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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 (pConclusion:The findings in the present study demonstrate a neuronal mechanism that may be of importance in the pathophysiology of sciatic pain in disc herniation.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Bone cement is sometimes used for vertebral body reconstruction following tumor removal. During such procedures, the polymerization of the methyl-metacrylate in the bone cement generates heat. Such temperature increase might cause damage to the nerve roots within the spinal canal. In the present study, pig cauda equina nerve roots were subjected to controlled temperature increases by means of a heat-generating probe. A temperature of 40°C applied for 5 min did not cause any changes in nerve root function. However, 70°C resulted in a complete block of nerve root function within 5 min. Histological nerve fiber damage was seen after exposure to 60°C and 70°C. The present study provides basic knowledge of heat-resistance properties of spinal nerve roots that might be directly applicable as guidelines for safety margins during surgical spine reconstruction procedures using bone cement.

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

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

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

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

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

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

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

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

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

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

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

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

Double level compression of nerve roots has been known to exist for a long time, but only in recent years the clinical significance of such conditions has been discussed. In the current study, nerve root function in double level compression was analyzed by measurements of tail muscle electromyogram, after stimulations of 1) the intermediate segment between the compression sites and 2) cranial to the two compression sites in a pig model. The electromyogram-amplitudes were more reduced after intermediate, than cranial stimulation. This difference was suggested to be based on an additional increasing threshold to elicit an action potential within the intermediate segment due to a nutritional deficit at this location. The results further stress the importance of local changes within the intermediate segment in double level nerve root compression.

Double-level nerve root compression had a greater effect on nerve impulse conduction, recorded as tail muscle action potential amplitude, than single-level compression at both 10 and 50 mm Hg. If the distance between the two compression sites at 10 mm Hg compression was increased from one vertebral segment (10 mm) to two vertebral segments (30 mm), the reduction of muscle action potential amplitude was further enhanced. These observations may give new insight into the pathophysiology of cauda equina compression in spinal disorders such as stenosis.

Neurophysiologic and neuropathologic changes were studied in rabbit hindlimbs after 2 hours of pneumatic tourniquet application with either 350 mmHg (n = 18) or 1,000 mmHg (n = 6) cuff inflation pressure. The toe spread reflex was decreased in 66% and absent in 33% of limbs 2 days after 350 mmHg compression, and was absent in all limbs after 1,000 mmHg compression. Compound motor action potential amplitudes (CMAPs), recorded from the abductor hallucis muscle, were significantly decreased with sciatic nerve stimulation 1 hour after 350 mmHg compression. CMAPs returned to baseline values one and two days later, however nerve conduction velocity (NCV) was still significantly decreased in the compressed sciatic nerves of these groups. In contrast, complete nerve conduction block, localized beneath the cuff's distal border, was observed two days after 1,000 mmHg compression, and NCV was still significantly decreased distal to the tourniquet zone. Using light and electron microscopy, scattered axonal degeneration, mild myelin damage, and normal nodes of Ranvier were observed two days after 350 mmHg tourniquet compression. Severe fiber damage and nodal obliteration were noted after 1,000 mmHg tourniquet compression. Although nodal invagination is probably not a significant pathogenic mechanism at clinically relevant tourniquet pressures and durations, functional abnormalities were induced by 2 hour, 350 mmHg tourniquet compression. Such changes probably correlate with clinical electromyographic abnormalities and delayed post-operative recovery following 'routine' extremity surgery using pneumatic tourniquets.

A well-controlled animal model is presented for the study of neuromuscular injury induced by a pneumatic tourniquet. This model comprises a curved tourniquet surrounded by a stiff exterior shell, both of which were specifically designed to fit the conical and oblong shape of the rabbit hindlimb. Computed tomographic imaging was used to assess transverse tissue displacement induced by tourniquet compression. The curved tourniquet/shell configuration occluded the distal arterial blood flow to the extremity at a significantly lower cuff inflation pressure than a straight tourniquet of equal width. The magnitude and distribution of tissue pressures in the subcutaneous and deep tissues beneath the tourniquet were similar to those recorded in previous human cadaver studies of tourniquet compression. This animal model will facilitate the quantitation and analysis of tissue injury induced beneath and distal to a pneumatic tourniquet. Such data can help define the critical pressure and time limits for the safe use of pneumatic tourniquets in extremity surgery.

