Propofol protects against blood-spinal cord barrier disruption induced by ischemia/reperfusion injury

Li-jie Xie, Jin-xiu Huang, Jian Yang, Fen Yuan, Shuang-shuang Zhang, Qi-jing Yu, Ji Hu,

1 Department of Anesthesiology, Liyuan Hospital of Tongji Medical College, Huazhong University of Science & Technology, Wuhan, Hubei Province, China

2 Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, China

Propofol protects against blood-spinal cord barrier disruption induced by ischemia/reperfusion injury

Li-jie Xie1, Jin-xiu Huang1, Jian Yang1, Fen Yuan1, Shuang-shuang Zhang1, Qi-jing Yu2, Ji Hu1,＊

1 Department of Anesthesiology, Liyuan Hospital of Tongji Medical College, Huazhong University of Science & Technology, Wuhan, Hubei Province, China

2 Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, China

How to cite this article:Xie LJ, Huang JX, Yang J, Yuan F, Zhang SS, Yu QJ, Hu J (2017) Propofol protects against blood-spinal cord barrier disruption induced by ischemia/reperfusion injury. Neural Regen Res 12(1):125-132.

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Graphical Abstract

Propofol attenuates disruption of the blood-spinal cord barrier following ischemia/reperfusion injury

orcid: 0000-0003-1175-752X (Ji Hu)

Propofol has been shown to exert neuroprotective e ff ects on the injured spinal cord. However, the e ff ect of propofol on the blood‐spinal cord barrier (BSCB) after ischemia/reperfusion injury (IRI) is poorly understood. Therefore, we investigated whether propofol could maintain the integrity of the BSCB. Spinal cord IRI (SCIRI) was induced in rabbits by infrarenal aortic occlusion for 30 minutes. Propofol, 30 mg/kg, was intravenously infused 10 minutes before aortic clamping as well as at the onset of reperfusion.en, 48 hours later, we per‐formed histological and mRNA/protein analyses of the spinal cord. Propofol decreased histological damage to the spinal cord, attenuated the reduction in BSCB permeability, downregulated the mRNA and protein expression levels of matrix metalloprotease‐9 (MMP‐9) and nuclear factor‐κB (NF‐κB), and upregulated the protein expression levels of occludin and claudin‐5. Our fi ndings suggest that propofol helps maintain BSCB integrity aer SCIRI by reducing MMP‐9 expression, by inhibiting the NF‐κB signaling pathway, and by maintaining expression of tight junction proteins.

nerve regeneration; spinal cord ischemia reperfusion injury; blood–spinal cord barrier; propofol; matrix metalloprotease-9; nuclear factor-κB; tight junction proteins; neural regeneration

Introduction

Spinal cord ischemia/reperfusion injury (SCIRI) is a devas‐tating complication of thoracoabdominal aortic interven‐tions, such as thoracoabdominal aortic aneurysm surgery and thoracoabdominal aortic repair surgery (LeMaire et al., 2012; Panthee and Ono, 2015). Several strategies have been used to improve spinal cord ischemic tolerance; however, the incidence of paraplegia remains high and poses a persistent threat to patients (Bischoff et al., 2011). Recent studies demonstrate that disruption of the blood‐spinal cord barrier (BSCB) plays an important role in SCIRI, and that maintain‐ ing its integrity can attenuate tissue damage (Lee et al., 2012; Li et al., 2014a). Barrier disruption leads to an increase in permeability, which may cause spinal cord edema, resulting in neuronal apoptosis and death (Sharma, 2005; Li et al., 2016).us, maintaining the integrity of the BSCB may im‐prove the prognosis for patients with SCIRI.

Propofol, a short‐acting intravenous anesthetic, widely used in clinical anesthesia, exerts neuroprotective e ff ects, and can protect the brain and spinal cord following isch‐emia/reperfusion injury (IRI) and improve neuronal func‐tion (Zhou et al., 2013; Sahin et al., 2015).e anestheticpossesses antioxidant, anti‐apoptotic and anti‐inflam‐matory actions (Fan et al., 2015). However, the e ff ects of propofol on BSCB disruption in IRI are unclear.

