Neuroprotective effect of hydrogen sulfide on acute cauda equina injury in rats

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The Spine Journal ■■ (2015) ■■–■■

Basic Science

Neuroprotective effect of hydrogen sulfide on acute cauda equina injury in rats Xingzhen Liu, MD1, Zhiyi Fu, MD*, Yujie Wu, MD1, Xiaopeng Hu, Jr, RN, Tong Zhu, Jr, RN, Chen Jin, Jr, RN Department of Orthopedics, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Rd, Shanghai 200011, China Received 10 March 2015; revised 30 September 2015; accepted 22 October 2015

Abstract

BACKGROUND: Hydrogen sulfide (H2S), as a novel gaseous messenger molecule, plays an important role in signal transduction and biological modulation. PURPOSE: In the present study the effect of H2S after compression injury of cauda equina was studied. STUDY DESIGN: The setting of this study is the laboratory investigation. METHODS: A total of 162 rats were randomly allocated into three groups: sham group, compression group, and H2S group. Cauda equina compression (CEC) injury in rats was induced by implanting silicone gels (10×1×1 mm) into the epidural spaces L5 and L6; laminectomy was performed at the L4 level of the vertebra in the sham-operated group. The experimental group was treated with sodium hydrosulfide intraperitoneally (20 µmol/kg body weight), whereas the compression and sham groups received equal volumes of physiological saline. Levels of malonaldehyde (MDA) and glutathione (GSH) were determined immediately before CEC surgery, 12 h, 24 h, 48 h, and 72 h after CEC surgery. Furthermore, hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferasemediated biotinylated UTP nick-end labeling (TUNEL) assay were performed 48 h after CEC. RESULTS: Hematoxylin and eosin staining showed that myelin sheath and the cauda equina fibers in the compression group were less compact and highly degenerated compared with the sham group, and that H2S treatment could improve the status. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling staining exhibited that decreased number of TUNEL positive cells was found in the H2S group than in the compression group. The level of MDA was increased in the sham and H2S groups compared with the compression group (p<.05, p<.01), whereas the level of GSH was decreased (p<.05, p<.01). CONCLUSIONS: With the above data, we conclude that H2S could reduce the oxidative stress and has neuroprotective effect in acute cauda equina syndrome. © 2015 Elsevier Inc. All rights reserved.

Keywords:

Cauda equina syndrome; Hydrogen sulfide; Oxidative stress effect; Malonaldehyde; Glutathione; Neuroprotective effect

Introduction FDA device/drug status: Not applicable. Author disclosures: XL: Nothing to disclose. ZF: Grant: National Natural Science (B, Paid directly to author). YW: Nothing to disclose. XH: Nothing to disclose. TZ: Nothing to disclose. CJ: Nothing to disclose. The disclosure key can be found on the Table of Contents and at www.TheSpineJournalOnline.com. This study was conducted according to the guidelines laid down in the Declaration of Helsinki. * Corresponding author. Department of Orthopedics, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Rd, Shanghai 200011, China. Tel.: +86 21 56691101; fax: +86 21 56691662. E-mail address: [email protected] (Z. Fu) 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.spinee.2015.10.046 1529-9430/© 2015 Elsevier Inc. All rights reserved.

Cauda equina syndrome (CES), a common clinical disease, results from dysfunction of the sacral and lumbar nerve roots in the vertebral canal [1]. It causes catastrophic consequences for the patients, including low back pain and radiculopathy, lower limb weakness, lower limb and saddle region sensory disturbance, and visceral impairment of bladder, bowel, and sexual function [2]. Cauda equina, unlike peripheral nerves, has a special anatomical location, which is the bridge of the spinal cord and peripheral nerves, connecting spinal cord neurons and dorsal root ganglion pseudounipolar neurons [3]. Different from peripheral nerves which are

