Distribution and tumor necrosis factor-alpha isoform binding specificity of locally administered etanercept into injured and uninjured rat sciatic nerve.

Tumor necrosis factor-alpha (TNF) is a pro-inflammatory cytokine that is implicated in the initiation of neuropathic pain. Locally administered TNF antagonist etanercept offers a promising new treatment approach to target neuropathic pain. Here we evaluate the distribution and binding specificity for TNF isoforms of locally administered etanercept into injured and uninjured rat sciatic nerve. Distribution and co-localization of etanercept and TNF in the injured and uninjured nerve was evaluated at 1, 24, 48 and 96 h after etanercept local application using immunohistochemistry. In addition, binding specificity of etanercept for TNF isoforms was analyzed using immunoblot assay system in nerve lysates. A new observation was that locally administered etanercept reached the endoneurium of the injured but not the uninjured nerve 1 h after its application and mainly co-localized with TNF-positive structures, morphologically similar to Schwann cells and macrophages. We further noticed that immunoblot analyses for etanercept demonstrated its preferential binding to transmembrane and trimer TNF isoforms. Finally, locally administered etanercept inhibited pain-related behaviors in a rat sciatic nerve crush model. We conclude that locally administered etanercept reaches the endoneurial space in the injured nerve and preferentially binds to transmembrane and bioactive trimer TNF isoforms to modulate neuropathic pain. Locally administered etanercept has potential as a targeted immunomodulating agent to treat local pathogenesis in neuropathic pain after peripheral nerve injury.

Effects of levobupivacaine and ropivacaine on rat sciatic nerve blood flow.

BACKGROUND: Ischaemia is one of the causative mechanisms of peripheral nerve injury, a documented complication of regional anaesthesia. Local anaesthetics per se and/or vasopressor adjuvants may account for changes in peripheral nerve blood flow. The aim of this study was to test the effects of levobupivacaine and ropivacaine in a rat sciatic nerve model with respect to local blood flow and histopathological changes. METHODS: Forty-eight female Sprague-Dawley rats were anaesthetized for left sciatic nerve exposure. After baseline nerve blood flow measurement with a laser Doppler flowmeter, 0.2 ml of one of the following solutions was applied topically to the nerve in a random fashion: saline 0.9%; lidocaine 10 mg ml(-1); levobupivacaine 2.5 mg ml(-1); levobupivacaine 5 mg ml(-1); levobupivacaine 7.5 mg ml(-1); ropivacaine 2 mg ml(-1); ropivacaine 7.5 mg ml(-1); and ropivacaine 7.5 mg ml(-1) plus epinephrine 5 microg ml(-1); all in saline 0.9%. Nerve blood flow was evaluated at 5-min intervals up to 30 min after local application of anaesthetic solution. Three animals per group were killed for histological evaluation 48 h later. Multiple one-way analyses of variance followed by Scheffé's post hoc test was used for statistical analysis. P<0.05 was considered significant. RESULTS: Local anaesthetics at all concentrations tested caused significant reduction in nerve blood flow. The combination of ropivacaine 7.5 mg ml(-1) plus epinephrine did not reduce nerve blood flow to a greater extent than ropivacaine 7.5 mg ml(-1) alone. Low concentrations of levobupivacaine (2.5 and 5 mg ml(-1)) reduced nerve blood flow to the same extent as lidocaine 10 mg ml(-1). No significant histological changes were observed at 48 h. CONCLUSION: Despite acute reductions in peripheral nerve blood flow, significant histopathological changes were not observed in this rat sciatic nerve model after topical application of levobupivacaine and ropivacaine at concentrations relevant to clinical practice.

Injury type-specific calcium channel alpha 2 delta-1 subunit up-regulation in rat neuropathic pain models correlates with antiallodynic effects of gabapentin.