Matrix metalloproteinase‐9 (MMP‐9) degrades the extra‐cellular matrix during vascular remodeling, cell migration, reproduction and embryonic development (Chaturvedi and Kaczmarek, 2014). MMP‐9 overexpression has been found in spinal cord injury and ischemic stroke (Kurzepa et al., 2014; Piao et al., 2014; Cai et al., 2015). MMP‐9 plays an important role in blood‐brain barrier (BBB) and BSCB damage follow‐ing IRI (Lee et al., 2015; Ren et al., 2015). Nobel et al. (2002) demonstrated that MMP‐9 regulates BSCB permeability and in fl ammation early aer spinal cord injury, and that block‐ing MMP‐9 activity decreases vascular permeability. Fang et al. (2013a, 2015) showed that MMP‐9 plays a role in the disruption of the BSCB, and that bone marrow stromal cells and dexmedetomidine can suppress the expression of MMP‐9. Nuclear factor‐kappa B (NF‐κB) is a transcription factor widely expressed in the nervous system. NF‐κB participates in the regulation of BBB and BSCB permeability (Lee et al., 2014; Li et al., 2014c; Liu et al., 2014), and its overexpression leads to an increase in BBB and BSCB permeability (Lee et al., 2014; Li et al., 2014c; Liu et al., 2014). Inhibiting NF‐κB expression reduces MMP‐2 and MMP‐9 expression (Li et al., 2012; Nguyen et al., 2014).

Tight junction proteins, including occludin and claudin‐5, play a vital role in maintaining the permeability of the cen‐tral nervous system capillary barrier (Na et al., 2015). Yang et al. (2015) reported that occludin and claudin‐5 expression levels are reduced aer acute stroke, along with an increase in permeability of the BBB. Similarly, disruption of tight junctions increases the permeability of the BSCB after spi‐nal cord ischemic injury, concomitant with a reduction in occludin and claudin‐5 levels (Lee et al., 2012; Fang et al., 2013a; Yu et al., 2015). Excessively high levels of MMPs, in particular MMP‐9, significantly perturb BSCB integrity by digesting basement membranes and tight junction proteins in endothelial cells (Rosenberg et al., 1998; Yang et al., 2007). Boroujerdi et al. (2015) recently found that MMP‐9 can de‐grade laminin and claudin‐5. Therefore, decreasing MMP‐9 expression should alleviate the disruption of tight junction proteins and help maintain the integrity of central nervous system capillaries. Interestingly, propofol inhibits the expres‐sion of MMP‐9 aer ischemic injury (Ji et al., 2015).ere‐fore, propofol might attenuate the loss of BSCB integrity by inhibiting MMP‐9 expression. In the present study, we inves‐tigated the e ff ects of propofol on the BSCB in a rabbit model of SCIRI, and we examined the levels of occludin, claudin‐5, MMP‐9 and NF‐κB.

Materials and Methods

Animals

This protocol was approved by the Ethics Committee of Huazhong University of Science & Technology, China and was in accordance with the Guide for the Care and Use of Laboratory Animals (U.S. National Institutes of Health pub‐lication No. 85‐23, National Academy Press, Washington DC, revised 1996). Thirty‐six healthy male Japanese white rabbits, 3 months of age, weighing 2.0—2.5 kg, were provided by the Experimental Animal Institute of Wuhan University, Wuhan, China (license No. SYXK (E) 2009‐0027). All rabbits were neurologically intact before anesthesia and housed in standard cages with free access to food and water and sepa‐rately housed aer surgery. Rabbits were randomly assigned to three groups (n= 12 per group) by means of the random number table, including sham, ischemia/reperfusion (I/R only) and propofol (I/R + P) groups.

