SPINE Volume 26, Number 8, pp 863–869
©2001, Lippincott Williams & Wilkins, Inc.
Selective Inhibition of Tumor Necrosis Factor-␣
Prevents Nucleus Pulposus-Induced Thrombus
Formation, Intraneural Edema, and Reduction of Nerve
Conduction Velocity
Possible Implications for Future Pharmacologic Treatment Strategies
of Sciatica
Kjell Olmarker, MD, PhD, and Björn Rydevik, MD, PhD
Study Design. The possibility to prevent nucleus pul-
posus-induced functional and structural nerve root injury
by selective tumor necrosis factor-␣ inhibition was as-
sessed in an experimental model in the pig spine.
Objective. The objective of the study was to evaluate
the role of tumor necrosis factor-␣ in the mediation of
nucleus pulposus-induced nerve injury by using selective
inhibition.
Summary of Background Data. The cytokine tumor
necrosis factor-␣ has been suggested to play a key role in
the nerve root injury induced by local application of nu-
cleus pulposus. However, previous studies have not been
able to distinguish the effects between tumor necrosis
factor-␣ and other disc-related cytokines because of the
use of nonspecific cytokine inhibition.
Methods. Autologous nucleus pulposus was har-
vested from a lumbar disc and applied to the porcine
sacrococcygeal cauda equina. The pigs were simulta-
neously treated with two selective tumor necrosis fac-
tor-␣ inhibitors (etanercept n ⫽ 8 and infliximab n ⫽ 5), a
heparin analogue (enoxaparin n ⫽ 5) or saline for control
(n ⫽ 5). After 7 days the nerve conduction velocity over
the application zone was determined and samples of the
exposed nerve roots were collected for light microscopic
evaluation.
Results. The two tumor necrosis factor-␣ inhibitors
prevented the reduction of nerve conduction velocity and
also seemed to limit the nerve fiber injury, the intracapil-
lary thrombus formation, and the intraneural edema for-
mation. However, treatment with enoxaparin did not
seem to be different from control regarding reduction of
nerve conduction velocity or histologic changes.
Conclusions. The data clearly indicate that tumor ne-
crosis factor-␣ is involved in the basic pathophysiologic
events leading to nerve root structural and functional
changes after local application of nucleus pulposus. The
study therefore provides a basic scientific platform with
potential clinical implications regarding the use of anti-
tumor necrosis factor-␣ medication as treatment in pa-
From the Department of Orthopaedics, Göteborg University, Gothen-
burg, Sweden.
Supported by grants from the Swedish Medical Research Council
(8685), the Swedish Medical Society, the Gothenburg Medical Society,
the Greta och Einar Asker Research Foundation, the Inga Britt and
Arne Lundberg Research Foundation, the Félix Neubergh Foundation,
the Handlanden Hjalmar Svenssons Foundation, and the SALUS Med-
ical Research Foundation.
Acknowledgment date: February 7, 2000.
First revision date: April 25, 2000.
Acceptance date: July 7, 2000.
Device status category: 1.
Conflict of interest category: 14.
_
tients with disc herniation and sciatica. [Key words:
spine,spinal nerve roots, intervertebral disc, herniated
disc, sciatica, cytokine, TNF-␣, nerve fiber] Spine 2001;26:
863– 869
The knowledge of the basic pathophysiologic events
leading to the symptoms of disc herniation has dramati-
cally increased during recent years.9,18,19,36,38 Thus, cur-
rent concepts regarding sciatica indicate that this condi-
tion is not the result only of mechanical deformation of a
lumbar nerve root. Instead, it seems that the nerve root
becomes sensitized by nucleus pulposus material that has
reached the epidural space because of a disc herniation
and that mechanical deformation of such a sensitized
root may induce pain.36,39 The cytokine tumor necrosis
factor-alpha (TNF-␣) has been suggested to play a key
role in mediating this sensitization, as well as other nu-
cleus pulposus-induced effects, such as edema formation,
intravascular coagulation, blood flow reduction, and
myelin splitting.36 TNF-␣ is known to be produced and
released from the chondrocyte-like cells of the nucleus
pulposus.36 Nonspecific cytokine inhibition by doxycy-
cline has been shown experimentally to block the nucleus
pulposus-induced effects on the nerve roots.36 It was also
recently shown that local application of TNF-␣ induces a
reduction of nerve conduction velocity similar to nucleus
pulposus, whereas application of other disc-derived cy-
tokines, such as interleukin-1-beta (IL-1␤) and interfer-
on-gamma (IFN-␥), does not.2
In the present study, we assessed if two selective
TNF-␣ inhibitors that are currently used clinically for the
treatment of Crohn’s disease and rheumatoid arthritis
could block the nucleus pulposus-induced reduction of
nerve root conduction velocity in an experimental setup.
