Eur Spine J DOI 10.1007/s00586-015-3926-x
ORIGINAL ARTICLE
Intervertebral disc degenerative changes after intradiscal injection of TNF-a in a porcine model Ran Kang1,2 • Haisheng Li1 • Kresten Rickers1 Steffen Ringgaard3 • Lin Xie2 • Cody Bu¨nger1
•
Received: 23 June 2014 / Revised: 31 March 2015 / Accepted: 1 April 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose To investigate whether exogenous tumor necrosis factor-a (TNF-a) will initiate a degenerative process in intervertebral disc in vivo. Methods Exogenous TNF-a in dosages of 50 and 100 ng in 50 lL Dulbecco’s Modified Essential Medium (DMEM) was injected into porcine lumbar discs; a third disc was injected only with 50 lL DMEM as a control. Magnetic resonance imaging (MRI) yielding T1- and T2-weighted images, T2-mapping, and post-contrast T1 images was performed and histology was studied as well. Results After 3 months, a significant decrease in T2 value calculated from T2-mapping MRI was observed in the annulus and nucleus of both groups injected with TNF-a along with a slight decrease in disc height and nucleus volumes in comparison to the control discs. No obvious visual differences among the groups were observed in the normal T1- and T2-weighted MRI images. Post-contrast T1 MRI showed increased annulus enhancement in both TNFa-injected groups compared to the control discs, while no enhancement difference was observed in the nucleus. Histological analysis showed degenerative changes with
& Ran Kang [email protected] & Lin Xie [email protected] 1
Orthopaedic Research Lab, Aarhus University, Building 1 A, Noerrebrogade 44, 8000 Aarhus C, Denmark
2
Department of Orthopedic Surgery, Jiangsu Province Hospital on Integration of Chinese and Western Medicine, 100th Shizi Street, Nanjing 210028, China
3
The MR Research Centre, Aarhus University Hospital, Skejby, 8000 Aarhus C, Denmark
annulus fissure, cell cluster, nucleus matrix loss, vascularization and interleukin-1b expression in the outer annulus of both TNF-a-injected discs, while no degenerative changes were observed in the control discs. Conclusions Intradiscal injection of exogenous TNF-a caused early stage disc degeneration in a porcine model. It may thus support the hypothesis of exogenic TNF-a being an important early pathogenetic factor in disc degeneration. Keywords Disc degeneration TNF-a MRI imaging and -mapping Post-contrast MRI Vascularization
Introduction The pathology of intervertebral disc degeneration (IDD) is currently not well understood. Tumor necrosis factor-a (TNF-a) is a cytokine that has been strongly linked to the pathogenesis of IDD: higher TNF-a expression is commonly found in patients’ degenerated discs [1]; TNF-a expression in discs induces such pathological changes in annulus tissue as angiogenesis [2] and sensory nerve ingrowth [3]. A recent study concluded that TNF-a has a role as a mediator in the disc degeneration process [4]. At the onset of disc degeneration, TNF-a is secreted by nucleus pulposus (NP) cells and annulus fibrosus (AF) cells, as well as by macrophages and T cells. It then triggers a range of pathogenic responses in the disc cells that promote autophagy, senescence, or apoptosis [4, 5]. These fundamental pathological changes subsequently result in disc herniation and neuropathic pain. Experimental in vitro studies of cell culture [6] and organ culture [5, 7, 8] have demonstrated that a culture medium containing TNF-a caused pathological changes in disc cell and -tissue. With the same mechanism, TNF-a in
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the serum or local tissue adjacent to the disc may also affects disc vitality in vivo, since it can transfer through the endplate [5, 7, 8]. This point could also to some extent be speculated on the basis of possible correlation between Modic changes and adjacent IDD. In discs with Modic changes, high expression of TNF-a has been found adjacent to the disc [9] and Modic changes have also been reported to correlate positively with IDD [10, 11]. Therefore, TNF-a in the area with Modic change might be one of the initiators of the adjacent IDD after its transfer through the endplate. TNF-a could raise to 532.6 pg/mL in serum in cerebral malaria patients higher than 46.42 pg/mL in health humans [12]. High level TNF-a in serum or local tissue causes many pathological changes in many organs [13, 14] and has implications for preventive measures. However, for intervertebral disc organ, whether exogenous TNF-a will initiate a degeneration process in a healthy disc in vivo has not been studied assertively. Thus we designed this study to investigate disc changes after intradiscal injection of TNFa with 50 or 100 ng per disc. Whether such high concentration of TNF-a could be reached by endplate transportation from serum or local vertebra is unknown, it is still important to study the disc changes if the TNF-a level is upgraded to a high level in an animal model. We hypothesized that exogenous TNF-a could initiate IDD.