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protected by multilayer of continuous connective tissue, including epineurium, nerve bundle membrane, and endoneurium, cauda equina is protected only by a layer of endoneurium [3]. Because of the lack of protective tissue, cauda equina is more vulnerable to mechanical compression injury. Timing surgical decompression, which, in a great extent, can reduce the further neurologic damage, is necessary for the treatment of CES [4]. Despite that, it is associated with limitations, which has no effect on the recovery of the function of damaged nerves. If the local pathologic factors cannot be improved in time after the compression of the cauda equina, it will turn into a vicious cycle, causing secondary injury, which includes not only the degeneration of cauda equina, but also the degeneration of dorsal root ganglion cells and neurons in the spinal cord, leading to permanent loss of neurologic functions [5]. Although the pathophysiological mechanism of cauda equina injury is not clear, it has been reported that oxidative damage and neuron cell apoptosis play an important role in the process of cauda equina injury [6]. Hydrogen sulfide (H2S), a transparent gas with the strong smell of rotten eggs, has historically been considered as a toxic and environmental hazard. However, H2S has recently received much interest as a novel gaseous messenger molecule after the finding of nitric oxide and carbon monoxide [7]. Hydrogen sulfide has been implicated in various physiological and pathologic processes, and plays a key role in signal transduction and biological activities, including vascular relaxation, angiogenesis, and apoptosis [7]. Moreover, H2S may function as a neuroprotectant against oxidative stress by enhancing the reducing activity in neurons [8,9]. In this study, we focused on investigating the protective effect of H2S on acute cauda equina injury in a rat model, which may have clinical implications in the drug treatment of CES. Materials and methods Animals Male Sprague-Dawley rats (weighing 200–250 g, aged 8–9 weeks) were purchased from the Experimental Animal Center of Second Military Medical University with License Number of SYXK (Shanghai) 2007–0003. The animal procedures used in this study were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. Rats were housed in the animal facility for free food and drink at 12-hour dark and light cycle. Animals were acclimatized for 1 week before experiment and were fasted 12 hours before surgery. Experiment design A randomized controlled animal experiment design was used. Cauda equina compression (CEC) model was accomplished as previously described [10]. Briefly, animals were anesthetized with 10% chloral hydrate (0.3 mL/100 g i.p.). After the injection of anesthesia, the animals were depilated on the dorsal spine and placed in prone position. A skin

incision was made over the spinal midline, and a laminotomy was performed with L4 lamina. Furthermore, the ligamentum flavum was removed between L5 and L6. A piece of silicone block (10×1×1 mm) was placed into the epidural space between the L5 and L6 vertebrae. Sham-operated rats underwent the same procedure except that the silicone was not placed. The rats’ bladders were pressed daily with artificial urination three times. To promote defecation and prevent urinary retention and obstruction, the abdominal cavities of the rats were massaged. The CEC model was validated by tail-flick assay in our preliminary study to test the pain and temperature at days 1, 3, and 7 after CEC surgery. Rats were randomly assigned into three groups, each comprising 54 rats; group 1 (H2S) received 20 µmol/kg sodium hydrosulfide (NaHS) (Sigma, USA) i.p., donor of H2S, 1 h before injury, and the other two groups (sham and compression) received equal volumes of physiological saline i.p. Hematoxylin and eosin staining Three groups each comprising two animals were anesthetized and used for histologic study 48 h after CEC surgery. The compressed part (L4–L6) of the cauda equina was removed, post-fixed overnight in 4% paraformaldehyde. The tissue was then paraffin-embedded, sectioned into 4 µm, and stained with hematoxylin and eosin (H&E) for histologic analysis. Measurement of malonaldehyde and glutathione The levels of malonaldehyde (MDA) and glutathione (GSH) were detected at time points of immediately before CEC surgery, 12 hours, 24 hours, 48 hours, and 72 hours after CEC surgery. The cauda equina tissue of L4–L6 from 10 animals in each group was homogenized in lysis buffer and centrifuged at 1,600 g for 10 minutes. The supernatant was then used for MDA and GSH measurements. The measurement was performed by lipid peroxidation MDA assay kit and GSH and glutathione oxidized assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Malonaldehyde can react with thiobarbituric acid, producing a red color substance. Glutathione is an important antioxidant, which plays a key role in anti-oxidative processes. In brief, the measurement was based on the absorbance of specific substrates. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling assay Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TUNEL) assay was performed to evaluate the cell apoptosis. The cauda equina tissue of L4–L6 from 2 rats in each group 48 h after CEC surgery was used for TUNEL assay. The assay was performed using TUNEL detection kit (Trevigen, Gaithersburg, USA) according to the manufacturer’s instructions. Briefly, the paraffin sections were deparaffinized, rehydrated, and treated with 3% H2O2 for