The first aim of the study was to investigate if selective
inhibition of only TNF-␣ could prevent the nucleus pul-
posus-induced reduction in nerve conduction velocity,
thereby further defining the role of TNF-␣ for inducing
the nucleus pulposus-related pathophysiologic effects on
the nerve roots. The second aim was to study if a possible
prevention of the nucleus pulposus-induced nerve con-
duction velocity reduction was linked to a corresponding
reduction of intraneural thrombus formation and
edema, both of which have been suggested to be part of
863
864 Spine • Volume 26 • Number 8 • 2001

the pathophysiologic events leading to the reduced nerve tion using the same dose 3 days after surgery. Five pigs received
conduction velocity. In addition to this second aim, we an intravenous infusion of 100 mg of Remicade (infliximab,
chose also to assess the same effects for an antithrom- Centocor B.V., Leiden, The Netherlands) at time of surgery.
botic drug acting through another route than TNF-␣ in Five pigs received 0.3 mL of Klexane (100 mg/mL, enoxaparin
an attempt to evaluate the role of TNF-␣ for inducing natrium, Rhône-Poulenc Rorer, Birkero 兾d, Denmark) subcuta-
neously behind the ears twice a day for 1 week. Five pigs re-
thrombosis formation.
ceived subcutaneous 0.3-mL saline injections behind the ears
twice a day for a week and these pigs served as a control group.
Materials and Methods
Twenty-three pigs (body weight approximately 25 kg) received Nerve Function. Seven days after the application, the pigs
an intramuscular injection of 20 mg/kg body weight of Ketalar were reanesthetized by an intramuscular injection of 20 mg/kg
(ketamine 50 mg/mL; Parke-Davis, Morris Plains, NJ), an in- body weight of Ketalar and an intravenous injection of 35
travenous injection of 20 mg/kg body weight of Hypnodil mg/kg body weight of Pentothal (thiopental sodium; Abbott
(methomidate chloride 50 mg/mL; AB Leo, Helsingborg, Swe- Laboratories, Chicago, IL). The pigs were ventilated on a res-
den), and 0.1 mg/kg body weight of Stresnil (azaperon 2 mg/ pirator. Anesthesia was maintained by an intravenous bolus
mL; Janssen Pharmaceutica, Beerse, Belgium). Anesthesia was injection of 100 mg/kg body weight of Chloralose (␣)-D-(⫹)-
maintained by additional intravenous injections of 2 mg/kg glucochloralose (Merck, Darmstadt, Germany) and by a con-
body weight of Hypnodil and 0.05 mg/kg body weight of tinuous supply of 30 mg/kg per hour of Chloralose. A laminec-
Stresnil. The pigs also received an intravenous injection of 0.1 tomy from the fifth sacral to the third coccygeal vertebra was
mg/kg of Stesolid Novum (diazepam, Dumex, Helsingborg, performed. The nerve roots were covered with Spongostane
Sweden) after surgery. (Ferrosan, Bagsvaerd, Denmark). Local tissue temperature was
Nucleus pulposus was harvested from the fifth lumbar disc continuously monitored and maintained at 37.5–38.0 C by
by a retroperitoneal approach.38 Approximately 40 mg of the means of a heating lamp.
