Neurochem Res DOI 10.1007/s11064-015-1756-1
ORIGINAL PAPER
Retinoic Acid Prevents Disruption of Blood-Spinal Cord Barrier by Inducing Autophagic Flux After Spinal Cord Injury Yulong Zhou1,2 • Binbin Zheng1,2 • Libing Ye2 • Hongyu Zhang2 • Sipin Zhu1,2 Xiaomeng Zheng2 • Qinghai Xia2 • Zili He1,2 • Qingqing Wang1,2 • Jian Xiao2 • Huazi Xu1
•
Received: 12 July 2015 / Revised: 6 October 2015 / Accepted: 30 October 2015 Ó Springer Science+Business Media New York 2015
Abstract Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB), which leads to infiltration of blood cells, inflammatory responses and neuronal cell death, with subsequent development of spinal cord secondary damage. Recent reports pointed to an important role of retinoic acid (RA), the active metabolite of the vitamin A, in the induction of the blood–brain barrier (BBB) during human and mouse development, however, it is unknown whether RA plays a role in maintaining BSCB integrity under the pathological conditions such as SCI. In this study, we investigated the BSCB protective role of RA both in vivo and in vitro and demonstrated that autophagy are involved in the BSCB protective effect of RA. Our data show that RA attenuated BSCB permeability and also attenuated the loss of tight junction molecules such as P120, b-catenin, Occludin and Claudin5 after injury in vivo as well as in brain microvascular endothelial cells. In addition, RA administration improved functional recovery of the rat model of trauma. We also found that RA could significantly increase the expression of LC3-II and decrease the expression of p62 both in vivo and in vitro.
Electronic supplementary material The online version of this article (doi:10.1007/s11064-015-1756-1) contains supplementary material, which is available to authorized users. & Jian Xiao [email protected] & Huazi Xu [email protected] 1
Department of Orthopaedics, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou 325000, China
2
School of Pharmacy, Key Laboratory of Biotechnology and Pharmaceutical Engineering, Wenzhou Medical University, Wenzhou 325035, China
Furthermore, combining RA with the autophagy inhibitor chloroquine (CQ) partially abolished its protective effect on the BSCB and exacerbated the loss of tight junctions. Together, our studies indicate that RA improved functional recovery in part by the prevention of BSCB disruption via the activation of autophagic flux after SCI. Keywords Blood-spinal cord barrier (BSCB) Retinoic acid (RA) Autophagy Spinal cord injury (SCI)
Introduction The blood-spinal cord barrier (BSCB) is primarily formed by highly specialized endothelial cells (ECs), which form a tight structural barrier due to the presence of well-developed tight junction (TJ) and adheren junction (AJ) proteins. The BSCB maintains an environment that allows neurons to function properly by protecting the spinal cord from toxins and pathogens [1–4]. The initial physical damage to the BSCB allows infiltration of macrophages and other immune cells to the area of injury, resulting in secondary injury in SCI, and the ‘programmed death’ of neurons and glia, leading to permanent neurological deficits [5]. Thus, preventing the BSCB disruption should be regarded as a potential approach for therapeutic interventions after SCI. Autophagy, a lysosome-dependent cellular degradation pathway, is an essential process for the maintenance of cellular homeostasis in the CNS, both under physiological conditions and pathological conditions [6, 7]. Recently, a number of researchers have focused on the role of autophagy in acute injuries such as traumatic brain injury (TBI) and SCI [8–10]. A growing body of studies focusing on autophagy in various cells (neuron, astrocytes, oligodendrocytes, and microglia) [11–14]. However, much less is
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known about either the mechanisms or the function of autophagy in ECs or BSCB. Retinoic acid (RA) the active metabolite of vitamin A is vital for embryonic development and maintenance of many organ systems in the adult is well established [15]. It is particularly crucial during development of the central nervous system [16–18]. More recently, a study pointed to an important role for RA in the induction of the BBB during human and mouse development [1, 19]. In addition, RA treatment also led to the increase in TJ proteins expression in brain ECs [1, 20]. Certain study has indicated retinoic acid induced BBB development under physiological conditions, however, it is unknown whether RA plays a role in maintaining BSCB integrity under the pathological conditions such as SCI. In the present study, we investigated the effect of RA and the involvement of autophagy in BSCB disruption after SCI.
Materials and Methods Reagents and Antibodies DMSO (Sigma-Aldrich) was used to dissolve non-water soluble reagents and as a vehicle control. Endothelial Cell Medium (ECM) which contains 500 ml of basal medium, 25 ml of fetal bovine serum (FBS, Cat. No. 0025), 5 ml of endothelial cell growth supplement (ECGS, Cat. No. 1052) and 5 ml of penicillin/streptomycin solution (P/S, Cat. No. 0503) were purchased from ScienCell Research Laboratories. Antibodies against Occludin, Claudin5, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p62 and microtubule-associated protein-1 light-chain 3 (LC3) were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-bcatenin and P120 were purchased from Abcam. All chemicals including RA were from Sigma Chemical Company. Spinal Cord Injury and RA Administration Adult female Sprague–Dawley rats (220–250 g) were purchased from the Animal Center of the Chinese Academy of Sciences in Shanghai, China. The protocol for animal care and use conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and was approved by the Animal Care and Use Committee of Wenzhou Medical University. All rats were fed under controlled temperature (21–23 °C), 12 h light and 12 h dark cycles and free access to food and tap water. Animals were deeply anaesthetized by an intraperitoneal injection of 10 % chloralic hydras (3.6 ml/ kg) [21–23] and the model of a T9 half-cut spinal cord
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column was made. Briefly, For each rat, the skin was incised along the mid line of back, and the vertebral column was exposed and a laminectomy was done at T9 vertebrae and moderate contusion injuries were performed using a vascular clip (30 g force, Oscar, China) for 2 min. Sham group rats received the same surgical procedure but sustained no injury. Post-operative cares involved manual urinary bladders empty twice daily until they return to bladder function and administration of cefazolin sodium (50 mg/kg, i.p.). Drug treatment was administered according to previous reports [24]. RA (Sigma) was diluted to a stock solution of 100 mg/mL in 100 % DMSO. To prevent oxidation, air above the aliquots was replaced with nitrogen, and stock aliquots were stored at -80 °C. All handling of RA occurred under a rayless condition. RA stock solution was further diluted in 100 % ethanol to a concentration of 2 mg/mL. RA was administered into the injured rats via intraperitoneal injection (15 mg/kg) immediately after spinal cord injury and then re-administered once a day for 2 weeks for behavioural tests. CQ was immediately administered into injured rats via intraperitoneal injection (50 mg/kg) together with RA or not. Equal ethanol and DMSO were administered for vehicle control. All animals showed no significant side effects resulting from drug treatment such as an increase in mortality or infectious diseases during these experiments. Cell Culture Primary cultures of human brain microvascular endothelial cells (HBMVEC) were purchased from ScienCell Research Laboratories. BMVEC cultures were expanded and maintained in Endothelial Cell Medium (ECM) which contains 500 mls of basal media, 25 ml of fetal bovine serum (FBS), 5 ml of endothelial cell growth supplement (ECGS,) and 5 ml of penicillin/streptomycin solution (P/S). They were then incubated in a humidified atmosphere containing 5 % CO2 at 37 °C. Endothelial cells were seeded on 6-well plates and were routinely cultured in ECM For OGD treatment, cells were rinsed once with warm (Gibco, containing 1000 mg/L glucose), and then refreshed with glucose-low DMEM containing RA (1 or 5 lM), or combined with CQ (100 lM), or CQ alone. Cells were then immediately placed in a sealed chamber (Thermo Scientific) loaded with mixed gas containing 5 % CO2 and 95 % N2 for 24 hs. All experiments were performed in triplicate. Measurement of BSCB Disruption The integrity of the BSCB was investigated with Evans blue dye extravasation according to previous reports [25] with minor modifications. At 1 day after SCI, 1 ml of 2 % EB dye (Sigma) solution in saline was injected
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intravenously into the tail vein. Three hours later, animals were anaesthetized and killed by intra-cardiac perfusion with saline. One centimeter of the spinal cord surrounding the T9 injury site was extracted and weighed, and snapfrozen in dry ice and then homogenized in a 50 % trichloroacetic acid solution. Samples (400 mg) were then homogenized in 400 lL of N, N-dimethylformamide (DMF) and incubated at 70 °C for 72 h. Samples were centrifuged at 18,000 rpm for 20 min twice. The supernatant was collected, aliquoted (200 ll) into a 96-well glass plate, and its fluorescence was quantified using a spectrophotometer at an excitation wavelength of 620 nm and an emission wavelength of 680 nm. Samples were normalized to the original sample weight, and EB concentration was calculated on the basis of a standard curve of EB in DMF (data reported as EB per spinal cord weight: lg/g). Locomotion Recovery Assessment Locomotor outcome after spinal cord contusion injury was conducted as previously described [23]. Two trained investigators who were blind to the experimental conditions scored the locomotion recovery in an open field, according to the Basso, Beattie and Bresnahan (BBB) scale. BBB is ranging from 0 indicative of no observed hindlimb movements, to a score of 21, representative of a normal ambulating rodent. Animals were placed individually on open fields and allowed to move freely for 5 min. The animals were evaluated every 2 days from day 0 to day 14 following the surgical procedure.