Peripheral nerves are often mechanically stretched in association with trauma to extremities, resulting in varying degrees of impairment of nerve function. However, little is known about the biomechanical properties of peripheral nerves and limits of stretching that the nerve may undergo before structural changes occur. Also, the injury pattern of nerves under stretching is poorly understood. In the present study, fresh rabbit tibial nerves (n = 18) were harvested. Nine nerves were stretched to failure in an INSTRON materials testing machine at a rate of 1 cm/min (strain rate of 0.5%/s). Load-deformation and stress-strain curves were determined. Histological examination by light microscopy of the stretched nerves as well as six normal control nerves and three clamped nonstretched control nerves was performed. The results show that the rabbit tibial nerves have an in situ strain of 11.0 +/- 1.5% and exhibit a nonlinear stress-strain relationship. After 20% strain, the curve becomes linear up to failure. The ultimate strain and tensile strength of the nerves were 38.5 +/- 2.0% and 11.7 +/- 0.7 MPa, respectively. At failure, the load dropped suddenly, but the specimens remained grossly intact. Histological analysis of the stretched nerves showed multiple ruptures of perineurial sheaths when compared to controls.

We describe the morphologic changes that follow division of the transverse carpal ligament in patients with carpal tunnel syndrome. Fifteen hands in 12 patients with carpal tunnel syndrome were studied with magnetic resonance imaging before operation and for 6 weeks after operation. Eight hands were studied at 8 months after operation. Carpal arch width, anterior displacement of the carpal canal contents, and carpal canal volume were measured by use of multiplanar reformation and three-dimensional reconstruction of magnetic resonance images. There was a 24.2 +/- 11.6% increase in carpal canal volume 6 weeks after carpal tunnel release (p less than 0.001). This difference persisted at 8-month follow-up. There was an anterior displacement of carpal canal contents 3.5 +/- 1.9 mm from its original position 6 weeks after operation (p less than 0.001). This palmar displacement persisted at the 8-month follow-up. There was no statistically significant increase in carpal arch width 8 months after carpal tunnel release. We believe that division of the transverse carpal ligament restores median nerve function by increasing the volume of the carpal canal. This volumetric increase results from an anterior displacement of the newly formed transverse carpal ligament and not from a widening of the bony carpal arch.

Compression applied to a peripheral nerve may easily interfere with intraneural blood flow. In the present experimental study, a vital microscopic technique was used to observe changes in intraneural microcirculation (intrafascicularly and extrafascicularly) when graded compression was applied to a rabbit's tibial nerve by a specially designed minicompression device. Interference with venular flow was observed already at a pressure of 20 to 30 mm Hg while arteriolar and intrafascicular capillary flow was impaired at about 40 to 50 mm Hg. At 60 to 80 mm Hg no blood flow could be observed in the nerve. Nerves observed 3 or 7 days after 2 hours of compression at 400 mm Hg showed no or very slow stagnant blood flow within the previously compressed segment. It is concluded that acute compression of nerve may cause persistent impairment of intraneural microcirculation due to mechanical injury to blood vessels.

Flexor tendon from the synovial sheath region of rabbits was resected and placed as a free body in a synovial knee joint recess. Structural changes of the surface of the graft caused by the instruments used to handle the tendon during the surgery (jewellers microforceps), was analysed by electron microscopy at various time intervals up to 6 weeks. The superficial coat of amorphous material, i.e. ground substance, on the surface of the tendon was dishevelled by the instruments. The injured areas were crowded with monocytes and macrophages during the first days. An increasing number of fibroblasts and mesothelial-like cells covered the damaged area in less than two weeks. The superficial coat of ground substance was not completely restored until after 3 weeks or more. The results obtained are discussed in relation to functional aspects.