Establishment of SCIRI model

Propofol intervention

Neurological assessment

Histological observation

All animals were euthanized by injection of pentobarbital (200 mg/kg) 48 hours aer IRI, and the spinal cord (L4—6) wasquickly removed.e spinal cord samples were fi xed with a 10% formalin solution for 24 hours, embedded in para ffin, and serial transverse sections (4‐μm‐thick) at the lumbo‐sacral level were obtained.e sections were dehydrated in ethanol and stained with hematoxylin and eosin to observe the histological changes. Neuronal injury was evaluated at 400× magnification by light microscopy (Olympus, Tokyo, Japan). When the cytoplasm was di ff usely eosinophilic and the structure was intact, the large motor neurons were con‐sidered to be necrotic or dead. When the cells demonstrated basophilic stippling (containing Nissl substance), the motor neurons were considered to be viable or alive.

Measurement of BSCB permeability

BSCB integrity was assessed by measuring the extravasa‐tion of vascular tracers into the spinal cord parenchyma (Fang et al., 2013b). Brie fl y, 48 hours aer IRI, Evans blue (EB) fl uorescence and content were used for the qualitative and quantitative examination of extravasation induced by BSCB disruption aer SCIRI, as previously described (Fang et al., 2015). 2% EB dye (10 mL/kg; Sigma‐Aldrich, St. Lou‐is, MO, USA) was slowly intravenously administered.e rabbits were anesthetized and perfused with 500 mL/kg saline through the leventricle aer the EB circulated for 1 hour, and the spinal cord (L4—6) was removed.e spinal cord tissue was fixed in 4% paraformaldehyde, sectioned at 10 mm thickness, and frozen and sealed in a dark con‐tainer before measurement of EB fl uorescence. EB staining was visualized using an Eclipse Ci fl uorescence microscope (Nikon, Tokyo, Japan). Thereafter, the spinal cord tissue was weighed and soaked in formamide for 24 hours (60°C), and then centrifuged. The absorbance of the supernatant was measured at 632 nm with a microplate reader (Bio Tek, Winooski, VT, USA).e content of EB in the spinal cord tissue was determined (quanti fi ed as μg/g) using a standard curve.

Real-time PCR

Rabbits were anesthetized with an overdose of pentobarbital, and the L4—6segments were rapidly collected for real‐time PCR 48 hours aer surgery. Total RNA was extracted using the TRIzol Kit (Life Technologies, Carlsbad, California, USA) and converted to first‐strand complementary DNA according to the manufacturer’s instructions. Quantitative real‐time PCR was performed using an ABI 7000 instrument (Applied Biosystems, Foster City, USA) and the SYBR Green Real‐time PCR Master Mix (Roche, Basel, Switzerland).e primer sequences were as follows: NF‐κB (p65) (204 bp): forward 5′‐AAT TAA CAG CGA CTA TCT GG‐3′, reverse 5′‐CTT CAC AAG CGT AAA CAT‐3′; MMP‐9 (132 bp): forward 5′‐TGC ACT GGG CTT GGA TCA CT‐3′, reverse 5′‐GGG TTG GGG TTA GGA CCA TAT AG‐3′; GAPDH (262 bp): forward 5′‐CCG CCC AGA ACA TCA TCC CT‐3′, reverse 5′‐GCA CTG TTG AAG TCG CAG GAG A‐3′.e thermocycling conditions were as follows: 50°C for 2 min‐utes (UDG incubation) and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. All reactions were performed in triplicate. Melting curve anal‐ysis was performed to ensure the speci fi city of quantitative PCR. Data analysis was performed using the 2–ΔΔCTmethod described by Livak and Schmittgen (Livak and Schmittgen, 2001), with GAPDH as the reference gene.

Western blot assay

Rabbits were anesthetized with an overdose of pentobarbital, and the L4—6segments were rapidly collected for western blot analysis 48 hours aer surgery to measure the expression lev‐els of MMP‐9, NF‐κB (p65), claudin‐5 and occludin. Brie fl y, the spinal cord tissues were homogenized, and total proteins were purified using cell and tissue protein extraction re‐agents according to the manufacturer’s instructions (Wuhan GuGe, Wuhan, Hubei Province, China). The samples were subjected to SDS‐PAGE, and the proteins were blotted onto a membrane and probed with the following antibodies: mouse anti‐NF‐κB (p65) polyclonal antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti‐MMP‐9 polyclonal antibody (1:1,000; Affinity, Zhenjiang, Jiangsu, China), goat anti‐claudin‐5 polyclonal antibody (1:500; Santa Cruz Biotechnology), rabbit anti‐occludin polyclonal antibody (1:1,000; Abcam, Cambridge, UK) and horseradish peroxidase (HRP)‐conjugated secondary antibodies (goat and donkey; 1:3,000; Wuhan GuGe).e membranes were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 30 minutes at room temperature, blocked and visualized by enhanced chemiluminescence. Semi‐quantitation of scanned films was performed using Quantity One soware (Bio‐Rad, Hercules, CA, USA).