Materials and methods Surgical procedure For this study we used seven Danish landrace female pigs (3 months old, weight approximately 35 kg). The experimental and animal care procedures were approved by the Danish Animal Welfare Committee. Under general anesthesia, the pigs were placed in supine position. A leftsided retroperitoneal approach was used to expose the lumbar spine. In three discs from L12 to L34 we injected 50 lL Dulbecco’s Modified Essential Medium (DMEM; 21063, Gibco, Invitrogen) containing either 50 or 100 ng TNF-a (Recombinant Swine TNF-a; GibcoÒ; 17.1 kDa) through a 26-gauge needle with a stop-marker (Fig. 1a) at a depth of 8 mm, which was confirmed in a pilot study to be the route depth of the NP center from the lateral anterial part. One disc injected with 50 lL DMEM without TNF-a served as a control. The injection was administered slowly, maintaining the pressure for 5 min. The needle was retracted to the annulus fibrosus and kept in this position for 2 min before removal [15]. Hemostasis was secured and the abdomen was closed in layers. The pigs were housed in separate boxes and fed with a standardized food recipe for 3 months.
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Fig. 1 a Using a 1 mL syringe with 26-gauge needle, the disc was injected with 50 lL dye; figure shows distribution of dye after 2 h; b overview of MRI images: no obvious difference among groups in T1-weighted (left), T2-weighted MRI (middle), but lower T2 value in nucleus exhibited by color-coded T2 mapping MRI (right)
Magnetic resonance imaging (MRI) procedure and evaluation Magnetic resonance imaging was performed under general anesthesia on a clinical Philips Achieva 1.5-T scanner (Philips Healthcare, Best, The Netherlands). Before TNF-a injection, a sagittal T1-weighted, T2-weighted, 3D T2weighted, and a sagittal T2-mapping scan (multi-echo spin echo; matrix, 248 9 248; field of view, 300 9 300 mm; repetition time, 1.0 s; 8 echoes with echo time, 15–120 ms (ms); number of excitations, 2) were performed. Three months after TNF-a injection, in addition to these above scans, post-contrast T1-weighted images were obtained at intervals of 0.5, 5, 10 min and then every 10 min for a total of 110 min after manual injection of 0.3 mmol/kg gadolinium (OMNISCANTM, Amersham Health AS, Oslo). All images were analyzed using custom programmed software written by one of the authors (SR). The change of disc height index (DHI) was expressed as percentage of DHI (% DHI) (% DHI = postoperative DHI/ preoperative DHI 9 100) [16, 17]. The change in NP volume was calculated as percentage of NP volume (% NP volume) in the middle axial position of the disc on T2weighted 3D images (% NP volume = postoperative NP volume/preoperative NP volume 9 100). T2 value within the AF and NP was calculated by fitting to an exponential T2 decaying equation [16]. An average of AF and NP T2 value was calculated according to the mean T2 value in the three middle slices using the following equation: T2
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value = (mean 1 9 area 1 ? mean 2 9 area 2 ? mean 3 9 area 3)/(area 1 ? area 2 ? area 3). In the post-contrast MRI images, the signal intensity (SI) within the anterior AF and the whole NP was calculated as percentage of contrast enhancement (% CE) (% CE = (SI post - SI pre)/SI pre 9 100); time-intensity curves were plotted according to % CE and each time point [18]. Histology The spine segments containing the experimental discs were removed at termination and fixed by 70 % ethanol for 7 days, dehydrated in a series of ethanol concentrations, and embedded in methyl methacrylate. Sections of 7 lm were stained with hematoxylin–eosin (HE) and toluidine blue for proteoglycan. The samples were graded for degeneration score (DS) using previously published histological 12-point scoring system [19] by judging: loss of demarcation between NP and AF, loss of proteoglycan from NP, presence and extent of fissures, cell cluster formation. Immunostaining of smooth muscle in vessels was also performed as previously described in the literature [20] with human alpha smooth-muscle actin n-terminal synthetic decapeptide primary antibody (R&D MAB1420), secondary biotinylated polyclonal goat anti-rabbit IgG (DAKO E0432), and tertiary antibody peroxidase-conjugated streptavidin (DAKO P0397). Immunostaining of interleukin-1b (IL-1b) used primary anti-IL-1b (ab193852), secondary biotinylated rabbit anti goat (DAKO 0466), and AEC (Sigma A9626) following the standard protocol. They were then examined using a photomicroscope (Olympus, Tokyo, Japan). Statistical analysis The data are expressed as mean ± standard deviation. Data of percentage DHI, NP volume, T2 value and DS were compared using one way-ANOVA analysis between the different interventions, and the Scheffe method was used to compare every two groups. Data of contrast enhancement were analyzed using two way-ANOVA (intervention 9 time point). Significance level was defined as P less than 0.05.
