A comparative scanning electron-microscopical study of endoneurial collagen around normal mouse nerve fibres,nerve fibres following crush injury and nerve fibres of the dystonic mouse mutant (dt/dt)

Summary The endoneurial collagen sheath around teased nerve fibres following crush injury was studied by scanning electron microscopy and compared with uninjured sciatic nerve fibres and with fibres from the dystonic mutant mouse. Following crush injury the endoneurial collagen became more abundant than seen in untreated nerve fibres and formed large, separate and longitudinally oriented bundles. However, by four weeks post injury the sheath regained a normal external appearance. Mutant nerve fibres were also associated with more than the usual amount of collagen, but the sheaths were more disorganised, with a marked disorientation and irregular aggregation of collagen, and these abnormalities were not confined to obviously degenerating or demyelinated regions of the fibres. The dystonic abnormalities of the endoneurial sheath may be important in the mechanism of the neuropathy.Medical Research Council, Radiobiology Unit, Harwell, Didcot, Oxon, OX11 ORD     

Effect of Treating Streptozotocin-Induced Diabetic Rats With Sorbinil,Myo-Inositol or Aminoguanidine on Endoneurial Blood Flow,Motor Nerve Conduction Velocity and Vascular Function of Epineurial Arterioles of the Sciatic Nerve

Previously we have demonstrated that diabetes causes impairment in vascular function of epineurial vessels, which precedes the slowing of motor nerve conduction velocity. Treatment of diabetic rats with aldose reductase inhibitors, aminoguanidine or myo-inositol supplementation have been shown to improve motor nerve conduction velocity and/or decreased endoneurial blood flow. However, the effect these treatments have on vascular reactivity of epineurial vessels of the sciatic nerve is unknown. In these studies we examined the effect of treating streptozotocininduced rats with sorbinil, aminoguanidine or myo-inositol on motor nerve conduction velocity, endoneurial blood flow and endothelium dependent vascular relaxation of arterioles that provide circulation to the region of the sciatic nerve. Treating diabetic rats with sorbinil, aminoguanidine or myo-inositol improved the reduction of endoneurial blood flow and motor nerve conduction velocity. However, only sorbinil treatment significantly improved the diabetes-induced impairment of acetylcholinemediated vasodilation of epineurial vessels of the sciatic nerve. All three treatments were efficacious in preventing the appropriate metabolic derangements associated with either activation of the polyol pathway or increased nonenzymatic glycation. In addition, sorbinil was shown to prevent the diabetes-induced decrease in lens glutathione level. However, other markers of oxidative stress were not vividly improved by these treatments. These studies suggest that sorbinil treatment may be more effective in preventing neural dysfunction in diabetes than either aminoguanidine or myoinositol.     

Scanning electron microscopy of isolated peripheral nerve fibres

Summary The surface morphology of normal myelinated nerve fibres prepared in different ways for scanning electron microscopy has been studied and compared with the surface features of similar fibres undergoing retrograde changes. Nodes of Ranvier, paranodal specializations, artefactual fractures of the myelin, and the endoneurial collagen sheaths are described. A regular pattern of elevations, usually with a pitted or depressed surface seen on normal myelinated fibres after certain preparative procedures are thought to be artefacts produced during preparation and to be related to the neurokeratin network.Alterations in the surface structure of fibres central to long-standing nerve transections include irregular protuberances, serial surface corrugations and large swellings, all associated with demyelination. Fibres that have undergone retrograde degeneration consist of endoneurial tubes with focal swellings occupied by macrophages or myelin debris, together with fine unmyelinated and small myelinated regenerating axons. Strict centrifugal progression of myelination of regenerating axons was not observed.We thank Mr. R. A. Willis for his collaboration and for taking the SEM photographs of normal nerve fibres, and the Cambridge Scientific Instrument Co. Ltd. for permission to reproduce the SEM photographs of experimental nerve fibres. We also thank Dr. A. Boyde for access to his SEM and for helpful comments on interpretation of the scanning electron micrographs, Prof. J. Z. Young, Dr. P. K. Thomas, and Dr. R. H. M. King for discussion, and Messrs. P. Reynolds and D. Gunn for photography.A grant from the Muscular Dystrophy Group of Great Britain is gratefully acknowledged.     

