Search

To investigate the effects on evoked thalamic neuronal activity of application of notochordal cells and chondrocyte-like cells derived from nucleus pulposus (NP) onto a dorsal root ganglion (DRG) and to compare these effects with a previously reported increased thalamic activity induced by NP.Nucleus pulposus was harvested from tail discs of adult rats and the disc cells were separated into two cell populations, notochordal cells and chondrocyte-like cells. The two cell populations were applied separately, or in combination, to the L4 DRG of anaesthetised female Sprague-Dawley rats during acute electrophysiological experiments. In control experiments, cell suspension medium was applied on the DRG. Recordings from the contralateral thalamus were sampled for 40 min while electrically stimulating the ipsilateral sciatic nerve at above Aδ-fibre thresholds.Application of notochordal cells resulted in a decrease in evoked thalamic activity within 10 min while chondrocyte-like cells did not induce any changes during the 40 min of recording. The difference in evoked thalamic activity 40 min after notochordal and chondrocyte-like cell application, respectively, was statistically significant. Neither an increased concentration of chondrocyte-like cells alone nor a combination of the two cell populations induced any changes in thalamic activity.Separate exposure of the DRG to the two NP-derived cell populations induced different effects on evoked thalamic activity, but none of the tested cell samples induced an increase in neuronal activity similar to that previously observed with NP. This indicates a high complexity of the interaction between NP and nervous tissue.

The P2X(3) receptor is a ligand-gated cation channel that is activated by extra cellular adenosine triphosphate (ATP) found in the dorsal root, trigeminal and nodose ganglia. It is one of the receptors transmitting nociceptive information of injuries and inflammation of the periphery by endogenous ATP released from damaged cells. The present study was performed in order to evaluate if there was an increased expression of P2X(3)-immunoreactivity in dorsal root ganglion (DRG) neurons after experimental disc herniation. There were four groups: exposure of the left L4 dorsal root ganglion and incision of the L4-L5 disc, exposure and slight displacement of the left L4 dorsal ganglion, sham exposure of the L4 dorsal root ganglion, and normal. Seven days after surgery, the DRG's were collected, sectioned and stained immunohistochemically for the P2X(3) receptor. The expression of P2X(3) increased significantly following incision of the L4-5 disc compared to the normal group. Sham surgery induced a minor, although statistically significant increase. Mechanical displacement did not induce any increased expression of the receptors. The study demonstrates that expression of the P2X(3)receptors in the DRG may be induced by local application of nucleus pulposus. This may increase our understanding of the pathophysiologic mechanisms related to disc herniation and sciatica.

The influence of ischemia on the permeability of the perineurium to protein tracers was studied in rabbit tibial nerve. Intravital ischemia was produced by a pneumatic cuff around the thigh and the effects of long-lasting permanent ischemia were investigated in postmortem experiments. The permeability was tested fluorescence microscopically by injections of albumin labelled with Evan's blue (EBA) around the nerve. The diffusion of EBA into the endoneurium of control tibial nerves was prevented by the perineurium. This “barrier” function of the perineurium resisted ischemia of even 24 hours’duration but after 48 hours of ischemia the tracer penetrated the perineurium and spread widely in endoneurial spaces. This diffusion barrier is therefore markedly resistant to ischemia and postmortem changes.

The mechanisms of apoptosis underlying a characteristic tissue reaction at the surface of the dorsal root ganglion (DRG) exposed to nucleus pulposus were studied in rats with special reference to the role of tumor necrosis factor-alpha (TNF).To study the characteristic tissue reaction at the surface of the DRG exposed to nucleus pulposus with special reference to the role of TNF.Nucleus pulposus cells are capable of producing TNF. Recently, local application of nucleus pulposus was shown to induce a characteristic tissue reaction at the DRG surface due to apoptosis.Recombinant TNF was applied to the DRG to mimic L4-L5 disc herniation in rats. The DRGs were resected 24 hours after surgery. Sections of the specimens were processed for immunohistochemistry using antisera to single-stranded DNA, Caspase 3, and TNF, and observed by light and electron microscopy.Typical apoptotic changes of the cell nuclei were observed in the DRG after application of TNF. The presence of single-stranded DNA, Caspase 3, and TNF further confirmed the occurrence of DRG cell apoptosis.TNF seemed to play a key role in induction of apoptosis of DRG cells, which resembled that induced by application of nucleus pulposus.

Study design Histologic changes in the dorsal root ganglion (DRG) and the nociceptive stimulation thresholds were studied in rats. Objective To examine the effects of tumor necrosis factor-alpha (TNF) with special reference to pain behavior and histology of the DRG. Summary of background data Recently, it was reported that local application of nucleus pulposus induces a characteristic tissue reaction at the surface of the DRG. However, to our knowledge, there have been no previous reports about the relationship between the histologic changes and pain behavior caused by cytokines. Methods Recombinant TNF was applied to the L4 DRG. Mechanical and thermal nociceptive thresholds were tested. The L4 DRG was sectioned and observed by light microscopy. Results After the application of 5 ng/microL TNF, significant differences were observed in mechanical and thermal stimulation thresholds. At the site of application of TNF, a characteristic a semilunar-shaped enlargement was observed. The average width of the part was significantly larger in the 5 ng/microL TNF application, as compared to the 0.5-ng/microL TNF application. Conclusions The higher concentration of TNF used induced allodynia and hyperalgesia responses. Because the region showing the histologic changes was significantly larger after application of the higher concentration of TNF, the reaction of the DRG may be related to pain.

STUDY DESIGN: The origin and the barrier properties of the characteristic reaction at the surface of the dorsal root ganglion (DRG) exposed to the nucleus pulposus was studied using Alcian-Blue staining, van Gieson staining, and the application of Evans Blue Albumin (EBA) complex in rats. OBJECTIVE: To study the origin and the barrier properties of the capsule, including the characteristic reaction, at the surface of the DRG exposed to the nucleus pulposus. SUMMARY OF BACKGROUND DATA: Local application of nucleus pulposus may induce a characteristic reaction at the surface of the DRG. This reaction histologically resembles an acute inflammatory reaction. However, it is not evident if this is a swelling of the DRG capsule, if it is located between the capsule and neurons of the DRG, or if it is only an attached nucleus pulposus. METHODS: Nucleus pulposus from the discs was obtained. The nucleus pulposus was smeared on glass slides. Alcian-Blue with hematoxylin and eosin staining was performed for each smear. Herniation of the nucleus pulposus was made in the L4-L5 disc in rats. The L4 DRGs were resected 3, 24, and 72 hours after surgery, and sectioned. The sections were processed for Alcian-Blue staining, van Gieson staining, and EBA complex infiltration. The sections were observed using light or fluorescent microscopy. RESULTS: Smear of nucleus pulposus was stained bright blue indicating mucins. A characteristic reaction, "inflammatory crescent," was confirmed at the surface of the DRG exposed to the nucleus pulposus. No mucins were observed in the crescent using Alcian-Blue. The results of van Gieson staining showed that the reaction started both inside and outside the elastic fiber layer, the DRG capsule, within 3 hours. The EBA complex was capable of infiltrating into the DRG capsule 24 hours after disc incision. CONCLUSIONS: The disintegrated capsule showed an increased permeability even for a large molecule as albumin, which indicates a possible entrance route for various substances induced by locally applied nucleus pulposus.

Application of nucleus pulposus and disc related cytokines in vitro on cultured dorsal root ganglion (DRG) cells.To study if tumor necrosis factor (TNF) and interleukin-1beta (IL-1beta) may induce similar inhibition of axonal outgrowth from cultured DRG cells as application of nucleus pulposus and to compare a new assessment method to previous data.Pro-inflammatory cytokines related to the intervertebral disc have been suggested to affect adversely neurons following local application, with implications for the nucleus pulposus-induced nerve injury seen in various studies. Nucleus pulposus is known to inhibit axonal outgrowth from cultured DRG cells, thereby indicating a neurotoxic potential. The mechanisms were not understood, but it was suspected that the effect was mediated by pro-inflammatory cytokines produced by the nucleus pulposus.DRG were harvested from newborn rats and put in culture. The axonal outgrowth was determined 24 hours after starting the culture. Twenty-four hours after exposing the cultured cells to nucleus pulposus, frozen nucleus pulposus, TNF, or IL-1beta, the axonal outgrowth was reassessed, and the outgrowth during the exposure time was calculated.Nucleus pulposus clearly reduced the axonal outgrowth. Also, application of TNF and IL-1beta reduced the outgrowth but not as pronounced as the nucleus pulposus. Frozen nucleus pulposus had no effects on the outgrowth. Overall, the data were similar regarding frozen and nonfrozen nucleus pulposus compared to a previous study.It was evident that the 2 studied cytokines inhibited the outgrowth of axons from cultured DRG cells, thus suggesting a neurotoxic potential. However, the inhibition was not as pronounced as for nucleus pulposus. These data may increase our understanding for cytokine induced nerve injury, with implications for future treatment strategies for such conditions.

