The inflammatory response in the regression of lumbar disc herniation

REVIEW Open Access The in lamma o y esponse in he eg ession o lumba disc he nia ion Ca la Cunha 1,2* , Ana J. Sil a 1,2 , Paulo Pe ei a 3,4,5 , Rui Vaz 1,3,4,5 , Raquel M. Gonçal es 1,2,6 and Má io A. Ba bosa 1,2,6 Abs ac Lumba disc he nia ion (LDH) is highly associa ed wi h in lamma ion in he con ex o low back pain. Cu en ly, in lamma ion is associa ed wi h ad e se symp oms ela ed o he s imula ion o ne e ibe s ha may lead o pain. Howe e , in lamma ion has also been indica ed as he main ac o esponsible o LDH eg ession. This appa en con o e sy places in lamma ion as a good p ognos ic indica o o spon aneous eg ession o LDH. This e iew add esses he molecula and cellula mechanisms in ol ed in LDH eg ession, including ma ix emodeling and neo ascula iza ion, in he scope o he clinical decision on conse a i e e sus su gical in e en ion. Based on he e idence, a special ocus on he in lamma o y esponse in he LDH con ex is gi en, pa icula ly in he monocy e/ mac ophage ole. The phenomenon o spon aneous eg ession o LDH, ex ensi ely epo ed in he li e a u e, is he e o e analyzed he e unde he pe spec i e o he modula o y ole o in lamma ion. Keywo ds: Low back pain, Spine, In e e eb al disc, Immunomodula ion, Mac ophages Low back pain and lumba disc he nia ion Lumba disc he nia ion (LDH) is a majo con ibu o o low back pain and a ec s a ound 9% o all people wo ld- wide, wi h a high associa ed economic bu den and a endency o inc ease as he popula ion ages [1]. LDH has been associa ed wi h dis up ion o he annulus ib osus (AF), ex usion o he nucleus pulposus (NP), and s imula- ion o ne e ibe s, leading o pain. Howe e , mo e e- cen ly, Rajaseka an e al. [2] sugges ed ha disc he nia ion is mo e commonly he esul o endpla e junc ion ailu e han AF up u e. He nia ed discs a e ound in 30–40% o asymp oma ic people by imaging diagnos ic ools [3]. The cu en ea men s o LDH, as well as o dege- ne a i e disc disease in gene al, can be di ided in o con- se a i e e sus su gical app oaches and he decision on which app oach o use is a iable and pa ien –clinician dependen . Symp oms o igina ed by LDH may disappea wi hou any su gical ea men and in some o hese pa- ien s his is accompanied by a educ ion o he size o disc he nia ion in imaging s udies. This phenomenon is known as spon aneous he nia eg ession, which may be pa ial o comple e. Clea ly, his e idence is indica i e o he pa amoun need o iden i y he mechanisms behind LDH eg ession and o de elop p edic i e me hods o de ec ion o his phenomenon in clinical p ac ice. Clinical e idence o spon aneous LDH eg ession Since he i s epo o spon aneous LDH eg ession [4] and o compu ed omog aphy (CT)-con i med spon aneous LDH eg ession [5], documen a ion o his phenomenon has become b oadly a ailable in he li e a u e. Imaging diagnos ic ools such as magne ic esonance imaging (MRI) ha e a cen al ole in con i ming LDH eg ession and, al hough con o e sial o a signi ican numbe o yea s, i is now widely ecognized ha la ge-sized and seques a ed LDH end o eg ess mo e han o he LDH sub ypes (Fig. 1). This eg ession may be pa ial o comple e. The le el mos commonly a ec ed by his phenomenon is L4–L5, which is also whe e LDH occu s mo e equen ly [6]. The main hypo hesis behind he ini ia ion o spon aneous LDH e- g ession has been desc ibed as he exposu e o he nia ed disc ma e ial o he epidu al ascula supply h ough he up u ed pos e io longi udinal ligamen (PLL) (Fig. 1). * Co espondence: [email p o ec ed] Ca la Cunha and Ana J. Sil a con ibu ed equally o his wo k. Raquel M. Gonçal es and Má io A. Ba bosa con ibu ed equally o his wo k. 1 i3S—Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do Po o, Rua Al edo Allen 208, 4200-135 Po o, Po ugal 2 INEB—Ins i u o de Engenha ia Biomédica, Uni e sidade do Po o, Rua do Campo Aleg e 823, 4150-180 Po o, Po ugal Full lis o au ho in o ma ion is a ailable a he end o he a icle © The Au ho (s). 