nucleus pulposus was applied to the sacrococcygeal cauda The cauda equina was stimulated by two E2 subdermal
equina through a midline incision and laminectomy of the first platinum needle electrodes (Grass Instrument Co., Quincy,
coccygeal vertebra in all pigs. Eight of the pigs received a sub- MA), which were connected to a Grass SD9 stimulator (Grass
cutaneous injection of 12.5 mg of Enbrel (etanercept, Immunex Instrument Co., Quincy, MA) and gently placed intermittently
Corp., Seattle, WA) at the time of surgery and a second injec- on the cauda equina, first 10 mm cranially and then 10 mm
caudally to the exposed area. The stimulation voltage was al-
ways kept at supramaximal strength and the stimulation dura-
Table 1. Histologic Changes tion was 1 ms. To ensure that only impulses from exposed
nerve fibers were registered, the nerve roots that left the spinal
NF Thrombus Edema canal between the cranial stimulation site and the application
zone were cut. A muscle action potential (MAP) was registered
Saline 0 ⫹⫹ 0
by two subdermal platinum needle electrodes, which were
⫹⫹ ⫹⫹ (⫹)
0 ⫹ 0 placed into the paraspinal muscles in the tail approximately 10
⫹⫹ ⫹⫹ ⫹⫹ mm apart. The MAP was visualized using a Power Macintosh
0 0 (⫹) G3 computer provided with Superscope software and Mac-
Enoxaparin ⫹ ⫹⫹ ⫹ Adios II A/D converter (GW Instruments, Sommerville, MA)
0 ⫹⫹ (⫹)
0 ⫹⫹ ⫹ together with a Grass P18 preamplifier (Grass Instrument Co.,
⫹ 0 ⫹ Quincy, MA). The separation distance between the first peaks
⫹⫹ ⫹⫹ ⫹⫹ of the MAPs from the two recordings was determined. This
Infliximab (⫹) 0 0 distance thus represented the time difference between the two
0 0 0
stimulation sites. The separation distance between the two
0 0 0
0 0 0 stimulation sites on the cauda equina was measured with cali-
0 ⫹ ⫹ pers. The nerve conduction velocity between the two stimula-
Etanercept 0 0 (⫹) tion sites could thus be calculated from these two measure-
0 ⫹⫹ ⫹⫹ ments. This procedure is reproducible and represents a
0 ⫹⫹ ⫹
0 0 0 functional measurement of the motor nerve fibers of the cauda
(⫹) 0 0 equina nerve roots34
0 0 0
⫹ 0 0 Histologic Assessment. The cauda equina was tied to a
0 0 0
wooden stick to avoid shrinkage artifacts and was fixed by
NF ⫽ Nerve fiber injury; 0 ⫽ no nerve fiber injury; (⫹) ⫽ occasional degener- immersion in Karnovsky’s mixture of formaldehyde and glu-
ated axons; ⫹ ⫽ minor area of a nerve root fascicle with degenerated axons;
⫹⫹ ⫽ minor area with degeneration in two or more fascicles or more than taraldehyde.17 Specimens for microscopy were obtained from
75% of the cross-sectional area of one fascicle. the exposed segments of the cauda equina. The specimens were
Thrombus ⫽ presence of thrombotic intraneural capillaries; 0 ⫽ no thrombotic dehydrated and embedded in Epon 812. Semithin sections (1
capillaries; (⫹) ⫽ increased number of blood cells in capillaries or only few
thrombotic minor capillaries; ⫹ ⫽ few thrombotic capillaries in one nerve root ␮m thick) were prepared and stained according to Richardson
fascicle; ⫹⫹ ⫽ thrombotic capillaries in more than one fascicle or a high et al.45 All sections were coded and analyzed regarding degree
number of thrombotic capillaries in one fascicle.
Edema ⫽ degree of intraneural edema formation; 0 ⫽ no edema; (⫹) ⫽
of nerve fiber injury, thrombus formation, and intraneural
tendency to increased interaxonal distance; ⫹ ⫽ mild edema in one fascicle; edema according to arbitrary scales (Table 1).
