European Spine Journal (2019) 28:1413–1422 https://doi.org/10.1007/s00586-019-05994-3

ORIGINAL ARTICLE

Molecular alterations of human lumbar yellow related to the process of intervertebral disk degeneration and stenosis

Delio E. Martins1,2,4 · Marcelo Wajchenberg2 · Juliana M. Veridiano3 · Thérèse R. Theodoro3 · Olga M. S. Toledo3 · Maria A. S. Pinhal1,3

Received: 2 March 2019 / Accepted: 30 April 2019 / Published online: 8 May 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract Purpose The objective of this study was to analyze the layers of yellow ligament in lumbar canal stenosis and disk herniation. Methods Eighteen were harvested from patients with lumbar spinal canal stenosis. Twenty-nine normal samples from lumbar spine disk herniation patients served as control. All surgical procedures were the same. Ligaments were stained in hematoxylin and eosin; picrosirius–hematoxylin for ; Weigert’s resorcin-fuchsin for elaunin, oxytalan and elastic fbers; and transmission electron microscopy. Immunohistochemistry was performed for Il-6; Il-10; and CD-31, PGP9.5. Results are described in means and standard error (mean ± SE), and all analyses adopted the signifcance level of P < 0.05. Results Spinal stenosis ligaments were 2.5 × thicker. Control superfcial ligaments presented a large number of thick, compact collagen fbers and a signifcant amount of oxytalan and mature elastic fbers. The deep layer presented a large number of mature elastic fbers. In the stenosis group, collagen was thinner and compacted in both layers. There was no diference in the interleukin profle among groups. The deep portion of the stenosis group presented a higher number of vessels and nerves. Conclusion Two layers compose the elastic system of the normal ligamentum favum, where the deep portion is mainly responsible for its elasticity (elaunin fbers), while its resistance depends on the concentration of oxytalan fbers, which are more present in the superfcial layer. Ligamentum favum in the stenosis samples presents more mononuclear infltrate and more degraded elastic fbers with a higher number of vessels in its deep portion. Graphical abstract These slides can be retrieved under Electronic Supplementary Material.

Key points Take Home Messages

Transmission Electron Microscopy images of elastic [Keywords: ligamentum flavum; spinal stenosis; elastic fibers; elaunin and collagen fibers of the ligamentum flavum of fibers; oxytalan fibers ] patients with disc herniation (A and B) and patients 1. The two layers of normal yellow ligament are different in with stenosis (C and D) stained with tanic acid. In A and B, elastic fibers of a disc herniation patient composition. 1. Anatomic and biological differentiation between layers of yellow presenting normal morphological appearance. In C and D, elastic fibers of a patient with stenosis, presenting ligament. changes in the core. Electron dense elastic fibers 2. The deep portion of ligamentum flavum is mainly responsible for (arrow); collagen fibers (C) Magnification: 20,000x (a / its elasticity while superficial portion is manly responsible for its c); 60,000x (b); 50,000x (d) 2. Deep portion of yellow ligament is the mainly responsible for resistance. elasticity. 3. Ligamentum flavum in the stenosis samples presents more 3. Deep portion of the ligament in stenosis patients has a higher mononuclear infiltrate and more degraded elastic fibers with a amount of vessels. higher number of vessels in its deep portion.

Martins DE, Wajchenberg M, Veridiano JM, Theodoro TR, de Toledo OMS, Pinhal MAS Martins DE, WajchenbergM, VeridianoJM, Theodoro TR, de Toledo OMS, Pinhal MAS Martins DE, WajchenbergM, Veridiano JM, Theodoro TR, de Toledo OMS, Pinhal MAS (2019) Molecular alterations of human lumbar yellow ligament related to the process of (2019) Molecular alterations of human lumbar yellow ligament related to the process of (2019) Molecular alterations of human lumbar yellow ligament related to the process of intervertebral disc degeneration and stenosis. EurSpine J; intervertebral disc degeneration and stenosis. EurSpine J; intervertebral disc degeneration and stenosis. EurSpine J;

