Can FES-rowing mediate bone mineral density in SCI: a pilot study.

STUDY DESIGN: A single case study. OBJECTIVES: To compare proximal tibia trabecular bone mineral density (BMD) of a participant with complete spinal cord injury (SCI), long-termed functional electrical stimulation-rowing (FES-R) trained, with previously reported SCI and non-SCI group norms. To estimate lower limb joint contact forces (JCFs) in the FES-R trained participant. SETTING: UK University and orthopaedic hospital research centre. METHODS: Bilateral proximal tibial trabecular BMD of the FES-R trained participant was measured using peripheral quantitative computerised tomography, and the data were compared with SCI and non-SCI groups. An instrumented four-channel FES-R system was used to measure the lower limb JCFs in the FES-R trained participant. RESULTS: Structurally, proximal tibial trabecular BMD was higher in the FES-R trained participant compared with the SCI group, but was less than the non-SCI group. Furthermore, left (184.7 mg cm(-3)) and right (160.7 mg cm(-3)) BMD were well above the threshold associated with non-traumatic fracture. The knee JCFs were above the threshold known to mediate BMD in SCI, but below threshold at the hip and ankle. CONCLUSION: As pathological fractures predominate in the distal femur and proximal tibia in chronic SCI patients, the fact that the FES-R trained participant's knee JCFs were above those known to partially prevent bone loss, suggests that FES-R training may provide therapeutic benefit. Although the elevated bilateral proximal tibial BMD of the FES-R participant provides circumstantial evidence of osteogenesis, this single case precludes any statement on the clinical significance. Further investigations are required involving larger numbers and additional channels of FES to increase loading at the hip and ankle.

Species, sex, size and male maturity composition of previously unreported elasmobranch landings in Kuwait, Qatar and Abu Dhabi Emirate.

This paper presents data from the first major survey of the diversity, biology and fisheries of elasmobranchs in the Persian (Arabian) Gulf. Substantial landings of elasmobranchs, usually as gillnet by-catch, were recorded in Kuwait, Qatar and the Emirate of Abu Dhabi (part of the United Arab Emirates), although larger elasmobranchs from targeted line fisheries were landed in Abu Dhabi. The elasmobranch fauna recorded was distinctive and included species that are undescribed, rare and have a highly restricted known distribution. Numerical abundance was dominated by sharks (c. 80%), of which carcharhinids were by far the most important. The milk shark Rhizoprionodon acutus and whitecheek shark Carcharhinus dussumieri together comprised just under half of all recorded individuals. Around 90% of recorded sharks were small (50-90 cm total length, L(T) ) individuals, most of which were mature individuals of species with a small maximum size (<100 cm L(T) ), although immature individuals of larger species (e.g. Carcharhinus sorrah and other Carcharhinus spp.) were also important. The most frequently recorded batoid taxa were cownose rays Rhinoptera spp., an undescribed whipray, and the granulated guitarfish Rhinobatos granulatus. The first size, sex and maturity data for a wide range of Gulf elasmobranch species are presented (including L(T) at 50% maturity for males of four shark species) and include some notable differences from other locations in the Indo-West Pacific Ocean. A number of concerns regarding the sustainability of the fishery were highlighted by this study, notably that most of the batoid species recorded are classed by the IUCN Red List as vulnerable, endangered, data deficient or not evaluated. Despite their considerable elasmobranch landings, none of the three countries sampled have developed a 'Shark Plan' as encouraged to do so under the FAO International Plan of Action: Sharks. Furthermore, Kuwait and Qatar currently report zero or no elasmobranch landings to the FAO.

Phylogenetic and ecological factors influencing the number and distribution of electroreceptors in elasmobranchs.

