rs - | SPINE Volume 23, Number 23, pp 2538-2543 819%, Lippincnt Willams & Wiking ® Tumor Necrosis Factor a and Nucleus-Pulposus-Induced Nerve Root Injury Kjell Olmarker, MD, PhD, and Karin Larsson, BSc ‘Study design. The effects of nucleus pulposus and var- ious treatments to block tumor necrosis factor « activity ‘were evaluated in an experimental setup using immuno- histochemistry and nerve conduction velocity recordings. Objectives. To assess the presence of tumor necrosis factor « in pig nucleus pulposus calls, and to see if block ‘of tumor necrosis factor a also blocks the nucleus-pulpo- sus-induced reduction of nerve root conduction velocity ‘Summary of Background Data. A meta-analysis of observed effects induced by nucleus pulposus revealed that these effects might relate to one specific cytokine — tumor necrosis factor «, Methods. Series-1: Cultured nucleus pulposus cells were stained immunohistologically with a monoclonal antibody for tumor necrosis factor a. Series-2; Nucleus pulposus was harvested from lumbar dises and applied to the sacrococeygeal cauda equina in 13 pigs autolo- ously. Four pigs received 100 mg of doxycycline intra vyenously; five pigs had a blocking monoclonal antibody to tumor necrosis factor « applied locally in the nucleus pulposus, and four pigs remained nontreated, forming a Control group. Three days after the application, the herve root conduction velocity was determined over the pplication zone by local electrical stimulation Results, Series-1: Tumor necrosis factor u was found to be present in the nucleus pulposus cells, Series-2: The selective antibody to tumor necrosis factor « lim- ited the reduction of nerve conduction velocity, al though in comparison with the control group this was not statistically significant. However, treatment with doxyoycline significantly blocked the nucleus-pulposus induced reduction of conduction velocity, Conclusion. For the first time, a specific substance, tumor necrosis factor a, has been linked to the nucleus- pulposus-induced effects of nerve raots after local ap plication. Although the effects of this substance may be ‘synergistic with those of ather similar substances, the data of the current study may be of significant impor- tance for the continued understanding of nucleus pulpo- sus" biologie activity, and of possible potential use for future strategies in managing sciatica. [Key words: cells From the Department of Orthopaedics, Sahlgrenska Universiey Hosp tal, Goteborg University, S413 45 Gorhenbuirg, Sweden, Supported by grants from the Swedish Medical Research Cour (8645, 9758), the Swedish Medical Society, the Gothenburg Medical Sosiety, the Greta och Finar Asker Research Foundation, the Carn Trygger Memorial Foundation, the higa Bint and Arne Lundberg Re search Foundation, the Felix Neubergh Foundation, the Handladen Hislmar Svensson Foundation, and the SALUS Medial Research 2538, disc herniation, intervertebral disc, low back pain, nerve root, nucleus pulposus, sciatica, tumor necrosis factor] Spine 1998,23:2538-2544 After being regarded in the past as just a biologically inactive tissue component compressing the spinal nerve root at disc herniation, the nucleus pulposus recently has been found highly active, inducing both structural and functional changes in adjacent nerve roots when applied epidurally.*°7%12 fe has thereby been established that autologous nucleus pulposus may induce axonal changes, a characteristic myelin injury,’ in sed vascular permeability,’ and intravascular co- agulation;?" and that membrane-bound structures or substances of the nucleus pulposus cells are responsible for these effects.°*” The effects also have been found to be efficiently blocked by methylprednisolone and cyclo sporin A A critical look at these data reveals that at least one cytokine relates to all of these effects —tumor necrosis factor a (TNFa). To assess if TNFa may be involved in the nucleus-pulposus-induced nerve root i jury, the authors assessed for the presence of TNFa in nucleus pulposus cells and studied to discover whether the nucleus:pulposus-induced effects could be blocked by doxyeycline and by a selective monoclonal antibody ‘™ Material and Methods Series-I: Presence of Tumor Necrosis Factor a in Pig Nucleus Pulposuss Cells, Nucleus pulposus from a total of lumbar and