Introduction
Spinal cord ischemia reperfusion (I/R) injury is well known as the most devastating complication in clinical thoracoabdominal aneurysm repair [
1]. One of the major pathological changes in I/R injury is the impairment of blood-spinal cord barrier (BSCB), which plays a critical role in maintaining homeostasis of the spinal cord. Similarly to the blood-brain barrier, BSCB consists of continuous capillary endothelial cells lining spinal microvessels, perivascular endfeet of astrocytes, pericytes, and tight junctions between adjacent endothelial cells and basement membrane [
2]. Astrocytes surround or closely associate with spinal capillaries. Reactive astrocytes can release chemical factors including inflammatory mediators to regulate blood-brain barrier permeability via interaction with the endothelium [
3]. The opening of tight junction pathways has generally been found to be responsible for the increase of endothelial permeability. Among the tight junction related proteins, Zonula occluden-1 (ZO-1) has been evaluated as a marker protein [
4]. Reduced expression of ZO-1 is closely related to BSCB breakdown in I/R injury [
5]. Disruption of BSCB function and structure following I/R injury leads to neurological deficit. Mounting evidence indicates that BSCB integrity can be a neuroprotection target for reducing spinal cord I/R injury [
5‐
9].
The receptor-mediated endocannabinoid system plays an important role in neuroprotection. The system consists of two main receptors, cannabinoid-1 receptor (CB1R) and cannabinoid-2 receptor (CB2R), endogenously-produced cannabinoids, and corresponding synthesizing and degrading enzymes [
10]. In the spinal cord, CB1R is expressed constitutively in neurons, and can be induced by reactive astrocytes; while CB2R is strongly expressed in response to cascading inflammation associated with ischemic episode, mainly in astrocytes and immune infiltrates [
11]. Our previous work showed that CB1R and CB2R were associated with BSCB disruption during spinal cord I/R injury [
12]. CB1R increases rapidly to peak within 2 h to 6 h after ischemia, and returns to baseline at 24 h postischemia. CB2R increases at 24 h after ischemia [
13,
14], and the hyperexpression can last for 28 days [
11]. Considering that the most serious BSCB disruption occurred at 48 h after spinal cord I/R injury [
15], we only aimed at CB2R in the current study. CB2R activation attenuates the cascading inflammation in hypoxia-ischemia events by downregulating reactive glial cells and reducing the release of inflammatory cytokines [
16‐
18]. Previous studies have demonstrated CB2R activation contributes to ameliorating blood-brain barrier damage during cerebral I/R injury in vitro and in vivo [
19‐
21]. CB2R agonist could modulate the BSCB permeability and the expression of tight junction proteins [
22]. However, the mechanisms involved in the protective function of CB2R activation in BSCB breakdown following spinal cord I/R injury are still largely unknown.
Various studies have reported that toll-like receptors 4 (TLR4) are strongly associated with the inflammatory responses after I/R injury and mediate the motor dysfunction and BSCB disruption [
7,
15,
23]. It has also been reported that brain ischemia induces TLR4 expressions in astrocytes, and that TLR4 activation leads to an astroglial polarization towards an inflammatory phenotype [
24]. Astrocytes contribute to blood-brain barrier damage through activation of endothelial matrix metalloproteinase 9 (MMP9) [
25]. MMP9 also influences BSCB integrity by degrading tight junction proteins, such as ZO-1, and modulate inflammation following tissue injury by facilitating immune cell infiltration [
5,
8,
26,
27]. Interestingly, recent findings reveal that the TLR4/MMP9 signal pathway could regulate tight junctions in blood-brain barrier dysfunction induced by ischemic stroke [
28]. Since exogenous activation of CB2R could inhibit TLR4 expression and hence have a significant effect on inflammatory responses in spinal cord injury [
16], we investigated the hypothesis that exogenous activation of CB2R may have the potential protective effects against neurological deficit and BSCB disruption during spinal cord I/R injury through modulation of the TLR4/MMP9 axis.