2018 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Cunha e al. A h i is Resea ch & The apy (2018) 20:251 h ps://doi.o g/10.1186/s13075-018-1743-4 MRI analyses ha allow p ecise quan i ica ions o he dec ease in he nia ion olume ha e shown ha spon an- eous LDH eg ession is mo e ela ed o he p esence o ansligamen ous ex ension and no so much o he ini- ial size o he nia ion. Ahn e al. analyzed longi udinally in ime 36 symp oma ic he nia ed lumba discs and showed ha 25 o hem dec eased in size and ha he mos equen he nia ion ypes ha educed in size we e seques e ed he nia ions (a e age dec eases o 17%, 48%, and 82% o he subligamen ous, ansligamen ous, and seques e ed he nia ion g oups, espec i ely). These e- sul s sugges ha he PLL up u e is mo e impo an han he ini ial size o he he nia [7]. In Takada e al.’s s udy [8], all cases o seques a ed discs we e comple ely esol ed a e 9 mon hs, whe eas ex uded discs we e only comple ely esol ed a e 12 mon hs. On he o he hand, disc p o usions showed li le o no signs o eg ession a e 12 mon hs, mos p obably because o he pa ien s’younge age and he abundance o collagen ibe s and chond ocy e-like cells om he NP in hese discs. The as e a e o adiog aphic esolu ion seen wi h seques a ed discs has been adi ionally associa ed wi h dehyd a ion and sh inkage, as he ee agmen is no longe supplied wi h nu ien s om he pa en disc [9,10]. O he au ho s pos ula e ha seques a ed discs, unlike o he LDH sub ypes, igge an in lamma o y e- sponse cha ac e ized by neo ascula iza ion and immune cell-media ed deg ada ion [11–13]. The subsequen inc ease in blood low a ound ee agmen s explains why he pe iphe y o disc seques a ions is enhanced wi h gadolinium (Gd) con as , which is measu ed by MRI im enhancemen . In ac , Au io e al. [11] p oposed ha seques a ed discs wi h highe le els o Gd die hylene iamine pen aace ic acid (Gd-DTPA) en- hancemen on MRI images se ed as p edic o s o hei highe eso p ion a e. In his s udy, MRI o he nia ed pa ien s was epea ed h oughou 12 mon hs showing ha a signi ican NP eso p ion occu ed un il 2 mon hs a e diagnosis and was mo e p onounced o e he 1-yea ollow-up pe iod. Highe eso p ion a es we e associa ed wi h highe baseline sco es o im enhancemen hickness, highe deg ee o he nia ed NP displacemen in he Komo i classi ica ion, and an age ange o 41–50 yea s. The hickness o im enhancemen was a s onge de e minan o spon- aneous eso p ion han i s ex en . The ex en o im enhancemen signi ican ly co ela ed wi h he deg ee o disc displacemen , being mos p onounced in he case o seques e s. Clinical decision on conse a i e e sus su gical in e en ion o LDH In o de o unde s and spon aneous LDH eg ession, i s p ognos ic ac o s, and i s p edic i e ou comes, la ge co- ho s ha e been conduc ed, namely he Maine Lumba Spine S udy [14], he Spine Pa ien Ou comes Resea ch Fig. 1 aSchema ic ep esen a ion o ypical L4–L5 he nia, wi h comp ession and possible up u e o pos e io longi udinal ligamen (PLL). b Human LDH agmen , ob ained om pa ien who unde wen mic odiscec omy a e in o med consen and e hics commi ee app o al om Cen o Hospi ala São João. cHis ological s aining o issue collec ed in (b), showing cell clus e s p oducing p o eoglycans (Alcian blue) embedded in a collagen ma ix (Pic osi ius ed). dLDH is cu en ly di ided in o ou sub ypes, acco ding o MRI, as bulging disc (mildes o m), p o usion, ex usion, and seques a ion, he se e es o m o LDH. P o eoglycan- ich nucleus pulposus in cen e is su ounded by collagen- ich concen ic ings o annulus ib osus. Scale ba s: (b) 3 cm, (c) 100 μm. Image c edi s: (a,d) used elemen s om Se ie Medical A ; (b, c) unpublished Cunha e al. A h i is Resea ch & The apy (2018) 20:251 Page 2 o 9 T ial (SPORT) [15], and he Hague Spine In e en ion P ognos ic S udy G oup [16], each one en olling hund eds o pa ien s su e ing om LDH. In e es ingly, all o hese s udies indica e ha ea ly su ge y achie es mo e apid elie o LDH symp oms han conse a i e ca e bu , in he long un, ou comes g adually become iden ical o conse a i e ea men . Many o he sys em- a ic s udies ha e consis en ly ob ained simila esul s [17–20]. Bu e mann [21] compa ed he LDH symp oms o pa ien s a e 6 weeks o conse a i e ea men (38 pa ien s) wi h hose who ecei ed epidu al injec ions (20 pa ien s) and ound ha bo h g oups had simila ou - comes, including he size o he nia ed NP. Despi e he e idence o spon aneous LDH eg ession, he e is s ill much deba e among clinicians conce ning he e icacy o conse a i e s nonconse a i e ea - men s. Ini ial managemen o pa ien s wi h he seques- a ed sub ype o LDH may be conse a i e due o he highe likelihood and as e a e o esolu ion in com- pa ison o he o he LDH sub ypes ( e iewed in [6,22]). Pa ien s wi h in ac able pain, neu ological de ici , o bowel o bladde dys unc ion, among o he associa ed ac o s, emain candida es o ea lie su gical in e en- ion. Howe e , i is s ill ha d o p edic which pa ien s a e mo e likely o bene i om conse a i e ea men and ha e highe p obabili y o spon aneous eg ession o he disc he nia ion. I is he e o e ema kably challen- ging o decide which pa ien s o submi o su ge y. Fu- u e la ge, p ospec i e, andomized ials a e equi ed o be e de e mine a se o speci ic clinical c i e ia, such as hose de ined by Chiu e al. [22], ha may p edic he su ge y ou come. On a mo e mechanis ic app oach, inding molecula ma ke s, possibly nonin asi e ones, such as sys emic ma ke s, o p edic he ou come o LDH would ha e a majo impac on he clinical decision. These app oaches a e only s a ing o eme ge, such as he s udy by Elkan e al. [23] in which high plasminogen ac i a o inhibi o 1, a ma ke o ib inolysis, analyzed in blood samples o pa ien s wi h LDH, was ai ly consis - en ly associa ed wi h poo LDH su ge y ou come. Mechanisms behind LDH spon aneous eg ession As al eady desc ibed, he spon aneous eg ession o IVD he nia ed issue is well documen ed clinically, bu he unde lying mechanisms emain unclea . To he bes o ou knowledge, h ee heo ies ha e been p oposed o explain he eso p ion o he nia ed ma e ial. The i s heo y p oposes ha he he nia ed disc agmen educes in size due o g adual dehyd a ion and sh inkage, which may explain he dec ease o signal in ensi y o he disc in he ollow-up MRI s udies [9,10]. The second hypo hesis sugges s ha ension applied by he PLL leads o e ac ion o he he nia ed disc agmen back in o he IVD space. This mechanism may explain he cases whe e he he nia ed disc has an in ac AF, bu no he cases wi h comple ely ex uded o mig a ed disc agmen s [5]. Onel e al. [24] showed ha unde a s a ic ac ion load o 45 kg, he he nia ed issue e ac ed in 21 ou o 30 LDH pa ien s, while in wo o hem he he nia ed issue has ac ually inc eased. The hi d heo y, he mos ex ensi ely s udied wi h p eclinical and clinical e idence o suppo i , is he g adual he nia eso p ion h ough enzyma ic deg ada ion and phagocy osis induced by an in lamma o y eac ion and neo ascula i- za ion [11–13]. This in lamma o y eac ion is supposed o be igge ed when he disc con en ex udes in o he epidu al space and is hen ecognized as o eign. Depending on each indi idual clinical condi ion, i is possible ha one speci ic mechanism o di e en combi- na ions o he h ee may ope a e in spon aneous eg es- sion o he he nia ed disc issue. The p i ileged immuni y o IVD The IVD is he la ges a ascula o gan in he human body and conside ed an immune-p i ileged si e [25]. The NP appea s o be pa icula ly isola