STUDY DESIGN: The time courses of nerve growth factor content and pain-related behavior were examined using experimental disc herniation models. OBJECTIVES: To investigate a relationship between nerve growth factor level and pain behavior. SUMMARY OF BACKGROUND DATA: An induction of nerve growth factor in the periphery is regarded as a major contributor of inflammatory hyperalgesia and neuropathic pain. However, it has not been clarified quantitatively whether disc herniation induces changes in nerve growth factor levels in the dorsal root ganglion in relation to pain-related behavior. METHODS: A total of 140 rats were used in this study. The animals had their left L4 nerve roots and associated dorsal root ganglion exposed and were equally divided into 4 groups: L4-L5 disc puncture, displacement of L4 nerve roots/dorsal root ganglion, the combination of disc puncture and displacement, and sham exposure. The content of nerve growth factor in the affected dorsal root ganglion was assessed by enzyme-linked immunosorbent assay as well as pain behavior during a postoperative 21-day period. RESULTS: Disc puncture resulted in nerve growth factor induction at postoperative day 3, but not apparent behavioral changes. Mechanical displacement induced nerve growth factor at postoperative day 1 and mechanical allodynia at postoperative day 3, respectively (P < 0.05). In the combination model, there were more pronounced changes in nerve growth factor induction and both mechanical and thermal threshold during 7 days after surgery (P < 0.05). CONCLUSIONS: These data suggest the possibilities that elevated nerve growth factor level is partly involved in pain behavior and further the combined model mimicking the clinical situation, which causes the marked neuronal responses, is helpful to advance the understanding of the mechanisms underlying sciatica due to lumbar disc herniation.

STUDY DESIGN: The possibility to prevent nucleus pulposus-induced structural changes of the dorsal root ganglion (DRG) by selective tumor necrosis factor-alpha (TNF-alpha) inhibition was assessed in an experimental model in the rat spine. OBJECTIVES: To evaluate the role of TNF-alpha in the mediation of nucleus pulposus-induced structural changes by using selective inhibition and to confirm the effect of TNF-alpha inhibitor at the point of histologic findings. SUMMARY OF BACKGROUND DATA: TNF-alpha is known to be released from the nucleus pulposus, and has been suggested to play a key role in chemical damage of the adjacent nerve tissue. The TNF-alpha inhibitor prevents the reduction of nerve conduction velocity and may limit the nerve fiber injury, intracapillary thrombus formation, and intraneural edema formation caused by nucleus pulposus. However, there is no report on the effect of the inhibitor regarding histologic findings and the appearance of the TNF-alpha in the DRG exposed to nucleus pulposus. METHODS: 1) Rats were treated with an intraperitoneal injection of infliximab. Nucleus pulposus from the disc was obtained 1, 3, 7, 14, and 21 days after the injection. The TNF-alpha-positive cells were observed using immunohistochemistry. 2) Disc herniation of the nucleus pulposus was made on the L4-L5 disc in rats. Two groups were treated with selective TNF-alpha inhibitor 1 day before or 3 hours after surgery. The other group received no TNF-alpha inhibitor. The L4 DRG was resected 1, 3, 7, 14, and 21 days after surgery. The specimens were processed for hematoxylin and eosin staining and immunohistochemistry using rabbit antisera to TNF-alpha. The histologic findings and TNF-alpha-positive cells were observed by light microscopy. RESULTS: 1) While positively stained immunoreactive TNF-alpha appeared between 7 and 21 days, no immunoreactive TNF-alpha was observed 1 and 3 days after injection in the nucleus pulposus. 2) The histologic changes of the DRG caused by nucleus pulposus were smaller in the infliximab treatment group than those in the nontreatment group. The number of immunoreactive TNF-alpha cells was high 1 and 3 days after surgery in the DRGs of disc herniation rats that were treated without an injection of the inhibitor, low on day 7 and 14, and very low on day 21 after surgery. No immunoreactive TNF-alpha was observed in the DRGs of the TNF-alpha inhibitor treatment groups on day 1, 3, and 21 after surgery. Weakly stained cells were sometimes observed in rats at day 7 and 14 after surgery. CONCLUSIONS: Infliximab may prevent the histologic damage induced by nucleus pulposus. When rats were given a single intraperitoneal injection of infliximab at the beginning of disc herniation, the histologic damage seemed to be reduced in comparison with the nontreated rats.

STUDY DESIGN: Distribution and appearance of tumor necrosis factor-alpha (TNF-alpha) in the dorsal root ganglion (DRG) exposed to experimental disc herniation were investigated using an immunohistochemical method in rats. OBJECTIVES: To study the distribution and appearance of TNF-alpha in the DRG following experimental disc herniation in rats. SUMMARY OF BACKGROUND DATA: Nucleus pulposus in the epidural space induces spinal nerve root injury not only by mechanical but also chemical mechanisms. Cytokines may play a key role in the chemical damage. There is, however, no report on the distribution and appearance of TNF-alpha in the DRG exposed to nucleus pulposus. METHODS: Nucleus pulposus from the discs was smeared on the glass slides and processed for immunohistochemistry by the avidin-biotinylated peroxidase complex technique using rabbit antisera to TNF-alpha in rats. A herniation of the nucleus pulposus was made by incision of the L4-L5 disc in rats. The L4 and L5 DRGs were resected 1, 3, 7, 14, and 21 days after surgery. The specimens were processed for immunohistochemistry using rabbit antisera to TNF-alpha. The TNF-alpha-positive cells were observed and counted using light microscopy. Distribution of the TNF-alpha products was compared on each day after surgery. RESULTS: A positive staining was seen in the cell bodies and in the matrix between the cells in the smeared nucleus pulposus. In the L4 DRG sections, the number of positive cells was significantly higher in the disc incision group than in the sham group at 1, 3, 7, and 14 days after surgery (P < 0.05). The positive cells showed a decrease in number day by day after surgery. On the contrary, in the L5 DRG, only a few positive cells were observed in the disc incision group after surgery. There was no statistically significant difference between disc incision and the sham groups at each day after surgery for the L5 DRGs. CONCLUSIONS: The immunoreactivity of TNF-alpha in the DRG directly exposed to nucleus pulposus increases during 2 weeks. A collapse of the positive cells was seen in the DRG directly exposed to the nucleus pulposus.

Recently, it was observed that local application of nucleus pulposus may induce a characteristic reaction at the surface of the dorsal root ganglion (DRG). This change was inflammatory in nature and occurred as early as 1 day after the application of nucleus pulposus. Herniation of the nucleus pulposus was surgically induced in the L4-5 disc in rats. The L4 DRGs were resected 3, 24, and 72 h after surgery and sectioned. The sections were processed for immunohistochemistry using antisera to the macrophage marker ED1 and observed using light microscopy. The appearance of macrophages was confirmed 3, 24, and 72 h after the surgery. Macrophages were mainly distributed in the epineurial space of the DRG 3 h after disc incision and also in the endoneurial tissue 24 and 72 h after disc incision. The immunoreactivity was significantly stronger at 24 and 72 h than at 3 h in the parts of the DRG without apparent changes in the disc incision series (p < 0.01). Within the epineurium adjacent to application of nucleus pulposus, the number was significantly higher at 3 h than at 24 and 72 h (p < 0.05). We conclude that experimental disc herniation with leakage of nucleus pulposus results in macrophage recruitment to the epineurium of the DRG 3 h after disc incision and to the endoneurium 24 and 72 h after disc incision.

Temporal distribution of intramembranous and endochondral bone formation was studied in experimental fracture defects in rats under different stability of fracture fixation and fracture environments. Animals were surgically treated with a specially developed external fixation construct: Group 1 had 42 rats with a 0-mm fracture gap with bone ends touching corresponding to an axial stiffness of 265.00 +/- 34.00 N/mm and Group 2 had 42 rats with a 2-mm fracture gap corresponding to an axial stiffness of 30.38+/- 2.07 N/mm. From each group, six animals were sacrificed at 4 days and 1, 2, 3, 4, 6, and 12 weeks. Qualitative histologic and morphometric analyses revealed that less fixation rigidity and increased fracture gap induces a later response of bone formation and greater endochondral bone formation leading to prolonged time for full ossification. Furthermore, in the early phase of fracture healing temporal distribution and histologic characteristics of periosteal and intramedullary bone formation are similar and not influenced by rigidity and fracture environment. Results also showed that if tissues associated with the intramedullary region are preserved, intramedullary bone formation is substantial. Finally, histologic data indicate that woven bone might be a prerequisite for the differentiation process of endochondral bone formation.

