Akira Onda

Epidural application of nucleus pulposus enhances nociresponses of rat dorsal horn neurons.

Study Design. An electrophysiologic study to examine responses of dorsal horn neurons in the rat L5 spinal cord to noxious stimuli after disc herniation or application of the nucleus pulposus to the L5 nerve root. Objectives. To investigate the pathogenic role of nucleus pulposus in the neural mechanism underlying sciatica and low back pain. Summary of Background Data. Application of the nucleus pulposus to the lumbar nerve root induced morphologic, vascular, and functional changes in the nerve root, suggesting that some factors in the nucleus pulposus may be implicated in the pathogenesis of chronic changes. However, it has not been studied whether the epidural application of nucleus pulposus enhances nociresponses of pain-processing neurons in the superficial dorsal horn of the spinal cord. Methods. Extracellular recordings were made from the L5 dorsal horn neurons in 20 Wistar rats. The wide-dynamic-range (WDR) neurons activated by electric stimulation of the ipsilateral footpad of hindpaw were selected, and their responses to noxious and innocuous stimulation were examined after L5–L6 disc herniation onto the L5 nerve root (Group A) and application of the autologous nucleus pulposus (Group B) or fat tissues (Groups C and D). Results. The herniation of the nucleus pulposus (Group A) and application of the autologous nucleus pulposus (Group B) to the nerve root remarkably enhanced responses of WDR neurons to noxious stimuli for hours, whereas application of fat tissue scarcely enhanced nociresponses (Groups C and D). Conclusions. Results suggest that somewhat pathogenic factors in the nucleus pulposus may have a crucial role in the induction of hyperalgesia. This may help to elucidate the reason why a severe pain is sometimes induced without a visually identified protrusion.

Distribution and Appearance of Tumor Necrosis Factor-α in the Dorsal Root Ganglion Exposed to Experimental Disc Herniation in Rats

Study Design. Distribution and appearance of tumor necrosis factor-alpha (TNF-&agr;) 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-&agr; 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-&agr; 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-&agr; 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-&agr;. The TNF-&agr;-positive cells were observed and counted using light microscopy. Distribution of the TNF-&agr; 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-&agr; 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.

Effects of neutralizing antibodies to tumor necrosis factor-alpha on nucleus pulposus-induced abnormal nociresponses in rat dorsal horn neurons.

Study Design. The effect of an anti–tumor necrosis factor alpha (anti-TNF&agr;) antibody on abnormal discharges caused by application of nucleus pulposus to the nerve root was investigated in an electrophysiologic study. Objectives. To assess whether inhibition of TNF&agr; can reduce nucleus pulposus–induced abnormal discharges. Summary of Background Data. It has been shown that TNF&agr;, a proinflammatory cytokine, is a key pathogenic factor in the development of nucleus pulposus–induced abnormal discharges as a pain sensation. However, the electrophysiologic mechanisms involved in sciatica after disc herniation still have not been elucidated. Methods. Extracellular activities of wide-dynamic-range neurons were assessed in 21 rats. Autologous nucleus pulposus harvested from the tail was applied to the L5 nerve root. The animals were simultaneously treated with antibodies to TNF&agr; (anti-TNF + nucleus pulposus group) and with phosphate-buffered saline (nucleus pulposus group). As a control (control group), a similar volume of muscle was applied to the nerve root with phosphate-buffered saline. Responses of wide-dynamic-range neurons to noxious and innocuous stimuli were examined for 2 hours. Results. Discharges evoked during noxious stimulation and discharges after withdrawal of stimulation in the nucleus pulposus group were significantly higher than those in the control group (P < 0.05). In the anti-TNF + nucleus pulposus group, discharges after withdrawal of stimulation were remarkably inhibited, as compared with those of the nucleus pulposus group (P < 0.05). However, evoked discharges during stimulation apparently were not inhibited. Responses to innocuous stimulation did not change throughout the measurements. Conclusions. These data indicate that application of TNF&agr; antibodies to the nerve root partially prevents the nucleus pulposus–induced abnormal nociresponses. Therefore, anti-TNF&agr; treatment may have a therapeutic effect on sciatica after lumbar disc herniation.