The anatomy and physiology of the nerve root complex in the lumbar spine are reviewed, with special reference to the effects of mechanical deformation of nerve roots in association with intervertebral disc herniation and spinal stenosis. Biomechanical aspects of nerve root deformation induced by compression are discussed. The functional changes induced by compression can be caused by mechanical nerve fiber deformation but also may be a consequence of changes in nerve root microcirculation, leading to ischemia and formation of intraneural edema. Nerve root compression can, by different neurophysiologic mechanisms, induce motor weakness and altered sensibility or pain. Intraneural edema and demyelination seem to be critical factors for the production of pain in association with nerve root compression.

The minimal pressure for impairment of fast anterograde axonal transport was determined in rabbit vagus nerve. Proteins, transported by fast anterograde axonal transport, were labeled by a microinjection of [3H]leucine into the nodose ganglion, and a small compression chamber was applied around the cervical vagus nerve. In this way the nerve was subjected to acute, graded compression. Compression at 20 mm Hg for 2 h as well as sham compression did not induce accumulation of axonally transported proteins at the level of compression. However, a pressure of 30 mm Hg for 2 h induced a block of axonal transport at the site of compression. The causes of the axonal transport block are discussed as well as the minimal pressure level in relation to pressures found in clinical nerve compression lesions.

Severed and subsequently sutured rabbit flexor tendons were kept free and isolated in the synovial cavity of the knee joint. In one series the tendon specimens were surrounded by a dialyzing membrane to avoid cell seeding from the synovial fluid. At different intervals of time over a period of 3 weeks, the tendons were studied morphologically with special reference to scanning electron microscopy. Adhesions were not observed and, with synovial fluid as the nutrient medium, the tendons showed an intrinsic ability to repair in the superficial layers, also bridging the suture gap. Moreover, cell seeding, mainly of macrophages, from the synovial fluid could be demonstrated on the very surface of the tendon. When this cell seeding was prevented, the fibroplasia in the superficial layer of the tendon did decrease slightly, but tendon cell morphology was that of active fibroblasts. The results support the concept that flexor tendons may show intrinsic fibroplasia when nourished by synovial fluid, while macrophages, mainly, of extrinsic origin, contribute to restoration of the tendon surface.

Surgical treatment of peripheral nerve lesions associated with intraneural fibrosis is sometimes extended to include internal neurolysis. This procedure is performed in order to release the individual nerve fascicles from interfascicular scar tissue which is believed to constrict nerve fibres and thereby interfere with their function and regenerative capacity. However, an internal neurolysis per se implies a significant trauma to the nerve and may induce microvascular damage and formation of new intraneural scar tissue. Considering this, such a procedure appears justified only when the preoperative intraneural fibrosis is more severe than the scarring which might be induced by the surgical procedure as such. In order to evaluate the tissue reactions following internal neurolysis an experimental investigation was carried out: internal neurolysis was performed on normal rabbit tibial nerve. After varying postoperative periods up to 6 months specimens of nerves were analysed. Reactive changes of connective tissue and myelin sheath lesions, indicating nerve fibre damage, were investigated in histological sections studied by light microscopy. Barrier function of perineurial membrane and endoneurial vessels was investigated by fluorescent microscopic tracing of locally applied or intravenously injected albumin labelled with Evans' blue. The results indicate that an experimental internal neurolysis per se may induce fibrosis in all layers of the nerve and may cause some nerve fibre damage. However, the barrier function of the perineurium and the endoneurial vessels seems to be generally well preserved. The findings are discussed in relation to indications for internal neurolysis.