Electroreception is found throughout the animal kingdom from invertebrates to mammals and has been shown to play an important role in prey detection, facilitating social behaviours, the detection of predators and orientation to the earth's magnetic field for navigation. Electroreceptors in elasmobranchs, the ampullae of Lorenzini, detect minute electric fields and independently process these stimuli, thereby providing spatial information to the central nervous system on the location of a source, often potential prey. The ampullae of Lorenzini are individually connected to a single somatic pore on the surface of the skin, with the spatial separation of each pore directly influencing how electrical stimuli are detected and processed. Pore abundance varies across taxonomic groups resulting in unique species-specific differences. The intricate distribution patterns created by the specific positioning of somatic pores on the head are, however, consistent within families, resulting in patterns that are identifiable at higher taxonomic levels. As elasmobranchs evolved, the electrosensory system became more complex and highly specialized, which is evident by a general trend of increasing pore abundance over time. The elasmobranch electrosensory system has evolved to operate efficiently under the environmental conditions of the particular habitat in which a species lives. For example, reduced pore abundance is evident in oceanic pelagic elasmobranchs, for whom visual cues are thought to be of great importance. Pore abundance and spatial distribution may be influenced by multiple factors including head morphology, phylogeny, feeding behaviour and habitat.

Changes in the structural and material properties of the tibia in patients with spinal cord injury.

STUDY DESIGN: A cross-sectional study. OBJECTIVES: To measure the change of structural and material properties at different sites of the tibia in spinal cord-injured patients using peripheral quantitative computerised tomography (pQCT). SETTING: Orthopaedic research centre (UK). METHODS: Thirty-one subjects were measured--eight with acute spinal cord injury (SCI), nine with chronic SCI and fourteen able-bodied controls. pQCT scans were performed at 2% (proximal), 34% (diaphyseal) and 96% (distal) along the tibia from the tibial plateau. Structural measures of bone were calculated, and volumetric bone mineral density (vBMD) was also measured at all three levels. Muscle cross-sectional area was measured at the diaphyseal level. RESULTS: Structurally, there were changes in the cortical bone; in the diaphysis, the shape of the cross-section changed to offer less resistance to AP bending, and the cross-sectional area of the cortical shell decreased both proximally and distally. There were corresponding changes in vBMD in the anterior aspect of the cortical diaphysis, as well as proximal and distal trabecular bone. Changes in muscle occurred more rapidly than changes in bone. CONCLUSION: There were clear changes of both structure and material at all three levels of the tibia in chronic SCI patients. These changes were consistent with specific adaptations to reduced local mechanical loading conditions. To assess fracture risk in SCI and also to monitor the effect of therapeutic interventions, the structure of the bone should be considered in addition to trabecular bone mineral density.

The relationship between physical activity and leg health in the broiler chicken.

1. The relationship between the physical activity and leg health of broiler chickens was assessed on a semi-commercial scale. 2. Three batches of birds (2128 per batch) were raised under two lighting regimes during the photoperiod; either a step-wise change of light intensity alternating between an illuminance of 200 and 10 lx or a constant illuminance of 10 lx. The activity of focal individuals (24 per batch) was observed at 2, 4 and 6 weeks of age, and leg health assessed weekly, based on gait score, the prevalence of burns on the hock and foot pad, and angulation and rotation of the leg at the intertarsal joint. Cortical bone density and thickness and area moments of inertia of the mid-physis tibiotarsus were measured post mortem at 6 weeks of age. 3. The step-wise change in light intensity did not affect overall performance, activity or leg health. 4. An individual bird's activity did not affect its gait score, the prevalence of hock burn or foot pad burn, cortical density or thickness or shape of the tibiotarsus. Sex of the bird was the only factor to affect significantly the area moment of inertia in the horizontal and vertical planes of the tibiotarsus, with females showing a lower moment of inertia for both. No variable had a significant effect on cortical density or thickness. Mean cortical density was low across all birds and may indicate that, when allowed to move freely as much or as little as they choose, broiler chickens do not exercise enough or do not perform the higher impact activities required to affect bone quality. 5. These findings imply that the activity of broiler chickens raised on a semi-commercial scale is unaffected by step-wise changes in light intensity and that other husbandry measures are needed to raise activity and hence improve leg health.

Isolation and characterization of 12 dinucleotide microsatellite loci in the anadromous fish Osmerus eperlanus (L.).

A subtractive hybridization method was used to isolate 12 dinucleotide microsatellite loci for the anadromous European smelt, Osmerus eperlanus (L.). Three to 17 (mean 8.08) alleles per locus were identified in the two populations screened, and observed heterozygosity ranged from 0.067 to 0.933. Loci in both populations showed significant deviations from Hardy-Weinberg expected frequencies. These 12 loci provide a good basis for investigation of O. eperlanus population structure.

In vitro and in vivo biological responses to a novel radiopacifying agent for bone cement.