Effects of phrenic nerve cooling on diaphragmatic function

The effects of phrenic nerve cooling at 0 degrees C on the nerve and diaphragmatic function were evaluated in dogs. Eleven dogs, anesthetized and mechanically ventilated, were studied. Left diaphragmatic function was assessed by recording the transdiaphragmatic pressure (Pdi) generated during electrical stimulation of the left phrenic nerve at different frequencies (0.5, 30, and 100 Hz). Phrenic nerve stimulations were achieved either directly by electrodes placed around the phrenic nerve above its pericardial course or by intramuscular electrodes placed close to the phrenic nerve endings. Electrical activity of the hemidiaphragm (Edi) was recorded and phrenic nerve conduction time (PNCT) was measured during direct phrenic stimulation. A transpericardial cooling of the nerve, at 0 degrees C, on a length of 1 cm, was performed during 30 min (group A, n = 7) or 5 min (group B, n = 4). After the cooling period, phrenic and diaphragmatic functions were assessed hourly for 4 h (H1-H4). Cooling the phrenic nerve produced a complete phrenic nerve conduction block in all dogs, 100 +/- 10 s after the onset of cold exposure. Conduction recovery time was longer in group A (11 +/- 7 min) than in group B (2 +/- 0.5 min) and PNCT remained increased throughout the study in group A. Furthermore, in group A, Pdi and Edi during direct phrenic stimulation were markedly depressed from H1 to H4. No change in these parameters was noted until H3 during intramuscular stimulation, time at which a significant decrease occurred. By contrast, Pdi and Edi from direct and intramuscular stimulations remained unchanged throughout the study in group B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic unconfined compression of articular cartilage under a cyclic compressive load

To study the effect of dynamic mechanical force on cartilage metabolism, many investigators have applied a cyclic compressive load to cartilage disc explants in vitro. The most frequently used in vitro testing protocol has been the cyclic unconfined compression of articular cartilage in a bath of culture medium. Cyclic compression has been achieved by applying either a prescribed cyclic displacement or a prescribed cyclic force on a loading platen placed on the top surface of a cylindrical cartilage disc. It was found that the separation of the loading platen from the tissue surface was likely when a prescribed cyclic displacement was applied at a high frequency.The purpose of the present study was to simulate mathematically the dynamic behavior of a cylindrical cartilage disc subjected to cyclic unconfined compression under a dynamic force boundary condition protocol, and to provide a parametric analysis of mechanical deformations within the extracellular matrix. The frequency-dependent dynamic characteristics of dilatation, hydrostatic pressure and interstitial fluid velocity were analyzed over a wide range of loading frequencies without the separation of the loading platen. The result predicted that a cyclic compressive force created an oscillating positive-negative hydrostatic pressure together with a forced circulation of interstitial fluid within the tissue matrix. It was also found that the load partitioning mechanism between the solid and fluid phases was a function of loading frequency. At a relatively high loading frequency, a localized dynamic zone was developed near the peripheral free surface of the cartilage disc, where a large dynamic pressure gradient exists, causing vigorous interstitial fluid flow.     

Urokinase plasminogen receptor and the fibrinolytic complex play a role in nerve repair after nerve crush in mice, and in human neuropathies

Remodeling of extracellular matrix (ECM) is a critical step in peripheral nerve regeneration. In fact, in human neuropathies, endoneurial ECM enriched in fibrin and vitronectin associates with poor regeneration and worse clinical prognosis. Accordingly in animal models, modification of the fibrinolytic complex activity has profound effects on nerve regeneration: high fibrinolytic activity and low levels of fibrin correlate with better nerve regeneration. The urokinase plasminogen receptor (uPAR) is a major component of the fibrinolytic complex, and binding to urokinase plasminogen activator (uPA) promotes fibrinolysis and cell movement. uPAR is expressed in peripheral nerves, however, little is known on its potential function on nerve development and regeneration. Thus, we investigated uPAR null mice and observed that uPAR is dispensable for nerve development, whereas, loss of uPAR affects nerve regeneration. uPAR null mice showed reduced nerve repair after sciatic nerve crush. This was a consequence of reduced fibrinolytic activity and increased deposition of endoneurial fibrin and vitronectin. Exogenous fibrinolysis in uPAR null mice rescued nerve repair after sciatic nerve crush. Finally, we measured the fibrinolytic activity in sural nerve biopsies from patients with peripheral neuropathies. We showed that neuropathies with defective regeneration had reduced fibrinolytic activity. On the contrary, neuropathies with signs of active regeneration displayed higher fibrinolytic activity. Overall, our results suggest that enforced fibrinolysis may facilitate regeneration and outcome of peripheral neuropathies.     

