Osseointegrated Percutaneous Prosthetic System for the Treatment of Patients With Transfemoral Amputation: A Prospective Five-year Follow-up of Patient-reported Outcomes and Complications.

INTRODUCTION: Direct skeletal attachment of prostheses has previously been shown to improve patient-reported outcome (PRO) measures of individuals with transfemoral amputation (TFA) at 2-year follow-up. This prospective study reports the outcomes at 5-year follow-up. METHODS: A total of 51 patients (55 legs) with TFA were included in a prospective study. Complications, success rate, and PRO measures were followed for 5 years. RESULTS: The cumulative fixture survival rate at 5 years was 92%, and the revision-free survival rate was 45%. Thirty-four patients had 70 superficial infections. Eleven patients had 14 deep infections. Fifteen patients had mechanical complications. Four fixtures were removed (ie, one deep infection and three loosening). PRO measures showed significant improvements including more use of the prosthesis, better mobility, fewer issues, and improved physical health-related quality of life (all P < 0.0001) compared with baseline. CONCLUSION: Individuals with TFA at 5-year follow-up had significant improvement in PRO measures, but increases in deep infections and mechanical complications are concerning.

Increase of sodium channels (nav 1.8 and nav 1.9) in rat dorsal root ganglion neurons exposed to autologous nucleus pulposus.

PURPOSE: It has been assumed that nucleus pulposus-induced activation of the dorsal root ganglion (DRG) may be related to an activation of sodium channels in the DRG neurons. In this study we assessed the expression of Nav 1.8 and Nav 1.9 following disc puncture. METHOD: Thirty female Sprague-Dawley rats were used. The L4/L5 disc was punctured by a needle (n=12) and compared to a sham group without disc puncture (n=12) and a naive group (n=6). At day 1 and 7, sections of the left L4 DRG were immunostained with anti-Nav 1.8 and Nav 1.9 antibodies. RESULT: At day 1 after surgery, both Nav 1.8-IR neurons and Nav 1.9-IR neurons were significantly increased in the disc puncture group compared to the sham and naive groups (p<0.05), but not at day 7. CONCLUSION: The findings in the present study demonstrate a neuronal mechanism that may be of importance in the pathophysiology of sciatic pain in disc herniation.

Local application of nucleus pulposus induces expression OF P2X3 in rat dorsal root ganglion cells.

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.

Local application of interleukin-6 to the dorsal root ganglion induces tumor necrosis factor-α in the dorsal root ganglion and results in apoptosis of the dorsal root ganglion cells.

STUDY DESIGN: The mechanisms of apoptosis behind the formation of tissue reactions at the surface of the dorsal root ganglion (DRG) exposed to the nucleus pulposus were studied with special reference to the role of interleukin-6 (IL-6), using electron microscopy and immunohistochemistry in rats. OBJECTIVE: To study the role of IL-6 on the DRG. SUMMARY OF BACKGROUND DATA: It has been reported that nucleus pulposus cells are capable to produce proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and IL-6. Recently, it was observed that local application of nucleus pulposus induced a characteristic tissue reaction at the surface of the DRG. This change was due to apoptosis of DRG neurons. However, the role of IL-6 is not known regarding the apoptosis of the DRG neurons. METHODS: Recombinant IL-6 was applied between the L4 DRG and the dura to mimic a disc herniation of the L4-L5 disc in rats. The L4 DRGs were resected 24 hours after surgery. The sections were processed for immunohistochemistry using antisera to TNF-α. Furthermore, the sections of the specimens were observed using light and electron microscopy to confirm the induced apoptosis of the DRG neurons. The sections were also processed for immunohistochemistry, using antisera to single-stranded DNA (ssDNA) and Caspase 3. RESULTS: TNF-α immunoreactivity was observed in the peripheral area of DRG at the site of the application of IL-6. Typical changes of the cell nuclei were observed in the DRG by light and electron microscopy, indicating the presence of apoptosis. The presence of ssDNA and Caspase 3 further enhanced the impression that there was apoptosis of the DRG neurons. CONCLUSION: IL-6 seemed to induce TNF-α at the surface of DRG exposed to IL-6 and to induce a characteristic reaction at the surface of the DRG. IL-6 may thus play an important role in nucleus pulposus-induced apoptosis of the DRG neurons as well as TNF-α.

Osseointegrated trans-femoral amputation prostheses: prospective results of general and condition-specific quality of life in 18 patients at 2-year follow-up.