thoracic discs was obtained from a pig used ft other purposes. The nucleus pulposus was washed once it Han’ F12 medium (Gibco BRL, Paisley, Scotland) and the centrifuged and suspended in 5 mi. of collagenase solution Ham's F12 medium (0.8 mg/mL, Sigma Chemical Coy St Louis, MO) for 40 minutes at 37°C in 25 emt tissue cule flasks, The separated mucleus pulposus cell pellets were st pended in DMEM/F12 1:1 medium (Gibco BRL, Paisley, St land) supplemented with 19% L-glutamine 200 moll. (Gib? BRL, Paisley, Scotland), 50 jxgimL gentamycine sulphate (Gibco BRL, Paisley, Scotland), and 10% fetal calf serum (Gibco BRL, Paisley, Scotland). The cells were eukuted at 37°C COs in air for 3 to 4 weeks and then cultured direct oon tssue-culture-treated lass slides (Becton Dickinson Company Labware, Franklin Lakes, Nf), Alter 5 days oa lass slides, the cells were fixed in situ by acetone for 10 mit — «4‘TNFa and NP-Induced Nerve Root Injury * Olmarker and Larsson 2539 ses, Once irrelevant antigens had been blocked by application {013% H,0, (Sigma Chemical Company, St.Louis, MO) for 30 minutes, and horse serum (ImmunoPure ABC, peroxidase tmovse IgG staining kit no, 32028; Pierce, Rockford, IL for 20 ‘minutes, the primary antibody (ant-pig INFa monoclonal pu ‘ied ancbody; Endogen, Cambridge, MA) was applied over: hight at 4, diluted at 1:10, 1:20, and 1:40. For control ovine serum albumin (BSA) (Intergen Company, New York, NY} suspended in phosphate-buffered saline (PBS) (Merck, Darmstadt, Germany) was applied in the same fashion. The text day the cells were washed with 1% BSA in PBS, and the secondary antibody (ImmunoPure ABC, peroxidase mouse IgG Saining kit nr.32028; Pierce, Rockford, IL) was applied for 30 tinates, To enhance this reaction, the cells were exposed to ‘Aviin-Biotin complex for an additional 30 minutes (Immuno Pare ABC, peroxidase mouse IgG staining it nr.32028; Pierce, Rockford, IL), The cells were then exposed to 20 mg of DAB {G4diaminobencidine tetrahydrochloride nr.D-5905; Sigma Chemical Company, St. Louis, MO) and 0.033 ml. of 3% 1,0, in 10 ml of saline for 10 minutes. The cells were washed inPBS, dehydrated in a scries of ethanol, mounted, and exam ined by light microscopy by an unbiased observer regarding the presence of a brown coloration chat indicated the presence of TNFa. Series-2: Neurophysiologic Evaluation, Thirceen pigs (body weight, 25-30 kg) received an intramuscular injection of 20mgkg body weight of Ketalar (ketamine 50 mg/mL; Parke Davis, Morris Plains, NJ), an intravenous (IV) injection of 4 mg/kg body weight of Hypnodil (methomidate chloride 5O mg mL; AB Leo, Helsingborg, Sweden), and 0.1 mg/kg, body ‘weight of Stresnil (azaperon 2 mg/ml ; Janssen Pharmaceutica, Beerse, Belgium). Anesthesia was maintained by additional TV injections of 2 mg/kg body weight of Hypnodil and 0.05 mg/kg body weight of Stresnil, The pigs also received an TV injection of 0.1 mg/kg of Stesolid Novum (diazepam; Dumex, Helsing borg, Sweden) after surgery. [Nucleus pulposus was harvested from the fifth lumbar dise bya retroperitoneal approach.*® Approximately 40 mg of the nucleus pulposus was applied to the sacrococcygeal cauda ‘equina through a midline incision and laminectomy of the frst coceygeal vertebra, Four pigs did not receive any treatment (no treatment group). Four other pigs received an IV infusion of 100 mg of doxycycline (Vibramycin; Pfizer Inc., New York, NY)in 100 mL of saline over a period of 1 hour. In five pigs, the rucleus pulposus was mixed before application with 100 ul. of 41.11 mg/ml. suspension of the anti-TNFa antibody used in series 1 Three days after the application, the pigs were reanesthe tized by an intramuscular injection of 20 mg/kg body weight of Ketalar and an IV injection of 35 mg/kg, body weight of Pento- thal (Thiopental sodium; Abbott Lab, Chicago, IL). The pigs were ventilated on a respirator. Anesthesia was maintained by an lV bolus injection of 100 ma/kg body weight of Chloralose (@-D(+)-gluco-chloralose (Merck, Darmstadt, Germany) and bya continuous supply of 30 mg/kg per hour of Chloralose. A laminectomy from the fourth sacral to the third coccygeal ver tebra was performed. The nerve roots were covered with Spon- ‘ostane (Ferrosan, Bagswaerd, Denmark), Local tissue temper- ature was continuously monitored and maintained at 37.5 