Statistical analysis

Data are expressed as the mean ± SD or median values (for neurologic assessments) and analyzed with SPSS so‐ware (version 19.0, IBM, Armonk, NY, USA). All variables were normally distributed. Groups were compared (except neurological assessment score) with one‐way analysis of variance (ANOVA), followed by Tukeypost hocanalysis. Neurological assessment scores were analyzed with the Kruskal‐Wallis test.Pvalues ＜ 0.05 were considered statis‐tically signi fi cant.

Results

Propofol improved neurological function after SCIRI

All rabbits showed normal motor function of the hind limbs before the induction of ischemia.e sham‐operated rabbits retained normal hind limb motor function throughout the study. The 30‐minute infrarenal aortic occlusion resulted in severe lower extremity neurologic de fi cits in the I/R and propofol groups. In the I/R group, most rabbits had low neu‐rological scores, while neurological scores in the propofol group were signi fi cantly higher than those in the I/R group (Figure 1).

Propofol improved the morphology of the spinal cord after I/R

Figure 1 Propofol improved motor function after SCIRI.

Figure 2 E ff ect of propofol on the morphology of the ischemia/reperfusion-injured spinal cord (hematoxylin-eosin staining, original magni fi cation, × 400).

Figure 3 Propofol attenuated BSCB damage after SCIRI.

Figure 4 Propofol suppressed the upregulation of NF-κB (p65) protein (A) and mRNA (B).

Hematoxylin‐eosin staining showed that in the sham group, neurons had a normal morphology. In contrast, in the I/R group, there were no normal‐looking neurons, and dead neurons with cytosolic vacuoles were apparent. In the propofol group, some normal neurons were visible (Figure 2).

Propofol decreased BSCB permeability after IRI

As shown in Figure 3, compared with the sham group, there was a signi fi cant increase in EB dye extravasation in the I/R group. Propofol treatment reduced extravasation following IRI, indicating that it helped maintain BSCB integrity.

Figure 5 Propofol inhibited MMP-9 protein (A) and mRNA (B) expression after spinal cord ischemia/reperfusion injury, as evaluated by western blot assay and real-time PCR.

Figure 6 Propofol inhibited the reduction in claudin-5 (A) and occludin (B) protein levels after ischemia/reperfusion injury, as assessed by western blot assay.

Propofol inhibited the expression of NK-κB (p65) in the I/R-injured spinal cord

Western blot analysis demonstrated that NF‐κB (p65) expression increased 48 hours after injury, and propofol significantly in‐hibited this upregulation (P＜ 0.05; Figure 4A). Real‐time PCR showed that propofol treatment signi fi cantly decreased NF‐κB (p65) mRNA expression aer SCIRI (P＜ 0.05; Figure 4B).

Propofol inhibited the upregulation of MMP-9 after SCIRIThe expression of MMP‐9 increased 48 hours after reper‐fusion, as assessed by western blot analysis, while propofol treatment signi fi cantly depressed the upregulation of MMP‐9 (P＜ 0.05). As shown in Figure 5A, in the I/R group, the expression level of MMP‐9 was remarkably increased. Sim‐ ilarly, real‐time PCR showed that propofol treatment sig‐ni fi cantly decreased mRNA expression of MMP‐9 (P＜ 0.05; Figure 5B).

Propofol suppressed the reduction in occludin and claudin-5 expression in the I/R-injured spinal cord

Western blot analysis indicated that the levels of occludin and claudin‐5 were decreased 48 hours after I/R injury in the I/R group (Figure 6A), while propofol suppressed this decrease in expression of occludin and claudin‐5 (P＜ 0.05; Figure 6B).