The pain mechanisms underlying radiculopathy due to disc herniation are still incompletely understood. This study assessed changes in brain-derived neurotrophic factor (BDNF) expression, a modulator of nociceptive information, in the dorsal root ganglion (DRG) and spinal cord dorsal horn following experimental disc herniation. Immunohistochemical analysis revealed an increase in percentage of BDNF-immunoreactive (IR) neurons profiles in the affected DRG and marked elevation in the BDNF-IR regions within both the superficial and deep layers at the corresponding spinal level with a peak at 3 days after nucleus pulposus (NP) application. These results thus show that herniated NP increases the BDNF production in the pain-processing neurons. Such changes can contribute to the development of inflammatory hyperalgesia.

We wished to establish a reproducible model for fracture fixation to be used in fracture healing research and therefore developed an external fixation construct and surgical procedure adapted to Sprague-Dawley rats. We evaluated the mechanical properties of the construct in brass rods and rat bone, in an Instron test machine with axial and transverse loading, and the in vivo performance. We found that the mechanical properties of the construct in brass rods were predictable and could be repeated in rat femora. In all tests, the axial load was about 10 times the transverse for the same degree of deformation. The stiffness among fixators was uniform. 1 mm pins caused about 50% less stiffness than 1.2 mm pins in axial loading of rat bone (p < 0.001) and brass rods (p < 0.001) as well as in transverse loading of brass rods (p < 0.001). Loosening of 1 or 2 screws that lock the pins to the fixator reduced stiffness by about 50% in axial loading of rat bone (p = 0.009) and brass rods (p = 0.05). A change in the distance between the bone surface and the fixator was linearly related to the stiffness in axial loading of rat bone (p < 0.001) and brass rods (p < 0.001) and in transverse loading of brass rods (p < 0.001). If the bone ends touched each other, the axial stiffness of the construct increased almost 10 times (265 N/mm), as compared to a fracture gap size of 2 mm (31 N/mm). In vivo experiments had a complication rate of less than 10% when we used 1.2 mm pins, 6 mm offset and rats weighing 350-450 g. Our method and device for experimental external fixation of rat femora are reliable and the findings are reproducible. These can be used in bone repair and fracture healing research.

The route by which an epidurally applied macromolecule might reach the endoneurial space of spinal nerve roots was assessed with light and electron microscopy in a pig model established to explore the pathophysiology of disk herniation. Horseradish peroxidase (HRP) dissolved in saline was infused epidurally. Animals were sacrificed after 5 minutes (n = 5) or 30 minutes (n = 5). Two control animals received only a saline infusion and were sacrificed after 30 minutes. Nerve root specimens were collected, fixed, and exposed to the HRP substrate, 3.3'-di-amino-benzidine (DAB). The distribution of HRP reaction product in the nerve tissue was studied with light and electron microscopy. In 5-minute specimens, HRP was found in epidural and intradural vessel walls. At the nerve root level, HRP was detected in meningeal membranes but was not seen in periaxonal space. In addition to engaging the outer cell layers of the dorsal root ganglion (DRG) capsule, HRP was detected as a gradient among the peripherally located nerve cell bodies and sometimes among the emerging afferent axons. The 30-minute group demonstrated similar findings. The results confirm that HRP can reach the periaxonal spaces of lumbar DRG within 5 minutes after epidural application. Although the transport mechanism is not fully understood, the DRG may constitute an anatomical location allowing epidurally applied macromolecules entrance to the endoneurial space, either by direct diffusion or via vascular transport. The demonstrated transport route may have implications in the pathophysiology of sciatica in conjunction with lumbar disc herniation.

It is known that 24 h or more after epidural application of autologous nucleus pulposus, functional and structural changes are established in adjacent nerve roots. It is, however, not known how soon after the application these changes appear. The aim of this study was to reduce the exposure duration to 3 h and to evaluate nerve function and histological changes in the nerve tissue during this time period. A total of 12 pigs was used. In ten pigs, autologous nucleus pulposus (NP) was applied epidurally on the cauda equina. Nerve function was then monitored for 3 h by measurements of muscle action potentials (MAP) in the tail muscles, following nerve root stimulation cranial to the exposed zone. In five of the ten pigs with NP application, nerve root compression to 50 mm Hg was added by means of an inflatable balloon. In two control animals, neither NP nor compression was applied. At the end point, nerve root specimens were harvested for histological assessment. No reduction of MAP amplitude was detected in any of the series. However, there was an epidural accumulation of leucocytes and mast cells, as well as minor axonal and Schwann cell changes in both the NP and NP+ compression series, as compared to the control series. Morphological changes in terms of an epidural inflammatory reaction and minor axonal and Schwann cell damage may thus be demonstrated within 3 h of NP application, with or without compression. However, there is no functional deterioration of the nerve roots detectable within this time period.

Study Design. Application of autologous nucleus purposus on nerve roots and treatment with local application of lidocaine in the pig. Objectives. Studies of the effects of lidocaine on nucleus pulposus-exposed nerve roots. of Background Data. Nerve root infiltration may improve radicular symptoms beyond the pharmacologic duration of local anesthetics, but the mechanisms for this effect are not known. Methods. Nucieus pulposus was harvested from a lumbar disc and placed onto the sacrococcygeai cauda equina in pigs. In Series 1, early lidocaine treatment of nucleus pulposus-induced nerve root injury, pigs received 2% lidocaine (n = 5) or saline (n = 5) before and after surgery. Nerve conduction velocity and histologic appearance were studied after 3 days. In Series 2, delayed lidocaine treatment of nucleus pulposus-induced nerve root injury, after 7 days 2% lidocaine was administered epidurally to nucleus pulposus-exposed (n = 4) and -nonexposed (n = 4) nerve roots. Nerve conduction velocity, muscle action potentials, and histologic appearance were assessed. Results. In Series 1, early treatment with lidocaine limited the reduction in nerve conduction velocity. The epidural inflammation was less in lidocaine treated animals. In Series 2, nerve conduction velocity was lower in nucleus pulposus-exposed animals than in nonex-posed animals, The initial reduction of nerve conduction velocity and muscle action potential was similar between the groups, but the recovery of muscle action potential was slower and less complete in nucleus pulposus-exposed nerve roots. There was minimal histologic nerve injury in both series and in both protocois. Conclusions. Early treatment with lidocaine may reduce nucleus pulposus-induced nerve root injury. Lidocaine induced a delayed recovery in nerve roots exposed to nucleus pulposus. Further studies are needed to clarify the therapeutic effects of nerve root infiltration and the pathophysiology of nucleus pulposus-induced nerve root injury.

In an attempt to evaluate whether experimental disc herniation can result in changes in walking pattern, presumably indicating pain, four groups of rats – sham (n = 5), disc puncture (NP, n = 5), displacement of nerve root and ganglion (DIS, n = 5), and the combination of disc puncture and displacement (NP + DIS, n = 6) – were assessed when walking in a Plexiglas corridor at day 2–14 after surgery. All surgical procedures were performed at the L4–5 level on the left side. Step length analysis showed that rats in the NP and DIS series had no difference between the legs initially, but a tendency towards slightly shorter left steps at day 8–12, whereas the animals in the NP + DIS series had slightly shorter right steps day 2–10. However, no statistically significant differences compared with the sham series could be detected for any of the groups. Nerve dysfunction on the operated side was only observed on one occasion in two rats and on 2 days in one rat, all from the NP + DIS series. Apparent limping was seen in three of the animals in the NP + DIS series and in one in the DIS series. Limping and nerve dysfunction only co-incided on one occasion out of a total of 13 observations of limping, suggesting that the limping was induced by pain and not neurologic deficit. In conclusion, the combination of epidural nucleus pulposus and displacement induced a limping whereas nucleus pulposus or displacment alone did not. Assessment of limping thus seemed to provide adequate information of presence of pain, but step length measurement did not provide any useful data. Although walking analysis may be a valuable assessment of sciatic pain, better modalities must be developed to analyze experimentally induced nerve root pain.