Claudin5 (1:800), P120 (1:1000), b-Catenin (1:1000), LC3II (1:1000), p62 (1:1000) at 4 °C for overnight. Then the membranes were washed with TBS for 5 min three times and incubated with secondary antibodies for 1 h at room temperature. Signals were visualized using the ChemiDicTM XRS ? Imaging System (Bio-Rad). Immunofluorescence Staining Spinal cord segments obtained from animals 1 day after surgery were cryoprotected in 4 % paraformaldehyde for 6 hs, then embedded in paraffin and sectioned into 5 lm slices. Sections were deparaffinized, rehydrated and washed twice for 10 min in PBS. For protein analysis in vitro, ECs were grown onto cover glasses in a 6-well plate. After treatment, the cells were fixed for 10 min using 4 % paraformaldehyde, then cells were washed with PBS for 2 min three times. Then sections and cells were incubated with 5 % albumin from bovine serum in PBS containing 0.1 % Triton X-100 in a 37 °C oven for 30 min. Then sections were incubated with antibodies against LC3II (1:400), CD31 antibody (1:100) at 4 °C for overnight. Sections were rinsed three times in PBS after primary antibody incubation and incubated with either fluorescent Alexa 568, 647 donkey anti-mouse/rabbit, or 488 goat antimouse/rabbit secondary antibody (1:500) for 1 h at room temperature. Sections were rinsed three times with PBS and incubated with 4,6-diamidino-2-phenylindole (DAPI) for 10 min and finally washed in PBS and sealed with a coverslip. All images were captured on a Nikon ECLIPSE Ti microscope (Nikon, Japan).
Western Blot Analysis Statistical Analysis Spinal cord tissue samples were removed 1 day after surgery, and rapidly stored at -80 °C for the western blotting. Briefly, total protein was prepared with a lysis buffer containing 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 % NP-40, 0.5 % deoxycholate, 0.1 % SDS, 10 mM Na2P2O7, 10 mM NaF, 1 m g/ml aprotinin, 10 mg/ml leupeptin, 1 mM sodium vanadate and 1 mM PMSF. For protein analysis in vitro, the endothelial cells (ECs) were washed twice with ice-cooled PBS and lysed in lysis buffer (25 mM Tris–HCl [pH 7.6], 150 mM NaCl, 1 % Nonidet P-40, 1 % sodium deoxycholate, and 0.1 % SDS) with protease and phosphatase inhibitors. Tissue and cells homogenates were incubated for 20 min at 4 °C, and centrifuged at 12,000 rpms, for 15 min at 4 °C. A 75 lg (in vivo) or 50 lg (in vitro) aliquot of proteins from each sample was separated by SDS-PAGE and then transferred onto a PVDF membrane (Bio-Rad). The membrane was blocked with 5 % non-fat milk in TBS with 0.05 % Tween 20 for 90 min, afterwards, the membranes were incubated with the specific antibodies against: Occludin (1:800),
Data are presented as the mean ± standard error of the mean (SEM) from three independent experiments. Statistical significance was examined using Student’s t test when there were two experimental groups. When more than two groups were compared, statistical evaluation of the data was performed using one-way analysis of variance (ANOVA) and Dunnett’s post hoc test. P values \ 0.05 were considered statistically significant.
Results RA Decreases of BSCB Permeability and Improves Locomotor Functional Recovery After SCI To determine whether RA attenuates the increases of permeability induced by SCI, the content of Evans Blue (EB) dye in the injured spinal cord tissue was investigated. We examined the effect of RA on BSCB permeability at 1 day
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after injury by EB assay (n = 4). As shown in Fig. 1a, b, SCI caused a marked increase in BSCB permeability of EB dyes compared with the sham groups, furthermore, RA treatment significantly reduced the increase of EB dye extravasation compared with SCI groups. In addition, the fluorescence intensity of EB in the injured spinal cord at 1 day post-SCI was higher than sham controls, and RA significantly reduced the fluorescence intensity (Fig. 1c, d). Qualitative analysis also shows (Fig. 1e) that the amount of EB dye extravasation in the injured spinal cord was higher than RA treated groups. Hindlimb locomotor functional recovery was then evaluated for 2 weeks after injury using the 21-point BBB rating scale. As shown in Fig. 1f, RA treatment significantly increased the hindlimb locomotor function 8 to 14 days after injury, compared with that observed in vehicle-treated controls (n = 5/group, 14 days, BBB rating scale, RA, 12.3 ± 0.85 vs. vehicle 6.8 ± 0.72; P \0.01). These data suggest that RA can effectively prevent BSCB disruption and promote functional improvement in locomotor activity after SCI.
Fig. 1 RA decreases the BSCB permeability and improves locomotor functional recovery after SCI. After SCI, rats were treated with RA and BSCB permeability was measured at 24 hs post-SCI by using EB dye (n = 4/group). a Representative whole spinal cords showing EB dye permeabilized into the spinal cord at 24 h. b Quantification of BSCB permeability data onto A by Image J software. c Representative confocal images of an EB extravasation in each group at 24 hs after
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RA Prevents the Loss of TJ and AJ Proteins After SCI It is well known that the TJ (Claudins and occludin) and AJ (b-catenin, P120) in the endothelial cells of blood vessels is involved in the integrity of BBB or BSCB. Thus, we next examined the expression of the TJ (Occludin, Claudin5) and AJ (b-catenin, P120) proteins in spinal lysates by western blot. As shown in Fig. 2, western blot results showed that (Fig. 2a) the levels of AJ (b-catenin, P120) were decreased at 1 day after SCI, as well as the TJ (Occluding, Claudin5) (Fig. 2c). Furthermore, these decreases were significantly attenuated in the RA-treated group (Fig. 2a, c). Analysis of the bands density of AJ (b-catenin, P120) and TJ (Occludin, Claudin5) proteins (Fig. 2b, d) showed that compared with the SCI group, levels of Occludin, Claudin5, b-catenin and P120 expression were higher in the RA group. These results imply that RA attenuated the increase of permeability by preventing the loss of TJ and AJ proteins after SCI.