⫹⫹ ⫽ pronounced edema in one fascicle or mild edema in two or more All functional and histologic analyses were performed in a
fascicles.
blinded fashion. A technician performed all the injections, and

Figure 1. Average nerve conduction velocity (NCV) for the three
experimental groups. The NCV was similar between the saline and
the enoxaparin groups and approximately 50 m/sec. Application of
retroperitoneal fat instead of nucleus pulposus in a previous study
have resulted in a NCV of 76 m/sec and has thus been considered
as the normal NCV in this experimental setup.38 Compared with the
normal NCV, the saline and enoxaparin groups thus displayed a
significant reduction. However, both the etanercept and the inflix-
imab groups showed NCVs close to the normal NCV. The results in
the etanercept series were statistically significantly different from
the results of both the saline and the enoxaparin groups (P ⬍ 0.02
for both comparisons). This was the case also for the infliximab
group (P ⬍ 0.02 for both comparisons).
the person performing both the functional and the histologic
analyses was unaware of the experimental protocol for each
specific pig. After the study was completed, the data were ar-
ranged into the four experimental groups. The nerve conduc-
tion velocity data were assessed statistically using ANOVA and
Fisher’s Protected Least Significant Difference test at 5%. The
experimental protocol for this study was approved by the local
Animal Ethics Research Committee.
Results
No infections or gross neurologic deficit were noted in
any of the animals. However, the animals in the enox-
aparin group all developed hematomas at the sites of
injection. So as not to reveal to which of the experimental
groups these pigs belonged to the person performing the
functional analyses, all pigs in the study had to be covered
by an operation sheath before this person could enter the
room to perform the exposure of the nerve roots.
Nerve Function
The nucleus pulposus-induced reduction in nerve con-
duction velocity was pronounced in the saline (control)
and in the enoxaparin groups (Figure 1), compared with
the normal, unaffected conduction velocity value seen
after application of retroperitoneal fat (76 ⫾ 11 m/sec) in
a previous study.38 In contrast, both the etanercept and
the infliximab groups displayed mean values of the nerve
conduction velocities close to normal. The results in the
etanercept series were statistically significantly different
from the results of both the saline and the enoxaparin
groups (P ⬍ 0.02 for both comparisons). This was also
TNF-␣ and Sciatica • Olmarker and Rydevik 865
the case for the infliximab group (P ⬍ 0.02 for both
comparisons).
Histologic Assessment
The data from the histologic evaluation are summarized
in Table 1. Nerve fiber injury seemed to be less pro-
nounced in the etanercept and the infliximab groups
compared with the enoxaparin and saline groups (Figure
2). Also, edema formation and intracapillary thrombus
formation seemed to be less pronounced for the etaner-
cept and the infliximab groups, although two cases in the
etanercept group displayed pronounced changes and one
case in the infliximab group displayed moderate changes
(Table 1).
Discussion
The data of the present study showed that selective inhi-
bition of the cytokine TNF-␣ by systemic treatment pre-
vented the nucleus pulposus-induced reduction of nerve
root conduction velocity. TNF-␣ inhibition also seemed
to partly block the nucleus pulposus-induced nerve fiber
injury, edema formation, and intracapillary thrombosis
formation.
Nucleus pulposus was described to exert direct effects
on nerve root function and structure for the first time in
1993.38 Various pathophysiologic mechanisms have
been described since then. The nucleus pulposus, in the
absence of a mechanical component, has then been
found to induce axonal degeneration,38 myelin edema,37
intravascular coagulation,19,32 increased vascular per-
meability,8,32 increased endoneurial fluid pressure,64 re-
duced intraneural blood flow,40 and reduction in nerve
root conduction velocity.38 The effects of the nucleus
pulposus have been found to be mediated by the cells of
the nucleus pulposus.20,33 Nucleus pulposus application
on the nerve roots has also been found to induce pain
behavioral changes,18 particularly in combination with
mechanical nerve root deformation.35,39
Recently, the cytokine TNF-␣ was suggested to be the
main substance mediating the nucleus pulposus-induced ef-
fects.36 TNF-␣ is a proinflammatory cytokine that is pro-
duced mainly by leukocytes but also by other cell types, for
instance, the nucleus pulposus cells.36 TNF-␣ is known to
induce axonal and myelin injury similar to that observed
after nucleus pulposus application,24,25,43,49,55 intravascu-
lar coagulation,30,57,61 and increased vascular permeabili-
ty.61 TNF-␣ is also known to be neurotoxic25,49,59,63 and to
induce pain behavioral changes12,52,60 as well as ectopic
nerve activity when applied in direct contact with a nerve.53
TNF-␣ has thereby attracted interest in the current context
since its physiologic effects on nerve roots seem well to
resemble those of nucleus pulposus.