STUDY DESIGN Nerve conduction velocity in pig nerve roots was assessed after application of various preparations of nucleus pulposus and control. OBJECTIVE To study whether cultured nucleus pulposus cells could reduce nerve conduction velocity after epidural application. SUMMARY OF BACKGROUND DATA It is known that nucleus pulposus applied epidurally may reduce the nerve conduction velocity of the adjacent nerve roots and that this reduction seems to be related to the cells of the nucleus pulposus. METHODS Nucleus pulposus cells and fibroblasts were cultured for 3 weeks, and various preparations were applied to the cauda equina in 29 pigs. The cells were always from the same animals from which they had been harvested. After 1 week, nerve conduction velocity was determined by local electrical stimulation. RESULTS Application of live fibroblasts and conditioned culture medium from the nucleus pulposus cell culture dishes did not induce significant reduction of conduction velocity, compared with application of dead fibroblasts, which served as control. However, application of live and dead nucleus pulposus cells induced significant reductions. CONCLUSIONS Application of nucleus pulposus cells reproduced the previously seen reduction in nerve conduction velocity induced by nonmodified nucleus pulposus. Because membranes of the nucleus pulposus cells had similar effects, it can be assumed that the effects are related to membrane-bound substances or structures.

Study design The effects on nerve tissue and blood vessels of locally applied chondroitinase ABC were studied in two experimental models using chymopapain and the vehicle of chondroitinase ABC for controls. Objectives To assess the effects of chondroitinase ABC on blood vessels and nerve tissue after local application. Summary of background data Chondroitinase ABC has been suggested for chemonucleolysis because it has a high specificity for nucleus pulposus matrix, which could mean a high efficiency in dissolving disc tissue combined with a low risk of side effects on other tissues. Methods Chondroitinase ABC or controls were injected intrathecally in the pig, and nerve conduction velocity and histologic changes were assessed after 7 days. The same substances were injected into the hamster cheek pouch and studied for 60 minutes for microvascular effects. The vehicle for the enzyme was used as a negative control and chymopapain in a therapeutic concentration served as a positive control. Results In all series there was a slight intrathecal fibrotic reaction that was most pronounced after chymopapain injection. The effects on nerve conduction velocity and nerve morphology were similar between chondroitinase ABC and its vehicle. Chymopapain induced a significant reduction in nerve conduction velocity and pronounced histologic changes. In the cheek pouch, chymopapain induced a stand-still of blood flow at the injection site, and microhemorrhage and macromolecular leakage from the vessels at the border of the injection site. Only a slightly reduced blood flow was occasionally found after injection of chondroitinase ABC and controls. Conclusions In agreement with the current literature, these observations indicate that chondroitinase ABC is safe regarding adverse effects on nerve tissue and blood vessels. The slight reduction in conduction velocity after intrathecal injection of chondroitinase ABC or its vehicle is most likely the result of surgical injury while releasing the nerve roots from the intrathecal fibrous adhesions. Such adhesions may be related to the laminectomy per se, and probably have no pathophysiologic significance.

Study Design. Ultrastructural changes were analyzed by transmission electron microscopy in nerve roots exposed to autologous nucleus pulposus experimentally. Objectives. To assess if ultrastructural changes were present in areas with no light microscopic changes in nerve roots exposed to autologous nucleus pulposus in a pig model. Summary of Background Data. Previous analyses have shown that there is focal nerve fiber damage in nerve roots exposed to autologous nucleus pulposus in the pig. These changes could not fully explain the reduction in nerve conduction velocity seen in the same nerve roots. In the present study, the parts of the nerve roots that did not display breakdown of axons or myelin sheaths at the light microscopic level were analyzed regarding ultrastructural changes. Methods. In a previous study, nucleus pulposus was harvested from a lumbar disc and placed epidurally onto the cauda equina at the sacrococcygeal level in pigs. Retroperitoneal fat was used as control. After 1, 3, and 7 days, the nerve roots were excised and processed for light microscopy. Parts of the nerve roots that appeared normal at the light microscopic level were further processed for the present electron microscopic examination. Results. Significant ultrastructural changes, such as expansion of the Schwann cell cytoplasm and intracellular edema with vesicular swelling of the Schmidt-Lanterman incisures, were observed in nerve fibers with normal axons. Although present after nucleus pulposus and control application, the changes were more pronounced after the application of nucleus pulposus. Conclusions. Epidural application of autologous nucleus pulposus without any pressure may induce not only nerve function impairment but also axonal injury and significant primary Schwann cell damage with vesicular swelling of the Schmidt-Lanterman incisures. However, because axonal and Schwann cell changes affected only part of the nerve fibers, further causes of the impaired nerve conduction need to be determined.

The pathophysiology of sciatica is not completely understood, although our understanding of its causes is increasing. Mechanical alterations combined with inflammatory changes lead to pain. Compression alters nerve root conduction and compromises the nutritional support of spinal nerve roots (through intrinsic and extrinsic vascularity and cerebral spinal fluid percolation). Mechanical forces can lead to intraneural damage and functional changes in nerve roots. Chemical and metabolic effects can create an inflammatory response. Varying causes of inflammation coupled with varying degrees of compression can occur anywhere along the cauda equina or spinal nerve root, including the dorsal root ganglia, and contribute to the pain response and neurologic deficits associated with sciatica.

Nucleus pulposus cells from the intervertebral disc have been shown to have inhibiting effects on neurite outgrowth in vitro. The nucleus pulposus consists of at least 2 cell populations, notochordal cells and chondrocyte-like cells. The aim of this study was to analyze the morphology of the neurites, from rat dorsal root ganglia (DRG) in a culture system, after exposure of these 2 cell populations. DRG from perinatal rats was harvested and placed in culture dishes for 24 h. Nucleus pulposus cells from donor rats were separated into 2 populations and applied to the DRG and neurite culture for a further 24 h and compared to control cultures exposed to culture medium without cells. The DRG and neurites were thereafter prepared for scanning or transmission electron microscopy (SEM/TEM). Descriptive SEM and TEM analyses and calculations of the neurite diameter were performed. The visual appearance after SEM and TEM preparation was similar in the three different culture conditions. However, there was a statistically significant reduction of the neurite diameter for the cultures exposed to notochordal cells compared to the cultures exposed to medium and chondrocyte-like cells (TEM preparation). Prominent and frequent pathologic abnormalities in peripheral nerve diseases have been observed with changes in axonal caliber. This study may suggest that a preserved small amount of notochordal cells, as seen in human adults, may play a role in clinical situations where nerve tissue is exposed to disc material, i.e. in disc herniation or degeneration.

Low doses of methotrexate (MTX) are safe and effective for treating adult and juvenile rheumatoid arthritis. However, because this powerful anti-inflammatory drug might negatively influence the healing of wounds and fractures, MTX administration is often stopped during surgical procedures. The present study assesses the effects of low- and high-dose MTX on early inflammatory processes and bone healing in an experimental model of fracture. Thirty male Sprague-Dawley rats were assigned to low- and high-dose MTX and control groups. A femur was cut using a reciprocating saw and a 2-mm fracture gap was made using a fixator. One or four weeks thereafter, macrophages were immunostained and new bone formation was histomorphometrically measured. Significantly less new bone was formed in the high-dose MTX, than in the control group (p< 0.01), whereas bone formation did not significantly differ between the low-dose MTX and control groups. These results suggested that a low dose of MTX does not affect the early process of endochondral bone formation during fracture healing, whereas a high dose might delay the progress of new periosteal bone formation. Although more macrophages were found in the groups treated with MTX, their impact on surrounding inflammatory processes remains unclear.

Compression of the spinal nerve roots may occur clinically at multiple levels at the same time; however, the basic pathophysiology of multi-level compression is largely unknown. In this study, the intraneural blood flow was analyzed continuously in the uncompressed segment between two compression balloons, with a pig used as an experimental model and a thermal diffusion method. At 10 mm Hg compression, there was a 64% reduction of total blood flow in the uncompressed segment compared with pre-compression values. Total ischemia occurred at pressures 10-20 mm Hg below the mean arterial blood pressure. After two-level compression at 200 mm Hg for 10 min, there was a gradual recovery of the intraneural blood flow towards the baseline. Recovery was less rapid and less complete after 2 h of compression. Double-level compression of the cauda equina can thus induce impairment of blood flow, not only at the compression sites, but also in the intermediate nerve segments located between two compression sites, even at very low pressures. These findings may have clinical importance in the understanding of the pathophysiology of multiple-level cauda equina compression.