SCI. d Quantification of the fluorescence intensity of EB in each group. e Quantification of the EB content of the spinal cord (lg/g). All data represent mean values ± SEM, n = 5. *P \ 0.01 versus vehicle controls. (F) BBB scores of the sham, SCI group and SCI rat treated with PBA group. *P \ 0.05 versus the SCI group, and **P \ 0.01 versus the SCI group, n = 5
Neurochem Res Fig. 2 RA prevents the loss of TJ and AJ proteins after SCI. a Representative western blots of AJ proteins b-catenin and P120 in the sham, SCI model and SCI model treated RA groups. b Quantification of the western blots data onto A. *P \ 0.01, **P \ 0.01 versus the SCI group, mean values ± SEM, n = 5. c Representative western blots of TJ proteins Occluding and Claudin5 in the sham, SCI model and SCI model treated RA groups. d Quantification of the western blots data onto C. *P \ 0.01, **P \ 0.01 versus the SCI group, mean values ± SEM, n = 5
Activation of Autophagic Flux is Involved in the Role of RA in Acute SCI It has been noted that autophagy activation is involved in SCI, however, until recently, their roles have not been clearly defined in impaired spinal cord, especially in BSCB. To investigate the role of RA in autophagy flux after acute SCI, western blots of LC3-II expression were performed at 1 day post-SCI in the SCI group and RA group. As shown in Fig. 3a, b, our results showed that the expression of LC3-II was increased in SCI group rats compared with the sham group, however, the expression of LC3-II was further increased in RA group compared with the SCI group. To gain further insight into the influence of RA on autophagic flux after SCI, western blotting of p62 expression was performed. As shown in Fig. 3a, c, the level of p62 was decreased at 1 day after SCI, and this decrease was further severe in the RA group rats. The double labelling immunofluorescence staining results of LC3-II (red) and CD31 (green) are in line with the western blot results (Fig. 3d). Taken together, these results demonstrate that RA is able to activate autophagic flux in ECs after acute SCI. Autophagy Inhibitor CQ Abolishes the BSCB Protective Effect of RA After SCI To further confirm that the activation of autophagic flux is important for the BSCB protective effect of RA in acute SCI, a classical autophagy inhibitor, CQ, was administered into injured rat via intraperitoneal injection (50 mg/kg) after SCI. First, the alterations of BSCB permeability by EB assay in sham, SCI, RA, RA combined with CQ, and CQ alone group rats were investigated. As shown in
Fig. 4a, b, compared with the SCI group the BSCB permeability of EB dye was decreased in RA group, and increased in RA and CQ co-treated group and CQ alone group rats. We next detected levels of TJ (Occludin, Claudin5) and AJ (b-catenin, P120) proteins in each group by western blots. Levels of TJ (Occludin, Claudin5) and AJ (b-catenin, P120) were significantly increased in RA group and these increases were abolished by CQ (Fig. 4c–f). These data indicate that inhibition of autophagy by CQ abolished the BSCB protective effect of RA after SCI. The levels of autophagy flux-associated proteins such as LC3-II and p62 in sham, SCI, RA, RA combined with CQ, and CQ alone group rats were then detected by western blot. The expression of LC3-II was further increased in RA and CQ co-treated group and CQ alone group compared with the RA group (Fig. 4g). The results also showed that the level of LC3-II in the RA and CQ co-treated group was significantly higher than that in CQ alone-treated group (Fig. 4h. P \ 0.05). In addition, as shown in Fig. 4g, i, the expression of p62 was significantly decreased in RA group, and this decrease was inhibited by CQ. Collectively, our results indicate that autophagy flux induced by SCI or RA can be effectively inhibited by CQ after SCI.
RA Protects OGD-Treated ECs by Inducing Autophagic Flux In Vitro To further confirm that the BSCB protective effect of RA is linked to the activation of autophagy flux in vitro, we applied OGD to ECs. ECs in OGD model were treated with RA (1 or 5 lM), or combined with CQ (100 lM), or CQ alone for 24 h. Western blot re sults showed that the levels of P120, b-catenin, Occludin, and Claudin5 were decreased
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Fig. 3 Activation of autophagic flux is involved in the role of RA in acute SCI. a Representative western blots of autophagy marker LC3II and p62 in the Sham group, SCI rats and treated with RA group. b, c Quantification of the western blots data onto A. *P \ 0.01, versus the SCI group. All data represent mean values ± SEM.
d Representative micrographs showing (original magnification 4009) double immunofluorescence with CD31 (endothelial cell marker, green) and LC3-II (red). Nuclei are labeled with DAPI (blue) in each group (Color figure online)
after treated with OGD for 24 h, compared with the control group (Fig. 5a, b). Whereas, RA 1 lM attenuated the decreases of P120 and Occludin (P \ 0.05), and RA 5 lM significantly attenuated these decreases in P120, b-catenin, Occludin, and Claudin5 induced by OGD. Although a slight increase in b-catenin and Claudin5 protein level in
RA 1 lM treatment group was observed (Fig. 5a, b), there was no statistical significance. Compared with the RA group cells, levels of Occludin, Claudin5, P120 and bcatenin expression were decreased in the RA and CQ cotreated group and in CQ alone group cells (Fig. 5a, b). Together, these results in vitro indicate that RA can
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Fig. 4 Autophagy inhibitor CQ abolishes the BSCB protective effect of RA after SCI. a Representative whole spinal cords showing EB dye permeabilized into the spinal cord at 24 hs post-SCI in the sham, SCI model and SCI model treated RA groups, RA compound with CQ, and CQ alone groups. b Quantification of BSCB permeability data onto A by Image J software. c–f Representative western blots and quantification data of TJ and AJ proteins Occludin, Claudin5, P120
and b-catenin in each group rats. *P \ 0.01, #P \ 0.01, versus SCI group. **P \ 0.01, ##P \ 0.01, versus treated RA groups. g–i Representative western blots and quantification data of autophagy marker LC3-II and p62 in each group. *P \ 0.01, versus SCI group. # P \ 0.01, versus RA compound with CQ groups (LC3-II), # P \ 0.01, versus treated RA groups (p62). All data represent mean values ± SEM
effectively prevent the loss of AJ and TJ proteins via the activation of autophagy flux in OGD treated ECs. Next we detected the levels of autophagy flux-associated proteins such as LC3-II and p62 in each group cells by western blot. As shown in Fig. 5c, compared with the OGD group cells, levels of LC3-II expression was increased in the RA group, and the expression of LC3-II was further increased in RA and CQ co-treated group and CQ alone group compared with the RA group
cells (Fig. 5c). The western blot results also showed that the level of LC3-II in the RA and CQ co-treated group was considerably higher than that in CQ alone-treated group cells. As shown in Fig. 5d, compared with the OGD group cells, levels of p62 expression were decreased in the RA group but this decrease was significantly inhibited in the RA and CQ co-treated group cells. Immunofluorescence staining result is consistent with the western blot result (Fig. 5e). These data suggest
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Neurochem Res b Fig. 5 RA protects OGD-treated ECs by inducing autophagic flux
in vitro. a, b Representative western blots and quantification data of TJ and AJ proteins Occludin, Claudin5, P120 and b-catenin in each group rats. *P \ 0.05, #P \ 0.05, &P \ 0.05 versus OGD group cells. ** P \ 0.01, ##P \ 0.01, versus treated RA (5 lM) group cells. c, d Representative western blots and quantification data of autophagy marker LC3-II and p62 in each group. *P \ 0.01, versus OGD cells. # P \ 0.01, versus RA compound with CQ cells (LC3-II); #P \ 0.01, versus treated RA cells (p62). All data represent mean values ± SEM. e Immunofluorescence staining of LC3-II (red) in ECs treated with OGD, RA or CQ, nuclei are labeled with DAPI (blue) (Color figure online)
that the RA can effectively induce autophagy flux in OGD treated ECs. Inhibition of Autophagy by 3-MA Attenuates the BSCB Protective Effect of RA In Vitro ECs in OGD model were treated with RA (5 lM), or combined with 3-MA (5 mM), or 3-MA alone for 24 h, then the change of TJ and AJ in ECs were detected. Western blot results showed that the levels of P120, bcatenin, Occludin and Claudin5 were increased after treated with RA, compared with the OGD group. At the same time, these increase in P120, b-catenin, Occludin, Claudin5 were significantly attenuated in the RA and 3-MA cotreated cells (Fig. 6a–c). As shown in Fig. 6d, f, the expression of LC3-II was further increased in RA group compared with the OGD group. On the contrary, a decrease of p62 expression was observed in RA group cells, and this decrease was partly inhibited in 3-MA group (Fig. 6d–e). The results were consistent with that of CQ.