Keywords Ligamentum favum · Spinal stenosis · Elastic fbers · Elaunin fbers · Oxytalan fbers

Electronic supplementary material The online version of this Introduction article (https​://doi.org/10.1007/s0058​6-019-05994​-3) contains supplementary material, which is available to authorized users. The thickening of the ligamentum favum (LF) and its bio- * Delio E. Martins chemical and morphological changes are considered one of [email protected] the most important causes of spinal stenosis [1]. The LF is Extended author information available on the last page of the article important for providing spinal stability in many postures

Vol.:(0123456789)1 3 1414 European Spine Journal (2019) 28:1413–1422 and maintaining a smooth surface on the posterior wall of sagittal T2-weighted (2890/58, 2; 256 × 192; 4 mm; 40 cm), the spinal canal and foramina [2]. LF is constituted of two coronal T2-weighted fat suppressed (2756/58, 2; 256 × 192; portions frmly adherent to each other. The superfcial layer 4 mm; 40 cm) and axial T2-weighted (2756/58, 2; 256 × 192; is a light-yellow structure, adjacent to the multifdus muscles 4 mm; 25 cm). and bigger than the deep component, while the deep layer is The thickness of the LF was measured at its midpoint a thin dark-yellow layer on the ventral side, adjacent to the using magnetic resonance imaging on T1-weighted axial spinal canal [3]. images, which was clearly seen as a low-signal intensity The elastic fber system consists of three diferent kinds mass just ventral to the vertebral lamina. All measures were of fbers. The oxytalan fber composed of parallel bundles done by a single investigator and performed at worksta- of microfbrils that serve as a guide for the deposition of a tions through the Carestream Vue Motion (PACS), version small amount of elastin, forming the elaunin fbers, and as 12.1.5.6529 Inc. 2009 system (Carestream Health, USA). more elastin adheres to those fbers, it becomes thicker and The average age of the disk herniation group was forms the mature elastic fber [4–6]. 40 ± 11.54 (range 19–57) with 18 males (62%); and in the The elasticity of the tissue depends on the concentration stenosis group, the average age was 67 ± 9.59 (range 56–83) of mature elastic fber and elaunin fbers, while its com- with 9 males (50%). pression resistance is related to oxytalan fbers [7]. A large The LF was cut sagittally and fxed with 4% paraformal- amount of oxytalan fbers has been reported as a marker of dehyde in 0.1 M PBS, pH 7.4, for 24 h. Samples were dehy- strong local mechanical stress in muscles [8]. Loss of LF drated in graded concentrations of ethanol and embedded elasticity could be responsible for allowing it to fold into the in parafn. Serial 5 μm coronal sections were made using spinal canal, thus promoting stenosis [1]. a manual microtome LEICA RM-2145-2245 (Leica, Nuss- This adherence between the layers has led the LF to be loch, Germany) and subjected to hematoxylin and eosin; treated as a single structure. However, the goal of this study picrosirius–hematoxylin for collagen analysis; Weigert’s was to determine whether there are diferences in thickness resorcin-fuchsin with previous oxidation for the detection between the layers and whether they difer in histomorphol- and analysis of elastic fbers; and transmission electron ogy, thus implying diferent function. An anatomical inves- microscopy (TEM). For and immunohistochem- tigation was therefore performed on LF layers obtained from istry, three slides from each sample were used for analysis. both spinal canal stenosis patients and lumbar disk hernia- All analyses were performed by the same investigator and tion patients. were blinded to groups. The histological sections were analyzed under a light microscope, the stained slides were analyzed, and images Methods were acquired using the Nikon Eclipse E800­ ® photomi- croscope (Nikon, Tokyo, Japan) and Nis-Elements® image After Institutional Review Board approval (897.222), 29 capture software (Nikon, Tokyo, Japan). This analysis was consecutive patients with lumbar disk herniation and 18 used for hematoxylin and eosin; picrosirius–hematoxylin and patients with lumbar spinal canal stenosis had their LF har- Weigert’s resorcin-fuchsin. vested during either a microdiscectomy procedure or decom- pression surgery after failure of conservative treatment. All Picrosirius–hematoxylin microdiscectomy procedures were performed by posterior approach using tubular retractors, and the LF was released Sections were deparafnized and hydrated in distilled water, from lamina using a curette. The two layers of the ligament stained with Sirius Red, dissolved in a 0.1% saturated pic- were removed. The frst dorsal layer was called superfcial, ric acid solution for 1 h at room temperature, rinsed with and the second ventral layer, which lies closer to the verte- distilled water and counterstained with hematoxylin for two bral canal, was called the deep portion. The decompression minutes. Sections were then dehydrated with increasing surgery was performed under direct vision. ethanol concentrations, diaphanized in xylene and mounted Spine MRI examinations were conducted as clinical rou- with Entellan (Merck, Darmstadt, Germany) [9]. tine on a 1.5-T magnet HDX (GE Healthcare, Milwaukee, USA) according to the departmental protocol: Subjects were Weigert’s resorcin‑fuchsin scanned in a supine position, and their leg extended using a multichannel spine dedicated coil. Examinations used the All elastic system fbers were stained after the tissue was following sequences: sagittal T2-weighted fat suppressed oxidized and prior to by Weigert’s resorcin-fuchsin. (TR/TE, 2756/58; number of excitations [NEX], 2; matrix, Sections were deparafnized and treated with a 10% potas- 256 × 192; thickness, 4 mm; feld of view [FOV], 40 cm), sium peroxymonosulfuric acid solution for 40 min [6] and sagittal T1-weighted (357/15; 2; 320 × 256; 4 mm; 40 cm), stained by Weigert’s resorcin-fuchsin method.