ed om he immune sys em o he hos , gi en i s posi ion be ween wo ca ilaginous endpla es and inside he dense collagen ib ous s uc u e o he AF. Addi ionally o his physiological ba ie , he IVD cells also ac i ely esis in asion by immune cells, due o he Fas ligand (FasL) exp ession, which is cha ac e is ic o immune-p i ileged si es [26]. FasL belongs o he umo nec osis ac o (TNF) amily and is known o induce apop- osis by binding o i s ecep o , Fas. While Fas is exp essed in a wide a ie y o cells, FasL exp ession is es ic ed o he su ace o cy o oxic T cells, na u al kille (NK) cells, umo cells, and s omal cells o some immune-p i ileged si es. In immune-p i ileged si es, FasL o s omal cells binds o Fas ecep o exp essed on immune cells and in il a ing cells. This ligand– ecep o binding induces apop osis o he in il- a ing immune cells, main aining he immune-p i ileged condi ion o he issue [27]. The immune-p i ileged en i onmen o he NP is he pilla o he heo y o he in- lamma o y eac ion behind LDH eso p ion. This heo y p oposes ha he ex usion o he NP issue in o he epidu al space e okes an au oimmune eac ion ha leads o he in il a ion o immune cells which will in e ac wi h IVD cells and sec e e a a ie y o molecules ini ia ing he he nia eso p ion p ocess [11,12,28]. Mac ophages as key playe s in LDH eg ession Mac ophages a e indica ed as he mos impo an im- mune playe s in he eso p ion p ocess o he nia ed discs. Nume ous s udies ha e ound by immunohis ochemis y he p esence o mac ophages in he nia ed IVD issue specimens [29–32]. These cells ha e he capabili y o ac i ely phagocy e he he nia ed issue and p ocess i in hei lysosomes illed wi h collagen-deg ading enzymes. Cunha e al. A h i is Resea ch & The apy (2018) 20:251 Page 3 o 9 Mac ophages also sec e e lysosomal enzymes by exocy- osis, which b eak down in e cellula subs ances such as he disc ma ix componen s p o eoglycans and collagens [12,33]. Fu he mo e, phagocy ic ac i i y o mac ophages was obse ed in su gically emo ed samples o he nia ed NP h ough elec on mic oscopy [12] and hese immune cells a e known o exp ess sca enge ecep o s, such as CD36, which ha e been cha ac e ized as he main espon- sible molecules o he phagocy osis o apop o ic cells, highligh ing he po en ial ole o mac ophages in he nia eso p ion [34]. In e es ingly, in LDH his ological samples, mac ophage phagocy osis was obse ed mo e o en in seques a ion sub ype LDH han subligamen ous ones [35], in acco dance wi h he clinical e idence showing ha seques e ed he nias a e mo e likely o eg ess. Ikeda e al. [29] also obse ed mo e equen ly he in il a ion o mac- ophages and neo ascula iza ion along he ma gins o he ansligamen ous ex uded disc ma e ial han in o he sub ypes o LDH. Monocy e ec ui men o IVD issue The exac mechanism by which monocy es a e ec ui ed o he IVD emains unclea , as e iewed p e iously [36]. Howe e , i is known ha he IVD endogenously in- cludes in lamma o y-like cells (i.e., cells wi h phagocy ic capaci y) and IVD cells a e able o p oduce in lamma- o y media o s [36], which may hemsel es con ibu e o ec ui o he immune cells o he he nia si e. In pa icu- la , monocy e chemoa ac an p o ein (MCP)-1, a CC chemokine ha con ibu es o he ac i a ion and ec ui - men o monocy es, has been ex ensi ely demons a ed o be p oduced by IVD cells [13,37–40]. In pa icula , Yoshida e al. [13] de eloped a he nia model in which IVD cells p oduced p oin lamma o y cy okines as an ini ial esponse o disc he nia ion. These cy okines s imula e he p oduc ion o MCP-1 by IVD cells, esul- ing in mac ophage in il a ion in he nia ed discs. The in il a ing monocy e-de i ed mac ophages also p oduce MCP-1, inc easing he monocy e ec ui men o he IVD [13]. Apa om mac ophages, plasmacy oid dend i ic cells ha e also been shown o be in ol ed in LDH e- so p