An experimental study investigating the effect of disc cells on neurite outgrowth in a rat dorsal root ganglion (DRG) culture system.To examine the effects of the 2 nucleus pulposus (NP) cell populations, notochordal cells (NC) and chondrocyte-like cells (CC) on neurite outgrowth from DRGs.NP consists of at least 2 cell populations, NC and CC. The cells in NP have been shown to be responsible for negative effects on neurite outgrowth in vitro and on nerve tissue in vivo. It is unknown whether 1 cell type or combinations of the 2 cell types are responsible for the reported effects.A total of 939 DRGs from newborn Sprague Dawley rats were harvested and placed in culture dishes. After 24 hours, the neurite outgrowth was measured. NP was harvested from tail discs of adult rats and the NP cells were separated into 2 populations, NC and CC. The cell populations were applied to the DRG culture in different cell concentrations and combinations, and compared to medium. After 24 hours of exposure, the neurite outgrowth was reassessed and expressed as the ratio between the outgrowth at 48 and 24 hours culture.NC in intermediate and high concentration and CC in high concentration induced a significant inhibition of the neurite outgrowth compared to culture medium. Further, one of the combinations (low NC and high CC concentration) resulted in a significant inhibition of the neurite outgrowth.The present study demonstrated negative effects of NP cells on nerve tissue culture explants. The combination of low NC and high CC concentrations may mimic the situation in humans, where we have an increased proportion of chondrocyte-like cells with age. The results from this study may provide a biologic explanation for the large variation of symptoms in disc herniation patients despite similar mechanical influence on nerve tissue.

This study determines the change in tension after an acute nerve lengthening, as would occur in peripheral nerve repair. Stress relaxation, a viscoelastic property, was studied with the use of 24 rabbit tibial nerves. The nerves were divided into three groups and were stretched 6%, 9%, or 12% beyond their original resting length. The mean 1-hour stress relaxations for the three groups were 48%, 34%, and 34%, respectively. Nerve stress relaxation was significantly greater at 6% strain than at 9% or 12% strain. These biomechanical findings have important clinical implications for nerve-stretch injury and for nerve repair.

The exact pathophysiologic mechanisms behind sciatica are incompletely known; however, compression of spinal nerve roots is known to be correlated to both pain and neural dysfunction in a segmental distribution of that specific nerve root. Compression per se may impair the transport of nutrients to the nerve tissue in such a way that affects the nerve root function. There also might be a local affect on nerve roots or root sleeves by substances leaking from the degenerated intervertebral discs.

Nerve root compression has been suggested as one important pathogenetic factor in low-back pain syndromes and sciatica. The underlying pathophysiologic mechanisms are, however, incompletely known, partly because of the lack of experimental data on this topic. In the present study, a model for experimental compression of the porcine sacrococcygeal cauda equina is presented. The model consists of surgical exposure of the cauda equina and compression of the cauda equina toward the ventral aspect of the spinal canal by an inflatable balloon fixed to the spine. This compression system was shown to have a high accuracy in pressure transmission from the balloon to the cauda equina. The gross and microscopic neural anatomy and the vascular anatomy of the porcine cauda equina were analyzed with light microscopic and ink-perfusion techniques. The porcine cauda equina showed a close anatomic resemblance to the human lumbosacral cauda equina. The presented model offers unique possibilities for experimental studies on nerve root compression injury because of the easy surgical exposure and the sufficient length of the nerve roots. In separate studies, this model, along with investigations of solute transport to the nerve tissue and of impulse propagation, has been used to analyze the effects of acute, graded compression on blood flow and edema formation in the cauda equina. The porcine cauda equina would also be particularly suitable for chronic compression studies because any neurologic deficit acquired would be restricted to the tail.

An autoimmune response to herniated nucleus pulposus has been proposed to constitute a pathophysiologic mechanism for inducing sciatica based on the fact that nucleus pulposus under normal conditions is excluded from the development of immunological tolerance. The manifestation of an autoimmune response comprises different steps starting with antigen capture, continuing with activation of T helper (T(H)) cells and ending with production of autoantibodies. Activated T(H) cells differentiate into either T(H)1 cells, predominately producing proinflammatory cytokines such as interferon gamma (IFNgamma) or a T(H)2 subset mainly producing anti-inflammatory cytokines such as interleukin-4 (IL-4). The aim of the present study was to examine if exposure of autologous nucleus pulposus (NP) to the immune system for 3 weeks is potent enough to prime T(H) cells to differentiate into T(H)2 cells. The study was performed in a pig model allowing the exposure of NP to the immune system. To assess the polarization of T(H) cells the intracellular production of IFNgamma and IL-4 was measured in T cells by using flow cytometry. The revealed predominant production of IL-4 together with low production of IFNgamma in T cells after NP exposure to the immune system indicates that nucleus pulposus may prime T(H) cells to develop into IL-4-producing T(H)2 cells after being exposed to the immune system, for example, in association with disc herniation.

Study design Assessment of activated T and B cells in a subcutaneous chamber filled with autologous nucleus pulposus using flow cytometry and immunohistochemistry. Objectives To examine if subcutaneously placed autologous nucleus pulposus may attract activated T and B cells in an animal model. Summary of background data Nucleus pulposus has been suggested to trigger an autoimmune response if exposed to the immune system, for example, in association with disc herniation. T-cell activation represents a hallmark in the generation of an autoimmune response, subsequently leading to the differentiation of B cells, but a causal association between the exposure of nucleus pulposus to the systemic circulation and T and B cell activation is still lacking. Methods Autologous nucleus pulposus was harvested from the intervertebral disc of 9 pigs and placed subcutaneously in perforated titanium chambers. In order to control for the effect of the titanium chamber, an additional empty chamber was placed subcutaneously in each pig. After 7 days, the pigs were killed and the chambers were harvested. Flow cytometry and immunohistochemistry were used for analysis of T-helper cells (CD4+), cytotoxic T cells (CD8+), and B cells (Igkappa) in the chamber exudates and T cells (CD45RC) in the remaining blood clot tissue of the chamber. Results As compared with the empty chambers, the proportion of activated T cells (CD4+ and CD8+) was significantly higher in the exudate of the nucleus pulposus filled chamber. The proportion of activated B cells expressing immunoglobulin kappa (Igkappa) was also significantly elevated in the exudate of the nucleus pulposus chambers. The analysis of the remaining chamber tissue revealed a significantly higher amount of T cells (CD45RC) in the nucleus pulposus chambers than in the empty chambers. Conclusions The present findings indicate that nucleus pulposus attracts activated T and B cells. However, since the cell population in the nucleus pulposus of young pigs may differ from that of adult humans, the obtained data may not be directly transferred to the human situation of a disc herniation. The observations in the present study may nevertheless explain some of the local tissue reactions occurring in association with disc herniation and nerve root involvement, thereby providing further insight into the pathophysiology of sciatica.

Study design The mechanisms behind the formation of a characteristic tissue reaction at the surface of the dorsal root ganglion (DRG) exposed to nucleus pulposus was studied with special reference to apoptosis using electron microscopy and immunohistochemistry in rats. Objectives To study the mechanism of the characteristic tissue reaction at the surface of the DRG exposed to nucleus pulposus. Summary of background data Recently, it was observed that local application of nucleus pulposus may induce a characteristic tissue reaction at the surface of the DRG. This change occurred as early as 1 day after the application of nucleus pulposus. METHODS.: Herniation of nucleus pulposus was created in the L4-L5 disc in rats. The L4 DRG were resected 3 and 24 hours after surgery. The sections of the specimens were observed using light and electron microscopy. The sections were processed for immunohistochemistry using antibodies to single-stranded DNA (ssDNA), caspase 3, and tumor necrosis factor alpha (TNF). Results There were typical changes of the cell nuclei observed by light and electron microscopy, especially those of the small-sized cells, in the DRG 24 hours after application of nucleus pulposus, indicating the presence of apoptosis. The presence of ssDNA, caspase 3, and TNF further enhanced the impression that there was apoptosis in the DRG. Nucleus pulposus induced apoptosis in the DRG at the site of application within as little as 24 hours. Conclusions Nucleus pulposus herniated from the disc induced apoptosis in at the surface of the DRG exposed to nucleus pulpous as early as 24 hours after exposure.

Different fracture fixation techniques and fracture environment influence bone formation in healing fractures. However, the influence on the development of biomechanical properties has not been clear described. We evaluated the influence of fracture fixation stability and fracture environment on mechanical properties in healing femoral fractures in rats.Animals were treated surgically with external fixation: 1 group (27 rats) with 0-mm fracture gap size with bone ends touching, corresponding to an axial stiffness of 265 (SD 34) N/mm, and a second group (27 rats) with 2-mm fracture gap size corresponding to an axial stiffness of 30 (SD 2.1) N/mm. From each group, 6-7 animals were killed at 2, 4, 6 and 12 weeks. Torsional test revealed a delay in torsional stiffness in fractures in group 2 compared to group 1. In group 2, the torsional stiffness of the contralateral femora was found to be greater at 12 weeks than the torsional stiffness in group 1.We found that during fracture healing, the development of torsional stiffness corresponds to the magnitude of endochondral ossification and late response of bone formation. A significantly increased torsional stiffness in the non-fractured leg of rats with delayed fracture healing was also found, possibly indicating a response to loading conditions or a systemic stimulation of bone mass.