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TEM between two groups. Results were compared and described in means and standard error (mean ± SE). All data were Small fragments of LF were fxed in 2.5% glutaraldehyde analyzed using the SPSS­ ® software (Statistical Package for and dissolved in 0.1 M sodium cacodylate bufer (pH 7.2) Social Sciences; SPSS, Chicago, IL, USA), version 17.0. containing 0.1% tannic acid for 2 h at room temperature, In all analyses, we adopted the signifcance level P < 0.05. followed by post-fxation in 1% osmium tetroxide for 1 h at 4 °C, and overnight block staining in 0.5% aqueous uranyl acetate. The samples were embedded in EPON resin, thin- Results sectioned in a Leica-UCT ultratome, double-stained with uranyl acetate and lead citrate and examined with a LEO In the disk herniation group, the LF thickness mean was 906 electron microscope. 1.68 ± 0.46 mm, while in the stenosis group, the thickness mean was 4.25 ± 0.92 mm. The distribution of levels is Immunohistochemistry shown in Table 1. Histological results from both groups show that the deep Slides with 3 µm sections were deparafnized and rehy- portion of the LF has a large number of fbrocytes. The col- drated. The recovery of the antigen was performed by warm- lagen fbers are very thick and compacted. A large number of ing the slides at 100 °C for 30 min in 10 mmol/L citrate mature elastic fbers are observed following the collagen fb- bufer, pH 6.0. Endogenous peroxidase was blocked with an ers (Fig. 1b, c). The superfcial portions of the LF of the her- aqueous solution of 3% hydrogen peroxide for 35 min. The niated group also present a large number of fbrocytes and a samples were incubated overnight at 4 °C with the primary large number of thick, compact collagen fbers (Fig. 1d, e). antibodies: anti-IL-6 (sc-130326) and anti-IL-10 (H-160/ The analysis of the elastic fbers of the LF of both groups sc-7888) (Santa Cruz Biotechnology, USA) in dilution using resorcin histochemistry observed mature and oxyta- 1:100; anti-PGP9.5 (ab27053) and anti-CD31 (ab28364) lan fbers in the superfcial portion (Fig. 2f), while TEM (Abcam, Cambridge, UK); and anti-VEGF-A (18077) allowed us to observe mature elastic fbers in the LF of ste- (Biorbyt, England). Finally, the slides were incubated with nosis patients and an irregular elastin core structure with a complex of streptavidin marked with peroxidase follow- light regions at the edges, similar to vacuoles, suggesting ing manufacturer’s instructions (LSAB­ ®) (DakoCytomation, degradation in both regions (Fig. 3c, d). Glostrup, Denmark). The sections were developed using Morphological results show that the deep portion of LF 3,3′-diaminobenzidine and counterstained with hematoxy- in patients with disk herniation presents a smaller proportion lin. The presence of a brown color was considered posi- of cells when compared to the stenosis group. The collagen tive for the respective molecules. The slides were analyzed fbers are thick, compact and oriented in several directions. with a TS100 Nikon Eclipse(r) light microscope to iden- Mature elastic fbers are seen in abundance (Fig. 2a–c). tify the areas that best represented the immune marking of In the LF of all patients with stenosis, the changes the molecules analyzed. In each case, quantifcation of the occurred in the deep portion of the ligament, which presents immune labeling was quantifed by a digital analysis. The a great number of cells, when compared to the herniated photomicrographs with 640 × 480 pixels were obtained from group, and a monocular infltrate in some areas (Fig. 2d). consecutive non-coincident felds with 400× magnifcation Yet, in the stenosis group, the histochemistry shows using a 4300 Nikon ­Coolpix® digital camera adjusted for collagen fbers that are thinner and less compact than the the same parameters. For analysis, ten diferent areas were chosen randomly and the evaluation was quantitative using ­ImageLab® (Softium ­Informatica®, Sao Paulo, Brazil), Table 1 Clinical features adjusted to a micrometric scale (µm) [10]. Digital quanti- Disk herniation Spinal stenosis fcation was expressed by the intensity of digital immune N staining (ItE) for each sample [10]. 29 18 Age (years) 40 ± 11.5 67 ± 9.59 Statistical analysis BMI (kg/m2) 26.3 ± 3.47 26.8 ± 3.73 Ligamentum favum thick- 1.68 ± 0.46 4.25 ± 0.92 ® ness (mm) Statistical analysis was performed using the Prisma5­ soft- Spine level ware (GraphPad Software, La Jolla, CA, USA). All vari- L3–L4 4 3 ables were considered nonparametric through the Kolmogo- L4–L5 14 15 rov–Smirnov test. Kruskal–Wallis test with the auxiliary test L5–S1 11 0 Dunn’s was applied to associations between all the groups, and Mann–Whitney test was used to analyze the associations BMI body mass index