Results All seven pigs tolerated the surgical procedure well. After 3 months, in general, there was no obvious difference between the discs injected with or without TNF-a viewed by normal T1- and T2-weighted images, however, lower T2 value was observed in the TNF-a injected discs in colorcoded T2-mapping image (Fig. 1b). Disc height, while
slightly decreased in both TNF-a-injected discs, showed no significant difference compared to the controls. NP volume was also decreased, especially in the discs with TNF-a 100 ng (P \ 0.05) (Fig. 2a). The T2 value of AF and NP in both TNF-a-injected discs was significantly lower than the controls (Fig. 2b). In post-contrast MRI images (Fig. 3a), the control discs exhibited no obvious enhancement dye in the annulus area, while in the course of time, uniform enhancement bands parallel to the endplate moved slowly towards the center in the nucleus; in both TNF-a-injected groups more enhancement was seen in the annulus, especially in the anterior annulus area with a pooling of contrast dye, while no obvious difference in the nucleus was observed in comparison to the controls. The corresponding time-intensity curve (Fig. 3b) showed increased enhancement in annulus in the TNF-a-injected discs (intervention P \ 0.01, time point P \ 0.01). No enhancement difference in the nucleus was found among groups (Fig. 3c). Histological images are shown in Fig. 4a–o. In both TNF-a-injected discs, degenerative changes were obvious; these included fissures, cell proliferation, and -cluster in the annulus, changes which did not occur in the control discs. We continued by applying specific staining for the disc ECM component proteoglycan. Nucleus pulposus had relatively less extracellular matrix and became loose in both TNF-a-injected discs, while the control disc had relatively dense matrix. According to a pervious published scoring system, the degree of IDD in both TNF-a-injected discs were both significantly higher than the control (Fig. 4p). Immunostaining of smooth muscle of vessels in the control disc showed almost no vessels inside the annulus, with more vessels in the annulus of both TNF-ainjected discs (Fig. 5a–f). Immunostaining of IL-1b in the control disc had almost no positive staining, with apparent positive staining in the annulus of both TNF-a-injected discs (Fig. 5g–l).
Discussion TNF-a synthesized near/in the disc area is a key mediator in the pathogenesis of IDD [4]. In several in vitro studies, exogenous TNF-a was used as an initiator of IDD [5, 7, 8]. In this study, we went further and confirmed that exogenous TNF-a administered by intradiscal injection initiates early IDD changes in vivo at 3 months. Intradiscal injection is often used to generate IDD. Small needle size has been recommended to avoid the effect of needle injection [21]. For delivering material into the nucleus, a spinal needle of smaller than 22 gauge and injection volume of less than 200 lL is recommended to prevent post-surgery leakage in a porcine model [22].
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Fig. 2 Data of percentage of disc height index (DHI) and nucleus pulposus (NP) volume and data of T2 value in annulus and nucleus (n = 7). a Percentage of DHI and NP volume changes in groups; b T2
value of annulus and nucleus changes in groups. Asterisk indicates significant difference between the intervened discs and the controls, P \ 0.05
Fig. 3 The serial T1-weighted post-contrast MRI images and timeintensity curves of annulus and nucleus (n = 7). a In the control, no obvious enhancement observed in the area of annulus, meanwhile in the nucleus, uniform enhancement bands parallel to the endplate slowly moved towards the center as time passed; in both TNF-ainjected groups, more enhancement was seen in the annulus,
especially in the anterior annulus area (arrow), with a pooling of contrast dye, while no obvious difference in the nucleus observed in comparison to the controls; b much more rapid enhancement in annulus of the TNF-a-injected discs in time-intensity curve (different intervention P \ 0.01, different time point P \ 0.01); c no enhancement difference in the nucleus among groups
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Fig. 4 Images of histology and degeneration score in different groups. Control disc: a overview of annulus; b regular structure in outer annulus; c regular inter annulus; d overview of nucleus matrix; e nucleus with relatively thick matrix. TNF-a 50 ng injected disc: f overview of annulus; g fissure in the outer annulus; h cell cluster at the inter annulus (arrows); i overview of nucleus matrix; j nucleus
with relatively less of matrix. TNF-a 100 ng injected disc: k overview of annulus; l fissure in the outer annulus; m cell cluster at the inter annulus (arrows); n overview of nucleus matrix; o nucleus with relatively less matrix. p degeneration score of each group (n = 7). Asterisk indicates significant difference between the intervened discs and the controls, P \ 0.05
Fig. 5 Immunostaining of smooth muscle in vessels in different groups: a, b control disc, almost no vessels inside of the annulus; c, d TNF-a 50 ng injected disc, vessels (arrows) in the annulus; e, f TNF-a 100 ng injected disc, vessels (arrows) in the annulus.