Experimental compression lesions of peripheral nerves were induced by applying pressure direct to exposed rabbit nerve trunks. A specifically designed compression chamber was used, enabling application of graded pressures (50--600 mmHg) to the nerves for various periods of time (15 min to 6 hours). After releasing the pressure, analyses were performed concerning the intraneural microvascular permeability and the barrier function of the perineurium. The method used was fluorescence microscopic tracing of intravenously injected or locally applied albumin labelled with Evans blue. The results indicate that a slight trauma to a nerve (e.g. 50 mmHg during 2 hours) induced an epineurial oedema by increasing the permeability of the epineurial vessels, which were more susceptible to compression trauma than the endoneurial vessels. Compression at higher pressure levels or of prolonged duration caused injury also to the endoneurial vessels, leading to intrafascicular oedema formation, which generally was most prominent at the edges of the compressed nerve segment. The perineurial barrier was remarkably resistant to compression trauma. Possible pathophysiological effects of various degrees of post-traumatic intraneural oedema formation are discussed.

1. Stretching of the tibial nerve cut 2 centimetres above the ankle has been the subject of an experimental study in rabbits. 2. The effects on intraneural microcirculation, on vascular permeability, and on the barrier function of the perineurium have been analysed with the aim of determining the extent to which a divided nerve can be stretched without interfering with the process of repair. 3. The results obtained may prove valuable for understanding basic mechanisms and for establishing certain important limitations when end-to-end suture of a nerve trunk is performed under some degree of tension in man.

Endoneurial fluid pressure measurements were made in the interstitial space of rat L5 dorsal root ganglia and in the corresponding sciatic nerves. Endoneurial fluid pressure was always higher in the ganglia than in the paired distal nerve. This proximodistal gradient in endoneurial fluid pressure may be the driving force responsible for the proximodistal convection of endoneurial fluid.

Compression injuries of spinal nerve roots are very common. However, the basic pathophysiology of these conditions is not fully understood. In this study a model is presented for experimental graded compression of the nerve roots of the pig cauda equina, including a technique for vital microscopic studies on the microcirculation flow of the intrinsic vessels of the nerve roots during compression. With this model critical pressure levels for compression-induced occlusion of the arterioles, capillaries, and venules of the intrinsic vasculature of the nerve roots were determined. The study showed that the average minimum pressure in the inflated balloon required to stop the flow in the arterioles was 127 mm Hg (SD = 18, n = 11), in the capillaries 40 mm Hg (SD = 6, n = 12), and in the venules 30 mm Hg (SD = 10, n = 12). The average mean arterial pressure (MAP) was 150 mm Hg (SD = 14, n = 12). There was a statistically significant correlation (p greater than 0.001) between the MAP and the pressure required to stop the flow in the arterioles (r = 0.83, n = 14), but no correlation between MAP and capillary (r = 0.09 NS, n = 20) or venular (r = -0.28 NS, n = 34) occlusion pressure. After the nerve roots had been compressed at 50 or 200 mm Hg for 10 min or 2 h, the recirculation started immediately. There was a hyperemia during the first 10 min, with a dilatation of the vessels, particularly the venules. In nerve roots exposed to compression for 2 h at either 50 or 200 mm Hg, an intraneural edema developed.(ABSTRACT TRUNCATED AT 250 WORDS)

Edema formation in spinal nerve roots of the pig cauda equina was studied following experimental compression at various pressure levels, durations, and rates of onset, using a fluorescence microscopic technique. The time-pressure thresholds for the occurrence of edema in the nerve roots were: following rapid onset of compression (0.05-0.1 seconds), 2 minutes at both 50 mm Hg and 200 mm Hg, and following slow onset of compression (the pressure was slowly increased during 15-20 seconds), 2 hours at 50 mm Hg and 2 minutes at 200 mm Hg. Generally, the edema formation was more pronounced after rapid than after slow onset of compression. The data in this study also indicate that intraneural edema might be more easily formed in nerve roots than in peripheral nerves after compression injury.