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Fig. 1 Photomicrographs of the deep layer of ligamentum favum (a–c) and the superfcial layer of ligamentum favum (d–f) of lumbar disk herniation patients (normal ligaments). In a and d, sections stained with hematoxylin and eosin. In b and e, sections stained with picrosirius–hematoxylin where the collagen fbers are red. In c and f, Weigert’s resorcin- fuchsin stained with previous treatment with oxone, where the mature elastic fbers are identi- fed in purple (arrowhead) and oxytalan fbers are identifed as black fbrils (arrow). Magnifca- tion: 200× (a and d) and 100× (b, c, e, f)

herniation group (Fig. 2e). Mature elastic fbers are present the superfcial and deep regions. Under TEM the elastic, in large quantities (Fig. 2f). oxytalan and collagen fbers are clearly visible (Fig. 4). In the superficial layer of stenotic patients, mature To investigate changes of infammatory cytokines in the elastic fbers and a large number of oxytalan fbers were LF of patients with stenosis compared to patients with disk observed distributed throughout this portion. Under light herniation, interleukin profles were analyzed. No difer- microscopy, no diference was found between the results ence was observed between the groups (Fig. 7). Further- of qualitative analysis of collagen and elastic fbers in the more, no alterations were observed when both superfcial ligament of both groups (Fig. 2). However, samples from and deep ligamentum were analyzed. patients with stenosis show the presence of a mononuclear VEGF is one of the most important proangiogenic fac- infltrate indicative of chronic infammation (Fig. 2d). tors, which stimulates vasculogenesis and angiogenesis. The elastic fbers of patients with disk herniation pre- However, the level of VEGF was not altered in the LF of sent mature elastic fbers with normal structure presenting patients with stenosis, as seen in Fig. 8. an elastin core surrounded by microfbrils (Fig. 3a, b). The CD31 is an integral membrane glycoprotein that might elastic fbers of patients with stenosis, in turn, present an be a useful marker for total blood vessels. Figure 5 shows elastin core with irregular borders, with clear spaces sug- a signifcant increase in the deep LF of stenosis patients gesting corrosion and degradation of the edges of the elas- compared to the deep LF of disk herniation patients. tin core (Fig. 3c, d). These changes were observed in both