The calcium channel alpha2delta-1 subunit is a structural subunit important for functional calcium channel assembly. In vitro studies have shown that this subunit is the binding site for gabapentin, an anticonvulsant that exerts antihyperalgesic effects by unknown mechanisms. Increased expression of this subunit in the spinal cord and dorsal root ganglia (DRG) has been suggested to play a role in enhanced nociceptive responses of spinal nerve-injured rats to innocuous mechanical stimulation (allodynia). To investigate whether a common mechanism underlies allodynic states derived from different etiologies, and if so, whether similar alpha2delta-1 subunit up-regulation correlates with these allodynic states, we compared DRG and spinal cord alpha2delta-1 subunit levels and gabapentin sensitivity in allodynic rats with mechanical nerve injuries (sciatic nerve chronic constriction injury, spinal nerve transection, or ligation), a metabolic disorder (diabetes), or chemical neuropathy (vincristine neurotoxicity). Our data indicated that even though allodynia occurred in all types of nerve injury investigated, DRG and/or spinal cord alpha2delta-1 subunit up-regulation and gabapentin sensitivity only coexisted in the mechanical and diabetic neuropathies. Thus, induction of the alpha2delta-1 subunit in the DRG and spinal cord is likely regulated by factors that are specific for individual neuropathies and may contribute to gabapentin-sensitive allodynia. However, the calcium channel alpha2delta-1 subunit is not the sole molecular change that uniformly characterizes the neuropathic pain states.

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.

Intramedullary osseointegration: development of a rodent model and study of histology and neuropeptide changes around titanium implants.

A rodent model has been developed to explore intramedullary osseointegration and the phenomena of osseoperception. Osseointegration with endosseous titanium implants is frequently used in oral surgery. More recently, intramedullary osseointegration has been used for direct skeletal anchoring of amputation prostheses, a procedure that provides for a stable prosthesis with improved perception. Experimental, commercially pure titanium rods with threaded ends were surgically implanted in the intramedullary space of 18 rat femurs, and were left in place for 8 wk. Microscopic and immunohistochemical observation of the titanium/bone interface at this time-point indicated successful osseointegration with normal remodeled bone adjacent to the fixture. Calcitonin gene-related peptide activity was upregulated during the process of bone remodeling, and there was no significant inflammatory reaction. There was new, normal bone adjacent to and fully occupying the space between fixture threads. Innervation also appeared normal in remodeled bone, as indicated by immunohistochemical observation of small nerve fibers with the antibody Protein Gene Product 9.5 (PGP 9.5). The model will be used further to explore intramedullary osseointegration and osseoperception in connection to clinical applications.

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 microL of 1000 microg/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 microL 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 microm. 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 cm H2O in the NP+PS (control) group and 1.8 +/- 0.4 cm H2O 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 cm H2O in the NP+PS group and 2.9 +/- 0.8 cm H2O 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.

Axonal transport of TNF-alpha in painful neuropathy: distribution of ligand tracer and TNF receptors.

Tumor necrosis factor-alpha (TNF-alpha) is a key player in peripheral nerve injury. In the inflammatory chronic constriction injury (CCI) model of sciatic neuropathy, upregulation of TNF-alpha mRNA and protein at the site of nerve injury has been associated with pain. We now report the distribution of endogenous TNF-alpha protein and its receptors along normal and CCI-injured sciatic nerves, and within the corresponding lumbar dorsal root ganglia (DRG). Using Western blotting, TNF-alpha was found to be distinctly increased at the injury site, as well as in the axons just distal to the corresponding DRG. The TNF-alpha signal between the injury site and DRG (midaxonal) was induced between 2 and 5 days post-CCI, suggesting activation of TNF-alpha axonal transport. Endogenous TNF-alpha was localized in small-diameter, presumably nociceptive, and large-diameter, presumably mechanoceptive, DRG sensory neurons in both normal and CCI animals. Intraneural microinjection of biotin-labeled TNF-alpha showed specific axonal uptake at the injection site, as detected by avidin-biotin-peroxidase histochemistry, and confirmed by co-localization with neurobiotin tracer. In control animals, fast retrograde transport of biotinylated TNF-alpha to both L4 and L5 DRG neurons was apparent 6 h following injection. TNF receptors TNFRI and TNFRII co-localized with biotinylated TNF-alpha tracer along the nerve trunk, suggesting that TNF-alpha transport may be receptor-mediated. In animals with CCI neuropathy, uptake of biotinylated TNF-alpha by neuronal soma was inhibited. Instead, there was signal accumulation in the axons immediately distal to the DRG, and TNFRI and RII were increased at this same anatomic location. These findings highlight a dynamic process of TNF-alpha protein and receptor regulation throughout the peripheral neural axis that bears on both the normal function of DRG neurons and the pathogenesis of painful neuropathies.