Exogenous Tumor Necrosis Factor-α Induces Abnormal Discharges in Rat Dorsal Horn Neurons

STUDY DESIGN An electrophysiologic study to examine effects of exogenous application of tumor necrosis factor-alpha (TNF-alpha ) activities and nociresponses of dorsal horn neurons in the spinal cord at L5. OBJECTIVES To investigate the role of TNF-alpha in the induction and development of hyperalgesia in neural mechanisms responsible for a radicular pain. SUMMARY OF BACKGROUND DATA TNF-alpha is found in the herniated disc and known to play a pivotal role in the development of inflammatory hyperalgesia; however, it is not known whether TNF-alpha causes abnormal discharge in the dorsal horn neurons and enhances nociresponse. METHODS Single-unit activities of neurons in the L5 superficial dorsal horn were extracellularly recorded, using 28 urethane-anesthetized rats. The wide dynamic range and nociceptive-specific neurons activated by stimulation of the hind paw were selected. Effects of exogenous TNF-alpha were examined regarding 1) spontaneous discharges of wide dynamic range and nociceptive-specific neurons, 2) responses of wide dynamic range neurons to noxious stimulation, and 3) morphologic changes in the dorsal root ganglion. RESULTS Application of TNF-alpha to the nerve root induced 1) a significant increase in spikes/sec in spontaneous discharges of wide dynamic range and nociceptive-specific neurons, 2) enhanced responses of wide dynamic range neurons to noxious stimulation, and 3) inflammatory changes in the ganglion. CONCLUSION These results suggest the possibility that TNF-alpha produced in the vicinity of nerve roots due to disc herniation might cause ectopic discharges in primary afferent fibers and thereby induce the prolonged excitation in pain-processing neurons responsible for radicular pain.

Infliximab Attenuates Immunoreactivity of Brain-derived Neurotrophic Factor in a Rat Model of Herniated Nucleus Pulposus

Study Design. The effect of infliximab, a chimeric monoclonal antibody to TNF-&agr;, 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-&agr; 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-&agr; in sciatica due to disc herniation and the possible therapeutic use of a TNF-&agr; inhibitor for this condition.

Selective Inhibition of Tumor Necrosis Factor-α Prevents Nucleus Pulposus-Induced Histologic Changes in the Dorsal Root Ganglion

Study Design. The possibility to prevent nucleus pulposus-induced structural changes of the dorsal root ganglion (DRG) by selective tumor necrosis factor-&agr; (TNF-&agr;) inhibition was assessed in an experimental model in the rat spine. Objectives. To evaluate the role of TNF-&agr; in the mediation of nucleus pulposus-induced structural changes by using selective inhibition and to confirm the effect of TNF-&agr; inhibitor at the point of histologic findings. Summary of Background Data. TNF-&agr; 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-&agr; 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-&agr; 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-&agr;-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-&agr; inhibitor 1 day before or 3 hours after surgery. The other group received no TNF-&agr; 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-&agr;. The histologic findings and TNF-&agr;-positive cells were observed by light microscopy. Results. 1) While positively stained immunoreactive TNF-&agr; appeared between 7 and 21 days, no immunoreactive TNF-&agr; 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-&agr; 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-&agr; was observed in the DRGs of the TNF-&agr; 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.

Prevention of Compartment Syndrome in Dorsal Root Ganglia Caused by Exposure to Nucleus Pulposus