A recent study confirmed the presence of TNF-␣ in
nucleus pulposus cells and that the nucleus pulposus-
induced effects were prevented by using a nonspecific
cytokine inhibitor.36 Because other cytokines such as
IL-1␤ and IFN-␥ were also blocked, it was not evident if
866 Spine • Volume 26 • Number 8 • 2001

Figure 2. Findings from the light microscopic assessment. A, The area surrounding the asterisk displays degenerated axons, whereas the
axons in the area located to the left has a more normal appearance. B, The nerve root fascicles have axons that are separated from each
other and are appearing more circular than usual, indicating the presence of intraneural edema. There are a lot of intraneural capillaries
with thrombi present in this section as indicated by arrows. C, Nerve root fascicles with axons that appear normal but with a considerable
number of thrombotic intraneural capillaries indicated by asterisks. D, Two extrathecal nerve roots. The upper nerve roots displays
moderate degeneration in the lower part and four thrombotic intraneural capillaries (black asterisks). The axons in the lower nerve root
have a more normal appearance. However, there are two thrombotized capillaries (black arrows) in this root. In the section there are also
two thrombotized capillaries in the epidural tissues outside the nerve tissue (white asterisks). Bar ⫽ 100 ␮m.

the absence of effects was based primarily on TNF-␣
inhibition or on a combined cytokine inhibition. How-
ever, another study demonstrated that local application
of TNF-␣ in the same experimental system in the pig
induced a significant reduction of nerve conduction ve-
locity whereas other disc-related, pro-inflammatory cy-
tokines such as IL-1␤ and IFN-␥ had minimal or no ef-
fects. 2 TNF- ␣ therefore seems to be of a certain
importance for the development of the nucleus pulposus-
induced effects, and one may therefore assume that selec-
tive inhibition of the cytokine might be of value in pa-
tients because it may thus prevent the nerve root injury
induced by a disc herniation.
One pathophysiologic effect that has been observed
after nucleus pulposus application is intravascular coag-
ulation.19,32 TNF-␣ may mediate its necrotic effects by
inducing coagulation via the endothelial cells.30,41,54
Such coagulation is the result of an increase of adhesion-
promoting molecules, such as intercellular adhesion mol-
ecule 1, endothelial leukocyte adhesion molecule, and
platelet activating factor on the surface of the endothelial
cells.1,7,15,29,47,62 This will thus lead to increased leuko-
cyte and platelet aggregation with a resulting thrombus
or embolus. Enoxaparin (heparin), which was used in the
present study, prevents coagulation by interfering with
the proteases in the coagulation cascade, such as IXa and
thrombin.3,6,26 This is thus a different coagulation mech-
anism than that initiated by TNF-␣. If the coagulation
were initiated by TNF-␣ alone, there would thus be no
reduction of the intracapillary thrombi after enoxaparin
treatment whereas anti-TNF-␣ treatment might prevent
this coagulation.