STUDY DESIGN: The effect of infliximab, a chimeric monoclonal antibody to TNF-alpha, on induction of brain-derived neurotrophic factor (BDNF) was examined using an experimental herniated nucleus pulposus (NP) model. OBJECTIVES: To investigate whether treatment of infliximab could attenuate an induction of BDNF, which functions as a modulator of pain, following NP application to the nerve root. SUMMARY OF BACKGROUND DATA: Evidence from basic scientific studies proposes that TNF-alpha is involved in the development of NP-induced nerve injuries. However, the therapeutic mechanisms of infliximab against pain have not been elucidated experimentally. METHODS: Twenty rats were used in this study. In the test groups, the animals underwent application of NP to the L4 nerve roots and received a single systemic (intraperitoneal) injection of infliximab at the time of surgery (Infli-0 group, n = 5) or at 1 day after operation (Infli-1 group, n = 5). As a control treatment, sterile water was administered intraperitoneally to 5 rats with NP application (NP group) and to 5 sham-operated rats (sham group). On day 3 after surgery, the L4 dorsal root ganglion (DRG) and L4 spinal segment were harvested and assessed regarding BDNF immunoreactivity. RESULTS.: Application of NP induced a marked increase of BDNF immunoreactivity in number in the DRG neurons and within the superficial layer in the dorsal horn compared with the sham group (P < 0.01). Infliximab treatment in the Infli-0 and Infli-1 groups reduced the BDNF induction in both DRG and spinal cord (P < 0.05). CONCLUSION: These findings indicate that infliximab attenuates the elevated BDNF levels induced by NP. The present study therefore further indicates the importance of TNF-alpha in sciatica due to disc herniation and the possible therapeutic use of a TNF-alpha inhibitor for this condition.

Application of nucleus pulposus to the spinal nerve and displacement of the adjacent nerve results in behavioral changes in rats. It has been reported that treatment with the tumor necrosis factor-alpha (TNFalpha) inhibitor, infliximab, significantly reduces spontaneous pain behavior in this animal model. However, there have been no reports of the effects of infliximab on mechanical or thermal hyperalgesia using this model. Disk incision and adjacent spinal nerve displacement were performed with (n = 6) or without (n = 6) injection of infliximab. A control group also underwent sham surgery (n = 6). The animals were tested for 3 days before and on days 1, 3, 7, 14, and 21 after surgery. Non-noxious mechanical thresholds were tested by determining the hind paw withdrawal response to von Frey hair stimulation of the plantar surface of the footpad with a touch stimulator. Thermal nociceptive thresholds were tested using a sensitive thermal testing device. While disk incision with displacement surgery rats showed mechanical and thermal hyperalgesia after surgery on the experimental side, neither rats treated with infliximab nor the sham operation controls showed these effects. Injection of infliximab seemed to prevent mechanical and thermal hyperalgesia caused by the combination of disk incision and nerve displacement.

STUDY DESIGN To analyze the effects of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma on cauda equina function and to define if any of these cytokines could induce nerve root dysfunction comparable with the situation with application of nucleus pulposus. SUMMARY OF BACKGROUND DATA Proinflammatory cytokines derived from the intervertebral disc have been suggested to mediate the nucleus pulposus-induced nerve root injury following local application of nucleus pulposus. However, it is not known if such cytokines may induce similar injury if applied separately. METHODS A total of 29 pigs were used. Nucleus pulposus was harvested from lumbar discs and applied to the sacrococcygeal cauda equina following laminectomy of the first coccygeal vertebra in seven pigs. Five pigs received 1.66 microg of tumor necrosis factor-alpha, five pigs received 0.85 microg of interleukin-1beta, and five pigs received 1.66 microg of interferon-gamma. Seven pigs received autologous fat for control. Nerve conduction velocity was studied by local electrical stimulation and recordings in the tail muscles 7 days after the application. RESULTS Application of nucleus pulposus and fat induced similar effects as seen in previous studies, with normal nerve conduction velocity for fat and a significant reduction for nucleus pulposus. Application of both interleukin-1beta and IFN-gamma induced slight reductions of nerve conduction velocity compared with fat, but they were not statistically significant. Tumor necrosis factor-alpha, however, induced a reduction of the velocity that was even more pronounced than for nucleus pulposus. CONCLUSION Based on previous observations and the data of the present study, one may conclude that tumor necrosis factor-alpha from nucleus pulposus cells seems to be intimately involved with the basic pathophysiologic events leading to both nerve root dysfunction and pain after local, epidural application of nucleus pulposus. One may therefore also suspect that pharmacologic inhibition of tumor necrosis factor-alpha may at least theoretically be considered in the clinical situation with disc herniation and sciatica.

STUDY DESIGN The effects of diclofenac and ketoprofen on nerve conduction velocity in experimental nerve root compression were evaluated in a setup using an established pig model. OBJECTIVE To assess the effects of two potent nonsteroidal antiinflammatory drugs, diclofenac and ketoprofen, in experimental nerve root compression. SUMMARY OF BACKGROUND DATA Compression of spinal nerve roots is recognized to be of major etiologic importance for several common spinal pain syndromes. Secondary inflammatory changes, induced by microvascular permeability changes and leakage of inflammatory mediators into the endoneural tissue, have been proposed as important for the induction of spinal nerve root injury by chronic compression. METHODS This study involved 21 pigs. An ameroid constrictor was used to induce compression. Seven pigs were treated with daily intramuscular injections of diclofenac 3 mg/kg for 7 days. Seven other pigs were treated with daily intramuscular injections of ketoprofen 4 mg/kg. For a control, seven pigs did not receive any drug treatment. After 7 days, the pigs were reanesthetized, and the nerve conduction velocity in the compressed nerve root segments was determined. RESULTS The nerve conduction velocity was significantly higher (P < 0.05, Student's t test) in the pigs treated with diclofenac (50 +/- 16 m/second) than in the untreated pigs (32 +/- 15 m/second). The nerve conduction velocity also was significantly higher (P < 0.05) in the pigs treated with ketoprofen (59 +/- 16 m/second) than in the untreated pigs. There were no significant differences in nerve conduction velocity between pigs treated with ketoprofen and those treated with diclofenac. CONCLUSIONS The findings indicate that intramuscular administration of diclofenac or ketoprofen, both potent antiinflammatory drugs, may reduce nerve root dysfunction induced by compression of spinal nerve roots in an experimental pig model.

Study design Nerve conduction velocity was measured in the pig cauda equina after local application of anulus fibrosus or in vitro/postmortem degenerated nucleus pulposus from the same pig. Objectives To analyze the effects of anulus fibrosus and degenerated nucleus pulposus on nerve conduction velocity. Summary of background data Previous studies on nucleus pulposus-induced effects on nerve roots have used normal, nondegenerated nucleus pulposus. Because both anulus fibrosus and degenerated nucleus pulposus are commonly seen in the clinical situation of disc herniation, the value of the previous work could be questioned. Methods Anulus fibrosus and nucleus pulposus were harvested using a retroperitoneal approach. The nucleus pulposus was degenerated artificially either by addition of sodium lactate with HCl added to form a pH of either 6.0 or 3.5 (in vitro degeneration), or by storing the nucleus pulposus at 4 C until a pH of 6.0 (postmortem degeneration) was reached. After epidural application, the nerve conduction velocity was determined at 7 days (anulus fibrosus) or 3 days (degenerated nucleus pulposus). Results Application of anulus fibrosus did not induce any reduction of nerve conduction velocity. In vitro and postmortem degenerated nucleus pulposus induced a reduction of nerve conduction velocity similar to that of normal nucleus pulposus. Conclusions Although only nerve function and not pain was assessed, it seems likely that previous experiments using normal nucleus pulposus may be relevant for evaluating the pathophysiologic mechanisms behind the nucleus pulposus-induced nerve root injury, also in a clinical perspective.