Iodixanol (IDX) and iohexol (IHX) have been investigated as possible radiopacification agents for polymethylmethacrylate (PMMA) bone cement, to replace the currently used barium sulphate and zirconia. IDX and IHX are both water-soluble iodine-based contrast media and for the last 20 years have been used extensively in clinical diagnostic procedures such as contrast media enhanced computed tomography, angiography and urography. One of the major reasons to remove the current radiopacifying agents is their well-documented cytotoxicity and their potential to increase bone resorption. Using in vitro bone resorption assays, the effect of PMMA particles plus IDX or IHX to induce osteoclast formation and lacunar resorption on dentine slices has been investigated. These responses have been compared with the in vitro response to PMMA particles containing the conventional radiopacifying agents, that is, barium sulphate and zirconia. In parallel, the in vivo reaction, in terms of new bone formation, to particles of these materials has been tested using a bone harvest chamber in rabbit tibiae. In vitro cell culture showed that PMMA containing IHX resulted in significantly less bone resorption than PMMA containing the conventional opacifiers. In vivo testing, however, showed no significant differences between the amounts of new bone formed around cement samples containing the two iodine-based opacifying agents in particulate form, although both led to fewer inflammatory cells than particles of PMMA containing zirconia. Our results suggest that a non-ionic radiopacifier could be considered as an alternative to the conventional radiopacifying agents used in biomaterials in orthopaedic surgery.

Water uptake and release from iodine-containing bone cement.

Water uptake and release characteristics of PMMA cement containing the water-soluble contrast media iohexol or iodixanol have been investigated. The water uptake study revealed that iohexol had the highest uptake of water (3.7%) and that iodixanol had an uptake close to that of Palacos R (2.3% and 1.9%). The curves obtained showed the materials to follow classic diffusion theory, with an initial linearity with respect to t(1/2) making it possible to calculate the diffusion coefficients. This showed iohexol to have the lowest diffusion coefficient, Palacos R the highest, and iodixanol close to that of Palacos R. The release study showed that more iohexol than iodixanol was released from the bone cement; the long-term release was above 25 microg/mL for iohexol compared to slightly above 10 microg/mL for iodixanol. A microCT investigation showed that the risk of developing an observable radiolucent zone is negligible.

Bone cement X-ray contrast media: a clinically relevant method of measuring their efficacy.

It is important to compare different contrast media used in bone cement according to their ability to attenuate X-rays and thereby produce image contrast between bone cement and its surroundings in clinical applications. The radiopacity of bone cement is often evaluated by making radiographs of cement in air at an X-ray tube voltage of 40 kV. We have developed a method for ranking contrast media in bone cement simulating the clinical situation, by (1) choosing the same X-ray tube voltage as used in clinical work, and (2) using a water phantom to imitate the effects of the patients' soft tissue on the X-ray photons. In clinical work it is desirable to have low radiation dose, but high image contrast. The voltage chosen is a compromise, because both dose and image contrast decrease with higher voltage. Three contrast media (ZrO(2), BaSO(4), and Iodixanol) have been compared for degree of "image contrast." Comparing 10 wt % contrast media samples at an X-ray tube voltage of 40 kV, ZrO(2) produced higher image contrast than the other media. However, at 80 kV, using a water phantom, the results were reversed, ZrO(2) produced lower image contrast than both BaSO(4) and Iodixanol. We conclude that evaluations of contrast media should be made with voltages and phantoms imitating the clinical application.

Ablation of bone, cartilage, and facet joint capsule using Ho:YAG laser.

OBJECTIVE: The objective of this study was to determine of the efficiency of holmium:YAG laser for bone ablation, compared to cartilage and soft tissue of the intervertebral foramen of the lumbosacral spine. BACKGROUND DATA: The holmium:YAG (Ho:YAG) laser has been used for ablation of bulging or prolapsed discs and also has the potential for decompression of the nerve root when there is narrowing of the foraminae (foraminoplasty). It is proposed that laser ablation of bone and ligament of the intervertebral foramen for nerve root decompression using the Ho:YAG laser is able to produce sufficient bone ablation without inducing significant thermal necrosis in surrounding tissues due to its short absorption length, which could result in significant clinical advantages. MATERIALS AND METHODS: Experiments were performed on samples of laminar bone, facet joint capsule, and cartilage for quantitative and qualitative determination of the effect of Ho:YAG ablation on tissue mass loss using a range of pulse energies from 0.5 to 1.5 J/P at 15 pulses/sec. RESULTS: The results showed a significant linear correlation between the mass loss and pulse energy, and between the mass loss and radiant exposure. Electron microscopy and histology showed that the Ho:YAG ablation resulted in a very sharp and clear border with little charring. Applying 0.01 k.J of total energy at two different settings (1.5 J/p, high power, and 0.5 J/p, low power) at 15 pulses/sec, the cross-sectional area/mm(2) of the ablated bone was measured, using light microscopy and the Scion Image analysis program. The ablated areas were 2.28 +/- 0.87 and 1.16 +/- 0.43 mm(2) at high and low power, respectively (p = 0.008).