Study Design. An experimental study to clarify the effects of pentoxifylline, as an anti-tumor necrosis factor-alpha therapy on endoneurial fluid pressure in the dorsal root ganglion using an animal model of herniated nucleus pulposus. Objectives. To investigate the effects of anti-tumor necrosis factor-alpha therapy to nucleus pulposus-induced nerve root/dorsal root ganglion changes. Summary of Background Data. It has been reported experimentally that application of nucleus pulposus into epidural space induces morphologic and functional changes in the nerve roots and induces compartment syndrome in the dorsal root ganglia. Tumor necrosis factor-alpha has been considered a key pathogenic factor in the initiation and maintenance of neuropathic pain states. Methods. A total of 11 adult, female Sprague-Dawley rats had their left L5 nerve roots and associated dorsal root ganglions exposed. Autologous nucleus pulposus was applied to the L5 nerve root just proximal to the dorsal root ganglion. A piece of Spongel (Yamanouchi Pharmaceutical Co., Tokyo) containing 20 &mgr;L of 1000 &mgr;g/mL pentoxifylline was applied with the nucleus pulposus (NP+PTX group). In control animals nucleus pulposus was applied with a piece of Spongel containing 20 &mgr;L of physiologic saline solution in a similar fashion (NP+PS group). Endoneurial fluid pressure was recorded with a servo-null micropipette system using glass micropipettes with tip diameters of 4 &mgr;m. Endoneurial fluid pressure in the dorsal root ganglion was measured before and 3 hours after application of test substances. After measurement of endoneurial fluid pressure, the nerve root and dorsal root ganglion were processed for histology and evaluated by light microscope. Results. Values of endoneurial fluid pressure before application of test substances were as follows: 2.4 ± 1.2 cmH2O in the NP+PS (control) group and 1.8 ± 0.4 cmH2O in the NP+PTX group. There was no statistically significant difference between these two pretreatment measurements. However, values of endoneurial fluid pressure after application were as follows: 8.6 ± 1.8 cmH2O in the NP+PS group and 2.9 ± 0.8 cmH2O in the NP+PTX group. Values of endoneurial fluid pressure in the NP+PTX group were significantly lower compared with the NP+PS group. Histologic examination consistently showed only a slight degree of edema evident in the NP+PTX group compared with the NP+PS group. Conclusion. Pentoxifylline, an anti-tumor necrosis factor-alpha drug, prevented the dorsal root ganglion compartment syndrome caused by topical application of nucleus pulposus. Anti-inflammatory cytokine therapy may become an effective treatment of sciatica due to disc herniation.

Changes in pain behavior and histologic changes caused by application of tumor necrosis factor-alpha to the dorsal root ganglion in rats.

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/&mgr;L 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/&mgr;L TNF application, as compared to the 0.5-ng/&mgr;L 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.

Nerve growth factor content in dorsal root ganglion as related to changes in pain behavior in a rat model of experimental lumbar disc herniation

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.

Effects of lidocaine on blood flow and endoneurial fluid pressure in a rat model of herniated nucleus pulposus.

STUDY DESIGN An experimental study was conducted to evaluate the effects of lidocaine on nucleus pulposus-induced pathophysiologic changes. OBJECTIVES To investigate the effects of lidocaine on blood flow in the hind paws and endoneurial fluid pressure in the dorsal root ganglia in a rat model of herniated nucleus pulposus, and to clarify the therapeutic mechanisms of nerve root infiltration. SUMMARY OF BACKGROUND DATA It has been shown experimentally that application of nucleus pulposus to the nerve roots increases endoneurial fluid pressure and decreases blood flow in the dorsal root ganglia and the corresponding hind paw. These changes are thought to be an important pathogenic mechanism associated with sciatica caused by disc herniation. Nerve root infiltration is one of the nonoperative effective therapies for radiculopathy caused by disc herniation. However, the therapeutic mechanisms still are unknown. METHODS For this study, 21 Sprague-Dawley rats were used. Autologous nucleus pulposus was applied to the nerve root with a piece of Spongel containing lidocaine (lido group) or physiologic saline solution (control group). In Series 1 of this study (Blood Flow in the Hind Paw), blood flow in the corresponding hind paws was monitored continuously using a laser Doppler flowmeter before application of the test solutions, and every 5 minutes thereafter for an additional 3 hours in both the control (n = 5) and lido (n = 5) groups. In Series 2 of this study (Endoneurial Fluid Pressure in the Dorsal Root Ganglion), endoneurial fluid pressure was recorded with a servo-null micropipette system using glass micropipettes before and 3 hours after application of the test solutions in both the control (n = 6) and lido (n = 5) groups. After measurements, dorsal root ganglia were assessed for histology. RESULTS In Series 1, blood flow in the corresponding hind paw in the control group showed significant reduction as compared with that of the Lido group, starting about 90 minutes after application (P < 0.01-0.05). Hind paw blood flow in the lido group did not show any reduction during measurements. In Series 2, the value of endoneurial fluid pressure in the lido group 3 hours after application was significantly lower than in the control group (P < 0.01). Interstitial (endoneurial) edema in the dorsal root ganglion in the lido group appeared to be qualitatively less than in the control group. CONCLUSIONS The data indicate that lidocaine reduces the pathophysiologic changes in the dorsal root ganglion and hind paws induced by nucleus pulposus. These effects of lidocaine may relate to the mechanisms underlying the therapeutic effects of nerve root infiltration.