Sciatic inflammatory neuritis (SIN): behavioral allodynia is paralleled by peri-sciatic proinflammatory cytokine and superoxide production.

We have recently developed a model of sciatic inflammatory neuritis (SIN) to assess how immune activation near peripheral nerves influences somatosensory processing. Administration of zymosan (yeast cell walls) around a single sciatic nerve produces dose-dependent low-threshold mechanical allodynia without thermal hyperalgesia. Low (4 microg) doses produce both territorial and extraterritorial allodynia restricted to the injected hindleg. In contrast, higher (40 microg) doses produce territorial and extraterritorial allodynias of both hindlegs, an effect not accounted for by systemic spread of the zymosan. The aim of these experiments was to determine whether these behavioral allodynias were correlated with immunological and/or anatomical changes in or around the sciatic nerve. These experiments reveal that zymosan-induced bilateral allodynia was associated with the following: (a) increased release of both interleukin-1beta and tumor necrosis factor-alpha from peri-sciatic immune cells; (b) increased release of reactive oxygen species from perisciatic immune cells; (c) no change in circulating levels of proinflammatory cytokine; (d) no apparent zymosan-induced influx of immune cells into the sciatic nerve from the endoneurial blood vessels; (e) mild edema of the sciatic, which was predominantly restricted to superficial regions closest to the peri-sciatic immune cells; and (f) no anatomic evidence of changes in either the ipsilateral saphenous nerve or contralateral sciatic nerve that could account for the appearance of extraterritorial or contralateral ("mirror") allodynia, respectively. No reliable differences were found when the low-dose zymosan was compared with vehicle controls. Taken together, these data suggest that substances released by peri-sciatic immune cells may induce changes in the sciatic nerve, leading to the appearance of bilateral allodynia.

Safety assessment of encapsulated morphine delivered epidurally in a sustained-release multivesicular liposome preparation in dogs.

We have shown that the epidural (EPI) delivery of morphine encapsulated in multivesicular liposomes (DepoFoam drug delivery system) produces a sustained clearance of morphine and a prolonged analgesia. We have sought to subsequently determine the likelihood of deleterious effects on local tissue of repetitive epidural injections of this encapsulated morphine preparation (C0401). Beagle dogs were prepared according to protocol approved by the Institutional Animal Care and Use Committee under volatile general anesthesia with chronic lumbar EPI catheters and subcutaneous injection ports. Male and female dogs (three groups) received a total of 4 EPI injections at 8-day intervals of 3 mL of C0401 (10 mg/mL morphine) (N = 6), DepoFoam vehicle (N = 6), or 0.9% sodium chloride (N = 6). Following EPI-C0401, but not saline or DepoFoam vehicle, there were transient (< 72 hr) decreases in food consumption, arousal, hindlimb muscle tone, and body temperature. Heart rate was unaltered, but there were modest decreases in blood pressure and respiratory rate, which persisted for 24-72 hr after C0401. No persistent changes in sensory/motor function, body weight, or stool/urine production were observed. Cerebrospinal fluid, blood chemistry, and urinalysis performed at surgery and on the day of sacrifice (24 hr after the last dose) were within normal ranges. Gross pathology at necropsy was unremarkable. Spinal histopathology findings were judged to be minimal (e.g., modest pericatheter inflammation and fibrosis) and present in all dogs. However, a statistical trend in the rank order of pathology scores was noted (Saline < DepoFoam vehicle < C0401). Repeated EPI injection of C0401 at the maximum dose that could be administered (30 mg) resulted in moderate, transient behavioral and physiological effects after each injection, consistent with morphine administration, and a modest effect on cord histopathology. This level of pathology is reflected in the lack of change observed in cerebrospinal fluid and lack of neurological findings. These results suggest that C0401 is without significant pathological effects at this dose after repeated epidural delivery in dogs.