The effect of irrigation on peak temperatures in nerve root, dura, and intervertebral disc during laser-assisted foraminoplasty.

BACKGROUND AND OBJECTIVE: The Holmium: YAG (Ho: YAG) laser has been used for the ablation of prolapsed discs but alternative techniques are available, and this application remains controversial. It also has potential for the decompression of nerve roots within narrowed foraminae with the technique of endoscopic laser foraminoplasty. Traditional methods of decompression necessitate a major surgical procedure with potential destabilisation of the lumbar spinal segment. Nevertheless, minimally invasive techniques are attractive only if serious complications can be avoided. This study reports the peak temperatures reached in surrounding tissues with and without saline irrigation. STUDY DESIGN/MATERIALS AND METHODS: Investigation of the hypothesis was carried out in excised sheep lumbar spines. T-type thermocouples were used for the measurement of tissue temperatures during laser ablation of nerve root foraminae. The temperature was assessed in the nerve root, dura mater, and disc space. RESULTS: The Ho: YAG laser was effective in widening the foraminae by approximately 1.5 mm with a total energy of 4.60 kJ. This was statistically significant in both vertical and horizontal directions (P < 0.0003 and P < 0.00005, respectively). The mean temperature of the nerve root, dura, and disc space during the procedure was 44 +/- 3.1 degrees C, 42.8 +/- 4.7 degrees C, and 41 +/- 3.4 degrees C respectively. There were transient high peaks seen in the temperature profiles. Using saline irrigation at 27 ml/minutes these temperatures were reduced to 34.1 +/- 1.8 degrees C (P = 0.0002), 34.9 +/- 1.5 degrees C (P = 0.002), and 37.2 +/- 1.2 degrees C (P < 0.014), for nerve roots, dura, and disc space respectively. CONCLUSIONS: Laser ablation of bone and ligament for nerve root decompression using the Ho: YAG laser may offer substantial advantages, but the risk of serious complication may only be avoided if the technique is combined with saline irrigation.

Identifying migratory Salmo trutta using carbon and nitrogen stable isotope ratios.

Many Salmo trutta populations consist of non-anadromous (freshwater-resident) brown trout and anadromous (sea-run migratory) sea trout. Although adult brown trout and sea trout can usually be identified using differences in size and body colouration, it is not possible to easily identify eggs/alevins as the progeny of brown trout or sea trout. In this study we show that delta(13)C and delta(15)N, measured using a continuous flow isotope ratio mass spectrometer (CF-IRMS), can accurately identify fish eggs as the progeny of freshwater-resident (delta(13)C(egg) = -25.7 +/- 1.9 per thousand,delta(15)N(egg) = 9.2 +/- 1.8 per thousand) or migratory (delta(13)C(egg) = -19.9 +/- 1.1 per thousand, delta(15)N(egg) = 14. 3 +/- 1.5 per thousand) adult female Salmo trutta. Case studies show that stable isotope analysis is a more reliable technique for distinguishing anadromous adult fish than differentiation using morphological characteristics. For example, stable isotope analysis of brown trout from Loch Eck, Scotland, revealed that some individuals possessed delta(13)C and delta(15)N signatures indicative of marine feeding despite visual identification as freshwater-resident fish. It is most likely that these fish are misidentified sea trout although it possible that these fish may be brown trout that have adopted an estuarine feeding strategy to avoid interspecific competition for food within Loch Eck with salmon, powan and Arctic charr. Most stable isotope studies of fish ecology use terminal tissue sampling to provide sufficient biological material for isotopic analysis; however, our study suggests that adipose fin tissue could provide a comparable measure of delta(13)C and delta(15)N. Such a strategy would be invaluable when studying the trophic ecology or migration patterns of fish of high conservation value.