Immunostaining of IL-1b in different groups: g, h control disc, almost no positive staining; i, j TNF-a 50 ng injected disc, positive staining (hollow arrows) in annulus area; k, l TNF-a 100 ng injected disc, positive staining (hollow arrows) in annulus area
In our study, we used a 26-gauge needle with 50 lL injection volume to minimize the effects caused by the delivery procedure. As our results show, we did not see any degenerative changes in the control discs, where only 50 lL DMEM was injected. Consisting with previous studies of inducing IDD model by enzymes, no IDD was
found in the control disc only injected small volume vehicle [17, 23, 24]. In our study, pro-inflammatory cytokine TNF-a were injected, instead of enzymes. In a previous in vitro study of bovine disc organ culture system, a 1-week exposure to 200 ng/mL TNF-a initiated the IDD process with non-
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recoverable catabolic shift [7]. Likewise, in an in vitro study of rat disc organ culture system, a cocktail of 100 ng/ mL TNF-a and 10 ng/mL IL-1b contained in a culture medium also led to IDD [5, 8]. Therefore, we established a dosage of 50 and 100 ng TNF-a, with corresponding concentration of 100 and 200 ng/mL in the nucleus (in our study, the nucleus volume of porcine lumbar disc was calculated at around 0.5 mL). TNF-a, a major pro-inflammatory cytokine, could accelerate the degradation of intervertebral disc extracellular matrix, causing an imbalance between catabolism and anabolism, leading to denaturalization of the disc tissue [4]. Exposure to TNF-a also stimulated interleukin, supporting and strengthening its role as an initiator and regulator of multiple proinflammatory cytokines in intact discs [7]. Consistent with these previous in vitro studies, the degenerative changes in our study showed that TNF-a could initiate IDD in vivo, confirmed by MRI and histological examinations. IDD is commonly characterized by MRI with reduced signal in T2-weighted images in NP, annulus collapse, and reduced disc height [25]. Such MRI techniques as quantitative MRI T2-mapping to analyze changes in disc matrix composition can also be used to reflect pathological changes of IDD in in vivo studies [24]. As the result showed, disc height and nucleus volume were decreased with both dosages of TNF-a intervention, and the T2 value calculated in quantitative MRI T2-mapping in the annulus and nucleus were both significantly decreased compared to the control discs. The histological changes of annulus fissure, cell cluster, and nucleus matrix loss confirmed the MRI-verified degenerative changes. TNF-a was reported to either induce or be affiliated with such pathological changes as angiogenesis [2] and sensory nerve ingrowth [3] in discs. In our study, we also found angiogenesis in the annulus of IDD; this was confirmed both by rapid enhancement in the annulus, detected by the post-contrast MRI scanning, and the histological study. The vascularization of IDD due to AF structural failure was previously described in the literature [18, 26, 27]. From these results, we could conclude that TNF-a caused IDD. On the other hand, no obvious visual changes were seen in the normal T1- and T2-weighted MRI. This may be explained by the fact that the TNF-a intervention in our study caused slight or earlystage disc degeneration which was attributable either to the small dosage or the short observation time. Further studies, with different observation durations and a larger dosage, are warranted. Furthermore, as disc degeneration is multifactorial, a single TNF-a application might not necessarily be capable of causing severe changes. Extending this experimental result to possible clinical relevance, this raises awareness that TNF-a in serum or local vertebra might affect disc vitality, if the TNF-a reaches the disc at a high enough concentration. Alcohol
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drinking [28], smoking [29], and a variety of diseases [13] may cause a rise in serum TNF-a level, which also reportedly has a correlation with myocardial infarction [30] and renal damage [13]. In the intervertebral disc area, serum TNF-a can penetrate through the endplate pathway; this has been tested by means of different molecular weight Firefly (61 kDa) and Renilla (38 kDa) luciferase in the disc organ culture system [8]. Therefore, there might be a correlation between IDD and the pro-inflammatory factor TNF-a in system serum or adjacent tissue. Accordingly, when there are high TNF-a levels in the serum, or in a local area near the disc such as in a vertebra exhibiting Modic change, such preventive measures as anti-inflammatory treatment [6] might be considered as a means of protecting the disc. Furthermore, this may also imply that disc regeneration treatments for IDD may require supplementary anti-inflammatory treatments. There are some limitations in the present study: We could not apply TNF-a in the serum and maintain a specific concentration for an extended period because of potential systematical damage to other organs. We injected TNF-a directly into the disc to simulate the situation in which the serum TNF-a has already transferred into the disc through the endplate. Because we were unable to monitor the concentration of TNF-a in the disc, we assumed that TNFa could remain in the disc and that in fact some of it might have diffused out of the disc. Moreover, whether TNF-a in serum or local vertebra could reach the disc at a high enough concentration as we applied to induce IDD need further investigations.
Conclusions Intradiscal injection of exogenous TNF-a caused early IDD changes in a porcine model. Exogenous TNF-a could be an initiator of disc degeneration. Acknowledgments We thank Anette Baatrup for histology technique support, Yufen Zhang for surgical assistance, and Chen Gan for MRI analysis. We gratefully acknowledge the funding from Velux (25906), Lundbeck, Gigtforeningen Foundation, Aarhus Spine Research Foundation, and the International Cooperation and Natural Science Foundation of Jiangsu province of China (BZ2011046, BL2012069, BK2012490). Conflict of interest
None.