Discussion The present study demonstrates that the neuroprotective effect of RA might be mediated in part by attenuating BSCB disruption via inducing autophagic flux after SCI. Our data showed for the first time that RA plays a role in maintaining BSCB integrity by preventing the loss of TJ and AJ proteins under pathological conditions such as SCI, and improves functional recovery after SCI. Furthermore, the present study also showed that autophagy induced by RA plays a further some significant role in BSCB disruption and prevents loss of TJ and AJ proteins. Based on our results, it is likely that autophagy may be a new target for protection BSCB integrity after SCI. Damage to the vasculature and breakdown of the BSCB is a universal consequence of SCI clinically as well as in animal models [4, 26]. The cellular and molecular mechanisms responsible for the long-term increase in BSCB permeability after SCI are numerous [27, 28]. Reactive
oxygen species (ROS) provide a common trigger for many downstream pathways that directly mediate blood-CNS barrier compromise such as oxidative damage, TJ modification and MMP activation [29, 30]. MMPs have been implicated in SCI in that plasma MMP-9 disrupted TJ proteins, rendering the BSCB leaky and allowing neurotoxic agents to enter the injured spinal cord [31]. Fluoxetine and valproic acid improved functional recovery in part by inhibiting matrix metalloprotease (MMP) activation and preventing BSCB disruption after SCI [5, 27]. Emerging evidence shows that inflammatory may also play a critical role in the blood-CNS barrier disruption in pathological conditions [32–34]. Salvianolic acid B inhibits the generation of inflammatory mediators in the injured spinal cord tissue, thereby decreasing BSCB disruption and eventually resulting in the alleviation of pathological changes caused by SCI [35]. Although many factors are known to contribute to BSCB disruption, the role of autophagy in the BSCB disruption and the regulation of the TJ proteins remains unknown, especially in acute SCI, and warrant further investigation. In the current study, we found that BSCB disruption and activation of autophagy are involved in the rat model of trauma and in vitro. Furthermore, we demonstrate for the first time that the autophagy induced by SCI plays a furthersome role in BSCB disruption in acute SCI. Thus our results firstly suggested that autophagy may be a new target for protection BSCB integrity. Retinoic acid (RA) is the active metabolite of vitamin A that is a key molecule in several physiological processes, such as reproduction, embryonic development, tissue remodeling, vision and immune function [15, 36]. The developmental defects resulting from vitamin A deficiency were well established, however, the teratogenic effects or apoptosis of retinoid overdose were also described in various tissues such as acute leukemia, glioma cells and even in some nerve fibroblasts [37–39]. In this study, RA (15 mg/kg) was administered by intraperitoneal injection after spinal cord injury. Our results also show that the expression of apoptosis protein caspase-12 was up-regulated at 1 day after SCI. Furthermore, RA treatment significantly decreased the expression of caspase-12 at 1 day after SCI (see Fig. S1). However, in the present study we did not definitely rule out the role of RA in caspase-12 expression. In the following study, signaling mechanisms by which RA inhibit apoptosis pathway will be focused on. The enzymes for RA synthesis, the retinaldehyde dehydrogenases (RALDHs) and RA-receptor (RAR), occurred by day 4 and peaked at days 8–14 following the SCI [40– 42]. Systemic administration of RA reduces early transcript levels of pro-inflammatory cytokines after experimental SCI [24]. Pharmacologic inhibition of RA-receptor activation during the differentiation of the murine BBB resulted in the leakage of a fluorescent tracer as well as
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Fig. 6 Inhibition of autophagy by 3-MA attenuates the BSCB protective effect of RA in vitro. a–c Representative western blots and quantification data of TJ and AJ proteins Occludin, Claudin5, P120 and b-catenin in each group rats. *P \ 0.05, #P \ 0.05, versus OGD group cells. &P \ 0.01, $P \ 0.01, versus treated RA (5 lM)
group cells. d–f Representative western blots and quantification data of autophagy marker LC3-II and p62 in each group. *P \ 0.05, versus OGD cells; #P \ 0.05, versus RA compound with 3-MA cells. ** P \ 0.01, versus OGD cells; ##P \ 0.01, versus treated RA cells. All data represent mean values ± SEM
serum proteins into the developing brain and reduced the expression levels of important BBB determinants [19], which indicate that RA as a developmental cue in the CNS vasculature is important for the induction of BBB properties in brain ECs. RA is important for the induction of BBB properties in brain ECs [19] and also leads to the increase in TJ proteins expression yielding a 4-fold increase in Occludin expression [20]. Retinoic acid induced BBB development is evident [1, 19, 43], however, it is unknown whether RA plays a role in maintaining BSCB integrity under the pathological conditions such as SCI. In this study, we demonstrated that the role of RA in SCI recovery is related to the prevention of BSCB disruption. We found that SCI caused a marked increase in BSCB permeability of EB dyes compared with the sham groups, furthermore, RA treatment significantly reduced the increase of EB dye extravasation compared with SCI groups. And the expression levels of TJ (Occludin, Claudin5) and AJ (b-catenin, P120) proteins decreased significantly after SCI. Exogenous RA treatment after SCI increased the levels of Occludin, Claudin5, b-catenin and P120. These results
indicate that RA also plays an important role in maintaining BSCB integrity under the pathological conditions such as SCI. In recent years, there is a good amount of evidence that moderate autophagy can protect ECs against cell injury under stressful circumstances [7, 44–48]. In an aneurismal subarachnoid hemorrhage (SAH) model, when autophagy was inhibited by 3-MA and wortmannin, the neurological scores were decreased, brain water content and BBB permeability were further aggravated compared with the SAH animals [49]. Upregulation of BMVEC autophagy has the potential to maintain BBB integrity after ischemia–reperfusion injury mediated by decreasing ROS production and increasing the expression of TJ protein ZO-1 [47]. Evaluating the above studies, the protective potential of autophagy in various ECs damage is evident [50, 51]. Many reports showed that RA exerted a cell-type dependent stimulatory effect on autophagy flux via Beclin-1 upregulation and inhibition of the mTOR pathway or by enhancing autophagosome maturation [36, 52, 53]. In the present study, Our study indicates that autophagy flux was
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stimulated after SCI and this stimulation was significantly exacerbated in the RA-treated group as compared with the vehicle-treated group both in vitro and in the SCI mice. Treatment of ECs with CQ, a lysosome specific inhibitor, or 3-MA, an autophagy specific inhibitor, suppressed the activation of autophagy flux in the RA-treated group. Effects of RA induction of autophagy flux after SCI were consistent with the previous reports. We thus assume the BSCB protective effect of RA might be related to its induction of autophagy flux after SCI. Current study showed RA prevented loss of TJ and AJ proteins by induction of autophagy flux, and inhibition of autophagy by CQ or 3-MA abolishes the BSCB protective effect of RA both in vivo and in vitro. To the best of our knowledge our group was the first to report the effect of exogenous RA on ECs and BSCB integrity by induction of autophagy flux after SCI. However, further work is needed to determine how autophagy may affect ECs survival and TJ and AJ proteins expression after SCI. In our following study, to further confirmed the precise mechanisms between autophagy and RA in BSCB disruption and TJ proteins regulation in acute SCI model, Beclin-1 and mTOR knockout rats should be applied, which may contributes to the evidences of the cross-talk between autophagy and RA in BSCB disruption and TJ proteins regulation in acute SCI. In conclusion, our research demonstrated that treatment with exogenous RA significantly attenuated BSCB permeability and degradation of TJ molecules such as P120, b-catenin, Occludin and Claudin5 at 1 day after injury, thus, improved functional recovery in SCI. RA significantly induced autophagic flux both in vivo and in vitro, furthermore, combination therapy with the autophagy inhibitor CQ partially abolished the BSCB protective effect of RA via exacerbating loss of TJ proteins both in vivo and in vitro. Based on the current results, RA may provide potential therapeutic interventions for preventing BSCB disruption after SCI. The present study lays the ground work for future translational confidence of RA in CNS diseases, especially the relations to the BSCB disruption. Acknowledgments This study was partially supported by a research Grant from the National Natural Science Funding of China (81302775, 81472165, 81200958, 81372112), Zhejiang Provincial Natural Science Foundation of China (LY14H090013, LY14H150010, LY14H170002), Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents (to J.X.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Compliance with Ethical Standards Conflict of interest
The authors declare no conflict of interest.