Geometrical dimensions of the lower lumbar vertebrae--analysis of data from digitised CT images.

The precise dimensions of the lumbar vertebrae and discs are critical for the production of appropriate spinal implants. Unfortunately, existing databases of vertebral and intervertebral dimensions are limited either in accuracy, study population or parameters recorded. The objective of this study is to provide a large and accurate database of lumbar spinal characteristics from 126 digitised computed tomographic (CT) images, reviewed using the Picture Archiving Communication System (PACS) coupled with its internal measuring instrumentation. These CT images were obtained from patients with low back pain attending the spinal clinic at the Hammersmith Hospitals NHS Trust. Measurements of various aspects of vertebral dimensions and geometry were recorded, including vertebral and intervertebral disc height. The results from this study indicated that the depth and width of the vertebral endplate increased from the third to the fifth lumbar vertebra. Anterior vertebral height remained the same from the third to the fifth vertebra, but the posterior vertebral height decreased. Mean disc height in the lower lumbar segments was 11.6 +/- 1.8 mm for the L3/4 disc, 11.3 +/- 2.1 mm for the L4/5, and 10.7 +/- 2.1 mm for the L5/S1 level. The average circumference of the lower endplate of the fourth lumbar vertebra was 141 mm and the average surface area was 1,492 mm2. An increasing pedicle width from a mean of 9.6 +/- 2.2 mm at L3 through to 16.2 +/- 2.8 mm at L5 was noted. A comprehensive database of vertebral and intervertebral dimensions was generated from 378 lumbar vertebrae from 126 patients measured with a precise digital technique. These results are invaluable in establishing an anthropometric model of the human lumbar spine, and provide useful data for anatomical research. In addition this is important information for the scientific planning of spinal surgery and for the design of spinal implants.

Nitric oxide in fracture repair. Differential localisation, expression and activity of nitric oxide synthases.

Our aim was to investigate whether nitric oxide synthase (NOS) isoforms, responsible for the generation of NO, are expressed during the healing of fractures. To localise the sites of expression compared with those in normal bone we made standardised, stabilised, unilateral tibial fractures in male Wistar rats. Immunostaining was used to determine the precise tissue localisation of the different NOS isoforms. Western blotting was used to assess expression of NOS isoform protein and L-citrulline assays for studies on NOS activity. Control tissue was obtained from both the contralateral uninjured limb and limbs of normal rats. Immunohistochemistry showed increased expression of endothelial NOS (eNOS) to be strongest in the cortical blood vessels and in osteocytes in the early phase of fracture repair. Western blot and image analysis confirmed this initial increase. Significantly elevated calcium-dependent NOS activity was observed at day 1 after fracture. Inducible NOS (iNOS) was localised principally in endosteal osteoblasts and was also seen in chondroblasts especially in the second week of fracture healing. Western blotting showed a reduction in iNOS during the early healing period. Significantly reduced calcium-independent NOS activity was also seen. No neuronal NOS was seen in either fracture or normal tissue. Increased eNOS in bone blood vessels is likely to mediate the increased blood flow recognised during fracture healing. eNOS expression in osteocytes may occur in response to changes in either mechanical or local fluid shear stress. The finding that eNOS is increased and iNOS reduced in early healing of fractures may be important in their successful repair.

Nitric oxide mediated vasoreactivity during fracture repair.

An experimental model of fracture healing has been used to investigate whether nitric oxide mediated vascular reactivity, determined using laser Doppler flowmetry, is present in bone after a fracture. Times corresponding to Days 0, 1, 3, 7, 14, and 28 after fracture were used to study the injured and contralateral limbs in response to bolus intravenous administration of nitric oxide inhibitor, N-nitro-L-arginine methyl ester, and nitric oxide stimulator, acetylcholine. N-nitro-L-arginine methyl ester administration (1 mumol/kg, 10 mumol/kg, and 100 mumol/kg) caused a dose dependent increase in systemic blood pressure in each of the assessment groups; however, there was no statistical difference between the groups. Doppler flow readings at the fracture site showed measurable changes in local vascular reactivity after drug administration. At Day 1 after fracture, the magnitude of unit change in vascular reactivity in response to N-nitro-L-arginine methyl ester (1 mumol/kg, 10 mumol/kg, and 100 mumol/kg) was significantly higher in the fractured limb compared with the contralateral limb and also when compared with other points of assessment. These results show that nitric oxide mediated vasoreactivity is present about a fracture site and is maximal in the early healing phase, before returning to basal levels as healing progresses. This is compatible with an initial restoration of blood flow at a fracture site by nitric oxide dependent vasodilation of preexisting blood vessels, followed by ingrowth of less nitric oxide dependent angiogenic vessels during the later phase of repair.