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ORIGINAL ARTICLE
Intervertebral disc degenerative changes after intradiscal injection of TNF-a in a porcine model Ran Kang1,2 • Haisheng Li1 • Kresten Rickers1 Steffen Ringgaard3 • Lin Xie2 • Cody Bu¨nger1
•
Received: 23 June 2014 / Revised: 31 March 2015 / Accepted: 1 April 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose To investigate whether exogenous tumor necrosis factor-a (TNF-a) will initiate a degenerative process in intervertebral disc in vivo. Methods Exogenous TNF-a in dosages of 50 and 100 ng in 50 lL Dulbecco’s Modified Essential Medium (DMEM) was injected into porcine lumbar discs; a third disc was injected only with 50 lL DMEM as a control. Magnetic resonance imaging (MRI) yielding T1- and T2-weighted images, T2-mapping, and post-contrast T1 images was performed and histology was studied as well. Results After 3 months, a significant decrease in T2 value calculated from T2-mapping MRI was observed in the annulus and nucleus of both groups injected with TNF-a along with a slight decrease in disc height and nucleus volumes in comparison to the control discs. No obvious visual differences among the groups were observed in the normal T1- and T2-weighted MRI images. Post-contrast T1 MRI showed increased annulus enhancement in both TNFa-injected groups compared to the control discs, while no enhancement difference was observed in the nucleus. Histological analysis showed degenerative changes with
& Ran Kang [email protected] & Lin Xie [email protected] 1
Orthopaedic Research Lab, Aarhus University, Building 1 A, Noerrebrogade 44, 8000 Aarhus C, Denmark
2
Department of Orthopedic Surgery, Jiangsu Province Hospital on Integration of Chinese and Western Medicine, 100th Shizi Street, Nanjing 210028, China
3
The MR Research Centre, Aarhus University Hospital, Skejby, 8000 Aarhus C, Denmark
annulus fissure, cell cluster, nucleus matrix loss, vascularization and interleukin-1b expression in the outer annulus of both TNF-a-injected discs, while no degenerative changes were observed in the control discs. Conclusions Intradiscal injection of exogenous TNF-a caused early stage disc degeneration in a porcine model. It may thus support the hypothesis of exogenic TNF-a being an important early pathogenetic factor in disc degeneration. Keywords Disc degeneration TNF-a MRI imaging and -mapping Post-contrast MRI Vascularization
Introduction The pathology of intervertebral disc degeneration (IDD) is currently not well understood. Tumor necrosis factor-a (TNF-a) is a cytokine that has been strongly linked to the pathogenesis of IDD: higher TNF-a expression is commonly found in patients’ degenerated discs [1]; TNF-a expression in discs induces such pathological changes in annulus tissue as angiogenesis [2] and sensory nerve ingrowth [3]. A recent study concluded that TNF-a has a role as a mediator in the disc degeneration process [4]. At the onset of disc degeneration, TNF-a is secreted by nucleus pulposus (NP) cells and annulus fibrosus (AF) cells, as well as by macrophages and T cells. It then triggers a range of pathogenic responses in the disc cells that promote autophagy, senescence, or apoptosis [4, 5]. These fundamental pathological changes subsequently result in disc herniation and neuropathic pain. Experimental in vitro studies of cell culture [6] and organ culture [5, 7, 8] have demonstrated that a culture medium containing TNF-a caused pathological changes in disc cell and -tissue. With the same mechanism, TNF-a in
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the serum or local tissue adjacent to the disc may also affects disc vitality in vivo, since it can transfer through the endplate [5, 7, 8]. This point could also to some extent be speculated on the basis of possible correlation between Modic changes and adjacent IDD. In discs with Modic changes, high expression of TNF-a has been found adjacent to the disc [9] and Modic changes have also been reported to correlate positively with IDD [10, 11]. Therefore, TNF-a in the area with Modic change might be one of the initiators of the adjacent IDD after its transfer through the endplate. TNF-a could raise to 532.6 pg/mL in serum in cerebral malaria patients higher than 46.42 pg/mL in health humans [12]. High level TNF-a in serum or local tissue causes many pathological changes in many organs [13, 14] and has implications for preventive measures. However, for intervertebral disc organ, whether exogenous TNF-a will initiate a degeneration process in a healthy disc in vivo has not been studied assertively. Thus we designed this study to investigate disc changes after intradiscal injection of TNFa with 50 or 100 ng per disc. Whether such high concentration of TNF-a could be reached by endplate transportation from serum or local vertebra is unknown, it is still important to study the disc changes if the TNF-a level is upgraded to a high level in an animal model. We hypothesized that exogenous TNF-a could initiate IDD.