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ORIGINAL PAPER
Retinoic Acid Prevents Disruption of Blood-Spinal Cord Barrier by Inducing Autophagic Flux After Spinal Cord Injury Yulong Zhou1,2 • Binbin Zheng1,2 • Libing Ye2 • Hongyu Zhang2 • Sipin Zhu1,2 Xiaomeng Zheng2 • Qinghai Xia2 • Zili He1,2 • Qingqing Wang1,2 • Jian Xiao2 • Huazi Xu1
•
Received: 12 July 2015 / Revised: 6 October 2015 / Accepted: 30 October 2015 Ó Springer Science+Business Media New York 2015
Abstract Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB), which leads to infiltration of blood cells, inflammatory responses and neuronal cell death, with subsequent development of spinal cord secondary damage. Recent reports pointed to an important role of retinoic acid (RA), the active metabolite of the vitamin A, in the induction of the blood–brain barrier (BBB) during human and mouse development, however, it is unknown whether RA plays a role in maintaining BSCB integrity under the pathological conditions such as SCI. In this study, we investigated the BSCB protective role of RA both in vivo and in vitro and demonstrated that autophagy are involved in the BSCB protective effect of RA. Our data show that RA attenuated BSCB permeability and also attenuated the loss of tight junction molecules such as P120, b-catenin, Occludin and Claudin5 after injury in vivo as well as in brain microvascular endothelial cells. In addition, RA administration improved functional recovery of the rat model of trauma. We also found that RA could significantly increase the expression of LC3-II and decrease the expression of p62 both in vivo and in vitro.
Electronic supplementary material The online version of this article (doi:10.1007/s11064-015-1756-1) contains supplementary material, which is available to authorized users. & Jian Xiao [email protected] & Huazi Xu [email protected] 1
Department of Orthopaedics, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou 325000, China
2
School of Pharmacy, Key Laboratory of Biotechnology and Pharmaceutical Engineering, Wenzhou Medical University, Wenzhou 325035, China
Furthermore, combining RA with the autophagy inhibitor chloroquine (CQ) partially abolished its protective effect on the BSCB and exacerbated the loss of tight junctions. Together, our studies indicate that RA improved functional recovery in part by the prevention of BSCB disruption via the activation of autophagic flux after SCI. Keywords Blood-spinal cord barrier (BSCB) Retinoic acid (RA) Autophagy Spinal cord injury (SCI)
Introduction The blood-spinal cord barrier (BSCB) is primarily formed by highly specialized endothelial cells (ECs), which form a tight structural barrier due to the presence of well-developed tight junction (TJ) and adheren junction (AJ) proteins. The BSCB maintains an environment that allows neurons to function properly by protecting the spinal cord from toxins and pathogens [1–4]. The initial physical damage to the BSCB allows infiltration of macrophages and other immune cells to the area of injury, resulting in secondary injury in SCI, and the ‘programmed death’ of neurons and glia, leading to permanent neurological deficits [5]. Thus, preventing the BSCB disruption should be regarded as a potential approach for therapeutic interventions after SCI. Autophagy, a lysosome-dependent cellular degradation pathway, is an essential process for the maintenance of cellular homeostasis in the CNS, both under physiological conditions and pathological conditions [6, 7]. Recently, a number of researchers have focused on the role of autophagy in acute injuries such as traumatic brain injury (TBI) and SCI [8–10]. A growing body of studies focusing on autophagy in various cells (neuron, astrocytes, oligodendrocytes, and microglia) [11–14]. However, much less is
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known about either the mechanisms or the function of autophagy in ECs or BSCB. Retinoic acid (RA) the active metabolite of vitamin A is vital for embryonic development and maintenance of many organ systems in the adult is well established [15]. It is particularly crucial during development of the central nervous system [16–18]. More recently, a study pointed to an important role for RA in the induction of the BBB during human and mouse development [1, 19]. In addition, RA treatment also led to the increase in TJ proteins expression in brain ECs [1, 20]. Certain study has indicated retinoic acid induced BBB development under physiological conditions, however, it is unknown whether RA plays a role in maintaining BSCB integrity under the pathological conditions such as SCI. In the present study, we investigated the effect of RA and the involvement of autophagy in BSCB disruption after SCI.
Materials and Methods Reagents and Antibodies DMSO (Sigma-Aldrich) was used to dissolve non-water soluble reagents and as a vehicle control. Endothelial Cell Medium (ECM) which contains 500 ml of basal medium, 25 ml of fetal bovine serum (FBS, Cat. No. 0025), 5 ml of endothelial cell growth supplement (ECGS, Cat. No. 1052) and 5 ml of penicillin/streptomycin solution (P/S, Cat. No. 0503) were purchased from ScienCell Research Laboratories. Antibodies against Occludin, Claudin5, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p62 and microtubule-associated protein-1 light-chain 3 (LC3) were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-bcatenin and P120 were purchased from Abcam. All chemicals including RA were from Sigma Chemical Company. Spinal Cord Injury and RA Administration Adult female Sprague–Dawley rats (220–250 g) were purchased from the Animal Center of the Chinese Academy of Sciences in Shanghai, China. The protocol for animal care and use conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and was approved by the Animal Care and Use Committee of Wenzhou Medical University. All rats were fed under controlled temperature (21–23 °C), 12 h light and 12 h dark cycles and free access to food and tap water. Animals were deeply anaesthetized by an intraperitoneal injection of 10 % chloralic hydras (3.6 ml/ kg) [21–23] and the model of a T9 half-cut spinal cord
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column was made. Briefly, For each rat, the skin was incised along the mid line of back, and the vertebral column was exposed and a laminectomy was done at T9 vertebrae and moderate contusion injuries were performed using a vascular clip (30 g force, Oscar, China) for 2 min. Sham group rats received the same surgical procedure but sustained no injury. Post-operative cares involved manual urinary bladders empty twice daily until they return to bladder function and administration of cefazolin sodium (50 mg/kg, i.p.). Drug treatment was administered according to previous reports [24]. RA (Sigma) was diluted to a stock solution of 100 mg/mL in 100 % DMSO. To prevent oxidation, air above the aliquots was replaced with nitrogen, and stock aliquots were stored at -80 °C. All handling of RA occurred under a rayless condition. RA stock solution was further diluted in 100 % ethanol to a concentration of 2 mg/mL. RA was administered into the injured rats via intraperitoneal injection (15 mg/kg) immediately after spinal cord injury and then re-administered once a day for 2 weeks for behavioural tests. CQ was immediately administered into injured rats via intraperitoneal injection (50 mg/kg) together with RA or not. Equal ethanol and DMSO were administered for vehicle control. All animals showed no significant side effects resulting from drug treatment such as an increase in mortality or infectious diseases during these experiments. Cell Culture Primary cultures of human brain microvascular endothelial cells (HBMVEC) were purchased from ScienCell Research Laboratories. BMVEC cultures were expanded and maintained in Endothelial Cell Medium (ECM) which contains 500 mls of basal media, 25 ml of fetal bovine serum (FBS), 5 ml of endothelial cell growth supplement (ECGS,) and 5 ml of penicillin/streptomycin solution (P/S). They were then incubated in a humidified atmosphere containing 5 % CO2 at 37 °C. Endothelial cells were seeded on 6-well plates and were routinely cultured in ECM For OGD treatment, cells were rinsed once with warm (Gibco, containing 1000 mg/L glucose), and then refreshed with glucose-low DMEM containing RA (1 or 5 lM), or combined with CQ (100 lM), or CQ alone. Cells were then immediately placed in a sealed chamber (Thermo Scientific) loaded with mixed gas containing 5 % CO2 and 95 % N2 for 24 hs. All experiments were performed in triplicate. Measurement of BSCB Disruption The integrity of the BSCB was investigated with Evans blue dye extravasation according to previous reports [25] with minor modifications. At 1 day after SCI, 1 ml of 2 % EB dye (Sigma) solution in saline was injected
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intravenously into the tail vein. Three hours later, animals were anaesthetized and killed by intra-cardiac perfusion with saline. One centimeter of the spinal cord surrounding the T9 injury site was extracted and weighed, and snapfrozen in dry ice and then homogenized in a 50 % trichloroacetic acid solution. Samples (400 mg) were then homogenized in 400 lL of N, N-dimethylformamide (DMF) and incubated at 70 °C for 72 h. Samples were centrifuged at 18,000 rpm for 20 min twice. The supernatant was collected, aliquoted (200 ll) into a 96-well glass plate, and its fluorescence was quantified using a spectrophotometer at an excitation wavelength of 620 nm and an emission wavelength of 680 nm. Samples were normalized to the original sample weight, and EB concentration was calculated on the basis of a standard curve of EB in DMF (data reported as EB per spinal cord weight: lg/g). Locomotion Recovery Assessment Locomotor outcome after spinal cord contusion injury was conducted as previously described [23]. Two trained investigators who were blind to the experimental conditions scored the locomotion recovery in an open field, according to the Basso, Beattie and Bresnahan (BBB) scale. BBB is ranging from 0 indicative of no observed hindlimb movements, to a score of 21, representative of a normal ambulating rodent. Animals were placed individually on open fields and allowed to move freely for 5 min. The animals were evaluated every 2 days from day 0 to day 14 following the surgical procedure.