ion [41]. Su gical ma e ial om ansligamen ous and subligamen ous seques a ions was analyzed by low cy ome y and plasmacy oid dend i ic cells we e ound o p edomina e o e mac ophages on ansligamen ous seques a ions, indica ing ha hese cells may be in ol ed in he ini ia ion o he immune esponse [41]. In lamma o y cascades in LDH-implica ing mac ophages Nume ous s udies analyzed immune media o s in LDH and especially implica ing mac ophages in LDH eg es- sion (Table 1). Shamji e al. [32] showed high exp ession le els o mac ophage p oduc s like IL-4, IL-6, IL-12, and in e e on gamma (IFN-γ) in he nia ed disc issue. O he s udies demons a ed ha IVD issue is also capable o spon aneously p oducing o he molecules, such as he chemokines IL-8 and MCP-1, he main unc ions o which a e chemo axis o mac ophages and angiogenesis [37]. Fu he mo e, Kang e al. [42] demons a ed ha he nia ed discs elease high le els o ma ix me allop o- einases (MMPs), ni ic oxide (NO), IL-6, and p os a- glandin E2 (PGE2) and his p oduc ion inc eases when he discs a e s imula ed wi h IL-1β, e idencing ha IVD cells a e biologically esponsi e o exogenous s imuli. Rega ding IL-6, i has al eady been demons a ed in i o ha i s p oduc ion is induced when IVDs and mac- ophages a e cocul u ed [43]. Mo e ecen ly, Takada e al. [44] showed ha cocul u ing IVDs and mac ophages up egula ed IL-8, PGE2, and cyclooxygenase 2 (COX-2). In bo h s udies, he men ioned biochemical media o s a e mainly p oduced by mac ophages. The la e s udy also showed ha a IVD au og a s induced ex ensi e mac ophage in il a ion in i o, inc easing he mRNA le els o TNF-α, IL-6, IL-8, and COX-2. TNF-αis e- qui ed o IL-6 and PGE2 p oduc ion, bu no o IL-8 p oduc ion, du ing IVD–mac ophage in e ac ion. Neu aliza ion o TNF-αand IL-8 may be a aluable he apy o pain ela ed wi h LDH [44]. O he s udies Table 1 Immune media o s implica ing mac ophages in LDH eg ession Immune media o Sample Species S udy IL-6, NO, PGE2, MMP-3, MMP-2/MMP-9 He nia ed IVD issue Human Kang e al., 1997 [42] IL-8, MCP-1 He nia ed IVD issue Human Bu ke e al., 2002 [37] IL-4, IL-6, IL-12, IFN-γHe nia ed IVD issue Human Shamji e al., 2010 [32] MMP-3, MMP-7 Cocul u e o IVD and mac ophages Mouse Ha o e al., 2000 [45] MMP-7, TNF-αCocul u e o IVD and mac ophages Mouse Ha o e al., 2000 [46] MMP-3, MMP-7, TNF-α, VEGF Cocul u e o IVD and mac ophages Mouse Ka o e al., 2004 [47] IL-6 Cocul u e o IVD and mac ophages Ra Takada e al., 2004 [43] IL-8, PGE2, COX-2 Cocul u e o IVD and mac ophages Ra Takada e al., 2012 [44] COX-2 cyclooxygenase 2, IFN-γin e e on gamma, IL in e leukin, IVD in e e eb al disc, LDH lumba disc he nia ion, MCP-1 monocy e chemoa ac an p o ein-1, MMP ma ix me allop o einase, NO ni ic oxide, PGE2 p os aglandin E2, TNF-α umo nec osis ac o alpha, VEGF ascula endo helial g ow h ac o Cunha e al. A h i is Resea ch & The apy (2018) 20:251 Page 4 o 9 ha e also analyzed he in e ac ion be ween mac ophages and IVD issue using cocul u es be ween mac ophages and chond ocy es o whole IVD. Ha o e al. demon- s a ed ha he p oduc ion o bo h MMP-3 and MMP-7 was s ongly up egula ed in IVD cell/mac ophage cocul- u e, MMP-3 being p oduced by bo h chond ocy es and mac ophages while MMP-7 is p oduced p edominan ly by mac ophages. Mo eo e , he au ho s also e ealed ha disc-de i ed MMP-3 is equi ed o he physical deg ada ion o disc issues and o mac ophage in il a- ion, which ul ima ely leads o he nia eso p ion [45]. In o he s udies, he same g oup demons a ed ha MMP-7 eleased by mac ophages con ibu es o he p ocess o he nia ed disc eso p ion h ough he elease o soluble TNF-α[46,47]. TNF-αis a po en induce o many MMPs, such as MMP-3, and also o ascula endo helial g ow h ac o (VEGF) ha is implica ed in he neo ascula iza ion o he nia ed discs [47,48]. The c ucial ole