Discussion

Propofol is widely used in clinical anesthesia, as well as seda‐tion (Hutchens et al., 2006). Propofol has protective e ff ects on some organs, such as the heart, kidney, lung and liver (Vasileiou et al., 2012; Shirakawa et al., 2014; Ge et al., 2015; Bellanti et al., 2016). Furthermore, the neuroprotection pro‐videdby propofol has been demonstrated to be e ffi cacious in a number of studies (Fang et al., 2015; Yue et al., 2015; Zhou et al., 2015; Hou et al., 2016). Neuroprotective effects of propofol have been reported in a spinal cord ischemia model and in a cerebral ischemia model (Zhou et al., 2013; Sahin et al., 2015; Yu and Yang, 2016). Propofol has antioxidant, an‐ti‐apoptotic and anti‐in fl ammatory actions.

The physical barriers between the blood and the central nervous system (BBB and BSCB) protect the central ner‐vous system from toxic and pathogenic agents in the blood. Indeed, disruption of the barrier aggravates damage to the central nervous system (Palmer, 2010). A study has shown that propofol treatment helps maintain BBB integrity in a rat model of focal cerebral IRI (Ji et al., 2015).

Our results demonstrate that propofol pre‐ and post‐treatment helps maintain BSCB integrity, and signi fi cantly improves both neurological and histological outcomes following ischemia/reperfusion. The expression levels of MMP‐9 and NF‐κB (p65) were increased in the IRI group, and claudin‐5 and occludin expression levels were de‐creased. Our fi ndings suggest that propofol might attenu‐ate disruption of the BSCB by inhibiting the expression of MMP‐9 and NF‐κB (p65) andby suppressing the reduction in claudin‐5 and occludin levels. Our study is of great sig‐ni fi cance for ischemia‐induced spinal cord injury in which disruption of BSCB integrity plays a major pathogenetic role.

NF‐κB, a key transcription factor, plays an important role in in fl ammatory responses and oxidative stress induced by I/ R (Yu et al., 2016). Propofol has been found to inhibit the in‐fl ammatory reaction by inhibiting NF‐κB activation during focal I/R, which may be one of the mechanisms of neuro‐protection (Feng et al., 2004). Indeed, overexpression of NF‐κB leads to BBB and BSCB dysfunction, including increased permeability (Li et al., 2014b; Liu et al., 2014). Ulbrich et al. (2016) found that propofol inhibits the NF‐κB signaling pathway. In the present study, we found that NF‐κB expres‐sion is signi fi cantly elevated aer ischemia/reperfusion, and that propofol treatment inhibited this increase.e high lev‐els of NF‐κB likely increased BSCB permeability.erefore, inhibition of the NF‐κB signaling pathway by propofol may help maintain BSCB integrity aer IRI.

MMP‐9, a potent regulator of inflammation, has an im‐portant role in acute in fl ammation (Liu et al., 2015). Over‐expression of MMP‐9 impairs the integrity of the BSCB aer IRI, likely by degrading tight junction proteins and basement membranes of endothelial cells (Yang et al., 2007). Numer‐ous studies have shown that BBB and BSCB dysfunction induced by stroke or I/R is associated with the upregulation of MMP‐9 (Noble et al., 2002; Xiang et al., 2012; Chaturvedi and Kaczmarek, 2014; Fang et al., 2015). MMP‐9 inhibitors, such as tissue inhibitor of matrix metalloproteinases (TIMPs) and BB‐1101, appear to help maintain BBB integrity (Cun‐ ningham et al., 2005).erefore, therapies that inhibit MMP‐9 expression might attenuate the disruption of the BBB and BSCB. Ji et al. (2015) found that propofol post‐treatment improves neurobehavioral dysfunction and attenuates BBB damage by decreasing MMP‐9 and aquaporin‐4 expression in a rat model of I/R‐induced focal cerebral injury. NF‐κB upregulates expression of matrix metalloproteinases, includ‐ing MMP‐2 and MMP‐9 (Yang et al., 2016). In our current study, MMP‐9 expression was substantially increased after IRI, and propofol reduced its expression. Concomitant with the increase in MMP‐9 expression, NF‐κB levels were increased in the I/R group.ese increases in MMP‐9 and NF‐κB expression were suppressed in the propofol group, indicating that propofol reduces MMP‐9 expression by in‐hibiting the expression of NF‐κB.