Claudin5 (1:800), P120 (1:1000), b-Catenin (1:1000), LC3II (1:1000), p62 (1:1000) at 4 °C for overnight. Then the membranes were washed with TBS for 5 min three times and incubated with secondary antibodies for 1 h at room temperature. Signals were visualized using the ChemiDicTM XRS ? Imaging System (Bio-Rad). Immunofluorescence Staining Spinal cord segments obtained from animals 1 day after surgery were cryoprotected in 4 % paraformaldehyde for 6 hs, then embedded in paraffin and sectioned into 5 lm slices. Sections were deparaffinized, rehydrated and washed twice for 10 min in PBS. For protein analysis in vitro, ECs were grown onto cover glasses in a 6-well plate. After treatment, the cells were fixed for 10 min using 4 % paraformaldehyde, then cells were washed with PBS for 2 min three times. Then sections and cells were incubated with 5 % albumin from bovine serum in PBS containing 0.1 % Triton X-100 in a 37 °C oven for 30 min. Then sections were incubated with antibodies against LC3II (1:400), CD31 antibody (1:100) at 4 °C for overnight. Sections were rinsed three times in PBS after primary antibody incubation and incubated with either fluorescent Alexa 568, 647 donkey anti-mouse/rabbit, or 488 goat antimouse/rabbit secondary antibody (1:500) for 1 h at room temperature. Sections were rinsed three times with PBS and incubated with 4,6-diamidino-2-phenylindole (DAPI) for 10 min and finally washed in PBS and sealed with a coverslip. All images were captured on a Nikon ECLIPSE Ti microscope (Nikon, Japan).
Western Blot Analysis Statistical Analysis Spinal cord tissue samples were removed 1 day after surgery, and rapidly stored at -80 °C for the western blotting. Briefly, total protein was prepared with a lysis buffer containing 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 % NP-40, 0.5 % deoxycholate, 0.1 % SDS, 10 mM Na2P2O7, 10 mM NaF, 1 m g/ml aprotinin, 10 mg/ml leupeptin, 1 mM sodium vanadate and 1 mM PMSF. For protein analysis in vitro, the endothelial cells (ECs) were washed twice with ice-cooled PBS and lysed in lysis buffer (25 mM Tris–HCl [pH 7.6], 150 mM NaCl, 1 % Nonidet P-40, 1 % sodium deoxycholate, and 0.1 % SDS) with protease and phosphatase inhibitors. Tissue and cells homogenates were incubated for 20 min at 4 °C, and centrifuged at 12,000 rpms, for 15 min at 4 °C. A 75 lg (in vivo) or 50 lg (in vitro) aliquot of proteins from each sample was separated by SDS-PAGE and then transferred onto a PVDF membrane (Bio-Rad). The membrane was blocked with 5 % non-fat milk in TBS with 0.05 % Tween 20 for 90 min, afterwards, the membranes were incubated with the specific antibodies against: Occludin (1:800),
Data are presented as the mean ± standard error of the mean (SEM) from three independent experiments. Statistical significance was examined using Student’s t test when there were two experimental groups. When more than two groups were compared, statistical evaluation of the data was performed using one-way analysis of variance (ANOVA) and Dunnett’s post hoc test. P values \ 0.05 were considered statistically significant.
Results RA Decreases of BSCB Permeability and Improves Locomotor Functional Recovery After SCI To determine whether RA attenuates the increases of permeability induced by SCI, the content of Evans Blue (EB) dye in the injured spinal cord tissue was investigated. We examined the effect of RA on BSCB permeability at 1 day
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after injury by EB assay (n = 4). As shown in Fig. 1a, b, SCI caused a marked increase in BSCB permeability of EB dyes compared with the sham groups, furthermore, RA treatment significantly reduced the increase of EB dye extravasation compared with SCI groups. In addition, the fluorescence intensity of EB in the injured spinal cord at 1 day post-SCI was higher than sham controls, and RA significantly reduced the fluorescence intensity (Fig. 1c, d). Qualitative analysis also shows (Fig. 1e) that the amount of EB dye extravasation in the injured spinal cord was higher than RA treated groups. Hindlimb locomotor functional recovery was then evaluated for 2 weeks after injury using the 21-point BBB rating scale. As shown in Fig. 1f, RA treatment significantly increased the hindlimb locomotor function 8 to 14 days after injury, compared with that observed in vehicle-treated controls (n = 5/group, 14 days, BBB rating scale, RA, 12.3 ± 0.85 vs. vehicle 6.8 ± 0.72; P \0.01). These data suggest that RA can effectively prevent BSCB disruption and promote functional improvement in locomotor activity after SCI.
Fig. 1 RA decreases the BSCB permeability and improves locomotor functional recovery after SCI. After SCI, rats were treated with RA and BSCB permeability was measured at 24 hs post-SCI by using EB dye (n = 4/group). a Representative whole spinal cords showing EB dye permeabilized into the spinal cord at 24 h. b Quantification of BSCB permeability data onto A by Image J software. c Representative confocal images of an EB extravasation in each group at 24 hs after
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RA Prevents the Loss of TJ and AJ Proteins After SCI It is well known that the TJ (Claudins and occludin) and AJ (b-catenin, P120) in the endothelial cells of blood vessels is involved in the integrity of BBB or BSCB. Thus, we next examined the expression of the TJ (Occludin, Claudin5) and AJ (b-catenin, P120) proteins in spinal lysates by western blot. As shown in Fig. 2, western blot results showed that (Fig. 2a) the levels of AJ (b-catenin, P120) were decreased at 1 day after SCI, as well as the TJ (Occluding, Claudin5) (Fig. 2c). Furthermore, these decreases were significantly attenuated in the RA-treated group (Fig. 2a, c). Analysis of the bands density of AJ (b-catenin, P120) and TJ (Occludin, Claudin5) proteins (Fig. 2b, d) showed that compared with the SCI group, levels of Occludin, Claudin5, b-catenin and P120 expression were higher in the RA group. These results imply that RA attenuated the increase of permeability by preventing the loss of TJ and AJ proteins after SCI.