In order to test our hypothesis, first, we studied the role of TLR4/MMP9 signal pathway by blocking the pathway with TLR4 inhibitor TAK-242, which could prevent TLR4 from interacting with its downstream targets [
29]. Next, we used CB2R agonist JWH-133 preconditioning with or without the CB2R antagonist AM630. JWH-133 is a potent CB2R agonist (Ki = 3.4 nM) displaying approximately 200-fold selectivity over CB1 receptor [
30,
31]. AM630 is a CB2R antagonist (Ki = 31.2 nM), which displays 165-fold selectivity over CB1 receptor [
32,
33].
Materials and methods
Experimental animals
Male Sprague-Dawley rats, weighting from 200 to 230 g, were purchased from the animal center of the China Medical University. The experimental procedures were approved by the Ethics Committee for Animal Experimentation of China Medical University and in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD).
Experimental protocol
Animals were randomly divided into one of five groups: Sham, I/R, I/R + TAK-242 (TAK), I/R+ JWH-133 (JWH), and I/R + JWH-133 + AM630 (JWH + AM) group,
n = 45 for each group. As described in previous studies, 1 h prior to sham surgery or ischemia, intraperitoneal administrations were performed with TAK-242 (3 mg/kg) for the TAK group [
34‐
36], JWH-133 (1 mg/kg) for the JWH group, JWH-133 (1 mg/kg), and AM630 (1 mg/kg) independently for the JWH + AM group [
21], or equivalent volume of vehicle (DMSO) for the sham and I/R groups. The descending aorta was exposed or occluded for 14 min, followed by reperfusion for 1, 2, 3, or 7 days. The animals were sacrificed with pentobarbital and their L4–6 segments of spinal cord were collected for BSCB disruption evaluation, western blot, gelatin zymography, and immunofluorescence staining. In each group, five rats at each time point were used for hindlimb locomotor functional assessment, spinal cord edema measurement, western blot, and gelatin zymography; five rats at each time point were used for evaluation of Evans blue (EB) extravasation; and the other five rats were used for immunofluorescence staining.
Surgical procedures
The spinal cord I/R model in rats was conducted according to the previously reported method [
37]. Briefly, the rats were anesthetized with intraperitoneal administration of 4% sodium pentobarbital (50 mg/kg). The rectal temperature was monitored and kept at 37 ± 0.5 °C with a heated operating table. Catheters were placed into the tail artery and the left carotid artery for detecting the distal and proximal arterial blood pressure, respectively. The descending aorta was exposed with a left lateral thoracotomy. After systemic heparinization (200 IU/kg), under direct visualization, descending aorta was occluded with a cross-clamp just distal to the left subclavian artery. A reduction of the distal blood pressure to less than 10 mmHg was confirmed as ischemia. The cross-clamp was removed after a 14-min ischemia, followed by reperfusion. All rats were allowed to recover in a 37 °C incubator for 4 h and were subsequently put in separate cages with free access to food and water. Manual bladder expression was performed twice daily until recovery of autonomic function.
Neurological assessment
The ability of rats to use their hindlimbs was investigated by an assessor blinded to the experimental protocol with Basso, Beattie, and Bresnahan (BBB) Locomotor Scale [
38] over 4 min on post-reperfusion 1, 2, 3, and 7 days. The scoring criteria included joint movement, paw placement, and coordination. The rats with normal motor function obtained a BBB score of 21points, while the rats losing motor function completely were scored 0 point.
BSCB disruption evaluation
The evaluation of BSCB integrity was performed with quantitative and qualitative measurements of EB extravasation and spinal cord edema. The content and fluorescence of EB were examined. Briefly, 60 min after the injection of EB dye (20 g/L; Sigma) at a dose of 10 ml/kg via the tail vein, the animals were anesthetized and then transcardially perfused with 0.9% saline containing 10 IU/ml heparin, until colorless perfusion fluid was obtained from the right atrium, to remove all EB dye that had not leaked into the interstitium. The L4–6 spinal cord tissue was weighed, then soaked with methanamide for 24 h (60 °C) and centrifuged. The fluorescent absorption of the supernatant was detected at 632 nm with a microplate reader (BioTek, Winooski, USA) and presented as EB amount per tissue weight (μg/g) with standardized curve. Furthermore, the spinal cord tissue was fixed with 4% paraformaldehyde. The transverse sections (10 μm) were visualized under a BX-61 (Olympus, JP) fluorescence microscope. EB dye was visualized as red under the green fluorescence excitation mode. The integrated optical density (IOD) of the positive fluorescence labeling in the lumbar spinal cord tissue was analyzed to quantify the EB extravasation (Image Pro Plus 6.0). The percentage of water content in the spinal cord tissue was calculated with a wet-dry method: (wet weight − dry weight)/wet weight × 100.