38.0°C by means of a heating lamp. The cauda equina was stimulated by two E2 subdermal platinum needle electrodes (Grass Instrument Company, Quincy, MA), which were connected to a Grass SD9 stimulator (Grass Instrument Company, Quincy, MA) and gently placed intermittently on the cauda equina, first 10-mm cranially and ‘then 10 mm caudally to the exposed area. To ensure that only impulses from exposed nerve fibers were registered, the nerve root that exited from the spinal canal between the two stimu lation sites was cut, An electromyogram (EMG) was registered by two subdermal platinum needle electrodes, which were placed into the paraspinal muscles inthe tail approximately 10, mm apart. This proceduce is reproducible and represents a functional measurement of the motor nerve fibers of the cauda equina nerve roots. The EMG was visualized using a Macin- tosh Ilci computer provided with Superscope software and Ma~ ‘Adios II A/D converter (GW Instruments, Sommerville, MA) together with a Grass P18 preamplifier (Grass Instrument ‘Company, Quincy, MA). The separation distance between the first peaks of the EMG from the two recordings was deter mined, and the separation distance between the two stimula- tion sites on the cauda equina was measured with calipers. The nerve conduction velocity between the two stimulation sites could thus be ea “The person performing the neurophysiologic analyzes was unaware of the experimental protocol for the individual ani mal. After the study was completed, the data were arranged in the three experimental groups, and statistical differences be- tween the groups were assessed by Student's t test. The exper- {mental protocol for this experiment was approved by the local animal research ethies committee. m Results Series-1: Presence of Tumor Necrosis Factor « in Pig ‘Nucleus Pulposus-Cells Examples showing the light microscopic appearance of, the stained glass slides can be seen in Figure 1. In the sections using BSA in PBS as “primary antibody” (con- trols), there was never any staining seen, ensuring that, there was no labeling and visualization of irrelevant an. tigens. When the anti-TNFa antibody was applied a 1:40 dilution, only a weak staining occurred. However, the staining increased with diminishing dilutions of the antibody. The staining was seen in the soma of the cells, and it was not possible to determine whether TNFa was located in the cytoplasm, on the cell surface bound to the cell-membrane, or both, lated from these two measurements. Series-2: Neurophysiologic Evaluation Application of nonmodified nucleus pulposus without any treatment induced a reduction in nerve conduction ‘elocity similar to that in previous studies (no treatment; Figure 2), whereas treatment with doxycycline com- pletely blocked this reduction (P< 0.01, Student's t test) Local application of anti-TNFa-antibody also induced a partial block of this reduction, although not as complete as did doxycycline and not statistically significant com pared with the no treatment group. 1m Discussion The data of the current study demonstrated that TNFa may be found in nucleus pulposus cells of the pig. If2540 _Spine + Volume 23 + Number 23 + 1998 Figure 1. Immunohistachemistry of nucleus pulposus cells regarding the presence of tumor necrosis factor « (TNFa). No brown staining 's seen in the control, which used bovine serum albumin in phosphato-buflered saline as the “primary antibody,” ensuring that nO irrelevant antigens were visualized. A clear staining of the nucleus pulposus cells was seen when they were labeled with a monoclonal anti-TNFa antibody that increased in intensity with increasing concentration. Bar — 100 ym TNFa was blocked by a selective monoclonal antibody, the nucleus-pulposus-induced reduction of nerve root conduction velocity was partially blocked, although this ‘was not statistically significant as compared with series of nontreated animals. However, if doxyeycline was used to inhibit TNFa, the reduction of nerve conduction ve- locity was significantly blocked. In recent years, it has been verified that local applica tion of autologous nucleus pulposus may injure the ad- jacent nerve roots. Thus, it has become evident that the nerve root injury seen at disc herniation may not be based solely on mechanical deformation of the nerve root, bur could also be induced by unknown “biochem: ical effects” related to the epidural presence of herniated nucleus pulposus. Although this new research field has generated many experimental studies, the mechanisms and substances inyolved are not fully known. It has been, seen that local application of autologous nucleus pulpo- sus may induce axonal injury," 4 characteri tic injury of the myelin sheath,”*84"-*2 a local increa of vascu tion, 1 permeability,” intravascular coagula- reduction of intrancural blood flow,*? and leuko- taxis."