SCI. d Quantification of the fluorescence intensity of EB in each group. e Quantification of the EB content of the spinal cord (lg/g). All data represent mean values ± SEM, n = 5. *P \ 0.01 versus vehicle controls. (F) BBB scores of the sham, SCI group and SCI rat treated with PBA group. *P \ 0.05 versus the SCI group, and **P \ 0.01 versus the SCI group, n = 5
Neurochem Res Fig. 2 RA prevents the loss of TJ and AJ proteins after SCI. a Representative western blots of AJ proteins b-catenin and P120 in the sham, SCI model and SCI model treated RA groups. b Quantification of the western blots data onto A. *P \ 0.01, **P \ 0.01 versus the SCI group, mean values ± SEM, n = 5. c Representative western blots of TJ proteins Occluding and Claudin5 in the sham, SCI model and SCI model treated RA groups. d Quantification of the western blots data onto C. *P \ 0.01, **P \ 0.01 versus the SCI group, mean values ± SEM, n = 5
Activation of Autophagic Flux is Involved in the Role of RA in Acute SCI It has been noted that autophagy activation is involved in SCI, however, until recently, their roles have not been clearly defined in impaired spinal cord, especially in BSCB. To investigate the role of RA in autophagy flux after acute SCI, western blots of LC3-II expression were performed at 1 day post-SCI in the SCI group and RA group. As shown in Fig. 3a, b, our results showed that the expression of LC3-II was increased in SCI group rats compared with the sham group, however, the expression of LC3-II was further increased in RA group compared with the SCI group. To gain further insight into the influence of RA on autophagic flux after SCI, western blotting of p62 expression was performed. As shown in Fig. 3a, c, the level of p62 was decreased at 1 day after SCI, and this decrease was further severe in the RA group rats. The double labelling immunofluorescence staining results of LC3-II (red) and CD31 (green) are in line with the western blot results (Fig. 3d). Taken together, these results demonstrate that RA is able to activate autophagic flux in ECs after acute SCI. Autophagy Inhibitor CQ Abolishes the BSCB Protective Effect of RA After SCI To further confirm that the activation of autophagic flux is important for the BSCB protective effect of RA in acute SCI, a classical autophagy inhibitor, CQ, was administered into injured rat via intraperitoneal injection (50 mg/kg) after SCI. First, the alterations of BSCB permeability by EB assay in sham, SCI, RA, RA combined with CQ, and CQ alone group rats were investigated. As shown in
Fig. 4a, b, compared with the SCI group the BSCB permeability of EB dye was decreased in RA group, and increased in RA and CQ co-treated group and CQ alone group rats. We next detected levels of TJ (Occludin, Claudin5) and AJ (b-catenin, P120) proteins in each group by western blots. Levels of TJ (Occludin, Claudin5) and AJ (b-catenin, P120) were significantly increased in RA group and these increases were abolished by CQ (Fig. 4c–f). These data indicate that inhibition of autophagy by CQ abolished the BSCB protective effect of RA after SCI. The levels of autophagy flux-associated proteins such as LC3-II and p62 in sham, SCI, RA, RA combined with CQ, and CQ alone group rats were then detected by western blot. The expression of LC3-II was further increased in RA and CQ co-treated group and CQ alone group compared with the RA group (Fig. 4g). The results also showed that the level of LC3-II in the RA and CQ co-treated group was significantly higher than that in CQ alone-treated group (Fig. 4h. P \ 0.05). In addition, as shown in Fig. 4g, i, the expression of p62 was significantly decreased in RA group, and this decrease was inhibited by CQ. Collectively, our results indicate that autophagy flux induced by SCI or RA can be effectively inhibited by CQ after SCI.
RA Protects OGD-Treated ECs by Inducing Autophagic Flux In Vitro To further confirm that the BSCB protective effect of RA is linked to the activation of autophagy flux in vitro, we applied OGD to ECs. ECs in OGD model were treated with RA (1 or 5 lM), or combined with CQ (100 lM), or CQ alone for 24 h. Western blot re sults showed that the levels of P120, b-catenin, Occludin, and Claudin5 were decreased
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Fig. 3 Activation of autophagic flux is involved in the role of RA in acute SCI. a Representative western blots of autophagy marker LC3II and p62 in the Sham group, SCI rats and treated with RA group. b, c Quantification of the western blots data onto A. *P \ 0.01, versus the SCI group. All data represent mean values ± SEM.
d Representative micrographs showing (original magnification 4009) double immunofluorescence with CD31 (endothelial cell marker, green) and LC3-II (red). Nuclei are labeled with DAPI (blue) in each group (Color figure online)
after treated with OGD for 24 h, compared with the control group (Fig. 5a, b). Whereas, RA 1 lM attenuated the decreases of P120 and Occludin (P \ 0.05), and RA 5 lM significantly attenuated these decreases in P120, b-catenin, Occludin, and Claudin5 induced by OGD. Although a slight increase in b-catenin and Claudin5 protein level in
RA 1 lM treatment group was observed (Fig. 5a, b), there was no statistical significance. Compared with the RA group cells, levels of Occludin, Claudin5, P120 and bcatenin expression were decreased in the RA and CQ cotreated group and in CQ alone group cells (Fig. 5a, b). Together, these results in vitro indicate that RA can
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Fig. 4 Autophagy inhibitor CQ abolishes the BSCB protective effect of RA after SCI. a Representative whole spinal cords showing EB dye permeabilized into the spinal cord at 24 hs post-SCI in the sham, SCI model and SCI model treated RA groups, RA compound with CQ, and CQ alone groups. b Quantification of BSCB permeability data onto A by Image J software. c–f Representative western blots and quantification data of TJ and AJ proteins Occludin, Claudin5, P120
and b-catenin in each group rats. *P \ 0.01, #P \ 0.01, versus SCI group. **P \ 0.01, ##P \ 0.01, versus treated RA groups. g–i Representative western blots and quantification data of autophagy marker LC3-II and p62 in each group. *P \ 0.01, versus SCI group. # P \ 0.01, versus RA compound with CQ groups (LC3-II), # P \ 0.01, versus treated RA groups (p62). All data represent mean values ± SEM
effectively prevent the loss of AJ and TJ proteins via the activation of autophagy flux in OGD treated ECs. Next we detected the levels of autophagy flux-associated proteins such as LC3-II and p62 in each group cells by western blot. As shown in Fig. 5c, compared with the OGD group cells, levels of LC3-II expression was increased in the RA group, and the expression of LC3-II was further increased in RA and CQ co-treated group and CQ alone group compared with the RA group
cells (Fig. 5c). The western blot results also showed that the level of LC3-II in the RA and CQ co-treated group was considerably higher than that in CQ alone-treated group cells. As shown in Fig. 5d, compared with the OGD group cells, levels of p62 expression were decreased in the RA group but this decrease was significantly inhibited in the RA and CQ co-treated group cells. Immunofluorescence staining result is consistent with the western blot result (Fig. 5e). These data suggest
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Neurochem Res b Fig. 5 RA protects OGD-treated ECs by inducing autophagic flux
in vitro. a, b Representative western blots and quantification data of TJ and AJ proteins Occludin, Claudin5, P120 and b-catenin in each group rats. *P \ 0.05, #P \ 0.05, &P \ 0.05 versus OGD group cells. ** P \ 0.01, ##P \ 0.01, versus treated RA (5 lM) group cells. c, d Representative western blots and quantification data of autophagy marker LC3-II and p62 in each group. *P \ 0.01, versus OGD cells. # P \ 0.01, versus RA compound with CQ cells (LC3-II); #P \ 0.01, versus treated RA cells (p62). All data represent mean values ± SEM. e Immunofluorescence staining of LC3-II (red) in ECs treated with OGD, RA or CQ, nuclei are labeled with DAPI (blue) (Color figure online)
that the RA can effectively induce autophagy flux in OGD treated ECs. Inhibition of Autophagy by 3-MA Attenuates the BSCB Protective Effect of RA In Vitro ECs in OGD model were treated with RA (5 lM), or combined with 3-MA (5 mM), or 3-MA alone for 24 h, then the change of TJ and AJ in ECs were detected. Western blot results showed that the levels of P120, bcatenin, Occludin and Claudin5 were increased after treated with RA, compared with the OGD group. At the same time, these increase in P120, b-catenin, Occludin, Claudin5 were significantly attenuated in the RA and 3-MA cotreated cells (Fig. 6a–c). As shown in Fig. 6d, f, the expression of LC3-II was further increased in RA group compared with the OGD group. On the contrary, a decrease of p62 expression was observed in RA group cells, and this decrease was partly inhibited in 3-MA group (Fig. 6d–e). The results were consistent with that of CQ.