Western blot analysis
The spinal cord tissue samples were extracted in RIPA lysis buffer, followed by centrifugation (12,000×g, 30 min, 4 °C). Protein concentration was measured with the BCA assay. Equal amounts of proteins (60 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The blots were blocked with 5% skim milk in Tris-buffered saline Tween (TBST) for 1 h at room temperature, and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-cannabinoid receptor II (1:500, Abcam 3561), rabbit polyclonal anti-ZO-1 antibody (1:500, Invitrogen 40-2200), rabbit polyclonal anti-TLR4 (1:500, Abcam 13556), rabbit polyclonal anti-MMP9 (1:500, Abcam 38898), rabbit polyclonal anti-MyD88 (1:500, Abcam 2064), rabbit polyclonal anti-NF-κB p-p65 (1:1000, Abcam 86299), rabbit polyclonal anti- NF-κB p65 (1:1000, Abcam 16502), or rabbit monoclonal anti-GAPDH (1:1000, Abcam 8245). After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Bioss, Beijing, China) for 2 h at room temperature. The bands were visualized by enhanced chemiluminescence (ECL Advance Kit; Bio-Rad). Protein expression was quantified by densitometric analysis (Image J). GAPDH was used as an internal loading control. The relative protein levels were normalized by the ratio of target protein to GAPDH.
Gelatin zymography
The MMP9 activity was determined with gelatin zymography assay. Equal amounts of proteins (60 μg) were separated by 10% SDS-PAGE containing 0.1% gelatin (Invitrogen). The gel was washed in renaturing buffer (2.5% Triton X-100) (Invitrogen) for 1 h at room temperature. Then it was incubated overnight at 37 °C in development buffer (Invitrogen). Finally, the gel was stained with 0.25% R-250 Coomassie blue for 2 h and destained in 2: 1 methanol: acetic acid solution for 2 h at room temperature. The brightness of the clear bands, where the gelatin was degraded by MMP9, was subjected to densitometric analysis (Image J).
Double immunofluorescence staining
The perivascular localization of CB2R, TLR4, MMP9, and reactive astrocytes were identified by double immunofluorescence labeling of CB2R, TLR4, MMP9, or reactive astrocytes marker glial fibrillary acidic protein (GFAP) with capillary endothelial cells marker CD31. Mouse monoclonal anti-CD31 [P2B1] (1:100, Abcam 24590) together with rabbit polyclonal anti-cannabinoid receptor II (1:500, Abcam 3561), rabbit polyclonal anti-TLR4 (1:100, Abcam 13556), rabbit polyclonal anti-MMP9 (1:100, Abcam 38898), or rabbit polyclonal anti-GFAP antibody (1:800, Abcam 7260) were used as primary antibodies to incubate the sections overnight at 4 °C, followed with corresponding secondary antibodies: Alexa 594-conjugated donkey anti-mouse IgG (1:200, Abcam 150108) and Alexa 488-conjugated donkey anti-rabbit IgG (1:200, Abcam 150073), each for 2 h at room temperature. The sections were imaged and analyzed by Leica TCS SP2 (Leica Microsystems, Buffalo Grove, IL, USA) laser scanning spectral confocal microscope. The average IOD of the positive fluorescence labeling in the gray matter of six transverse sections with ×40 magnification, at 140 μm intervals from 500 μm rostral and caudal to the center of the L4-6 spinal cord in each rat (n = 5 in each group), was analyzed to quantify the target protein expression (Image Pro Plus 6.0). Quantification of the perivascular protein expression is represented as the average IOD ratio of perivascular target protein to corresponding CD31 in six transverse sections at ×100 magnification in each rat (n = 5 in each group). Three blood vessels in the gray matter of each section were randomly selected by an assessor blinded to the experimental protocol.