Main problem. Nucleus pulposus and/or chronic compression can induce spinal nerve root injury. Inflammation has been proposed as having major importance in the pathophysiologic mechanisms involved in the induction of such injuries. Corticosteroids, potent anti-inflammatory drugs, have been demonstrated to reduce nucleus pulposus-induced spinal nerve root injury. The aim of the present study was to assess the effects of two potent nonsteroidal anti-inflammatory drugs (NSAIDs), diclofenac and ketoprofen, in experimental nucleus pulposus-induced spinal nerve root injury in a pig model. Methods. Eighteen pigs were included in the study. Autologous nucleus pulposus was harvested from a lumbar disc and applied locally around the first sacral nerve root after a partial laminectomy of the first and second sacral vertebrae. Six pigs were treated with daily intramuscular injections of diclofenac, 3 mg/kg body weight, for 7 days. Six other pigs were treated with daily intramuscular injections of ketoprofen, 4 mg/kg body weight, for 7 days. As controls, six pigs received injections with physiologic saline. After 7 days, the pigs were reanesthetized and the nerve conduction velocity over the exposed nerve root area was determined. Results. The nerve conduction velocity was significantly higher in pigs treated with diclofenac than in the saline-treated controls, (57±6 m/s vs 38±18 m/s, P

STUDY DESIGN The possibility to prevent nucleus pulposus-induced functional and structural nerve root injury by selective tumor necrosis factor-alpha inhibition was assessed in an experimental model in the pig spine. OBJECTIVE The objective of the study was to evaluate the role of tumor necrosis factor-alpha in the mediation of nucleus pulposus-induced nerve injury by using selective inhibition. SUMMARY OF BACKGROUND DATA The cytokine tumor necrosis factor-alpha has been suggested to play a key role in the nerve root injury induced by local application of nucleus pulposus. However, previous studies have not been able to distinguish the effects between tumor necrosis factor-alpha and other disc-related cytokines because of the use of nonspecific cytokine inhibition. METHODS Autologous nucleus pulposus was harvested from a lumbar disc and applied to the porcine sacrococcygeal cauda equina. The pigs were simultaneously treated with two selective tumor necrosis factor-alpha 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-alpha inhibitors prevented the reduction of nerve conduction velocity and also seemed to limit the nerve fiber injury, the intracapillary thrombus formation, and the intraneural edema formation. 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 necrosis factor-alpha 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-alpha medication as treatment in patients with disc herniation and sciatica.

Although it is well established that nucleus pulposus cells may induce structural and functional changes in adjacent nerve roots when placed epidurally, it is not known whether this is due to direct neurotoxic effects or whether the nerve roots are affected indirectly by reduction of nutrition and inflammatory/immunologic mechanisms. In the present study we assessed the effects of various tissues on cultured dorsal root ganglions from newborn rats. Nucleus pulposus was found to have a toxic effect on the axons by blocking axonal outgrowth, but no similar effects on the nerve cell bodies (extra-ganglionic nerve cell density, nerve cell arborisation) were found as compared to the series with only culture medium. Sterile water for 1 or 24 h (positive controls) induced significant effects by all four criteria, whereas medium without nerve growth factor, fat and frozen nucleus pulposus had no statistically significant effects. The study thus showed that there are direct axonotoxic effects induced by the nucleus pulposus, and since frozen nucleus pulposus did not have any effects, it may be assumed that the mechanisms are related to substances produced by the nucleus pulposus cells. The presented model allows for future studies on the neurotoxic properties of nucleus pulposus cell-derived candidate substances.

STUDY DESIGN Measurement of changes in cerebrospinal fluid concentrations of nerve tissue markers, total proteins, and immunoglobulin after compression of nerve root or application of nucleus pulposus in a pig model. OBJECTIVES To assess whether compression or application of nucleus pulposus to spinal nerve roots may cause increased levels of cerebrospinal fluid markers of nerve tissue damage and total proteins, and whether synthesis of immunoglobulins may be induced in cerebrospinal fluid. SUMMARY OF BACKGROUND DATA Previous studies have reported that there seems to be a relationship between elevated cerebrospinal fluid total protein concentrations, nerve tissue markers, clinical findings, and compression of the nerve root evaluated by radiologic changes in patients with sciatica. METHODS Subjects included 41 pigs, including 5 control animals. In two groups of experimental animals (n = 7; n = 5), an ameroid constrictor was slid onto the S1 nerve root. In two other groups (n = 7; n = 5), nucleus pulposus harvested from the L2-L3 disc was applied to the S1 nerve root. Two sham animal groups (n = 7; n = 5) underwent the same laminectomy. Twenty-one pigs underwent reoperation after 1 week, and 15 pigs after 4 weeks. A syringe was used to remove 3 mL of cerebrospinal fluid at L4-L5. Concentrations of total proteins, the light subunit of the neurofilament protein, S-100 protein, neuron-specific enolase, and glial fibrillary acidic protein were measured, and the presence of oligoclonal bands (immunoglobulins) were assayed in cerebrospinal fluid. RESULTS The pigs with compressed S1 nerve root had considerably higher neurofilament protein and total protein concentrations in their cerebrospinal fluid than the-control animals (P < 0.001 and P < 0.01, respectively) or the sham animals (P < 0.001 and P < 0.05) in the 1-week experiment. Nucleus pulposus did not induce a significant increase in concentrations of the different protein markers. The presence of oligoclonal bands in cerebrospinal fluid in the experimental groups did not differ between the control and sham animals. CONCLUSIONS The neurofilament protein and total protein concentrations in cerebrospinal fluid may have diagnostic importance in cases wherein clinical findings are not clearly related to the radiologic changes and vice versa. These protein markers also may be useful tools in different experimental models.

STUDY DESIGN Compression onset rate, anatomic aspects, and morphologic and neurophysiologic effects in spinal nerve roots were studied in a nerve root compression model in pigs. OBJECTIVES To analyze the compression onset rate by measuring the gradual reduction of the inner diameter of the constrictor, the motor nerve conduction velocity by electromyography, the morphologic changes by light microscopy, and the gross and vascular anatomy by dissection and ink injections, respectively, in a model for experimental chronic nerve root compression. SUMMARY OF BACKGROUND DATA Chronic nerve root compression is recognized to be related to back pain syndromes, including sciatica. Various aspects of morphologic and physiologic changes have been studied previously in models for acute compression and chronic nerve root irritation, but a controlled, graded chronic nerve root compression model has not been described. METHODS An ameroid constrictor was applied around a spinal nerve root just cranial to the dorsal root ganglion. The inner diameter of this constrictor gradually becomes reduced. After 1 week or 4 weeks, electromyographic measurements were performed, and tissue samples were harvested for histologic analyses. The gross and vascular anatomy of the pigs' spinal nerve roots were studied by dissection and ink injections. RESULTS There was a statistically significant decrease in the nerve conduction velocity in compressed compared with noncompressed spinal nerve roots after 1 week and after 4 weeks. The ameroid constrictors induced nerve fiber damage, endoneural hyperemia, bleeding, and inflammation at the compression zone. There was often a severe reduction in the number of myelinated fibers after 4 weeks. CONCLUSION A model for controlled, chronic, partial nerve root injury using a gradual compression-onset constrictor is presented. This model allows for induction of a controlled graded chronic nerve root injury and can be used for research on basic pathophysiologic mechanisms and on the effects of various interventions on nerve root injury development.

Study design Autologous nucleus pulposus was modified and applied to the cauda equina in pigs. Histology and neurophysiology were assessed after 7 days. Objectives To assess if alterations of the nucleus pulposus would change the degree and distribution of the nerve injury induced by autologous nucleus pulposus. Summary of background data It was reported recently that nucleus pulposus may induce structural and functional changes in nerve roots after epidural application. The basic mechanisms causing these changes are not fully understood. Methods Nucleus pulposus was harvested from lumbar discs and submitted to either of three treatments; 37 C for 24 hours (n = 5), -20 C for 24 hours (n = 5), or digestion by hyaluronidase for 24 hours (n = 6). In two additional pigs, nucleus pulposus was applied just after harvest as a control to verify previous observations. After 7 days, nerve conduction velocity was recorded, and specimens were processed for blinded light microscopic assessment. Results When nucleus pulposus was applied just after harvest, or when it had been kept at 37 C or digested by hyaluronidase for 24 hours, there was a significant reduction in nerve conduction velocity similar to previous observations. When nucleus pulposus had been kept at -20 C for 24 hours, however, there was no reduction in conduction velocity. There were no apparent differences between the groups at the histologic assessment. Staining of the nucleus pulposus showed that the cells in the nucleus pulposus exposed to -20 C were lysed, whereas the cells in the nucleus pulposus treated by the two other methods were mainly unaffected. Conclusions Because freezing of the nucleus pulposus probably kills the cells but does not affect other components, one may assume that the biologic effects induced by the nucleus pulposus may be related to its cell population.