Are subjective clinical findings and objective clinical tests related to the motion characteristics of low back pain subjects?

Many authors have demonstrated that low back pain sufferers have impaired spinal motion. However, it is not known to what degree the patient's signs and symptoms influence this impairment in motion. The aim of this study was therefore to conduct an explorative study to investigate the relationship between low back pain patients' signs and presenting symptoms and objective measurements of motion. One hundred thirty-eight patients with low back pain completed a questionnaire detailing their signs and symptoms and underwent a full clinical examination. A potentiometric motion analysis system of known accuracy and repeatability was used to measure their spinal motion during a flexion-extension test. The effects of a variety of subjective and clinical findings on the motion characteristics were examined, which revealed that certain factors, such as diagnosis, pain characteristics, symptom severity, and level of disruption, were related to the resulting motion measurements. The strength of these relationships were not as strong as anticipated (accounting for 16-45% of the variability). This suggests that routine objective assessments cannot be used as a substitute for a thorough physical and subjective examination.

Endothelium-dependent vasodilatation produced by the L-arginine/nitric oxide pathway in normal and ischemic bone.

We used an experimental model of the perfused isolated rabbit tibia to investigate the vasodilatation produced by nitric oxide in the circulation of bone. Tibiae were perfused at a constant flow rate while the perfusion pressure was monitored continuously. Perfusion pressure was raised by the addition of noradrenaline to the perfusate, and dose responses were measured for bolus doses of acetylcholine and sodium nitroprusside. N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, was then added to the perfusate at a concentration of 10(-4) M, and the dose responses to acetylcholine and sodium nitroprusside were repeated. Measurements were performed on groups of bones after 0, 6, 12, and 24 hours of normothermic ischemia (n 5, 4, 6, and 9, respectively). Both acetylcholine and sodium nitroprusside produced significant vasodilatation after 0 and 6 hours' ischemia, but no significant response was observed after 12 or 24 hours of ischemia. The vasodilatation produced by acetylcholine was significantly attenuated when L-NAME was added to the perfusate, but the vasodilatation produced by sodium nitroprusside remained unchanged. These findings confirm endothelial production of NO by stimulation of muscarinic receptors on the endothelial cells in bone and indicate that vasodilatation via the L-arginine/NO pathway remains viable for 6 hours after normothermic ischemia.

Aseptic loosening of total hip replacement. Macrophage expression of inducible nitric oxide synthase and cyclo-oxygenase-2, together with peroxynitrite formation, as a possible mechanism for early prosthesis failure.

Aseptic loosening is a major cause of failure of total hip arthroplasty. The adverse tissue response to prosthetic wear particles, with activation of cytokine and prostanoid production, contributes to bone loss around the implants. We have investigated the possibility that inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) are expressed in macrophages in the pseudomembrane at the bone-implant interface, thereby contributing to the periprosthetic bone resorption. We also assessed whether peroxynitrite, a nitric oxide (NO)-derived oxidant associated with cellular injury, is generated in the membrane. Enzymatic activity of iNOS was measured using the arginine-citrulline assay technique and prostaglandin E2 (PGE2), as an indicator of COX-2 activity, was measured using an enzyme immunoassay. Cellular immunoreactivity for iNOS, nitrotyrosine (a marker of peroxynitrite-induced cellular injury) and COX-2 was assessed by quantitative peroxidase immunocytochemistry while immunofluorescence methods were used for subsequent co-localisation studies with CD68+ macrophages. The presence of calcium-independent iNOS activity and PGE2 production was confirmed in the homogenised interface membrane. Immunocytochemistry showed that periprosthetic CD68+ wear-debris-laden macrophages were the most prominent cell type immunoreactive for iNOS, nitrotyrosine and COX-2. Other periprosthetic inflammatory and resident cell types were also found to immunolocalise nitrotyrosine thereby suggesting peroxynitrite-induced protein nitrosylation and cellular damage not only in NO-producing CD68+ macrophages, but also in their neighbouring cells. These data indicate that both iNOS and COX-2 are expressed by CD68+ macrophages in the interface membrane and peroxynitrite-induced cellular damage is evident in such tissue. If high-output NO and peroxynitrite generation were to cause macrophage cell death, this would result in the release of phagocytosed wear debris into the extracellular matrix. A detrimental cycle of events would then be established with further phagocytosis by newly-recruited inflammatory cells and subsequent NO, peroxynitrite and prostanoid synthesis. Since both NO and PGE2 have been implicated in the induction and maintenance of chronic inflammation with resulting loss of bone, and peroxynitrite in the pathogenesis of disease states, they may be central to the pathogenesis of aseptic loosening.

