Pain Markers and Epidural Fibrosis Caused by Repeated Spinal Surgery in Sprague–Dawley Rats

Epidural brosis is one of the aetiologies of pain following spinal revision surgery. However, roles of epidural brosis caused by repeated spinal surgery and pain-related proteins in causing the post spinal surgery syndrome remain unknown. In this study, using a rat spinal surgery epidural brosis and adhesion model, we evaluate and investigated the relationship between pain marker and epidural brosis caused by repeated spinal surgery in Sprague-Dawley rats.


Background
Each year, 1 million people worldwide undergo lumbar disc surgeries for disc herniation and spinal stenosis, making it one of the most common treatments for spinal diseases [1][2][3][4]. However, occasionally, despite appropriate decompression, the outcomes of such spinal surgeries are not necessarily correlated with the clinical outcomes. This may be because of the development of epidural brosis and adhesions, which is a normal reaction during healing following spinal surgery [5]. However, epidural brosis causes pulling, stretching or compression of the associated nerve root or dura mater and can lead to persistent back and leg pain; this is known as the post laminectomy syndrome, failed back syndrome or post spinal surgery syndrome [6,7]. Epidural brosis and adhesion are inevitable following spinal surgery, and despite advancements in surgical techniques, some patients continue to suffer from recurrent postoperative pain [1]. It is di cult to expect good outcomes even when repeated surgery is performed to eliminate epidural brosis and adhesions. In addition, such repeated surgeries may lead to dural tears, nerve root injuries and excessive bleeding [8].
Neuronal injury and post-injury regeneration progress as neural peptides and signal transduction molecules are expressed in the dorsal root ganglion (DRG) and spinal dorsal horn. In this regard, mitogenactivated protein kinases (MAPKs) are attracting much attention [9,10]. MAPKs are serine/threonine protein kinases that conventionally comprise extracellular signal-regulated kinases 1/2 (ERK1/2), p38 and c-Jun N-terminal (JNK) [11]. MAPKs are activated by diverse extracellular stimuli, such as hormones and growth factors, and they transduce extracellular stimuli to intracellular transcriptional and posttranscriptional responses [9,12]. Increasing evidence has shown that MAPKs play important roles in the induction and maintenance of chronic pain [13][14][15]. However, the roles of epidural brosis caused by repeated spinal surgery and pain-related proteins expressed in the spinal dorsal horn in causing the post spinal pain syndrome remain unknown.
In the present study, we evaluated the extent of epidural brosis by measuring dural thickness following repetitive surgery using a previously established rat model of laminectomy and investigated the association of MAPK expression in the spinal dorsal horn with post spinal surgery syndrome or chronic pain syndrome development.

Animals
In this study, a total of 45 male Sprague-Dawley rats (age, 8 weeks) were obtained from Orient Bio Inc company (Korea). All animal experiments were approved by the Institutional Review Board of St. Mary`s Hospital of Catholic University (CMCDJ-AP-2012-017). The animals were randomly divided into three groups; group A (n = 15) underwent a single spinal laminectomy; group B (n = 15) underwent two laminectomies repeated at an interval of 3 weeks; and group C (n = 15) underwent three laminectomies repeated at intervals of 3 and 6 weeks ( Table 1). One more trial after 3 + 3 weeks 9 weeks Surgery All animals were intraperitoneally anaesthetised with 40 mg/kg ketamine hydrochloride (Ketamine 50®; YUHAN, Korea) and 5 mg/kg Rompun injection (Rompun®; BAYER, Korea). After shaving the lower back, the surgical site was sterilised with povidone. The sterilised site was then covered with sterile surgical sutures, and the lumbosacral fascia and paraspinous muscles were dissected, followed by L4-6 laminectomy ( Fig. 1). During surgery, the animal's body temperature was maintained using a warm pad. At the end of the experimental period, the animals were euthanized using CO 2 gas. Using knife, the entire laminectomy site was excised according to the spinal cord levels; step by step under a dissecting microscope (Nikon, SMZ800N, Japan); the site was dissected using a micro-rongeur and micro-curette for the removal of muscle and other tissues. After the removal of the surgical site, half of the area was xed in 10% formalin solution and the remaining half was stored at − 80 °C in a cryotube for protein extraction.

Measurement of dural thickness
The xed specimen was axially cut through from the upper L4 to lower L6 levels to isolate the laminectomy site. After decalci cation and dehydration, para n blocks were prepared, and 4-µm-thick section of the laminectomy site were cut and stained with hematoxylin. Slides were evaluated in a blinded manner by a histologist who analysed dural thickness. Dural thickness was measured at 3 points; the rst sample was harvested from the midpoint of the laminectomy defect, the second sample was obtained 2 mm from the right side of the rst sample, and the third sample was obtained 2 mm from the left side of the rst sample [16]. Mean dural thickness was considered for statistical evaluation.