Dietary hardness, loading behavior, and the evolution of skull form in bats

The morphology and biomechanics of the vertebrate skull reflect the physical properties of diet and behaviors used in food acquisition and processing. We use phyllostomid bats, the most diverse mammalian dietary radiation, to investigate if and how changes in dietary hardness and loading behaviors during feeding shaped the evolution of skull morphology and biomechanics. When selective regimes of food hardness are modeled, we found that species consuming harder foods have evolved skull shapes that allow for more efficient bite force production. These species have shorter skulls and a greater reliance on the temporalis muscle, both of which contribute to a higher mechanical advantage at an intermediate gape angle. The evolution of cranial morphology and biomechanics also appears to be related to loading behaviors. Evolutionary changes in skull shape and the relative role of the temporalis and masseter in generating bite force are correlated with changes in the use of torsional and bending loading behaviors. Functional equivalence appears to have evolved independently among three lineages of species that feed on liquids and are not obviously morphologically similar. These trends in cranial morphology and biomechanics provide insights into behavioral and ecological factors shaping the skull of a trophically diverse clade of mammals.     

Mechanical functioning of peripheral nerves: linkage with the “mushrooming” effect

Biomechanical properties of nerve have been studied extensively. All neural matrix tissues have been suggested to be the main load-bearing component. Based on the ultrastructure it has been proposed that the architecture of the epineurium allows some degree of extensibility of the nerve. A role of the perineurium could be to withstand the positive endoneurial pressure. The hypothesis is that the mechanical behaviour of nerves is dependent on an interaction between the core swelling pressure and restraint by the outer sheath. Loss of this balance will alter that behaviour. To test this, rat sciatic nerves were subjected to mechanical loading at in vivo and ex vivo tension. Retraction of nerve segments was measured after excision and after incubation at 37°C or freezing. Swelling properties of the nerve were measured by immersion in water or PBS (phosphate buffer solution) with intact or opened epineurium. Results showed a significant decrease in strength and stiffness with an increase in strain of the nerve after excision, compared to in vivo. Retraction was on average 11%. Freezing or incubation at 37°C did not alter retraction. The swelling properties of the nerve demonstrated a significant difference between intact and opened epineurium and similar results for water and PBS, indicating that epineurium is a constraint and that the nerves are underhydrated. The proposed model for the intact nerve is a continuous connective tissue tube surrounding and constraining an inner swelling pressure of the neural core. Loss of integrity of the nerve has detrimental effects on its biomechanical properties.Preliminary forms of this project were presented at the 1st UK Tissue & Cell Engineering Society meeting, Hammersmith Hospital, RPMS, London, 5 July 1999, and at the 7th FESSH, Barcelona, Spain, 22 June 2000. Support was partly given by EU grant QLK3-CT-1999-00625: NEURO-TEC     

Cardiomyocytes from late embryos and neonates do optimal work and striate best on substrates with tissue-level elasticity: metrics and mathematics

In this review, we discuss recent studies on the mechanosensitive morphology and function of cardiomyocytes derived from embryos and neonates. For early cardiomyocytes cultured on substrates of various stiffnesses, contractile function as measured by force production, work output and calcium handling is optimized when the culture substrate stiffness mimics that of the tissue from which the cells were obtained. This optimal contractile function corresponds to changes in sarcomeric protein conformation and organization that promote contractile ability. In light of current models for myofibillogenesis, a recent mathematical model of striation and alignment on elastic substrates helps to illuminate how substrate stiffness modulates early myofibril formation and organization. During embryonic heart formation and maturation, cardiac tissue mechanics change dynamically. Experiments and models highlighted here have important implications for understanding cardiomyocyte differentiation and function in development and perhaps in regeneration processes.     

Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.     

Sural nerve oxygen tension in diabetes.

Peripheral nerve oxygen tensions were assessed in vivo by using microelectrodes to measure endoneurial oxygen tension in exposed sural nerve. In 11 diabetic patients with chronic sensorimotor neuropathy the mean endoneurial oxygen tension was 39.7 (SD 10.2) mm Hg. In all but one patient compared with none of four non-neuropathic subjects the mean nerve oxygen tensions were below dorsal foot vein values. This unphysiological state may have a role in the aetiology of diabetic neuropathy.     