Injury to dorsal root ganglia alters innervation of spinal cord dorsal horn lamina involved in nociception.

STUDY DESIGN: A study of the relation between the development of mechanical allodynia and the reorganization of primary afferent terminals in the sensory lamina of the rat spinal cord dorsal horn after partial dorsal root ganglion injury in rats. OBJECTIVES: To investigate the pathologic mechanisms of mechanical allodynia after partial dorsal root ganglion injury. SUMMARY OF BACKGROUND DATA: After experimental peripheral nerve injury causing neuropathic pain, myelinated afferent fibers sprout into lamina II of the dorsal horn. This lamina is associated with nociceptive-specific neurons that generally are not stimulated by myelinated fiber input from mechanical receptors. These morphologic changes are suggested to have significance in the pathogenesis of chronic mechanical allodynia, although it is not known whether this kind of morphologic change occurs after dorsal root ganglion injury. METHODS: After partial dorsal root ganglion crush injury, the mechanical force causing footpad withdrawal was measured with von Frey hairs, and myelinated primary afferents were labeled with cholera toxin B subunit horseradish peroxidase, a selective myelinated fiber tracer that identifies transganglionic synapses. RESULTS: After partial dorsal root ganglion injury, mechanical allodynia developed in the corresponding footpad within 3 days and persisted throughout the experimental period. At 2 and 4 weeks after the injury, B subunit horseradish peroxidase-positive fibers, presumably myelinated afferents, were observed to be sprouting into lamina II of the dorsal horn on the injured side, but not on the contralateral control side. CONCLUSIONS: Morphologic change in spinal cord dorsal horn lamina II occurs after partial dorsal root ganglion injury. This change may have significance in the pathogenesis of chronic mechanical allodynia after partial dorsal root ganglion injury.

Upregulation and interaction of TNFalpha and gelatinases A and B in painful peripheral nerve injury.

Chronic constriction injury (CCI) to peripheral nerve causes a painful neuropathy in association with a process of axonal degeneration and endoneural remodeling that involves macrophage recruitment and local increase in extracellular proteases and tumor necrosis factor alpha (TNF-alpha). Cell surface activation of TNF-alpha from its transmembrane precursor, as well as sequestration of TNF-alpha receptors II and I, is performed by the zinc-dependent endopeptidase family of matrix metalloproteinases (MMPs). Among TNF-alpha-converting MMPs, basal lamina degrading gelatinases are thought to play a role in sciatic nerve injury. In the present study, we determined the forms of TNF-alpha involved in the development of CCI neuropathy in rats, using Western blot analysis, and the temporal correlation of TNF-alpha and TNFRI protein profiles with gelatinases activity at the site of peripheral nerve injury. We observed two peaks in TNF-alpha protein during the first week of CCI that correspond to previously reported peaks in painful behavior. We propose that the first peak at 6 h post-CCI is due to the local expression of the cytotoxic transmembrane 26 kDa TNF-alpha protein released by resident Schwann cells, mast cells and macrophages. This peak in TNF-alpha protein expression corresponds to an increase in gelatinase B (MMP-9) activity, which is greatly upregulated as early as 3 h following CCI to rat sciatic nerve. The second peak occurs at 5 days post-CCI, and may represent TNF-alpha protein released by hematogenously recruited macrophages. This peak is marked by the increase in active soluble 17 kDa TNF-alpha and by gelatinase A (MMP-2) upregulation. These observations suggest that there is a pathogenic role for the TNF-alpha-converting function of MMP-2 in painful CCI neuropathy. We conclude that severe nerve injury induces MMPs, TNF-alpha and TNFRI, which interactively control the privileged endoneurial environment and the pathogenesis of the painful neuropathies associated with the macrophage-dependent processes of Wallerian degeneration.

2000 Volvo Award winner in basic science studies: Exogenous tumor necrosis factor-alpha mimics nucleus pulposus-induced neuropathology. Molecular, histologic, and behavioral comparisons in rats.