Immunohistochemical analysis
Formalin-xed tissues were sectioned to prepare the slides. The prepared slides were dehydrated in xylene and graded ethanol, immersed in citrate buffer and boiled for 10 min using an electronic rage. Immunostaining was performed following the ABC kit manual. After blocking, the slides were incubated with the following primary antibodies at 4 °C overnight: phosphorylated ERK1/2 (1:400; #4370, Cell Signaling Technology, Inc. USA), phosphorylated p38 (1:1600; #4511, Cell Signaling Technology, Inc. USA) and phosphorylated JNK (1:100; #9251, Cell Signaling Technology, Inc. USA). Subsequently, the slides were incubated with a secondary antibody at room temperature for 1 h. All slides were developed using the ImmPACT™ NonaRED™ peroxidase substrate (Sk-4805; Vector Laboratories Inc., USA), mounted and observed under a microscope. Three elds of sections from each tissue sample were imaged at 200 × to quantify the percent p-ERK-positive, p-p38-positive and p-JNK-positive cells.

Statistical analysis
All data are presented as mean and standard deviation. Correlations were analysed using Student's twotailed and paired t-tests. Statistical Package for the Social Sciences v.12.0 software (Chicago, IL, USA) was used for data analysis. P < 0.05 was considered signi cant.

Epidural brosis following repeated laminectomy
Epidural brosis was indirectly analysed by measuring the dural thickness. Thickness was 6.363 ± 1.911 µm in group A, 13.238 ± 2.123 µm in group B and 19.4 ± 2.115 µm in group C (Fig. 2). The thickness in group A was signi cantly lower than that in groups B and C (p < 0.05), and the thickness in group B was signi cantly lower than that in group C (p < 0.05). Therefore, repetitive laminectomy seemingly increases the dural thickness. In other words, repetitive spinal surgery alone increases epidural brosis.
ERK1/2 protein expression in the spinal cord following repeated laminectomy In western blotting, total-ERK expression was similar across groups, but compared with group A, group B showed a 1.04-fold increase and group C showed a 0.89-fold increase in expression.
Phosphorylated ERK expression was in the lowest in group A; group B showed 1.77-fold increased expression compared with group A, and group C showed the highest expression, with a 2.42-fold increased expression compared with group A (Fig. 3A and B). In addition, phosphorylated ERK expression was signi cantly different between groups A and C and between groups B and group C (p < 0.05).
Immunohistochemical staining revealed that phosphorylated ERK1/2 was expressed in the spinal dorsal horn in group A with primary surgery, in group B with secondary surgery and in group C with tertiary surgery. Moreover, phosphorylated ERK1/2 expression gradually increased with the number of surgeries, and expression in group B and C was signi cantly different from that in group A (p < 0.05) (Fig. 6A and  B).
These results indicate that phosphorylated ERK1/2, which is considered to be involved in causing neuropathic pain, is overexpressed following repeated laminectomy.
p38 protein expression in the spinal cord following repeated laminectomy In western blotting, total-p38 expression showed a decreasing trend with the number of laminectomies. Group A showed the highest expression, while groups B and C showed 0.81-fold and 0.57-fold decreases compared with group A; expression in groups A and C was signi cantly different (p < 0.05).
Phosphorylated p38 showed an increasing trend with the number of laminectomies. Group A showed the lowest expression, and groups B and C showed a 1.17-fold and 1.33-fold increased expression compared with group A; expression in groups A and C was signi cantly different (p < 0.05).
This decrease in total-p38 expression and increase in phosphorylated p38 expression indicate that epidural brosis occurs due to repetitive laminectomy and that phosphorylated p38 is involved in this process ( Fig. 4A and B).
Immunohistochemical staining revealed that phosphorylated p38 was expressed in group A with primary surgery, in group B with secondary surgery and in group C with tertiary surgery. Moreover, phosphorylated p38 expression gradually increased with the number of surgeries, as con rmed by the results of western blotting (Fig. 6A). Moreover, compared with group A, groups B and C were showed signi cantly increased expression (p < 0.05) (Fig. 6B) Therefore, phosphorylated p38, which is considered to be involved in neuropathic pain, is overexpressed following repeated laminectomy.
JNK protein expression in the spinal cord following repeated laminectomy Western blotting results revealed that total-JNK expression was the lowest in group A, while groups B and C showed 1.64-fold and 1.24-fold increases in expression compared with group A, and the expression in groups A and group B was signi cantly different (p < 0.05). Expression increased in group B but decreased in group C.
Phosphorylated JNK expression was the lowest in in group A, and compared with group A, groups B and C showed 1.62-fold and 1.43-fold increased expression; expression in groups A and B was signi cantly different (p < 0.05) (Fig. 5A and B).
Immunohistochemical staining revealed that phosphorylated JNK expression was the lowest in group A. Compared with group A, groups B and C showed increased expression (p < 0.05). However, phosphorylated JNK expression in the spinal dorsal horn was higher in group B than in group C, as con rmed by the results of western blotting (Fig. 6A and B).