Materials and methods Surgical procedure For this study we used seven Danish landrace female pigs (3 months old, weight approximately 35 kg). The experimental and animal care procedures were approved by the Danish Animal Welfare Committee. Under general anesthesia, the pigs were placed in supine position. A leftsided retroperitoneal approach was used to expose the lumbar spine. In three discs from L12 to L34 we injected 50 lL Dulbecco’s Modified Essential Medium (DMEM; 21063, Gibco, Invitrogen) containing either 50 or 100 ng TNF-a (Recombinant Swine TNF-a; GibcoÒ; 17.1 kDa) through a 26-gauge needle with a stop-marker (Fig. 1a) at a depth of 8 mm, which was confirmed in a pilot study to be the route depth of the NP center from the lateral anterial part. One disc injected with 50 lL DMEM without TNF-a served as a control. The injection was administered slowly, maintaining the pressure for 5 min. The needle was retracted to the annulus fibrosus and kept in this position for 2 min before removal [15]. Hemostasis was secured and the abdomen was closed in layers. The pigs were housed in separate boxes and fed with a standardized food recipe for 3 months.
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Fig. 1 a Using a 1 mL syringe with 26-gauge needle, the disc was injected with 50 lL dye; figure shows distribution of dye after 2 h; b overview of MRI images: no obvious difference among groups in T1-weighted (left), T2-weighted MRI (middle), but lower T2 value in nucleus exhibited by color-coded T2 mapping MRI (right)
Magnetic resonance imaging (MRI) procedure and evaluation Magnetic resonance imaging was performed under general anesthesia on a clinical Philips Achieva 1.5-T scanner (Philips Healthcare, Best, The Netherlands). Before TNF-a injection, a sagittal T1-weighted, T2-weighted, 3D T2weighted, and a sagittal T2-mapping scan (multi-echo spin echo; matrix, 248 9 248; field of view, 300 9 300 mm; repetition time, 1.0 s; 8 echoes with echo time, 15–120 ms (ms); number of excitations, 2) were performed. Three months after TNF-a injection, in addition to these above scans, post-contrast T1-weighted images were obtained at intervals of 0.5, 5, 10 min and then every 10 min for a total of 110 min after manual injection of 0.3 mmol/kg gadolinium (OMNISCANTM, Amersham Health AS, Oslo). All images were analyzed using custom programmed software written by one of the authors (SR). The change of disc height index (DHI) was expressed as percentage of DHI (% DHI) (% DHI = postoperative DHI/ preoperative DHI 9 100) [16, 17]. The change in NP volume was calculated as percentage of NP volume (% NP volume) in the middle axial position of the disc on T2weighted 3D images (% NP volume = postoperative NP volume/preoperative NP volume 9 100). T2 value within the AF and NP was calculated by fitting to an exponential T2 decaying equation [16]. An average of AF and NP T2 value was calculated according to the mean T2 value in the three middle slices using the following equation: T2
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value = (mean 1 9 area 1 ? mean 2 9 area 2 ? mean 3 9 area 3)/(area 1 ? area 2 ? area 3). In the post-contrast MRI images, the signal intensity (SI) within the anterior AF and the whole NP was calculated as percentage of contrast enhancement (% CE) (% CE = (SI post - SI pre)/SI pre 9 100); time-intensity curves were plotted according to % CE and each time point [18]. Histology The spine segments containing the experimental discs were removed at termination and fixed by 70 % ethanol for 7 days, dehydrated in a series of ethanol concentrations, and embedded in methyl methacrylate. Sections of 7 lm were stained with hematoxylin–eosin (HE) and toluidine blue for proteoglycan. The samples were graded for degeneration score (DS) using previously published histological 12-point scoring system [19] by judging: loss of demarcation between NP and AF, loss of proteoglycan from NP, presence and extent of fissures, cell cluster formation. Immunostaining of smooth muscle in vessels was also performed as previously described in the literature [20] with human alpha smooth-muscle actin n-terminal synthetic decapeptide primary antibody (R&D MAB1420), secondary biotinylated polyclonal goat anti-rabbit IgG (DAKO E0432), and tertiary antibody peroxidase-conjugated streptavidin (DAKO P0397). Immunostaining of interleukin-1b (IL-1b) used primary anti-IL-1b (ab193852), secondary biotinylated rabbit anti goat (DAKO 0466), and AEC (Sigma A9626) following the standard protocol. They were then examined using a photomicroscope (Olympus, Tokyo, Japan). Statistical analysis The data are expressed as mean ± standard deviation. Data of percentage DHI, NP volume, T2 value and DS were compared using one way-ANOVA analysis between the different interventions, and the Scheffe method was used to compare every two groups. Data of contrast enhancement were analyzed using two way-ANOVA (intervention 9 time point). Significance level was defined as P less than 0.05.