o MMP-7 in he ini ia ion o he nia ed disc eso p ion esul ed in he de elopmen o a ecombinan human MMP-7 in adiscal he apy, which is in phase I/II clinical ials in he Uni ed S a es. This he apy a oids he side e ec s associa ed wi h su ge y, such as ne e issue damage [49]. Neo ascula iza ion in LDH eso p ion Al oge he , hese s udies p o ide e idence o a mecha- nism o LDH eso p ion associa ed wi h a cascade o in- lamma ion, ma ix emodeling, and angiogenesis. Howe e , only ew s udies ha e speci ically add essed neo ascula iza ion. Usually, ew blood essels exis in he ma u e IVD. Howe e , p oli e a ion o new essels has al eady been demons a ed a he ma gin o he he nia ed issue and his is hough o be a majo de e minan o spon a- neous eg ession o LDH [11,50]. Se e al molecula me- dia o s ha e been sugges ed o be in ol ed in he neo ascula iza ion o LDH, including TNF-α, VEGF, and basic ib oblas g ow h ac o (bFGF). As men ioned ea lie , TNF-αcan p omo e he exp ession o VEGF, which plays an essen ial ole in he o ma ion o new blood essels, and some s udies con i med he p esence o VEGF and VEGF ecep o s in human LDH issue [48,51]. Also, he in e ac ion be ween mac ophages and disc issue leads o gene a ion o in lamma o y cy okines, which a e known o be in ol ed in he induc ion o angiogenesis. Ha o e al. obse ed an in- c ease in he mRNA and p o ein le els o VEGF when mac ophages we e in con ac wi h disc issue compa a i ely o he exp ession le els when hey we e cul u ed alone. This enhancemen o he VEGF le els was appa en ly media ed by a TNF-α-dependen pa hway since his e ec was ab oga ed by he use o aTNF-αneu alizing an ibody [48]. Using MRI in humans, he p esence o capilla ies in ading he he nia and monocy e-de i ed mac ophages mig a ing ou o hese capilla ies has been demons a ed [12]. Addi ionally, in an expe imen al model in abbi s, bFGF, a ac o known o s imula e mi ogenesis and chemo axis o ib oblas s and capilla y endo helial cells, as well as o s imula e angiogenesis, p omo ed he eso p ion o disc ma e ial. In his s udy, ea - men wi h bFGF led o an inc ease in he numbe o newly o med essels wi h a consequen highe in il- a ion o in lamma o y cells (mac ophages, lym- phocy es, and ib oblas s), which con ibu ed o he eso p ion p ocess [52]. These s udies ein o ce he impo ance o he c oss alk be ween angiogenesis and in lamma ion, which ul i- ma ely leads o LDH eg ession. Con ibu ion o o he immune esponse media o s An au oimmune eac ion is a mo e complex han he me e mac ophage ec ui men and neo ascula iza ion so a desc ibed. Indeed, apa om mac ophages, o he immune cells a e p esen in LDH (Fig. 2). The comple e unde s anding o he immune esponse associa ed wi h LDH eg ession has bene i ed o a g ea ex en om in- i o animal models. Mo e han in- i o e idence o LDH eso p ion, al eady ho oughly clinically demon- s a ed, animal models ha e been employed o un a el he unde lying mechanisms o IVD eg ession. We ha e shown spon aneous eg ession o LDH in a a IVD le- sion model [53]. In his wo k, IVD lesion was induced by ei he 21-G o 25-G needle punc u e and we ha e ound ha he size o he he nia o med was p opo - ional o he needle gauge used. In bo h cases, he nias signi ican ly diminish in olume om 2 o 6 weeks pos inju y. Also, we ound ha he numbe o CD68 + mac o- phages wi hin he he nia as well as cell apop osis wi hin he issue we e bo h p opo ional o he he nia olume. Using he same model, we u he con i med ha he numbe o CD68 + mac ophages in he he nia was p o- po ional o i s size and hypo hesized ha only a ce ain numbe o mac ophages will be ec ui ed and ac i a ed pe a ea o he nia, keeping he issue homeos asis. Mo eo e , we ha e ound ha he sys emic ansplan - a ion o a bone ma ow MSC esul ed in a signi ican educ ion in he size o he he nias o med 2 weeks pos