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Fig. 2 Photomicrographs of ligamentum favum of patients with disk with picrosirius–hematoxylin where the collagen fbers are red. In c herniation (control)—(a–c) and patients with stenosis (d–f). In a and and f, sections stained with Weigert’s resorcin-fuchsin with oxone d, sections stained with hematoxylin and eosin. In d, note the mono- where mature elastic fbers are identifed in purple. Magnifcation: nuclear infltrate in the delimited area. In b and e, sections stained 200× (a and d) and 100× (b, c, e, f)

It is very important to emphasize that it was crucial to normal ligament. Components of the collagen and elastic divide the LF into two groups (superfcial and deep) to show system focusing on oxytalan, elaunin and mature elastic fb- the diference in the CD31 expression between stenosis and ers were analyzed. The presence of interleukins, vessels and herniation tissues, since no change had been observed when nerves was also investigated through immunohistochemistry. total LF was analyzed (Fig. 9). In the superfcial layer of the ligament of herniated patients The PGP9.5 antibody reacts with human neuroendocrine (normal ligament), we found a large amount of thick, com- marker. Expression of PGP9.5 is highly specifc to neurons pact collagen fbers, a signifcant amount of oxytalan fbers and to cells of the difuse neuroendocrine system and their and mature elastic fbers, while in the deep layer, only a tumors. In order to determine whether there is an increase in large amount of mature elastic fbers was observed. In the the expression of PGP9.5 as a function of pain nociceptive stenosis group, we observed collagen that was thinner and mechanisms related to the pathophysiology of stenosis and compacted in both layers of the ligament, with the deep herniation, the PGP9.5 antibody was used. Figure 6 shows no portion of the ligament presenting a monocular infltrate in signifcant diference in PGP9.5 expression. some areas. However, there was no diference in the inter- Although no significant statistical difference could be leukin profle among the groups. In the deep portion of LF, detected, it was possible to see in the samples from herniated a higher number of vessels and nerves were observed in the group that there were no alterations on the number of vessels stenosis group. and nerves, as well as there was no degradation of elastic fb- The functions of LF have been debated over the years. It ers, while in the samples collected from patients with stenosis was considered the only ligament of the posterior spine that there were a higher number of vessels and nerves and addition- had any restrictive efect and that this provided the vertebral ally the samples had signifcantly degradation of elastic fbers. canal a smooth dorsal covering in all positions of the spine [11]. However, it has been demonstrated that LF accounts for a portion of the disk pressure and that its elasticity prevents Discussion it from protruding into the spinal canal [12]. Studies in rats demonstrate that elasticity depends on the We analyzed the two leafets of LF from individuals with concentration of the mature and elaunin elastic fbers [13]. and without disease in the ligament separately. Samples with Bearing in mind that there are diferences between rodents ligament disease were an average of 2.5 × thicker than the and humans, but at the same time observing that we analyze