This is the first report on prospective outcome for individuals treated with bone-anchored trans-femoral amputation prostheses (OI-prostheses) using the method of osseointegration. The aim was to analyze general and condition-specific health related quality of life (HRQL) at 2-year follow-up as compared to the preoperative situation. The study population consists of the first 18 consecutively treated patients (8 male/10 female, mean age 45 years) in a clinical investigation with amputations mainly caused by trauma and tumour. At inclusion the mean time since the amputation was 15 years (10 months - 33 years). Two self-report questionnaires were answered preoperatively and at follow-up: the SF-36 Health Survey (SF-36) and the Questionnaire for persons with a Transfemoral Amputation (Q-TFA). At follow-up 17/18 patients used the OI-prosthesis; one did not due to pain and loosening of the implant. Four of the scales of the SF-36 (Physical Functioning, Role Functioning Physical, Bodily Pain and Physical Component Score) and all four scores of Q-TFA (Prosthetic Use, Prosthetic Mobility, Problems and Global Health) were statistically significantly improved at follow-up showing superior general physical HRQL, increased prosthetic use, better prosthetic mobility, fewer problems and a better global amputation situation. Thus, osseointegrated prostheses represent a promising development in the rehabilitation of individuals with transfemoral amputation and increase their quality of life.

The role of tumor necrosis factor-alpha in apoptosis of dorsal root ganglion cells induced by herniated nucleus pulposus in rats.

STUDY DESIGN: 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). OBJECTIVE: To study the characteristic tissue reaction at the surface of the DRG exposed to nucleus pulposus with special reference to the role of TNF. SUMMARY OF BACKGROUND DATA: 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. METHODS: 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. RESULTS: 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. CONCLUSION: TNF seemed to play a key role in induction of apoptosis of DRG cells, which resembled that induced by application of nucleus pulposus.

Production of tumor necrosis factor-alpha from porcine nucleus pulposus cells at various time points in cell culture under conditions of nutritional deficiency.

Nucleus pulposus (NP) in the epidural space induces spinal nerve damage not only by mechanical but also chemical mechanism. NP has been shown to be capable of producing tumor necrosis factor-alpha (TNF). TNF may play key roles in the NP-induced chemical damage. One of the main pathways to reach the avascular NP is diffusion from the blood supply of the vertebral body through the cartilage endplate. On disk herniation, when NP moves to the epidural space, the distance from the endplate to the herniated NP are longer in the herniated disk than in the intact disk. That is, it seems more difficult to receive adequate nutritional supply from the endplate in the sequestrated type. However, there have been only a few reports of the appearance of TNF in NP. The present study was performed to investigate TNF production in porcine NP under conditions of nutritional deficiency. NP cells were cultured and processed for immunohistochemistry using antisera to TNF, and for ELISA to measure TNF production. The latter was compared longitudinally. The immunoreactivity increased over time. On the other hand, the results of ELISA showed a peak in TNF production 12h, and lower amounts 1 day and 2 days after application of PBS. These observations may suggest that a nutritional deficit is a possible turn-on switch for TNF up-regulation in the NP.

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/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.

Nucleus pulposus-induced apoptosis in dorsal root ganglion following experimental disc herniation in rats.

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.

Effects of lumbar sympathectomy on pain behavioral changes caused by nucleus pulposus-induced spinal nerve damage in rats.

It has been suggested that lumbar sympathectomy can reduce pain behavior, including mechanical allodynia and thermal hyperalgesia, caused by ligation of the spinal nerve. One well-characterized model, which involves application of nucleus pulposus to the spinal nerve and displacement of the adjacent nerve, shows behavioral changes in rats. However, there have been no previous reports regarding sympathectomy performed in this model. Disk incision and adjacent spinal nerve displacement were performed with (n=6) or without (n=6) sympathectomy. Sham surgery was also performed with (n=6) or without (n=6) sympathectomy. The animals were tested for 3 days before surgery 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 using a touch stimulator. Thermal nociceptive thresholds were tested using a sensitive thermal-testing device. While rats in the disk incision with displacement surgery group showed allodynia and hyperalgesia after surgery on the experimental side, sympathectomized animals did not. No allodynia was observed in the sham groups. Sympathectomy seemed to prevent the pain behavioral changes caused by the combination of disk incision and nerve displacement.

Incision of the intervertebral disc induces disintegration and increases permeability of the dorsal root ganglion capsule.

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.

Effects of selective tumor necrosis factor-alpha inhibition to pain-behavioral changes caused by nucleus pulposus-induced damage to the spinal nerve in rats.

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.

Disc related cytokines inhibit axonal outgrowth from dorsal root ganglion cells in vitro.

STUDY DESIGN: Application of nucleus pulposus and disc related cytokines in vitro on cultured dorsal root ganglion (DRG) cells. OBJECTIVES: 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. SUMMARY OF BACKGROUND 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

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-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.

Selective inhibition of tumor necrosis factor-alpha 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-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.

Distribution and appearance of tumor necrosis factor-alpha in the dorsal root ganglion exposed to experimental disc herniation in 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.

Macrophage appearance in the epineurium and endoneurium of dorsal root ganglion exposed to 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.

Effects of fracture fixation stability on ossification in healing fractures.

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.