STUDY DESIGN: This study tested the hypothesis that the 17-kDa form of tumor necrosis factor-alpha is the pathophysiologic agent expressed by herniated nucleus pulposus in vivo that is primarily responsible for the histologic and behavioral manifestations of experimental sciatica associated with herniated lumbar discs. OBJECTIVE: The authors determined the molecular weight and concentration of active tumor necrosis factor-alpha in rat herniated disc and used exogenous tumor necrosis factor-alpha at the same molecular weight to study its neuropathologic effect on rat nerve root and dorsal root ganglion preparations in vivo. SUMMARY OF BACKGROUND DATA: Expressed by herniated nucleus pulposus in culture, tumor necrosis factor-alpha causes neuropathologic injury in nerve roots and neuropathic pain states in which mechanical allodynia is seen in response to peripheral stimuli. METHODS: Western blotting was used to identify the molecular weight of the operative tumor necrosis factor-alpha protein form, and measures of optical density were used for semiquantitative determination of concentration. Plastic-embedded nerve roots and dorsal root ganglion were used for neuropathologic evaluation, and von Frey stimulation was used to quantify mechanical allodynia. RESULTS: The 17-kDa form of tumor necrosis factor-alpha is expressed by herniated nucleus pulposus at a concentration of approximately 0.48 ng per herniated rat lumbar disc. Exogenous tumor necrosis factor-alpha applied in vivo to rat nerve roots produced neuropathologic changes and behavior deficits that mimicked experimental studies with herniated nucleus pulposus applied to nerve roots. CONCLUSIONS: The data reinforce other evidence that tumor necrosis factor-alpha is involved in mechanisms of neuropathic pain.

Preclinical toxicity screening of intrathecal adenosine in rats and dogs.

BACKGROUND: Intrathecally administered adenosine receptor agonists have antinociceptive effects in animals, suggesting that intrathecal adenosine might provide analgesia in humans. The authors performed preclinical neurotoxicity studies to define the safety of intrathecally administered adenosine in rats and dogs. METHODS: Eighteen rats with long-term intrathecal catheters received daily injections of saline or 100 microg adenosine for 4 days and were observed for general behavior and thermal nociception before being killed on day 6. Nine beagle dogs were prepared with long-term, lumbar intrathecal catheters and infused continuously with saline or adenosine, 2.4 mg/day for 48 h, then 7.2 mg/day for 26 days. Animals were then anesthetized and perfused with preservative and their spinal cords were examined systematically. RESULTS: No disturbances in neurologic function were detected in either animal species. intrathecal adenosine caused transient sedation in rats and increased muscle tone in dogs, resolving with continued exposure to drug. Neither adenosine-nor saline-treated rats or dogs showed acute thermal analgesia. Adenosine groups did not differ from saline groups regarding histopathology, although a moderate fibrotic and inflammatory reaction was noted in both, and protein concentrations in cerebrospinal fluid were increased in both. CONCLUSION: The current study in rats and dogs failed to provide behavioral or histologic evidence of neurotoxicity from intrathecal administration of adenosine. This provides evidence for the presumption of safety of adenosine in this dose range, and supports phase I safety trials of acute intrathecal adenosine administration in humans.

Intermittent reperfusion fails to prevent posttourniquet neurapraxia.

This study examined the effects of intermittent reperfusion on peripheral nerve function. Rabbits were randomized to undergo 4 hours of 350 mm Hg tourniquet compression to a hind limb either continuously, interrupted by a single 10-minute reperfusion interval after 2 hours, or interrupted by 10 minutes of reperfusion after each hour. A control group had the tourniquet applied for 4 hours but it was never inflated. The animals were examined clinically for neuromuscular dysfunction and the structure and function of the peripheral nerves were evaluated 1 week after tourniquet compression. Animals that underwent compression had a foot drop and decreased toe-spread reflex. There was greater intraneural edema and slower nerve conduction velocity in nerve segments that were directly compressed by the tourniquet but no apparent abnormalities in segments distal to the tourniquet. Intermittent reperfusion failed to diminish the clinical, structural, or functional consequence of the neurologic injury.