Results All seven pigs tolerated the surgical procedure well. After 3 months, in general, there was no obvious difference between the discs injected with or without TNF-a viewed by normal T1- and T2-weighted images, however, lower T2 value was observed in the TNF-a injected discs in colorcoded T2-mapping image (Fig. 1b). Disc height, while
slightly decreased in both TNF-a-injected discs, showed no significant difference compared to the controls. NP volume was also decreased, especially in the discs with TNF-a 100 ng (P \ 0.05) (Fig. 2a). The T2 value of AF and NP in both TNF-a-injected discs was significantly lower than the controls (Fig. 2b). In post-contrast MRI images (Fig. 3a), the control discs exhibited no obvious enhancement dye in the annulus area, while in the course of time, uniform enhancement bands parallel to the endplate moved slowly towards the center in the nucleus; in both TNF-a-injected groups more enhancement was seen in the annulus, especially in the anterior annulus area with a pooling of contrast dye, while no obvious difference in the nucleus was observed in comparison to the controls. The corresponding time-intensity curve (Fig. 3b) showed increased enhancement in annulus in the TNF-a-injected discs (intervention P \ 0.01, time point P \ 0.01). No enhancement difference in the nucleus was found among groups (Fig. 3c). Histological images are shown in Fig. 4a–o. In both TNF-a-injected discs, degenerative changes were obvious; these included fissures, cell proliferation, and -cluster in the annulus, changes which did not occur in the control discs. We continued by applying specific staining for the disc ECM component proteoglycan. Nucleus pulposus had relatively less extracellular matrix and became loose in both TNF-a-injected discs, while the control disc had relatively dense matrix. According to a pervious published scoring system, the degree of IDD in both TNF-a-injected discs were both significantly higher than the control (Fig. 4p). Immunostaining of smooth muscle of vessels in the control disc showed almost no vessels inside the annulus, with more vessels in the annulus of both TNF-ainjected discs (Fig. 5a–f). Immunostaining of IL-1b in the control disc had almost no positive staining, with apparent positive staining in the annulus of both TNF-a-injected discs (Fig. 5g–l).
Discussion TNF-a synthesized near/in the disc area is a key mediator in the pathogenesis of IDD [4]. In several in vitro studies, exogenous TNF-a was used as an initiator of IDD [5, 7, 8]. In this study, we went further and confirmed that exogenous TNF-a administered by intradiscal injection initiates early IDD changes in vivo at 3 months. Intradiscal injection is often used to generate IDD. Small needle size has been recommended to avoid the effect of needle injection [21]. For delivering material into the nucleus, a spinal needle of smaller than 22 gauge and injection volume of less than 200 lL is recommended to prevent post-surgery leakage in a porcine model [22].
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Fig. 2 Data of percentage of disc height index (DHI) and nucleus pulposus (NP) volume and data of T2 value in annulus and nucleus (n = 7). a Percentage of DHI and NP volume changes in groups; b T2
value of annulus and nucleus changes in groups. Asterisk indicates significant difference between the intervened discs and the controls, P \ 0.05
Fig. 3 The serial T1-weighted post-contrast MRI images and timeintensity curves of annulus and nucleus (n = 7). a In the control, no obvious enhancement observed in the area of annulus, meanwhile in the nucleus, uniform enhancement bands parallel to the endplate slowly moved towards the center as time passed; in both TNF-ainjected groups, more enhancement was seen in the annulus,
especially in the anterior annulus area (arrow), with a pooling of contrast dye, while no obvious difference in the nucleus observed in comparison to the controls; b much more rapid enhancement in annulus of the TNF-a-injected discs in time-intensity curve (different intervention P \ 0.01, different time point P \ 0.01); c no enhancement difference in the nucleus among groups
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Fig. 4 Images of histology and degeneration score in different groups. Control disc: a overview of annulus; b regular structure in outer annulus; c regular inter annulus; d overview of nucleus matrix; e nucleus with relatively thick matrix. TNF-a 50 ng injected disc: f overview of annulus; g fissure in the outer annulus; h cell cluster at the inter annulus (arrows); i overview of nucleus matrix; j nucleus
with relatively less of matrix. TNF-a 100 ng injected disc: k overview of annulus; l fissure in the outer annulus; m cell cluster at the inter annulus (arrows); n overview of nucleus matrix; o nucleus with relatively less matrix. p degeneration score of each group (n = 7). Asterisk indicates significant difference between the intervened discs and the controls, P \ 0.05
Fig. 5 Immunostaining of smooth muscle in vessels in different groups: a, b control disc, almost no vessels inside of the annulus; c, d TNF-a 50 ng injected disc, vessels (arrows) in the annulus; e, f TNF-a 100 ng injected disc, vessels (arrows) in the annulus.