lesion and ha he numbe o B lymphocy es su oun- ding he he nia inc eased [54]. In ano he s udy, a abbi he nia ion model was de eloped, consis ing o he in o- duc ion o a needle up o pene a ing he PLL and hen physically comp essing o ex ude he NP o he epidu al space [13]. The he nia ed discs spon aneously educed in size g adually up o 12 weeks pos su ge y. In il a ing cells, mainly composed o mac ophages, we e obse ed om day 3. Immunohis ochemically, IVD cells in he Cunha e al. A h i is Resea ch & The apy (2018) 20:251 Page 5 o 9 he nia ed discs p oduced TNF-αand IL-1βon day 1, ollowed by MCP-1 on day 3. Ne e heless, mos o he cu en in- i o LDH models a e au o ansplan a ion models in which IVDs a e ansplan ed in o subcu aneous o do sal epidu al spaces o he animals a e laminec omy, wi h he scope o speci ically analyzing he associa ed immune esponse. This is because i is s ill con o e sial whe he he in lamma o y esponse o he hos is ini ia ed simply by exposu e o s uc u al elemen s and compounds ha a e p esen in he disc cell memb ane and ma ix, by di ec con ac o he NP ma e ial wi h he immune sys em ia an au oimmune esponse, o whe he i is seconda y o an au o- immune esponse. I has been shown p e iously ha subcu aneously ansplan ed NP cell su i al was e- duced in associa ion wi h an immunological eac ion, by ansplan ing NP issue in o Lewis a s and in o NOD mice, and ha NK cells and mac ophages we e p esen a ound he ou g own NP issues bu no T cells we e ound [55]. Ano he s udy implan ed au ologous NP subcu aneously in pigs and ac i a ed T cells (CD4 + and CD8 + ) we e ound in he exuda es in conside able numbe , as well as ac i a ed B cells ex- p essing immunoglobulin kappa (Igκ). The esul s showed ha NP a ac s ac i a ed T and B cells [56]. Wha mos o hese s udies show is ha he immune sys em is able o espond o he in ac heal hy NP issue. To analyze whe he in lamma ion occu s in esponse o compounds sec e ed om iable cells in he NP o whe he in lamma ion simply equi es exposu e o s uc- u al cell o ma ix componen s, Rand e al. [57] used a mouse model in which he animal was exposed o disc issue con aining iable NP and AF cells, o disc issue con aining iable AF cells, o o disc issue wi h no iable cells. The h ee issue p epa a ions we e inse ed in o he igh lowe pe i oneal ca i y. The de i alized is- sue was in ended o assess a possible e ec o chemical i i an s ha may be p esen in he disc and ha may in- duce in lamma ion. Mac ophage ec ui men occu ed o e he cou se o 1, 2, and 7 days pos inju y and only a e exposu e o iable disc issue bu no a e expos- u e o de i alized disc componen s [57], indica ing ha mac ophage ec ui men occu ed only in esponse o cell cues. Mos o he e idence o he in lamma o y eac ion a ound spon aneous he nia eg ession has been collec ed om animal s udies. Despi e his, some s udies mos ly in ol ing immunohis ochemis y analysis o clinical samples ha e con i med he esul s ob ained in animal expe imen a ion. The p esence o T and B lymphocy es on isola ed human he nia ed discs was u he con i med by he same g oup [58]. Also, his ological analyses o human he nia ed discs e ealed he p esence o in il a ed T cells [59]. Fu he mo e, in human he - nia ed issue, lymphocy es we e ound o be h ee imes mo e abundan in seques a ed he nias han in ex u- sions, while no o he in lamma o y cells we e seen in p o usions apa om mac ophages [60]. In ano he s udy, he in lamma o y in il a e has been cha ac e ized by immunos aining in po ions o he nia ed discs which unde wen su ge y o LDH. None o he 38 samples exp essed he immunopheno ypic ma ke s o he lymphocy e (CD20, CD45RO, CD4, CD8, TCR), ma u e monocy e (CD33), o dend i ic cell (CD1a, CD80, CD86, S100). Howe e , an abundan in il a ion o CD68 + cells ha lacked CD33 wi h a iable amoun s o CD11b, CD11c, and