Effect of various nerve decompression procedures on the functions of distal limbs in streptozotocin-induced diabetic rats: further optimism in diabetic neuropathy

It is known that diabetic neuropathy is the result of endoneurial edema caused by various biochemical reactions triggered by hyperglycemia. This sequence of events can cause cessation of circulation at the perineurial level, or the tough layer, which is not resilient enough to spread intraneural pressure. Internal and external limiting structures create a double crush phenomenon to the nerve structure. Decompression of the nerve trunk at separate levels is one of the adjuncts to the overall treatment plan for diabetic neuropathy. In this study, the right sciatic nerves of 30 rats with streptozotocin-induced diabetes were used; three groups were created. In the control group, the sciatic nerves were explored and dissected only. In group II, tarsal tunnel release was performed and accompanied by epineurotomy of the sciatic nerve and its peroneal and tibial extensions. In group III, in addition to the procedures performed in group II, perineural sheaths, exposed through the epineurotomy sites at both the peroneal and tibial nerves, were incised for decompression of the fascicles. Improvement in diabetic neuropathy was evaluated by using footprint parameters. The last print length values, estimated according to the 38-month measurements, were 26.1 +/- 0.12 mm in the control group, 23.2 +/- 0.07 mm in group II, and 22.2 +/- 0.1 mm in group III. The toe spread and intermediate toe spread values of the groups were parallel to improvements in print lengths throughout the study. The best improvement was observed in the perineurotomy group. Finally, an electron microscopic study revealed variable degenerative changes in all groups, but they were milder in groups II and III. This experimental study reveals that adding internal decompression to external release doubled the effect in reducing derangement in the sciatic nerves of the rats and, in the authors' opinion, offers cause for further optimism in the treatment of diabetic neuropathy.     

Muscle cells in a nerve trunk of a frog muscle

Summary Three muscle fibers were identified by electron microscopy within a nerve of a frog muscle. They resembled extrafusal muscle fibers but were located in an endoneurial rather than in an endomysial compartment. To call these endoneurial muscle fibers the obvious continuation of extrafusal fibers of a muscle spindle is certainly unwarranted; to label these fibers ectopic and to let the matter rest there is probably an understatement of sorts.     

Regulation of arterial pressure: role of pressure natriuresis and diuresis

The importance of the renal pressure natriuresis and diuresis mechanisms in long-term control of body fluid volumes and arterial pressure has been controversial and difficult to quantitate experimentally. Recent studies, however, have demonstrated that in several forms of chronic hypertension caused by aldosterone, angiotensin II (AngII), vasopressin, or norepinephrine and adrenocorticotropin, increased renal arterial pressure is essential for maintaining normal excretion of sodium and water in the face of reduced renal excretory capability. When renal arterial pressure was servo-controlled in these models of hypertension, sodium and water retention continued unabated, causing ascites, pulmonary edema, or even complete circulatory collapse within a few days. Apparently, other mechanisms for volume homeostasis, such as the various natriuretic and diuretic factors that have been postulated, are not sufficiently powerful to maintain fluid balance in the absence of increased renal arterial pressure when renal excretory function is reduced in these forms of hypertension. The intrarenal mechanisms responsible for pressure natriuresis and diuresis are not entirely clear, but they seem to involve small increases in glomerular filtration rate and filtered load as well as reductions in fractional reabsorption in proximal and distal tubules. During chronic disturbances of arterial pressure additional factors, especially changes in AngII and aldosterone formation, act to amplify the effectiveness of the basic renal pressure natriuresis and diuresis mechanisms in regulating arterial pressure and body fluid volumes.     

Effects of anterior offsetting of humeral head component in posteriorly unstable total shoulder arthroplasty: Finite element modeling of cadaver specimens

A novel technique of “anterior offsetting” of the humeral head component to address posterior instability in total shoulder arthroplasty has been proposed, and its biomechanical benefits have been previously demonstrated experimentally. The present study sought to characterize the changes in joint mechanics associated with anterior offsetting with various amounts of glenoid retroversion using cadaver specimen-specific 3-dimensional finite element models. Specimen-specific computational finite element models were developed through importing digitized locations of six musculotendinous units of the rotator cuff and deltoid muscles based off three cadaveric shoulder specimens implanted with total shoulder arthroplasty in either anatomic or anterior humeral head offset. Additional glenoid retroversion angles (0°, 10°, 20°, and 30°) other than each specimen׳s actual retroversion were modeled. Contact area, contact force, peak pressure, center of pressure, and humeral head displacement were calculated at each offset and retroversion for statistical analysis. Anterior offsetting was associated with significant anterior shift of center of pressure and humeral head displacement upon muscle loading (p<0.05). Although statistically insignificant, anterior offsetting was associated with increased contact area and decreased peak pressure (p > 0.05). All study variables showed significant differences when compared between the 4 different glenoid retroversion angles (p < 0.05) except for total force (p < 0.05). The study finding suggests that the anterior offsetting technique may contribute to joint stability in posteriorly unstable shoulder arthroplasty and may reduce eccentric loading on glenoid components although the long term clinical results are yet to be investigated in future.     