Immunostaining of IL-1b in different groups: g, h control disc, almost no positive staining; i, j TNF-a 50 ng injected disc, positive staining (hollow arrows) in annulus area; k, l TNF-a 100 ng injected disc, positive staining (hollow arrows) in annulus area
In our study, we used a 26-gauge needle with 50 lL injection volume to minimize the effects caused by the delivery procedure. As our results show, we did not see any degenerative changes in the control discs, where only 50 lL DMEM was injected. Consisting with previous studies of inducing IDD model by enzymes, no IDD was
found in the control disc only injected small volume vehicle [17, 23, 24]. In our study, pro-inflammatory cytokine TNF-a were injected, instead of enzymes. In a previous in vitro study of bovine disc organ culture system, a 1-week exposure to 200 ng/mL TNF-a initiated the IDD process with non-
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recoverable catabolic shift [7]. Likewise, in an in vitro study of rat disc organ culture system, a cocktail of 100 ng/ mL TNF-a and 10 ng/mL IL-1b contained in a culture medium also led to IDD [5, 8]. Therefore, we established a dosage of 50 and 100 ng TNF-a, with corresponding concentration of 100 and 200 ng/mL in the nucleus (in our study, the nucleus volume of porcine lumbar disc was calculated at around 0.5 mL). TNF-a, a major pro-inflammatory cytokine, could accelerate the degradation of intervertebral disc extracellular matrix, causing an imbalance between catabolism and anabolism, leading to denaturalization of the disc tissue [4]. Exposure to TNF-a also stimulated interleukin, supporting and strengthening its role as an initiator and regulator of multiple proinflammatory cytokines in intact discs [7]. Consistent with these previous in vitro studies, the degenerative changes in our study showed that TNF-a could initiate IDD in vivo, confirmed by MRI and histological examinations. IDD is commonly characterized by MRI with reduced signal in T2-weighted images in NP, annulus collapse, and reduced disc height [25]. Such MRI techniques as quantitative MRI T2-mapping to analyze changes in disc matrix composition can also be used to reflect pathological changes of IDD in in vivo studies [24]. As the result showed, disc height and nucleus volume were decreased with both dosages of TNF-a intervention, and the T2 value calculated in quantitative MRI T2-mapping in the annulus and nucleus were both significantly decreased compared to the control discs. The histological changes of annulus fissure, cell cluster, and nucleus matrix loss confirmed the MRI-verified degenerative changes. TNF-a was reported to either induce or be affiliated with such pathological changes as angiogenesis [2] and sensory nerve ingrowth [3] in discs. In our study, we also found angiogenesis in the annulus of IDD; this was confirmed both by rapid enhancement in the annulus, detected by the post-contrast MRI scanning, and the histological study. The vascularization of IDD due to AF structural failure was previously described in the literature [18, 26, 27]. From these results, we could conclude that TNF-a caused IDD. On the other hand, no obvious visual changes were seen in the normal T1- and T2-weighted MRI. This may be explained by the fact that the TNF-a intervention in our study caused slight or earlystage disc degeneration which was attributable either to the small dosage or the short observation time. Further studies, with different observation durations and a larger dosage, are warranted. Furthermore, as disc degeneration is multifactorial, a single TNF-a application might not necessarily be capable of causing severe changes. Extending this experimental result to possible clinical relevance, this raises awareness that TNF-a in serum or local vertebra might affect disc vitality, if the TNF-a reaches the disc at a high enough concentration. Alcohol
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drinking [28], smoking [29], and a variety of diseases [13] may cause a rise in serum TNF-a level, which also reportedly has a correlation with myocardial infarction [30] and renal damage [13]. In the intervertebral disc area, serum TNF-a can penetrate through the endplate pathway; this has been tested by means of different molecular weight Firefly (61 kDa) and Renilla (38 kDa) luciferase in the disc organ culture system [8]. Therefore, there might be a correlation between IDD and the pro-inflammatory factor TNF-a in system serum or adjacent tissue. Accordingly, when there are high TNF-a levels in the serum, or in a local area near the disc such as in a vertebra exhibiting Modic change, such preventive measures as anti-inflammatory treatment [6] might be considered as a means of protecting the disc. Furthermore, this may also imply that disc regeneration treatments for IDD may require supplementary anti-inflammatory treatments. There are some limitations in the present study: We could not apply TNF-a in the serum and maintain a specific concentration for an extended period because of potential systematical damage to other organs. We injected TNF-a directly into the disc to simulate the situation in which the serum TNF-a has already transferred into the disc through the endplate. Because we were unable to monitor the concentration of TNF-a in the disc, we assumed that TNFa could remain in the disc and that in fact some of it might have diffused out of the disc. Moreover, whether TNF-a in serum or local vertebra could reach the disc at a high enough concentration as we applied to induce IDD need further investigations.
Conclusions Intradiscal injection of exogenous TNF-a caused early IDD changes in a porcine model. Exogenous TNF-a could be an initiator of disc degeneration. Acknowledgments We thank Anette Baatrup for histology technique support, Yufen Zhang for surgical assistance, and Chen Gan for MRI analysis. We gratefully acknowledge the funding from Velux (25906), Lundbeck, Gigtforeningen Foundation, Aarhus Spine Research Foundation, and the International Cooperation and Natural Science Foundation of Jiangsu province of China (BZ2011046, BL2012069, BK2012490). Conflict of interest
None.
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