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Fig. 3 Transmission electron microscopy images of elastic and collagen fbers of the ligamentum favum of patients with disk herniation (a, b) and patients with stenosis (c, d) stained with tanic acid. In a and b, elastic fbers of a disk hernia- tion patient presenting normal morphological appearance. In c and d, elastic fbers of a patient with stenosis, present- ing changes in the elastin core. Electron dense elastic fbers (arrow); collagen fbers (C) Magnifcation: 20,000× (a, c); 60,000× (b); 50,000× (d)

diferent tissues that are subject to distinct biomechani- must be done comparing LF from cervical, thoracic and even cal forces, we observed that the deep layer of the LF has a upper levels of the lumbar spine. Another limitation is the higher concentration of these fbers than the superfcial layer, use of LF from lumbar disk herniation patients instead of suggesting that this small portion of the ligament is mainly cadavers or trauma patients without degenerative disease. responsible for the elastic properties of the LF. While the An important point that must be emphasized is that the tissue compression resistance is dependent upon the con- LF from patients with lumbar spinal canal stenosis has irreg- centration of oxytalan fbers [13], we demonstrated that the ular elastin core edges with vacuoles, suggesting degrada- superfcial or dorsal layer of the LF has the highest concen- tion, which may impact elasticity and could explain why the tration of oxytalan fbers. LF bulge into the spinal canal in the standing position even Consequently, the highest concentration of oxytalan fbers if normal in thickness [16]. may be related to the increase in compression strength of the Despite stenosis, it is well known that intervertebral disk superfcial LF, since oxytalan fbers support more compres- herniation pain is a result of spinal nerve infammation and sion than elastic and elaulinic fbers. swelling caused by the pressure of the herniated disk. The Reports vary since around 60% of the patients with lum- stenosis process narrows the spinal canal (central stenosis) bar disk herniation have a thick LF and around 59% or them and presses on the spinal cord and can also cause infamma- exhibit marked scarring with fbrous [14], tion and weakness. The increased thickness of the LF is a while other reports show slight fbrotic changes with aging common feature of patients with spinal stenosis, while it is but without alterations with disk herniation [15]. Although not a characteristic of patients with herniation. in this study we have used LF from patients with lumbar disk The role of infammatory mediators in the pathophysi- herniation as controls, the LF of all these samples has nor- ology of herniation and stenosis is still controversial. Sev- mal thickness and was considered normal ligaments. Sample eral fndings suggest that the infammatory response occurs size was a limitation of this study, and additional studies in the early stage of disk herniation and stenosis, but the

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Fig. 4 Transmission electron microscopy images of elastic and collagen fbers of the super- fcial portion of the ligamentum favum of patients with disk herniation stained with tanic acid. Electron dense elastic fb- ers (arrow); collagen fbers (C), microfbrils (OX). Magnifca- tion: 20,000× (a, c); 60,000× (b, d)

Fig. 5 Immunohistochemistry quantifcation of CD31 in favum liga- (right). In both groups, tissues were removed from the superfcial mentum tissues. The results represent the intensity of expression of and deep region of the ligamentum favum. The values represent the CD31. a Digital quantifcation from tissues of the ligamentum favum mean and standard error of the intensity of expression (ItE) obtained obtained after surgery in patients with herniated disk (gray bars) or as described in methods. The lines indicate mean and standard error with stenosis (white bars) and b images from immunohistochem- value of CD31 expression in each group (P* = 0.0167; nonparametric istry reaction in patients with herniated disk (left) or with stenosis Mann–Whitney test)