TNF-␣ may be inhibited in various ways. First, the
synthesis may be blocked by, for instance, corticoste-

roids, IL-10 or TGF-␤, or by drugs such as cyclosporin,
pentoxifylline, or chlorpromazine.4,23,31,44 This is a non-
specific inhibition and would not be clinically desirable,
both due to the low specificity and the side effects related
to these drugs. Another way to inhibit TNF-␣ would be
to block the activation of its membrane-bound inactive
form. TNF-␣ is activated by enzymatic cleavage by a
matrix metalloproteinase, also previously called TNF-␣
converting enzyme.5,27,46 Those enzymes, also known as
matrix metalloproteinases, may be blocked using non-
specific inhibitors that inactivate the enzyme by attach-
ing to the metal ion of the enzyme, usually Mg.11,14,42,48
However, this would not be recommended either in the
clinical situation because this is a nonspecific inhibition
and also because these inhibitors usually have antibiotic
effects, which in this circumstance would be considered
as side effects. The best way to limit the effects of TNF-␣
would instead be to use either soluble TNF-␣ receptors
or specific inactivating antibodies. Both these types of
drugs are now commercially available and currently
evaluated for other TNF-␣-mediated diseases, such as
Crohn’s disease and rheumatoid arthritis.13,21,28,56,58
Soluble TNF-␣ receptors (etanercept) and selective anti-
bodies (infliximab) were both used at therapeutic con-
centrations in the present study.
The present study showed that selective TNF-␣ inhi-
bition can prevent the nucleus pulposus-induced effects
on nerve root function and may limit the structural in-
jury induced by nucleus pulposus. This indicates that
TNF-␣ has a key role in the pathophysiology of nucleus
pulposus effects on nerve roots, and that one may well
consider trying the use of an anti-TNF-␣ substance for
pharmacologic treatment of sciatica. The physiologic pa-
rameters assessed in this study relate more to nerve dys-
function than pain, but there are studies clearly indicat-
ing that TNF-␣ is involved in neurogenic pain generation
as well.12,16,51,60 In particular, local application of nu-
cleus pulposus seems to induce an accumulation of Na⫹
channel protein in axons, rendering the nerve root sen-
sitized to mechanical deformation.10 The combination of
a nucleus pulposus-sensitized nerve root and mechanical
component has been found to induce pain in experimen-
tal models.35,39 This also correlates to clinical observa-
tions that intentional mechanical deformation of a nu-
cleus pulposus-exposed nerve root, during surgery under
local anesthesia, reproduces the sciatic pain whereas de-
formation of a nonexposed, nonsensitized root does
not.22,50 One may therefore suspect that, in some patient
groups, instead of eliminating the mechanical compo-
nent by surgery, another approach would be to prevent
the nerve root sensitization by pharmacologic means in
early phases of the disease.
This study also indicated that histologic changes such
as nerve fiber injury, thrombus, and edema formation
were less pronounced after anti-TNF-␣ treatment. It
might have been desirable to have performed a more
detailed assessment of, for instance, the percentage of
thrombotic vessels in each exposed nerve root. This
TNF-␣ and Sciatica • Olmarker and Rydevik 867
would have enabled a more precise description and also
allowed for statistical analysis. However, such analysis
would have required specially designed staining tech-
niques for vessels and could not be performed with the
light microscopic evaluation employed in this study. This
study nevertheless suggests that intraneural thrombus
formation and edema development may be involved in
the pathophysiology of nucleus pulposus-induced nerve
injury, which thus supports this hypothesis. These data
also support the assumption that the intravascular coag-
ulation and thrombus formation are initiated by TNF-␣
because enoxaparin treatment did not seem to have any
potential in reducing the thrombus formation. The key
role of TNF-␣ for inducing the nucleus pulposus-induced
nerve root injury is thus further indicated by this study.
In conclusion, the data of the present investigation
demonstrated that selective inhibition of TNF-␣ may ef-
ficiently prevent the nucleus pulposus-induced reduction
in nerve root conduction velocity and also partly reduce
the nerve fiber injury, intracapillary thrombus forma-
tion, and intraneural edema development. These basic
science data thus create a platform with potential clinical
implications regarding the use of anti-TNF-␣ medication
as treatment in patients with disc herniation and sciatica.
Key Points
● The data further supports the assumption that
TNF-␣ is a key player in the initiation processes of
the nucleus pulposus-induced nerve root injury.
● Selective inhibition of TNF-␣ efficiently blocked
the nucleus pulposus-induced changes in this ex-
perimental setting.
● In the present protocol, treatment was initiated
at the same time as the injury, which is never fea-
sible in the clinical setting.
● Carefully conducted clinical trials must therefore
be performed to establish the efficacy and therapeu-
tic windows for such treatment before providing
any clinical recommendations.
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