Myelinated afferents sprout into lamina II of L3-5 dorsal horn following chronic constriction nerve injury in rats.

In order to investigate the consequences of chronic constriction injury (CCI) to nerve, we explored the relationship between the development of mechanical allodynia and the reorganization of primary afferent terminals in the sensory lamina of the rat spinal cord dorsal horn. Following sciatic CCI neuropathy, mechanical allodynia developed in the corresponding footpad within two weeks and persisted throughout the experimental period which extended for an additional two weeks. The neuropathy of the sciatic injury includes extensive Wallerian-like degeneration of myelinated fibers but relative sparing of unmyelinated fibers. We observed that there was no significant change in the dorsal horn termination of unmyelinated C fibers in lamina II of the dorsal horn, using nerve injections of wheat germ agglutin-horseradish peroxidase for transganglionic axonal tracing of these fibers from the nerve injury site, and no evidence of sprouting into adjacent lamina. In contrast, myelinated afferent fibers were observed to be sprouting into lamina II of the dorsal horn, as indicated by cholera toxin beta-subunit-horseradish peroxidase retrograde axonal tracings. This region of the dorsal horn is associated with nociceptive-specific neurons that are not generally associated with myelinated fiber input from mechanical and proprioceptive receptors. As previously suggested in nerve transection and crush injuries, and now demonstrated in CCI neuropathy, these morphological changes may have significance in the pathogenesis of chronic mechanical allodynia.

Acute effects of nucleus pulposus on blood flow and endoneurial fluid pressure in rat dorsal root ganglia.

STUDY DESIGN: An experimental study to elucidate the initial factors in the pathogenesis of lumbar pain caused by disc herniation. OBJECTIVE: To evaluate the effects of autologous nucleus pulposus on blood flow and endoneurial fluid pressure in dorsal root ganglia. SUMMARY OF BACKGROUND DATA: Human sciatica is known to be associated with compression of lumbar nerve roots and dorsal root ganglia by herniated intervertebral discs. Recently, it has been shown that application of nucleus pulposus to nerve roots induces injury and pain-related behavior in experimental animals. In this study, the authors hypothesized that nucleus pulposus applied to a nerve root would cause increased intraneural edema and reduced blood flow in the corresponding dorsal root ganglia. Studies in peripheral nerves have shown that these initial pathophysiologic disturbances initiate complex events that exacerbate nerve injury and cause pain. METHODS: A total of 29 adult female Sprague-Dawley rats weighing 200 to 250 g had their left L5 nerve roots and associated dorsal root ganglia exposed. Autologous nucleus pulposus was harvested from the tail and applied to the L5 nerve root just proximally to the dorsal root ganglia (nucleus pulposus group). For control, the same volume of muscle was harvested from the surgical area in the back and applied similarly to the neural tissue (control group). Blood flow was continuously monitored using a laser Doppler flow probe for 3 hours (n = 10) or 4 hours (n = 8) in animals with indwelling cannulas for measurement of systemic arterial pressure. Endoneurial fluid pressures were recorded with a servonull micropipette system using glass micropipettes with tip diameters of 4 microns. Endoneurial fluid pressure in the dorsal root ganglia was measured before and 3 hours after application of nucleus pulposus (n = 7) or muscle (n = 4). After measurement of blood flow and endoneurial fluid pressure, the nerve root and dorsal root ganglia were processed for histology and evaluated by light microscope. RESULTS: Blood flow in the nucleus pulposus group was reduced by 10% to 20% from the initial value after 3 to 4 hours. This reduction was statistically significant compared with that of the control group (P < 0.01). Endoneurial fluid pressure was initially 2.6 +/- 1.2 cm H2O in the nucleus pulposus group, and 2.1 +/- 0.6 cm H2O in the control group. Three hours after application, endoneurial fluid pressure was 7.5 +/- 4.6 in the nucleus pulposus group (P > 0.05), and 2.0 +/- 0.8 in the control group (P > 0.05). Edema was the principal pathologic finding seen consistently in the nerve roots and in many of the associated dorsal root ganglia from animals treated with nucleus pulposus. CONCLUSION: Application of nucleus pulposus to nerve root increased endoneurial fluid pressure and decreased blood flow in the dorsal root ganglia. This study's acute observations in the dorsal root ganglia may thus help to explain why disc herniations without compression of neural tissue are sometimes painful because similar pathologic findings are observed after only nucleus pulposus application to the nerve root. The authors further suggest that exposure of nerve roots to nucleus pulposus may establish a "compartment syndrome" in the dorsal root ganglia.