Tight junctions between cerebral microvascular endo‐thelial cells are the functional and structural basis of the BBB and are responsible for blocking the free movement of compounds from the blood into the parenchyma (Zlokov‐ic, 2008). Cerebral IRI leads to tight junction disruption, allowing numerous compounds to cross the BBB (Jiao et al., 2011). Occludin and caudin‐5, the major tight junc‐tion‐associated proteins, play a critical role in connecting the membrane with cytoskeletal proteins. The expression of occludin and claudin‐5 are significantly decreased af‐ter SCIRI (Fang et al., 2013a, 2015; Yu et al., 2015), which increases the permeability of capillaries and aggravates spinal cord injury. MMP‐9 proteolytically digests occlu‐din and caudin‐5, resulting in barrier dysfunction (Fang et al., 2013a; Li et al., 2014b; Boroujerdi et al., 2015; Yu et al., 2015). In our present study, the reduction in occludin and caudin‐5 levels paralleled the increase in MMP‐9 ex‐pression in the I/R group, consistent with previous studies showing that MMP‐9 degrades claudin‐5 and occludin aer SCIRI.

There are a number of limitations of our study. First, we assessed BSCB integrity at only one time point—48 hours after reperfusion. Previous studies demonstrate that BSCB dysfunction worsens 48 hours aer SCIRI (Li et al., 2014c). Hence, we selected this time window for this study. How‐ever, in future studies, a wider observation window would help better evaluate the protective e ff ects of propofol on the BSCB aer SCIRI. Second, we used a rabbit model. Experi‐ments using other animal models are necessary to con fi rm the fi ndings. In addition, the high dose of propofol used in the current study might impair the respiratory and circula‐tory systems (Simons et al., 2013; Meng et al., 2016). Indeed, clinically, propofol is not suitable for patients su ff ering from heart diseases. At present, although the protective e ff ects of propofol against SCIRI have been clari fi ed in animal models (Zhou et al., 2013; Fan et al., 2015; Yang et al., 2015), no clin‐ical study has examined whether propofol protects against BSCB dysfunction in humans.

Our findings show that intravenous infusion of propofol maintains the integrity of the BSCB aer SCIRI, laying the foundation for further research. Future studies should exam‐ine the e ff ectiveness of a combination of drugs and treatmentstrategies. Recently, propofol injection combined with bone marrow mesenchymal stem cell transplantation was shown to improve electrophysiological function of the hind limb and attenuate spinal cord injury in a rat model (Wang et al., 2015). Moreover, a more detailed examination of the cellular and molecular mechanisms is also warranted (Ning et al., 2014; Li et al., 2015).

In summary, propofol pre‐ and post‐treatment attenuates BSCB damage induced by I/R and ameliorates neurobehav‐ioral de fi cits by maintaining levels of occludin and claudin‐5, downregulating MMP‐9, and inhibiting the NF‐κB signaling pathway. Our findings should assist in the development of novel therapeutic strategies for SCIRI.

Author contributions:LJX, JXH, JY and SSZ participated in the experimental procedures and data process. LJX, JXH and FY were in charge of western blot assay and statistics. LJX and SSZ were in charge of real-time PCR and statistics. JY and QJY were in charge of neurological and histological assessment. LJX and JH were responsible for data analysis and experimental results. LJX, JXH and JH designed the study and wrote the paper. All authors approved the fi nal version of this paper.

Con fl icts of interest:None declared.

Plagiarism check:This paper was screened twice using CrossCheck to verify originality before publication.

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Copedited by Patel B, Copper C, Xie LJ, Yu J, Li CH, Li JY, Song LP, Zhao M

10.4103/1673-5374.199004

Accepted: 2016-11-21

＊Correspondence to: Ji Hu, hbwhly01@163.com.