was blinded to the animals’ conditions.

4.11 Brain edema

To study the edema in the injured brain, brain water content was measured using the wet-dry method (Rosenberg et al., 1998). After separation of the rat brain tissues, the brains were divided into ipsilateral and contra-lateral hemispheres and quickly weighed the wet weights. Then the samples were placed in a 100℃ oven for 48 h and weighted the dry weights. The percentage of brain water content (%) was calculated as [(wet weight - dry weight)/(wet weight)] × 100%.

4.12 Statistical Analyses

All the data were expressed as the mean ± standard deviation and analyzed using SPSS 18.0 software. Statistical analyses of the time course of FJB, Real-Time PCR and western blot data were performed using one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test for comparisons between each TBI group and the sham group. The immunofluorescence staining data were analyzed using Student’s t-test. Statistical comparisons among the remaining data were analyzed using one-way ANOVA followed by Tukey’s post hoc test to compare data from multiple groups. ANOVA’s F-value together with relative degrees of freedom was presented as F (df1, df2). p< 0.05 was considered to be a statistically significant difference.

Acknowledgements
This work was supported by the Gusu Health Training Projects(GSWS2019076); Suzhou "Science and Technology Xingwei" Youth Science and Technology Project (KJXW2018057); Zhangjiagang Science and Technology Pillar Program (ZKS1712, ZKS1829); and Zhangjiagang Hospital of Traditional Chinese Medicine Youth Natural Science Foundation Project (ZZYQ1802). Declaration of Competing Interests
The authors have no potential competing interest to disclose.

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