Prosaptide prevents hyperalgesia and reduces peripheral TNFR1 expression following TNF-alpha nerve injection.

This study demonstrated that hyperalgesia resulting from an intraneural injection of the cytokine tumor necrosis factor-alpha (TNF) was prevented by preemptive administration of a single dose of the prosaptide TX14(A) (200 microg/kg). TX14(A) is a synthetic 14-mer peptide with neurotrophic and cytoprotective activities. Efforts to elucidate TX14(A) antagonism of hyperalgesia concentrated on determining the effect of TX14(A) on the up-regulation of the 55 kDa TNF receptor (TNFR1) at the nerve injury site. It has been previously shown that TNFR1 expression is upregulated following nerve injury and parallels the display of nociceptive behavior. In our experiments, TNFR1 was decreased at the TNF nerve injection site in TX14(A)-treated rats when compared to vehicle-treated or control peptide-treated rats. Light microscopic evaluation of nerve injury site tissue displayed qualitatively similar neuropathology in both treatment groups during the time of peak hyperalgesia (day 3), but appeared more normal than untreated nerves at day 7 (histological scoring, mean +/-s.d., 3.7+/-0.57 for TX14(A)-treated and 5.67+/-0.5 for control peptide-treated). These results suggest that TX14(A) decreased nociceptive behavior by attenuating both TNFR1 upregulation and Schwann cell activation in response to TNF injection. This prosaptide neurotrophin may also moderate nerve degeneration or promote regeneration. It is not known whether TX14(A) also acts rostral to the lesion site.

Prosaptide prevents paclitaxel neurotoxicity.

Paclitaxel (Taxol), a chemotherapeutic agent used to treat breast and ovarian tumors, has been reported to induce a predominantly sensory neuropathy. Co-treatment with neurotrophic factors and paclitaxel has been proposed for preventing or reversing paclitaxel-induced peripheral neuropathy. Prosaposin, the precursor of saposins A, B, C and D was recently identified as a neurotrophic factor and was reported to facilitate nerve regeneration in vivo. Peptides (prosaptides) encompassing the neurotrophic sequence located in the saposin C domain, have neurotrophic activity similar to the holoprotein (O'Brien et al. 1995). In the present study, we investigated the effect of a 14-mer prosaptide, TX14(A), or a 22-mer prosaptide, 769P, on paclitaxel-induced neutrotoxicity in vitro and in vivo. Paclitaxel treatment (1 microM) decreased cell viability of both PC12 and Schwann cells. TX14(A) (10 nM) prevented paclitaxel-induced loss of cell viability in PC12 cells but not in Schwann cells. Systemic injections (i.p.) of paclitaxel (1.2 mg/kg/day) given five times per week for three weeks (cumulative dose 18 mg/kg) or given every third day (25, 12.5 and 12.5 mg/kg) for 10 ten days (cumulative dose 50 mg/kg) in adult rats induced thermal hypoalgesia that was not accompanied by morphological changes in the sciatic nerve or changes of nerve conduction velocity. Co-administration of paclitaxel with prosaptides (cumulative dose 3 or 10 mg/kg) prevented paclitaxel-induced thermal hypoalgesia. In the short-term high dose study, paclitaxel treated rats lost 10% of their body weight, had reduced erythrocyte counts, hematocrit and hemoglobin concentrations which were not prevented by treatment with prosaptide. TX14(A) did not diminish paclitaxel cytotoxicity of breast cancer cells in vitro. These findings suggest that prosaptide prevents the neurotoxic effects of paclitaxel while not interfering with its anti-tumor activity.