For double staining of astrocytic marker with CB2R, TLR4, and MMP9, mouse monoclonal anti-GFAP antibody (1:800, Abcam 10062) together with rabbit polyclonal anti-cannabinoid receptor II (1:500, Abcam 3561), rabbit polyclonal anti-TLR4 (1:100, Abcam 13556), or rabbit polyclonal anti-MMP9 (1:100, Abcam 38898) were used as primary antibodies to incubate overnight at 4 °C, followed with corresponding secondary antibodies as mentioned above. The colocalization of target proteins with reactive astrocytes was quantified by the average IOD of the colocalization fluorescence labeling in the gray matter of six transverse sections with ×100 magnification in each rat (n = 5 in each group) (Image Pro Plus 6.0).
Statistical analysis
The values were presented as means ± SEM and statistically analyzed with the SPSS 20.0 software. The data collected over time among the groups were compared by repeated measures ANOVA with time as within subject factor, group as between subject factor, followed by Bonferroni post-hoc tests. The other data were compared with Student’s t test or one-way ANOVA followed by Newman-Keuls post hoc analysis. Values of P < 0.05 were considered statistically significant.
Discussion
The present study showed several novel outcomes: (1) CB2R agonist JWH-133 attenuated BSCB disruption, spinal cord edema, and improved neurological function after spinal cord I/R injury. (2) JWH-133 prevented downregulation of ZO-1 and upregulation of perivascular reactive astrocytes following I/R injury. (3) The beneficial effects of JWH-133, at least in part, involved the TLR4/MMP9 pathway.
Currently, hypothermia and cerebrospinal fluid drainage are clinically effective for prevention and treatment of spinal cord ischemic injury in clinical thoracoabdominal aneurysm repair. The medical options available for spinal cord protection are extremely limited. Modulation of the receptor-mediated endocannabinoid system can produce neuroprotective effects during spinal cord injury [
11,
39]. Our study revealed that administration of CB2R agonist JWH-133 improved motor function and preserved BSCB integrity over the 7-day period of observation in a spinal cord I/R rat model, which could be blocked by CB2R antagonist AM630, corresponding with the previous study [
40,
41]. In the current study, we utilized the rat spinal cord I/R injury model with 14-min occlusion of descending aorta [
37], which is much closer to clinical thoracoabdominal aneurysm repair than I/R rat model conducted with transient occlusion of abdominal aorta [
22] and the murine spinal cord contusion model [
16].
One of the mechanisms via which CB2R agonist treatment may contribute to preservation of BSCB integrity is modulation of inflammatory responses following spinal cord injury [
16]. TLR4 and MMP9 are the important mediators in inflammation-induced BSCB breakdown [
5,
15,
28]. TLR4 elevation induces astrocytes polarization [
24]. The reactive astrocytes around microvessels activate endothelial MMP9 [
25] or release inflammatory mediators such as cytokines IL-1β, IL-6, and TNFα to regulate blood-brain barrier permeability in inflammation [
42]. The MMP-9 activation is responsible for the degradation of tight junction proteins and the opening of tight junctions [
34], which leads to the increase of endothelial permeability [
3]. In the current investigation, we were able to show that exogenous activation of CB2R prevented upregulation of TLR4/MMP9 expression after I/R injury, and proportionally prevented perivascular activation of astrocytes and downregulation of ZO-1, followed by improvement of BSCB permeability and neurological function, which were also found in rats with TAK-242 administration. Therefore, it can be assumed that exogenous activation of CB2R alleviates spinal cord I/R injury by regulating TLR4/MMP9 expression.
The myeloid differentiation factor 88 (MyD88) is the central adaptor protein of TLR4 signal pathway, and facilitates the transduction of downstream signaling molecules, such as NF-κB, to regulate the expression of proinflammatory cytokines and chemokines [
43‐
45]. In addition, it has been suggested that the activation of MMP9 is modulated by NF-κB phosphorylation [
28,
46]. To confirm that the exogenous activation of CB2R in the context of I/R injury directly inhibit TLR4 signaling to modulate MMP9 activity, we further analyzed the expression levels of MyD88 and NF-κB phosphorylationin response to the JWH-133 treatment, as well as MMP9 activity by gel zimography. We found JWH-133 pretreatment prevented the activation of MyD88/NF-κB and MMP9 following I/R injury, suggesting that the exogenous activation of CB2R inhibited TLR4/MyD88/NF-κB signaling to reduce MMP9 activity. Previous studies have reported that intrathecal injection of specific siRNAs targeting MMP9 to reduce its activity and expression could improve BSCB integrity in spinal cord I/R injury [
8,
47,
48].