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Fig. 1. Protective effect of hydrogen sulfide (H2S) on cauda equina in rats. Magnification ×400 (hematoxylin and eosin staining, ×400). (Left) Sham group: Cauda equina nerve tissue was dense and regular, with complete myelin sheath, no axon swelling. (Middle) Compression group: Cauda equina nerve tissue was sparse, with the presence of demyelination, and partial axons and myelin sheath swelling. (Right) H2S group: Cauda equina nerve tissue was tight, with little axon swelling and demyelination. Scale bar=50 µm.

group. The H2S group showed compact cauda equina fibers, little axon swelling, and demyelination (Fig. 1). The result shows that H2S could, at least partly, protect myelin in the spinal cord and the cauda equina fibers in CEC rats.

20 minutes at room temperature. After that, sections were used for TUNEL staining. Statistical analysis Statistical analysis was performed using Statistical Product and Service Solutions 19.0 software (IBM SPSS, Chicago, IL, USA). All data were expressed as mean±SD of n determinations. Differences between groups were calculated using one-way analysis of variance. A statistically significant difference was determined when the p-value was less than .05.

H2S decreased the level of MDA and increased the level of GSH To examine the effect of H2S on oxidative stress, the expression levels of MDA and GSH were determined in the cauda equina of the sham, compression, and H2S group. As shown in Table 1, increased expression of MDA at 12, 24, 48, and 72 time points post-operation was seen in the cauda equina of the compression group in comparison with that of the sham group. The H2S group showed significantly decreased level of MDA compared with the compression group at all time points (p<.05). The level of GSH was significantly lower in the compression group than in the sham group; however, treatment with H2S could induce significantly the expression of GSH at 12, 24, 48, and 72 time points post-operation compared with the compression group (p<.05) (Table 2).

Results Silicone-block induced CEC After CEC surgery, artificial massage on rat bladder was done three times a day to help empty the urine until the recovery of bladder voiding reflex. No rat died during that time. H2S decreased the CEC damage The integrity of myelin in the spinal cord was detected by H&E staining. The sham group showed compact cauda equina fibers, integrated myelin, and axons. Myelin sheath and the cauda equina fibers in the compression group were less compact and highly degenerated compared with the sham

H2S reduced cell apoptosis The effect of H 2 S on cell apoptosis was determined by TUNEL assay. Compared with the sham group, the

Table 1 Effect of H2S on MDA levels in cauda equina Time point of MDA (nmol/mg) Treatment group

Dose (µmol/kg)

0h

12 h

24 h

48 h

72 h

Sham group Compression group H2S group

— — 20

1.543±0.803 1.653±0.734 1.429±0.678

1.650±0.624 3.874±1.128* 2.515±0.869†

1.676±0.842 4.916±1.220* 2.980±1.139†

1.494±0.618 5.960±1.202* 3.222±0.972†

1.747±0.721 6.649±1.244* 3.798±1.185†

H2S, hydrogen sulfide; MDA, malonaldehyde. Compared with sham group: *p<.01. Compared with compression group: †p<.01.

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4 Table 2 Effect of H2S on GSH levels in cauda equina

Time point of GSH (nmol/mg) Treatment group

Dose (µmol/kg)

0h

12 h

24 h

48 h

72 h

Sham group Compression group H2S group

— — 20

30.115±3.776 31.652±4.621 31.826±3.341

33.269±5.957 23.340±3.741* 28.758±3.315‡

33.032±5.375 19.334±4.122* 25.499±4.644‡

30.934±4.194 16.232±3.643* 21.810±4.788‡

32.747±4.477 12.767±4.855* 18.368±4.052†

H2S, hydrogen sulfide; GSH, glutathione. Compared with sham group: *p<.01. Compared with compression group: †p<.05, ‡p<.01.

compression group showed more TUNEL positive cells, which were dark brown stained in the nuclei under microscopy. However, a decreased number of TUNEL positive cells were found in the H2S group than in the compression group (Fig. 2). The apoptotic index (AI) was calculated from the ratio of TUNEL positive cells divided by the total cells in five randomly selected fields under microscopy. The compression group showed significantly higher AI than the sham group (p<.01), while the H2S treated group resulted in significantly lower AI compared with the compression group (p<.01). These results show that H2S treatment could reduce cell apoptosis in the CEC model. Discussion Cauda equina syndrome may result in bladder and bowel dysfunction, reduced sensation in the saddle area, or sexual dysfunction, causing catastrophic consequences for the patients and their family [1]. Recent evidence has shown that neuronal apoptosis caused by oxidative stress plays an important role in the onset and pathogenesis of several prominent central nervous system diseases [11,12]. Oxidative stress leads to secondary neurologic damage and dysfunction, including the normal neuronal tissues that were not damaged in the primary injury [13]. Oxidative stress refers to the imbalance between the formation of oxidative substances and the innate antioxidants. Oxidative substances are often reactive oxygen species (ROS), myeloperoxidase, nitric oxide,