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Fig. 6 Immunohistochemistry quantifcation of PGP9.5 in favum nosis (right). In both groups, tissues were removed from the superf- ligamentum tissues. The results represent the intensity of expression cial and deep region of the favum ligament. The values represent the of PGP9.5. a Digital quantifcation from tissues of the favum liga- mean and standard error of the intensity of expression (ItE) obtained mentum obtained after surgery in patients with herniated disk (gray as described in methods. The lines indicate mean and standard error bars) or with stenosis (white bars) and b images from immunohisto- value of PGP9.5 expression in each group (P = 0.5962; nonparametric chemistry reaction in patients with herniated disk (left) or with ste- Kruskal–Wallis test) increase in infammatory cytokines is transient. Indeed, that occurs in the development of the disease. In summary, most studies failed to demonstrate infammatory cytokines according to the results of the present study, the two layers increased during chronic disk herniation samples or stenosis. of normal LF are diferent in composition, the deep portion Our results corroborate such data, since Interleukin-6 (IL- being mainly responsible for its elasticity. LF in the steno- 6), a pro-infammatory cytokine, as well as Interleukin-10 sis samples presents more mononuclear infltrate and more (IL-10), an anti-infammatory cytokine, was not changed in degraded elastic fbers with a higher number of vessels in the yellow ligament when patients with intervertebral disk its deep portion. herniation and stenosis were compared. In this study, a few potential sample errors inherent to Acknowledgements We thank Nelson Astur (Hospital Israelita Albert Einstein) for his assistance in collecting the samples. The authors are the nature of the work must be remembered such as the age grateful to Professor Elia Garcia Caldini for his suggestions. Part of of patients that may interfere with elastic fbers and col- this work has been carried out in the Electron Microscopy Laboratory lagen features, as well as the fact that the ligaments have of Faculdade de Medicina da USP. been removed from diferent levels of the lumbar spine. Although most came from the L4–L5 level (Table 1), it was Compliance with ethical standards not assessed whether there was a diference between lumbar levels. Conflict of interest The authors declare that they have no competing In this study, we also did not evaluate which type of inner- interest. vation is present in the LF, but it was possible to demonstrate an increase in vessels and nerves in the deep region of LF Appendix from patients with stenosis, suggesting that LF could have some function in the proprioception of the spine. Therefore, LF may present some function in the physiopathology of the See Figs. 7, 8 and 9. stenosis beyond the compression caused by the thickening

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Fig. 7 Digital quantifcation of IL-6 and IL-10 in favum ligamen- represent the mean and standard error of the intensity of expression tum tissues. The results represent the intensity of expression of inter- (ItE) obtained as described in methods. The statistical analysis was leukins − 6 (a) and − 10 (b) in tissues of the ligamentum favum performed using Kruskal–Wallis test with the auxiliary test Dunn’s to obtained after surgery in patients with herniated disk (gray bars) or associations between all the groups and Mann–Whitney test to ana- with stenosis (white bars). In both groups, tissues were removed from lyze associations between two groups. Results described in means the superfcial and deep region of the favum ligament. The values and standard error (IL-6, P = 0.9225 and IL-10, P = 0.6407)

Fig. 9 Immunohistochemistry quantifcation of CD31 in favum liga- Fig. 8 Digital quantifcation of VEGF-A in favum ligamentum tis- mentum tissues. The results represent the intensity of expression of sues. The results represent the intensity of expression of VEGF-A in CD31 in patients with herniated disk (gray bars) or with stenosis tissues of the ligamentum favum obtained after surgery in patients (white bars) without diferentiating them into superfcial and deep with herniated disk (gray bars) or with stenosis (white bars). In both portions. The values represent the mean and standard error of the groups, tissues were removed from the superfcial and deep region intensity of expression (ItE) obtained as described in methods. The of the favum ligament. The values represent the mean and standard lines indicate mean and standard error value of CD31 (P = 0.7431; error of the intensity of expression (ItE) obtained as described in nonparametric Mann–Whitney test) methods. The statistical analysis was performed using Kruskal–Wal- lis test with the auxiliary test Dunn’s to associations between all the groups and Mann–Whitney test to analyze associations between two groups. Results described in means and standard error (P = 0.7277)

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Afliations

Delio E. Martins1,2,4 · Marcelo Wajchenberg2 · Juliana M. Veridiano3 · Thérèse R. Theodoro3 · Olga M. S. Toledo3 · Maria A. S. Pinhal1,3

1 Universidade Federal de Sao Paulo – UNIFESP, R. 3 Faculdade de Medicina do ABC – FMABC, Av. Lauro Sena Madureira, 1500 ‑ Vila Clementino, Sao Paulo, Gomes, 2000 – Vila Sacadura Cabral, Santo Andre, SP 04021‑001, Brazil SP 09060‑870, Brazil 2 Hospital Israelita Albert Einstein, Av. Albert 4 Universidade Anhembi Morumbi, R. Dr. Almeida Lima, Einstein,627/701 – Morumbi, Sao Paulo, SP 05652‑900, 1134, Sao Paulo, Brazil Brazil

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