Multi-factorial analysis of variables influencing the bone loss of an implant placed in the maxilla: Prediction using FEA and SED bone remodeling algorithm

The aim of this study was to investigate the interactions of implant position, implant–abutment connection and loading condition influencing bone loss of an implant placed in the maxilla using finite element (FE) analysis and mathematical bone remodeling theory. The maxilla section contours were acquired using CT images to construct FE models containing RS (internal retaining-screw) and the TIS (taper integrated screwed-in) implants placed in SC (along the axis of occlusal force) and RA (along the axis of residual ridge) positions. The adaptive strain energy density (SED) algorithm was combined with FE approach to study the preliminary bone remodeling around implant systems under different load conditions. The simulated results showed that the implant position obviously influenced the bone loss. An implant placed in the RA position resulted in substantially increased bone loss. Implant receiving a lateral load slightly increased bone loss compared with an axial load. The implant type did not significantly influence bone loss. It was found that buccal site suffered the most bone loss around the implant, followed by distal, lingual and mesial sites. The implant position primarily influenced bone loss and it was found most obviously at the buccal site. Implant placed along the axial load direction of a proposed prosthesis could obtain less bone loss around the implant. Attaining proper occlusal adjustments to reduce the lateral occlusal force is recommended in implant–bone–prosthesis system. Abutments of internal engagement with or without taper-fit did not affect the bone loss in the surrounding bone.     

Adipose-derived stromal cell transplantation for treatment of stress urinary incontinence

We aimed to investigate the application of adipose-derived stromal cells in the treatment of stress urinary incontinence (SUI). Animal models of stress urinary incontinence were established with Sprague-Dawley female rats by complete cutting of the pudendal nerve. Rat adipose-derived stromal cells were isolated, cultured and successfully transplanted into animal models. Effects of stem cell transplantation were evaluated through urodynamic testing and morphologic changes of the urethra and surrounding tissues before and after transplantation. Main urodynamic outcome measures were measured. Intra-bladder pressure and leak point pressure were measured during filling phase. Morphologic examinations were performed. Transplantation of adipose-derived stem cells significantly strengthened local urethral muscle layers and significantly improved the morphology and function of sphincters. Urodynamic testing showed significant improvements in maximum bladder capacity, abdominal leak point pressure, maximum urethral closure pressure, and functional urethral length. Morphologic changes and significant improvement in urination control were consistent over time. It was concluded that periurethral injection of adipose-derived stromal cells improves function of the striated urethral sphincter, resulting in therapeutic effects on SUI. Reconstruction of the pelvic floor through transplantation of adipose-derived cells is a minimally invasive and effective treatment for SUI.     

Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells

Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Cre-recombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation.     

RABBIT SCIATIC NERVE FASCICLE AND''ENDONEURIAL''PREPARATIONS FOR IN VITRO STUDIES OF PERIPHERAL NERVE GLUCOSE METABOLISM

Abstract— Suitable preparations for in vitro studies of the composite glucose and energy metabolism of peripheral nerve axons and Schwann cells have not been available. Methods are described for the preparation and incubation of a defined segment of a rabbit sciatic nerve fascicle, free of epineurial contamination, but with an intact perineurial membrane; removing the perineurium provides in addition an‘endoneurial’preparation. Conditions were selected for incubating each preparation with glucose that maintained stable P-creatine and ATP concentrations and a stable rate of O₂ uptake; under these conditions the preparations retained an unaltered EM appearance during a 2-h incubation. Glucose diffusion into the endoneurial compartment of the fascicle is restricted, possibly by the perineurial membrane, and a higher medium glucose concentration (20 mM) was required to maintain a steady state of energy metabolism in this preparation than in the‘endoneurial’preparation, which was incubated with 5 mwglucose. The‘endoneurial’preparation required 0.50 mm -myoinositol in the medium to prevent a decrease in tissue free myoinositol and a slow decrease in O₂ uptake, which occurred when it was omitted. Under the incubation conditions selected the glucose concentrations in the‘endoneurial’preparation and in the endoneurial compartment of the fascicle were reasonably similar, and the preparations had similar rates of respiration, similar estimated rates of glucose utilization, and similar relative rates of respiration and lactate production. The preparations derive the major fraction of their energy requirements from respiration. Their rates of O₂ uptake are 60% higher than the previous indirect estimate of O₂ uptake in whole rabbit tibial nerve in situ. Constant rates of incorporation of ¹⁴C from [U-¹⁴C]glucose into CO₂ and total lipid were observed in the‘endoneurial’preparation after a 15-min equilibration period. The preparations reported provide suitable tools for in vitro studies of peripheral nerve metabolism not previously available.