and other ROS [14]. If the cells cannot remove the oxidative substances successfully, they may be damaged [15]. Therefore, the capability of cells to effectively remove oxidative substances and reduce oxidative stress has received more interest. A previous study demonstrated that simvastatin exhibited a neuroprotective effect in a cauda equina model through its anti-inflammatory and anti-nociceptive properties [6]. In addition, H2S has been recently recognized for its antioxidant properties. Endogenous H2S, formed from l-cysteine mainly by two enzymes, cystathionine β-synthase and cystathionine γ-lyase, as a novel gasotransmitter and modulator, is associated with physiological regulation and the pathogenesis of several disorders. The production of H2S, which is a small molecule that can travel through cell membranes without specific transporters, is regulated through endogenous synthetic enzymes and the metabolic pathways in vivo [16]. Hydrogen sulfide can, alone or in synergy with other molecules, regulate physiological or pathologic functions. Evidence showed that physiological concentration of H2S could facilitate the induction of hippocampal longterm potentiation [17]. Hydrogen sulfide could also act as an endogenous vasorelaxant factor that hyperpolarizes membrane potential of vascular smooth muscle cells in a KATP channel-dependent manner [18]. The H2S donor NaHS has been shown to exhibit antioxidant property in several pathologic conditions such as myocardial and smooth muscle ischemic or reperfusion injury [7]. Kesherwani et al. have showed that H2S had therapeutic effect on acute compression

Fig. 2. Effect of H2S on cauda equina cell apoptosis in rats. Magnification ×200 (TUNEL staining, ×200). (Left) Sham group: The number of positive cells was less in the spinal cord. (Middle) Compression group: Abundant positive cells were detected in the spinal cord. (Right) H2S group: The number of positive cells was significantly lower in the H2S group than that in the compression group. Scale bar=100 µm.

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injury of the spinal cord [19]. Furthermore, Campolo et al. have found that H2S accelerated the recovery from experimental spinal cord injury [20]. Also, Yin et al. demonstrated that H 2 S exerts anti-necrotic, anti-inflammatory, and neuroprotective function after cerebral ischemia-reperfusion in rats [21]. In the pathogenesis of CES, the body will produce many kinds of ROS, resulting in excessive oxidation state of the body. Cauda equina compression does not always cause CES, but once damaged, function of the saddle area, bowel, and bladder is difficult to recover, indicating that irreversible neuronal cell damage occurs in a short time. Cell apoptosis occurs as with the progression of pathologic changes after CEC, which is closely related with inflammation responses [22,23]. Oxidative stress is one of the important mechanisms for inflammation induced damage [24–29]. Malonaldehyde is the final product in lipid peroxidations, which is a process with free radicals in which unsaturated lipids convert to lipid peroxides. Malonaldehyde, which could cross-link with proteins and nucleic acids, leading to cytotoxicity, shows lipid peroxidation level and is a marker of increased oxidative stress, indirectly reflecting the extent of oxidative damage. Glutathione, a non-protein biothiol in cells, plays a variety of important roles as an antioxidant, cellular protectant, regulatory signaling molecule, and in the maintenance of intracellular redox state. The concentration of GSH is an important indicator to measure the antioxidant capacity of cells [30,31]. In this study, we evaluated whether H2S had a neuroprotective role on CES. Our results showed that H2S could improve the pathologic changes in the CEC model. Hydrogen sulfide treatment significantly decreased the level of MDA and increased that of GSH, suggesting that H2S could reduce the damage induced by oxidative stress. The generation of ROS, and subsequent oxidative stress, could lead to apoptosis of neuronal cells, further resulting in dysfunction of the nervous system [32]. The number of apoptosis cells was significantly lower after H2S treatment in cauda equina detected by TUNEL assay, indicating the role of H2S in reducing cell apoptosis. Methylprednisolone (MP), a corticosteroid, is a widely used clinical drug for the treatment of acute nervous damage by its anti-inflammatory effect or by inhibiting hydrolysis and peroxidation reactions [33,34]. Its therapeutic effect has been widely recognized, but it is very expensive and may bring serious side effects. High dose of MP may cause cardiac arrhythmia or cardiac arrest. Even a regular dose of MP can result in mental disorders, gastrointestinal ulcers, infections, high blood sugar, high blood pressure, intraocular pressure, electrolyte disorders, endocrine disorders, femoral necrosis, and other complications. Although NaHS has not been clinically used, the results on animal experiments show it has therapeutic advantages. Furthermore, another major advantage of NaHS application is its easy manipulation because of the gaseous property. Therefore, at the site of accident and during ambulatory transportation, patients with cauda equina injury can be treated conveniently with NaHS at the early stage