Study design Intramuscular pressure and blood flow of the back muscles were evaluated topographically during posterior lumbar spine surgery. The topographic damage of the back muscle after surgery was studied. Objective To investigate the relationship between intramuscular pressure or blood flow during posterior lumbar surgery and the back muscle injury after surgery. Summary of background data Latrogenic back muscle injury in an animal and human model has been reported previously. Changes of intramuscular pressure and blood flow during surgery might be related to the muscle injury. No previous study on this issue has been published. Methods The contact pressure between the retractor blade and muscle tissue was monitored in 10 pigs during posterior surgery of the lumbar spine. On one side, intramuscular pressure at 5, 10, and 20 mm lateral to the retractor and on the other side blood flow of the back muscle at 5 and 20 mm during surgery were measured. Histologic changes of the back muscle at 5, 10, and 20 mm to the midline were evaluated 3 hours after surgery. Results The contact pressure decreased exponentially with time. Intramuscular pressure 5 mm lateral to the retractor was 114 +/- 31 mm Hg and was significantly higher than at 10 mm and 20 mm. Blood flow markedly decreased during surgery and recovered incompletely after releasing the retractor at 5 mm and 20 mm lateral to the retractor. Blood flow at 5 mm was significantly lower than at 20 mm throughout surgery. The muscle damage 3 hours after surgery was more severe near the retractor blade. Conclusions The back muscles were exposed to pathophysiologic condition by a retractor during posterior lumbar spine surgery. External compression by a retractor increases intramuscular pressure to levels that impede local muscle blood flow. The muscle degeneration after surgery could be explained by direct mechanical damage and by the increased intramuscular pressure of muscle tissue by the retractor.

Study Design. Lumbar intertransverse process arthrodesis using recombinant human bone morphogenetic protein-2 was performed in a previously established rabbit model for posterolateral spinal fusion and compared with fusions achieved using autogenous bone graft. Objectives. To qualitatively compare different recombinant human bone morphogenetic protein-2 dosages and carriers and to determine the efficacy of recombinant human bone morphogenetic protein-2 as a bone graft substitute to produce lumbar intertransverse process fusion in a validated rabbit model for posterolateral spinal fusion. of Background Data. Autogenous bone was considered the most successful bone graft material used for spinal arthrodesis. Problems with its use may occur in 25-30% of patients and prompted the search for and investigation of bone graft substitutes and osteoinductive growth factors, such as bone morphogenetic proteins. Recombinant human bone morphogenetic protein-2 was used successfully in orthotopic sites to generate bone in animal mandibular and long bone defect models. Methods. Posterolateral intertransverse process arthrodeses were performed at L5-L6 in 56 rabbits using recombinant human bone morphogenetic protein-2 or autogenous bone graft. Rabbits were killed either 5 weeks later to qualitatively compare fusions achieved using different recombinant human bone morphogenetic protein-2 dosages and carriers or 4 weeks later to compare the efficacy of recombinant human bone morphogenetic protein-2 in achieving spinal fusion compared with using autogenous bone graft. Inspection, manual palpation, radiography, histology, and biomechanic testing were used to assess the fusion. Results. All rabbits implanted with recombinant human bone morphogenetic protein-2 achieved solid spinal fusion by manual palpation and were fused radiographically, whereas only 42% of the autograft control fusions were solid. More mature fusions with greater trabecular bone formation were shown radiographically and histologically in rabbits implanted with the high-dose recombinant human bone morphogenetic protein-2 than with the low-dose recombinant human bone morphogenetic protein-2. Fusions achieved using recombinant human bone morphogenetic protein-2 delivered in the collagen carrier were more remodeled and homogeneous compared with using recombinant human bone morphogenetic protein-2 delivered in autograft ± collagen carrier. Fusions achieved with recombinant human bone morphogenetic protein-2 were biomechanically stronger and stiffer than fusions achieved using autogenous bone graft. Conclusions. Recombinant human bone morphogenetic protein-2 successfully and reliably achieved lumbar intertransverse process fusion in a validated rabbit model for posterolateral spinal fusion. Radiographically and histologically, greater and more rapid bone formation, consolidation, and remodeling were shown with recombinant human bone morphogenetic protein-2 compared with autogenous bone graft. Fusions achieved with recombinant human bone morphogenetic protein-2 were biomechanically stronger and stiffer than autograft fusions.

Study Design. The inflammatogenic properties of nucleus pulposus were assessed in two experimental models previously used for screening of inflammatogenic properties of other substances. This study was performed to assess the inflammatogenic properties of nucleus pulposus in models previously screened for other substances. Summary of Background Data. Previous experimental studies, as well as clinical observations, have indicated that inflammatory mechanisms may constitute an important pathogenetic component in sciatica due to herniation of the nucleus pulposus. Methods. In the first experimental series, autologous nucleus pulposus and retroperitoneal fat were placed in perforated titanium chambers, which were placed subcutaneously in pigs, together with an empty chamber as sham. After 7 days, the number of leukocytes in the chambers was determined. In the second experimental series, the microvascular reactions were studied by vital microscopy of the hamster cheek-pouch after local injections of suspensions of homologous nucleus pulposus and homologous subcutaneous fat. Macromolecular extravascular leakage was studied by fluorescence microscopy using FITC-dextran as a tracer. Results. The leukocyte ratio betwee fat control and sham was 0.9±0.6 and between nucleus pulposus and sham 2.4±0.7. The nucleus pulposus thus attracted significantly more leukocytes than fat. Injection of nucleus pulposus suspension induced thrombosis formation and pronounced leakages of macromolecules in a majority of the injection sites. However, injection of vehicle and fat suspension in the cheek-pouch only resulted in minor vital microscopic changes. Conclusions. Nucleus pulposus demonstrated inflammatogenic properties as indicated by leukotaxis and an increase of vascular permeability. It was not clear, however, if these reactions were induced by substances from the nucleus pulposus per se or from substances being liberated from other as a response to an interaction with components of the nucleus pulposus

Study design The possibility of epidurally applied substances reaching the intraneural capillaries of the spinal nerve roots and cauda equina was assessed in the pig sacrococcygeal spine. Methods The presence of Evans blue-labelled albumin in intraneural capillaries after epidural application for 1, 10, or 30 minutes was studied with fluorescence microscopy. Ink angiography was used to determine whether there were any direct communicating vessels between the epidural vein plexus and the intraneural capillaries. Results Evans blue-labelled albumin was present in the intraneural capillaries 1 minute after epidural application. Microangiography demonstrated small venules that connected the epidural vein plexus and the intraneural capillaries. Conclusions The results of this study demonstrated a rapid transport route between the epidural space and the intraneural capillaries. The results suggest that nucleus pulposus material, as well as epidurally applied substances, such as local anesthetic drugs or epidurally injected corticosteroids, may have a rapid, direct transport route to the axons of the spinal nerve roots. The demonstrated transport route also may be related to the mechanisms behind epidural anesthesia and spinal nerve root infiltration.

Study Design. The effects of intervention by intravenous injection of methylprednisolone to reduce the nerve root injury after epidural application of autologous nucleus pulposus was studied in an experimental model on the pig cauda equina in 20 animals. Methods. Nucleus pulposus was harvested from a lumbar disc. After lowering the pH of the nucleus pulposus to 3.5 it was placed onto the sacrococcygeal cauda equina. Fifteen of the pigs received a single intravenous injection of 30 mg/kg methylprednisolone, 5 minutes, 24 hours, or 48 hours, respectively, after the application. After 7 days, the nerve conduction velocity was determined, and biopsies of the cauda equina was examined by lightmicroscopy. Results. In the live pigs that did not receive any methylprednisolone treatment, nerve conduction velocity was reduced, whereas it was normal in the pigs treated 5 minutes and 24 hours after nucleus pulposus application. In pigs treated after 48 hours, nerve conduction velocity was reduced only slightly. At the light microscopic level, significant changes occurred in all series. Conclusions. This study indicates that the nucleus pulposus-induced effects on nerve function in an experimental pig model may be reduced dramatically by high-dose methylprednisolone administration within 24–48 hours after epidural application of autologous nucleus pulposus. The light microscopic changes were probably not significant for the nerve function. Instead, a morphologic explanation on a subcellular level should probably be sought.

Epidural application of autologous nucleus pulposus in pigs, without mechanical nerve root compression, induced a pronounced reduction in nerve conduction velocity in the cauda equina nerve roots after 1-7 days, compared to epidural application of retroperitoneal fat in control experiments. Histologically, the nerve fiber injury was more pronounced after application of nucleus pulposus than after control tissue application. The results demonstrate that nucleus pulposus may induce nerve tissue injury by mechanisms other than mechanical compression. Such mechanisms may be based on direct biochemical effects of nucleus pulposus components on nerve fiber structure and function and microvascular changes including inflammatory reactions in the nerve roots.