Discussion The present study demonstrates that the neuroprotective effect of RA might be mediated in part by attenuating BSCB disruption via inducing autophagic flux after SCI. Our data showed for the first time that RA plays a role in maintaining BSCB integrity by preventing the loss of TJ and AJ proteins under pathological conditions such as SCI, and improves functional recovery after SCI. Furthermore, the present study also showed that autophagy induced by RA plays a further some significant role in BSCB disruption and prevents loss of TJ and AJ proteins. Based on our results, it is likely that autophagy may be a new target for protection BSCB integrity after SCI. Damage to the vasculature and breakdown of the BSCB is a universal consequence of SCI clinically as well as in animal models [4, 26]. The cellular and molecular mechanisms responsible for the long-term increase in BSCB permeability after SCI are numerous [27, 28]. Reactive
oxygen species (ROS) provide a common trigger for many downstream pathways that directly mediate blood-CNS barrier compromise such as oxidative damage, TJ modification and MMP activation [29, 30]. MMPs have been implicated in SCI in that plasma MMP-9 disrupted TJ proteins, rendering the BSCB leaky and allowing neurotoxic agents to enter the injured spinal cord [31]. Fluoxetine and valproic acid improved functional recovery in part by inhibiting matrix metalloprotease (MMP) activation and preventing BSCB disruption after SCI [5, 27]. Emerging evidence shows that inflammatory may also play a critical role in the blood-CNS barrier disruption in pathological conditions [32–34]. Salvianolic acid B inhibits the generation of inflammatory mediators in the injured spinal cord tissue, thereby decreasing BSCB disruption and eventually resulting in the alleviation of pathological changes caused by SCI [35]. Although many factors are known to contribute to BSCB disruption, the role of autophagy in the BSCB disruption and the regulation of the TJ proteins remains unknown, especially in acute SCI, and warrant further investigation. In the current study, we found that BSCB disruption and activation of autophagy are involved in the rat model of trauma and in vitro. Furthermore, we demonstrate for the first time that the autophagy induced by SCI plays a furthersome role in BSCB disruption in acute SCI. Thus our results firstly suggested that autophagy may be a new target for protection BSCB integrity. Retinoic acid (RA) is the active metabolite of vitamin A that is a key molecule in several physiological processes, such as reproduction, embryonic development, tissue remodeling, vision and immune function [15, 36]. The developmental defects resulting from vitamin A deficiency were well established, however, the teratogenic effects or apoptosis of retinoid overdose were also described in various tissues such as acute leukemia, glioma cells and even in some nerve fibroblasts [37–39]. In this study, RA (15 mg/kg) was administered by intraperitoneal injection after spinal cord injury. Our results also show that the expression of apoptosis protein caspase-12 was up-regulated at 1 day after SCI. Furthermore, RA treatment significantly decreased the expression of caspase-12 at 1 day after SCI (see Fig. S1). However, in the present study we did not definitely rule out the role of RA in caspase-12 expression. In the following study, signaling mechanisms by which RA inhibit apoptosis pathway will be focused on. The enzymes for RA synthesis, the retinaldehyde dehydrogenases (RALDHs) and RA-receptor (RAR), occurred by day 4 and peaked at days 8–14 following the SCI [40– 42]. Systemic administration of RA reduces early transcript levels of pro-inflammatory cytokines after experimental SCI [24]. Pharmacologic inhibition of RA-receptor activation during the differentiation of the murine BBB resulted in the leakage of a fluorescent tracer as well as
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Fig. 6 Inhibition of autophagy by 3-MA attenuates the BSCB protective effect of RA in vitro. a–c Representative western blots and quantification data of TJ and AJ proteins Occludin, Claudin5, P120 and b-catenin in each group rats. *P \ 0.05, #P \ 0.05, versus OGD group cells. &P \ 0.01, $P \ 0.01, versus treated RA (5 lM)
group cells. d–f Representative western blots and quantification data of autophagy marker LC3-II and p62 in each group. *P \ 0.05, versus OGD cells; #P \ 0.05, versus RA compound with 3-MA cells. ** P \ 0.01, versus OGD cells; ##P \ 0.01, versus treated RA cells. All data represent mean values ± SEM
serum proteins into the developing brain and reduced the expression levels of important BBB determinants [19], which indicate that RA as a developmental cue in the CNS vasculature is important for the induction of BBB properties in brain ECs. RA is important for the induction of BBB properties in brain ECs [19] and also leads to the increase in TJ proteins expression yielding a 4-fold increase in Occludin expression [20]. Retinoic acid induced BBB development is evident [1, 19, 43], however, it is unknown whether RA plays a role in maintaining BSCB integrity under the pathological conditions such as SCI. In this study, we demonstrated that the role of RA in SCI recovery is related to the prevention of BSCB disruption. We found that SCI caused a marked increase in BSCB permeability of EB dyes compared with the sham groups, furthermore, RA treatment significantly reduced the increase of EB dye extravasation compared with SCI groups. And the expression levels of TJ (Occludin, Claudin5) and AJ (b-catenin, P120) proteins decreased significantly after SCI. Exogenous RA treatment after SCI increased the levels of Occludin, Claudin5, b-catenin and P120. These results
indicate that RA also plays an important role in maintaining BSCB integrity under the pathological conditions such as SCI. In recent years, there is a good amount of evidence that moderate autophagy can protect ECs against cell injury under stressful circumstances [7, 44–48]. In an aneurismal subarachnoid hemorrhage (SAH) model, when autophagy was inhibited by 3-MA and wortmannin, the neurological scores were decreased, brain water content and BBB permeability were further aggravated compared with the SAH animals [49]. Upregulation of BMVEC autophagy has the potential to maintain BBB integrity after ischemia–reperfusion injury mediated by decreasing ROS production and increasing the expression of TJ protein ZO-1 [47]. Evaluating the above studies, the protective potential of autophagy in various ECs damage is evident [50, 51]. Many reports showed that RA exerted a cell-type dependent stimulatory effect on autophagy flux via Beclin-1 upregulation and inhibition of the mTOR pathway or by enhancing autophagosome maturation [36, 52, 53]. In the present study, Our study indicates that autophagy flux was
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stimulated after SCI and this stimulation was significantly exacerbated in the RA-treated group as compared with the vehicle-treated group both in vitro and in the SCI mice. Treatment of ECs with CQ, a lysosome specific inhibitor, or 3-MA, an autophagy specific inhibitor, suppressed the activation of autophagy flux in the RA-treated group. Effects of RA induction of autophagy flux after SCI were consistent with the previous reports. We thus assume the BSCB protective effect of RA might be related to its induction of autophagy flux after SCI. Current study showed RA prevented loss of TJ and AJ proteins by induction of autophagy flux, and inhibition of autophagy by CQ or 3-MA abolishes the BSCB protective effect of RA both in vivo and in vitro. To the best of our knowledge our group was the first to report the effect of exogenous RA on ECs and BSCB integrity by induction of autophagy flux after SCI. However, further work is needed to determine how autophagy may affect ECs survival and TJ and AJ proteins expression after SCI. In our following study, to further confirmed the precise mechanisms between autophagy and RA in BSCB disruption and TJ proteins regulation in acute SCI model, Beclin-1 and mTOR knockout rats should be applied, which may contributes to the evidences of the cross-talk between autophagy and RA in BSCB disruption and TJ proteins regulation in acute SCI. In conclusion, our research demonstrated that treatment with exogenous RA significantly attenuated BSCB permeability and degradation of TJ molecules such as P120, b-catenin, Occludin and Claudin5 at 1 day after injury, thus, improved functional recovery in SCI. RA significantly induced autophagic flux both in vivo and in vitro, furthermore, combination therapy with the autophagy inhibitor CQ partially abolished the BSCB protective effect of RA via exacerbating loss of TJ proteins both in vivo and in vitro. Based on the current results, RA may provide potential therapeutic interventions for preventing BSCB disruption after SCI. The present study lays the ground work for future translational confidence of RA in CNS diseases, especially the relations to the BSCB disruption. Acknowledgments This study was partially supported by a research Grant from the National Natural Science Funding of China (81302775, 81472165, 81200958, 81372112), Zhejiang Provincial Natural Science Foundation of China (LY14H090013, LY14H150010, LY14H170002), Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents (to J.X.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Compliance with Ethical Standards Conflict of interest
The authors declare no conflict of interest.
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