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of CES. Even though the effect of NaHS is similar to MP, it will reduce the side effects and the cost for the patients with acute neurologic injury. One of the limitations in this study is the small sample size and lack of statistical analysis with regard to H&E and TUNEL staining. In the future study, these design defects should be modified with the use of a larger sample size. In this study, we observed that H2S could ameliorate the pathologic changes of CEC injury in rats, reduce the oxidative stress, and decrease neuronal cell apoptosis, showing a role in neuroprotection. Our animal experiment shows that H2S has a therapeutic effect on the CES, but its pathophysiological, cytologic, immunologic mechanisms, and its pharmacologic effects need to be studied. Acknowledgments This research is supported by the National Natural Science Foundation of China (grant #81400997). References [1] Gardner A, Gardner E, Morley T. Cauda equina syndrome: a review of the current clinical and medico-legal position. Eur Spine J 2011;20:690–7. [2] Todd NV. Cauda equina syndrome: findings on perineal examination. Br J Neurosurg 2013;27:852. [3] Wall EJ, Cohen MS, Massie JB, Rydevik B, Garfin SR. Cauda equina anatomy. I: Intrathecal nerve root organization. Spine 1990;15:1244–7. [4] Qureshi A, Sell P. Cauda equina syndrome treated by surgical decompression: the influence of timing on surgical outcome. Eur Spine J 2007;16:2143–51. [5] Hung-Kai WR, Chang MC, Feng SW, Wang ST, Liu CL, Chen TH. Progressive growth of arachnoid cysts with cauda equina syndrome after lumbar spine surgery. J Chin Med Assoc 2013;76:527–31. [6] Shunmugavel A, Martin MM, Khan M, Copay AG, Subach BR, Schuler TC. Simvastatin ameliorates cauda equina compression injury in a rat model of lumbar spinal stenosis. J Neuroimmune Pharmacol 2013;8:274–86. [7] Guo W, Kan JT, Cheng ZY, Chen JF, Shen YQ, Xu J, et al. Hydrogen sulfide as an endogenous modulator in mitochondria and mitochondria dysfunction. Oxid Med Cell Longev 2012;2012:878052. [8] Kimura Y, Kimura H. Hydrogen sulfide protects neurons from oxidative stress. FASEB J 2004;18:1165–7. [9] Umemura K, Kimura H. Hydrogen sulfide enhances reducing activity in neurons; neurotrophic role of H2S in the brain? Antioxid Redox Signal 2007;9:2035–41. [10] Watanabe K, Konno S, Sekiguchi M, Kikuchi S. Spinal stenosis: assessment of motor function, VEGF expression and angiogenesis in an experimental model in the rat. Eur Spine J 2007;16:1913–18. [11] Head E, Rofina J, Zicker S. Oxidative stress, aging, and central nervous system disease in the canine model of human brain aging. Vet Clin North Am Small Anim Pract 2008;38:167–78. [12] Lehtinen MK, Bonni A. Modeling oxidative stress in the central nervous system. Curr Mol Med 2006;6:871–81. [13] Jia Z, Zhu H, Li J, Wang X, Misra H, Li Y. Oxidative stress in spinal cord injury and antioxidant-based intervention. Spinal Cord 2012;50:264–74. [14] Yousuf S, Atif F, Kesherwani V, Aqrawal SK. Neuroprotective effects of tacrolimus (FK-506) and cyclosporin (CsA) in oxidative injury. Brain Behav 2011;1:87–94.

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