Discussion
Epidural brosis-a common complication of lumbar disc surgery-causes repeated radicular pain or back pain due to compression of the exposed dura and nerve roots [17]. Recently, some surgeon tries to approach with indirect decompression in revision surgery to avoid these scar formations and incidental durotomy complications [18].
Post-laminectomy epidural brosis is well known, and Turkoglu et al. [7] have identi ed the mechanism of action of etanercept after inducing spinal epidural brosis in a rat model post laminectomy. Similarly, Alkalay et al. [19] have demonstrated that a post-laminectomy epidural brosis model could be used to prevent epidural brosis of bioplastic materials. In addition, Kurt et al. [20] have used a post-laminectomy epidural brosis model to compare the effects of waxed paper and Gore-Tex on the prevention of postlaminectomy epidural brosis. Therefore, in the present study, we constructed a rat model of postlaminectomy epidural brosis. But repeated multiple spinal surgery model is not existing in animal study.
As this reason, we evaluated multiple spinal surgery rat model to evaluation pain marker expression and relationship between dural thickness and surgery time.
In animal models of neuropathic pain caused by peripheral nerve injury, neuropathic pain was not completely manifested at an early stage (i.e. 0-3 days post-lesion), but it was well developed at a later stage (i.e. 7-21 days post-lesion) [19]. In addition, in a partial sciatic nerve ligation model, mechanical allodynia was well established in the affected hind paw at 3 weeks post-lesion [9,21]. Therefore, in this study, the reoperation interval was set as 3 weeks. Furthermore, in our experiments, laminectomy was repeated once, twice or thrice, and it was con rmed that epidural brosis progressed as the number of repeated surgeries increased. These ndings suggest that repeated spinal surgeries increase dural thickness and which in turn causes neuropathic pain. Therefore, repetitive spinal surgery may increase epidural brosis.
Increasing studies on MAPKs have uncovered their roles in the generation of chronic central neuropathic pain due to spinal cord injury [22][23][24]. In addition, MAPKs such as ERK and p38 have been reported to contribute to dorsal horn hyperexcitability in a peripheral neuropathic pain model [25][26][27][28]. Thus, in the present study, we evaluated MAPK expression in the spinal cord following repetitive surgery.
The ERK/MAPK pathway plays an important role in cell proliferation and differentiation. Additionally, the activation of ERK/MAPK signalling contributes to the pain response of the dorsal horn and dorsal root ganglia following in ammation and/or nerve injury [10]. These data suggest that the MAPK family is actively involved in the pain-related processes [9]. Zhuang et al. [28] have suggested that ERK acts on neurons, microglia and astrocytes via spinal nerve ligation as well as contributes to mechanical allodynia in a neuropathic pain model. Likewise, in our study, ERK was expressed following laminectomy, and the protein expression of phosphorylated ERK gradually increased with the number of repetitions of surgery, suggesting that ERK contributes to pain development due to epidural brosis.
Phosphorylated p38/MAPK induction by nerve injury mainly occurs in the spinal dorsal horn and dorsal root ganglia, which has been extensively studied in terms of the initiation and maintenance of neuropathic pain [9]. In addition, the phosphorylated forms of ERK 1/2 and p38 are reportedly upregulated in similar regions of the spinal cord in injured rats, which induced mechanical allodynia [22]. These ndings suggest that activated ERK1/2 and p38 regulate changes in nociceptive reactivity in peripheral nerve injury models [29][30][31]. In our experiments, phosphorylated ERK and p38 were expressed in the spinal dorsal horn. Furthermore, in the present study, the expression of phosphorylated ERK and p38 was upregulated as the number of repeated surgeries increased, suggesting that p38 and ERK contribute to pain development due to epidural brosis.
In neuropathic pain, the role of JNK is lesser known than those of ERK and p38, with only few studies having been conducted. Zhuang et al. [15] have reported that JNK acts on the sensory nerves and astrocytes to develop and maintain neuropathic pain. However, Crown et al. [22] have reported that increases in expression of activated forms of ERK1/2 and p38 but not JNK are correlated with the expression of at-level mechanical allodynia following spinal cord injury. In our study, JNK was expressed in the spinal dorsal horn following laminectomy; however, its expression level did not gradually increase with the number of repeated surgeries. Moreover, protein expression level of phosphorylated JNK did not increase with the number of repeated surgeries. Therefore, JNK may not contribute to pain development due to epidural brosis.
Repetitive spinal surgery was stimulated by the spinal dorsal horn, resulting in increased ERK1/2 and p38 expression. Thus, neuropathic pain is likely induced by epidural brosis, and ERK1/2 and p38 are the potential pain-related factors.

Conclusions
This study was the rst to analyse the association between pain markers and epidural brosis due to repeated spinal surgery in rats. Repeated spinal surgeries seemingly increase dural thickness, ultimately leading to epidural brosis. In addition, repeated spinal surgeries increased expressions of pain markers such as ERK and p38, indicating that pain increased with the repeated surgeries. However, the DRG was not focussed upon in this study because repeated surgery does not de ne the anatomical region of the DRG. Thus, DRG function should be evaluated and pain behaviour should be tested through further animal studies.