Sensory and sympathetic innervation of the vertebral endplate in patients with degenerative disc disease.

We obtained intervertebral discs with cartilage endplates and underlying cancellous bone at operation from patients with degenerative disc disease and then used immunohistochemical techniques to localise the nerves and nerve endings in the specimens. We used antibodies for the ubiquitous neuronal protein gene product 9.5 (PGP 9.5). Immunoreactivity to neuropeptide Y was used to identify autonomic nerves and calcitonin gene-related peptide (CGRP) and substance P to identify sensory nerves. Blood vessels were identified by immunoreactivity with platelet-endothelial cell-adhesion molecule (CD31; PECAM). In a control group with no known history of chronic back pain, nerve fibres immunoreactive to PGP 9.5 and neuropeptide Y were most closely related to blood vessels, with occasional substance P and CGRP immunoreactivity. In patients with severe back pain and markedly reduced disc height, proliferation of blood vessels and accompanying nerve fibres was observed in the endplate region and underlying vertebral bodies. Many of these nerves were immunoreactive to substance P or CGRP, and in addition, substance P- and CGRP-immunoreactive nociceptors were seen unrelated to blood vessels. Quantification by image analysis showed a marked increase in CGRP-containing sensory nerve fibres compared with normal control subjects. We speculate that a chemotactic response to products of disc breakdown is responsible for the proliferation of vascularity and CGRP-containing sensory nerves found in the endplate region and vertebral body adjacent to degenerate discs. The neuropeptides substance P and CGRP have potent vasodilatory as well as pain-transmitting effects. The increase in sensory nerve endings suggests increase in blood flow, perhaps as an attempt to augment the nutrition of the degenerate disc. The increase in the density of sensory nerves, and the presence of endplate cartilage defects, strongly suggest that the endplates and vertebral bodies are sources of pain; this may explain the severe pain on movement experienced by some patients with degenerative disc disease.

Clearance of albumin by cortical bone and marrow.

Male Sprague-Dawley rats, approximately 300 g, were anesthetized. The jugular vein and carotid artery were cannulated and the left femoral vein was exposed. The femoral vein was then ligated, and 2 minutes later albumin, labeled with 125I, was injected. The albumin was allowed to equilibrate in the vascular system for 3 minutes, and then 1 group of animals was euthanized. Further groups of animals were euthanized after 15, 30, and 60 minutes. Tibias were removed from both legs together with samples of muscle and liver. The tibias were separated into diaphyseal cortex, marrow, and proximal and distal ends. All tissue samples and terminal blood samples were counted for radioactivity, and volumes of distribution were calculated. In cortical bone the volume distribution increased from an initial value of 12.9 +/- 1.1 microL/g to 18.6 +/- 2.5 microL/g at 30 minutes in the control leg (mean +/- standard error of the mean, n = 5). In the congested leg, the volume of distribution increased from 10.6 +/- 0.6 to 19.6 +/- 2.1 microL/g during the same time interval. The difference in rate of increase between the congested and control leg was not statistically significant. Albumin seemed to equilibrate within 3 minutes within the marrow. The data are consistent with the hypothesis that macromolecular transport in the interstitial fluid of cortical bone is coupled to convectional flow from endosteal to periosteal surface. This data may help to understand mechanisms by which venous hypertension has been shown to stimulate bone growth and fracture repair, and also the distribution of bone density changes observed after prolonged exposure to microgravity.