CD40 was obse ed, likely ep esen ing a p ocess o di e en ia ion om monocy es o mac o- phages [61]. Fig. 2 Rep esen a i e p oposed mechanism o LDH eso p ion. Bo h he nia ed IVD issue and mac ophages p oduce umo nec osis ac o alpha (TNF-α), monocy e chemoa ac an p o ein (MCP)-1, ma ix me allop o einases (MMPs), in e leukin (IL)-6, IL-8, p os aglandin E2 (PGE2), cyclooxygenase 2 (COX2), and ni ic oxide (NO), which con ibu e o he in lamma o y eac ion and eso p ion o he he nia ed issue. Vascula endo helial g ow h ac o (VEGF) induces blood essel ing ow h and neo ascula iza ion, which suppo immune cell mobiliza ion o he nia si e. Inse : in a model o IVD he nia ion, CD68 + mac ophages localized wi hin he nia (delimi a ed by dashed line), which include a blood essel (a ow). Scale ba : 100 μm. Image used elemen s om Se ie Medical A ; inse : unpublished Cunha e al. A h i is Resea ch & The apy (2018) 20:251 Page 6 o 9 Conclusions LDH spon aneous eso p ion is well documen ed clini- cally and in p eclinical s udies. Spine su geons a e becoming inc easingly awa e o his phenomenon and many ecognize he use ulness o conse a i e ea men o LDH and ad ise pa ien s acco dingly. Di e en o ms o nonsu gical ea men s should be exhaus ed be o e conside ing su ge y in acu e s ages o LDH, unless conse a i e ea men is con aindica ed o easons such as neu ological de ici and in ole able pain despi e adminis a ion o adequa e pain medica ions. I is clea ha he in lamma o y esponse ha occu s associa ed wi h LDH is c ucial o i s spon aneous e- so p ion. The e o e, in lamma ion in his speci ic clinical con ex is a good p ognos ic indica o and should no be hal ed. S ill, i is exac ly an in lamma o y esponse ha causes a ha m ul e ec on he adjacen ne e oo s, causing pain. The con ol o he in lamma o y eac ion in his se ing is an impo an challenge when ea ing pa ien s wi h LDH. The combina ion o knowledge om he biological mechanisms behind LDH eso p ion and he de ailed pe sonalized diagnosis will be he de e min- an o ailo ea men o each indi idual pa ien and may ul ima ely lead o educ ion in cos s o he heal h sys em. Abb e ia ions AF: Annulus ib osus; IVD: In e e eb al disc; LDH: Lumba disc he nia ion; MSC: Mesenchymal s em cells; NP: Nucleus pulposus Acknowledgemen s The igu es used elemen s om Se ie Medical A , p o ided by Se ie , licensed unde a C ea i e Commons A ibu ion 3.0 unpo ed license (h p:// sma .se ie .com). The au ho s decla e no po en ial con lic o in e es s. Funding This wo k was inanced by p ojec “Bioenginee ed The apies o in ec ious diseases and issue egene a ion”(NORTE-01-0145-FEDER-000012), suppo ed by No e Po ugal Regional Ope a ional P og amme (NORTE 2020), unde he PORTUGAL 2020 Pa ne ship Ag eemen , h ough he Eu opean Regional De elopmen Fund (ERDF), by FEDER/COMPETE 2020 (POCI), Po ugal 2020, and by Po uguese unds h ough FCT/MCTES in he amewo k o he p ojec "Ins i u e o Resea ch and Inno a ion in Heal h Sciences" (POCI-01- 0145-FEDER-007274). Cunha C and Gonçal es RM acknowledge FCT by hei pos doc ellowship (SFRH/BDP/87071/2012) and FCT In es iga o G an (IF/ 00638/2014), espec i ely. Sil a AJ acknowledges he ellowship unde he amewo k o he p ojec No e-01-0145-FEDER-000012. A ailabili y o da a and ma e ials Da a sha ing no applicable o his a icle as no da ase s we e gene a ed o analyzed du ing he cu en s udy. Au ho s’con ibu ions CC, AJS, RMG, and MAB concei ed he s udy. CC, AJS, PP, RV, RMG, and MAB w o e he manusc ip . All au ho s ead and app o ed he inal manusc ip . E hics app o al and consen o pa icipa e No applicable. Consen o publica ion No applicable. Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Publishe ’sNo e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a ilia ions. 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