* Te also has been observed that the nucleus pulposus-related effects may be blocked efficiently by methylprednisolone™ and cyclosporin A,? and slightly less efficiently by indomethacin’ and lidocain.®? Further- more, it has been understood that the effects are med ated by the nucleus pulposus cells,” particularly by sub: stances or structures bound to the cell membranes.* When these data are considered critically, it becomes evident that atleast one specific cytokine could be related to these observed effects— TNFa, Tumor necrosis factor @ may induce nerve injury,?*"™455°" mainly seen as 2 characteristic myelin injury that closely: resembles the nucleus-pulposus-induced myelin — inj ry PASE SEENESES Te also may induce an increase it vascular permeability"’*** and initiate coagula- tion." Furthermore, TNFa may be blocked by st roids**2'°"°% and cyclosporin A.! 5567.8 Howevet the blocking effect on TNEa is not so pronounced byTNFa: and NP-Induced Nerve Root Injury + Olmarker and Larsson 2541 Nev (mis:S0) 100 Saaanana 90 80;—— 70 60 50: 40 30+— 20: 10 ee Fgure 2. It is known that application of retroperitoneal fat, con- Sideted as @ contro, for 3 days will result in a conduction velocity, f+ § m/sec.* In the current study, nerve conduction velocity \was reduced in the nontreated group in which normal, autologous rucleus pulposus was applied. Local application of a blocking ‘monoclonal antibody of TNFa to the nucleus pulposus limited this ‘eduction, although the velocity was not statistically diferent from the velocity of the nontreated serias. However, intravenous treat- ment by doxycycline resulted in a mean conduction velocity that, was near that observed after application of retroperitoneal fat as, described earlier NSAIDs,!*1720 Recently, it was observed that local application of nucleus pulposus may induce pain-related behavior in fats, particularly thermal hyperalgesia.” *” Tumor ne crosis factor a also has been found related to such pain: behavioristic changes as thermal hyperalgesia,!”"**"°°"° and also to neuropathies in general.'°"*""""" However, no studies have assessed the possible presence of TNFa in the cells of the nucleus pulposus. To assess whether TNFa could be related to the ob- served nucleus-pulposus-induced reduction in nerve root conduction velocity, the authors first had to analyze and see if there was TNFa in the nucleus pulposus cells. ‘The data clearly demonstrated that TNFa was present in these cells (see Figure 1). Tumor necrosis factor a is pro daced as precursor (pro-TNF) bound to the membrane, and its activated by cleavage from the cell membrane by 2 zine-dependent metalloendopeptidase (TNFa converting enzyme, TACE).© 1168"? may relate well to experimental findings in which appli: cation of the mere cell membranes of autologous nucleus pulposus cells induced nerve conduction velocity reduc tion, which indicated thar the effects were mediated by a membrane-bound substance.”* and very low or the opposite by lido- This therefore Second, the effects of the TNFa had to be blocked in a controlled manner. The authors thus first chose to add the same selective antibody used for immunohistochem istry in series 1, which is known also to block the effects of TNFa, to the nucleus pulposus before application ‘Also, they chose to treat the pigs with doxycycline, which is known to block TNFa.2*7"**2°% However, due to the low pH of the doxycycline preparation, it was de~ cided to treat the pigs by IV injection instead of local addition to the nucleus pulposus because nucleus pulpo: sus ata low pH has been found to potentiate the effects of the nucleus pulposus."*”* The data regarding nerve conduction velocity showed that the reduction was completely blocked by the doxy- cycline treatment, and that the nerve conduction velocity in this series was near that observed after application of a control substance (retroperitoneal fat) from a previous study.’ Application of the anti-TNFa-antibody to the nucleus pulposus also partially blocked the reduction in velocity, but not as much as with doxycycline, and the velocity in this series was not statistically different from, the velocity in the series with nontreated animals, the wide deviation of the data. The fact that the anti-TNF antibody treatment only partially blocked the nucleus-pulposus-induced reduc- tion of nerve conduction velocity and the high standard deviation of the data could probably have at least (wo different explanations. First, in looking at the specific data in this group we find that the nerve conduction velocity was low in two animals (mean, 37.5 m/sec) and high in three animals (mean, 81.3 m/sec). There are thus ‘wo groups of distinctly different data in the anti-TNFa, treatment series. This will account for the high standard, deviation and might imply that the blocking effect was, sufficient in three animals and insufficient in two animals. The lack of effects in these animals could be based simply on the insufficient amount of antibodies in relation to, TNFa molecules. Thus, if higher dose of the antibody had been used, the TNFa effects would have been blocked even in these animals. Such a scenario could then theoretically imply that TNFa alone is responsible for the observed nucleus-pulposus-induced effects, and that this could not be verified experimentally due to the amount of antibody being too low. Second, it also is known that tetracyclines such as doxyeycline and minocycline may block a number of cytokines and other substances. For instance, they may block IL-1,!28°* IFNz,2” NO-synthetase,! and metallo prot °5* Particularly I-1 and IF to act synergistically with TNFa and to be neurotoxic. !01*'*1956? These substances also are blocked by steroids and cyclosporin A, which corre sponds well with the previous observations on nucleus pulposus-induced nerve root injury showing that the nu- cleus-pulposus-induced effects may be blocked by these substances.**” Therefore, the possibility also may be considered that a selective block of TNFa may not be re knownOO = CClti(‘“‘=:S 2542 Spine + Volume 23 + Number 23 + 1998 | sufficient to block completely the nucleus-pulposus~ induced effects on nerve function, and that simultaneous block of other synergistic substances is necessary as well Thus, this scenario, on the other hand, implies that TNFais not solely responsible for the nucleus-pulposu: induced effects, and that other synergistic substance which also are blocked by doxycycline, are necessary Tumor necrosis factor a may have various pathophys iologic efects. It may have direct effects on tissues such as nerve tissue and blood vessels. It may trigger other cells to produce other pathogenic substances, and it perhaps triggers release of more TNFa, both by inflammatory cells and also by Schwann cells locally in the nerve tis sue.°° Thus, there is reason to believe that even low amounts of TNFa may be suificient to initiate these pro: cesses, and that there is a local recruitment of cytokine- producing cells and a subsequent increase in production and release of other cytokines as well as TNFa. Tumor necrosis factor a may therefore act as the “ignition key” of the pathophysiologic processes and play an important role for the initiation of the pathophysiologic cascade behind the nucleus-pulposus-induced nerve injury In conclusion, although the exact role of TNFa can not be fully understood from the experimental setup, it can be concluded that for the first time a specific sub, stance, TNFa, has been linked to the nucleus-pulposus induced nerve root injury, probably potentiated by other substances that also are blocked by doxycycline, such as IL-1, IFNr, and NO-synthetase. This new information may be of significant importance for the continued un- derstanding of nucleus-pulposus-induced nerve injury. It ‘may raise the question as to the potential future clinical use of pharmacologic interference with TNFa and re lated substances, such as doxycycline o metalloprotein ase inhibitors, for the management of sciatica. References 1. Amin AR, Attur MG, Thakker GD, et al. 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