However, matrix metalloproteinases (MMPs) play a dual role by acutely disrupting the tight junction proteins in the blood-brain barrier and chronically promoting angiogenesis [
49]. Recent study showed MMPs promote the activation and migration of astrocytes to form protected zone and reduce the cerebral infarct volume after ischemic stroke [
50]. It has been reported that in central nervous system injuries, there are at least two types of reactive astrocytes A1 and A2, one type being helpful and the other harmful [
51]. In the present study, we did not identify the reactive astrocytes as A1 or A2, but found that the reactive astrocytes are involved in the destruction of BSCB.
This study confirmed perivascular localization of CB2R, TLR4, and MMP9 by double immunofluorescence labeled with capillary endothelial cells marker CD31. Moreover, the expression of TLR4/MMP9 and reactive astrocytes along spinal microvessels after I/R injury were all reduced by CB2R activation. These findings revealed the close apposition of CB2R, TLR4, MMP9, reactive astrocytes, and spinal microvessels, which providing geographic evidence for CB2R activation modulating perivascular expression of TLR4/MMP9 and reactive astrocytes to preserve tight junctions and endothelial integrity. Moreover, the findings also indicated that reactive astrocytes may be the key player in the CB2R/TLR4/MMP9 axis. So, we further detected the colocalization of CB2R, TLR4, and MMP9 with reactive astrocytes. As expected, CB2R, TLR4, and MMP9 colocalized with reactive astrocytes after I/R injury, and JWH-133 treatment significantly decreased the astrocytic TLR4/MMP9 expression compared to I/R group, which may provide evidence for JWH-133 working on reactive astrocytes to regulate TLR4/MMP9 expression. Previous studies have shown that CB2R is located in glial cells, and its expression increases in response to neuroinflammation [
11,
14,
52,
53]. Both glial TLR4 and MMP9 pathways are involved in the inflammation following I/R injury [
8,
15]. So, we may also look into microglia or perivescular fibroblasts to prove the specificity of cell type that JWH-133 works on in the further research.
CB2R activation showed the beneficial effects on spinal cord I/R injury throughout the entire observation period in this study, which was consistent with the high expression of CB2R after injury. We evaluated the expression of CB2R in the spinal cord tissue, which showed a sustained increase from day 1 to day 7 after injury. Previous studies also suggested that the ischemic injury induced a delayed increase of CB2R expression at 24 h after injury [
13,
14], and the increased expression of CB2R could last for 28 days after spinal cord injury [
11]. The endocannabinoids act through CB1 and CB2 cannabinoid receptors to display a protective response involved in spontaneous recovery up to 90 days after spinal cord injury [
39]. Therefore, a longer observation time is essential to evaluate the effects of CB2R activation on the secondary injury and the spontaneous recovery after spinal cord I/R injury.
Considering that prevention is better than treatment, we tried pretreatment in the current study, expecting the protective effects before acute ischemic injury and secondary inflammatory injury. The findings, as expected, showed that pretreatment with CB2R agonist JWH-133 reduced spinal I/R injury, which is similar to those previous reports [
22,
54]. However, post injury administration is also clinically relevant due to some unpredictable spinal cord injuries. Yu et al. indicated that 48 h post-stroke treatment with a CB2R agonist did not efficiently suppress brain damages, while preconditioning of the CB2R agonist significantly reduced the infarct volume [
55]. A similar report has also showed that CB2R agonists, given either before or 10 min after the beginning of ischemia, reduced the microglial activation, the area of infarction, and neurological symptoms [
17], suggesting that early treatment with CB2R agonists may attenuate ischemic cerebral injury. In our previous study, we observed the disrupted BSCB at 4 h after reperfusion [
12], which may aggravate injury, resulting in paraplegia. Treatment with a selective CB2R agonist 1 h post spinal cord contusion injury also revealed beneficial effects [
16]. Therefore, we predict that post injury administration of JWH-133 before BSCB disruption could alleviate BSCB leakage and neurological deficit following I/R injury, which needs further studies to confirm.
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